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Prescott JB, Liu KJ, Lander A, Pek NMQ, Jha SK, Bokelmann M, Begur M, Koh PW, Yang H, Lim B, Red-Horse K, Weissman IL, Loh KM, Ang LT. Metabolically purified human stem cell-derived hepatocytes reveal distinct effects of Ebola and Lassa viruses. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.02.17.638665. [PMID: 40027809 PMCID: PMC11870522 DOI: 10.1101/2025.02.17.638665] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/05/2025]
Abstract
Ebola and Lassa viruses require biosafety-level-4 (BSL4) containment, infect the liver, and cause deadly hemorrhagic fevers. The cellular effects of these viruses, and whether different families of hemorrhagic-fever viruses elicit similar effects, remain fundamental questions in BSL4 virology. Here, we introduce a new metabolic selection approach to create nearly-pure hepatocytes from human pluripotent stem cells, killing non-liver cells by withholding essential nutrients. Unexpectedly, Ebola and Lassa exerted starkly different effects on human hepatocytes. Ebola infection activated the integrated stress response (ISR) and WNT pathways in hepatocytes in vitro and killed them, whereas Lassa did not. Within non-human primates, Ebola likewise infected hepatocytes and activated ISR signaling in vivo . In summary, we present a single-cell transcriptional and chromatin accessibility roadmap of human hepatocyte differentiation, purification, and viral infection.
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Gilglioni EH, Bansal M, St-Pierre-Wijckmans W, Talamantes S, Kasarinaite A, Hay DC, Gurzov EN. Therapeutic potential of stem cell-derived somatic cells to treat metabolic dysfunction-associated steatotic liver disease and diabetes. Obes Rev 2025:e13899. [PMID: 39861937 DOI: 10.1111/obr.13899] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/25/2024] [Revised: 10/22/2024] [Accepted: 12/04/2024] [Indexed: 01/27/2025]
Abstract
Developments in basic stem cell biology have paved the way for technology translation in human medicine. An exciting prospective use of stem cells is the ex vivo generation of hepatic and pancreatic endocrine cells for biomedical applications. This includes creating novel models 'in a dish' and developing therapeutic strategies for complex diseases, such as metabolic dysfunction-associated steatotic liver disease (MASLD) and diabetes. In this review, we explore recent advances in the generation of stem cell-derived hepatocyte-like cells and insulin-producing β-like cells. We cover the different differentiation strategies, new discoveries, and the caveats that still exist regarding their routine use. Finally, we discuss the challenges and limitations of stem cell-derived therapies as a clinical strategy to manage metabolic diseases in humans.
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Affiliation(s)
- Eduardo H Gilglioni
- Signal Transduction and Metabolism Laboratory, Université libre de Bruxelles, Brussels, Belgium
| | - Mayank Bansal
- Signal Transduction and Metabolism Laboratory, Université libre de Bruxelles, Brussels, Belgium
| | | | - Stephanie Talamantes
- Signal Transduction and Metabolism Laboratory, Université libre de Bruxelles, Brussels, Belgium
| | - Alvile Kasarinaite
- Institute for Regeneration and Repair, Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK
| | - David C Hay
- Institute for Regeneration and Repair, Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK
| | - Esteban N Gurzov
- Signal Transduction and Metabolism Laboratory, Université libre de Bruxelles, Brussels, Belgium
- WELBIO Department, WEL Research Institute, Wavre, Belgium
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3
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Zhang L, Deng Y, Bai X, Wei X, Ren Y, Chen S, Deng H. Cell therapy for end-stage liver disease: Current state and clinical challenge. Chin Med J (Engl) 2024; 137:2808-2820. [PMID: 39602326 DOI: 10.1097/cm9.0000000000003332] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2024] [Indexed: 11/29/2024] Open
Abstract
ABSTRACT Liver disease involves a complex interplay of pathological processes, including inflammation, hepatocyte necrosis, and fibrosis. End-stage liver disease (ESLD), such as liver failure and decompensated cirrhosis, has a high mortality rate, and liver transplantation is the only effective treatment. However, to overcome problems such as the shortage of donor livers and complications related to immunosuppression, there is an urgent need for new treatment strategies that need to be developed for patients with ESLD. For instance, hepatocytes derived from donor livers or stem cells can be engrafted and multiplied in the liver, substituting the host hepatocytes and rebuilding the liver parenchyma. Stem cell therapy, especially mesenchymal stem cell therapy, has been widely proved to restore liver function and alleviate liver injury in patients with severe liver disease, which has contributed to the clinical application of cell therapy. In this review, we discussed the types of cells used to treat ESLD and their therapeutic mechanisms. We also summarized the progress of clinical trials around the world and provided a perspective on cell therapy.
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Affiliation(s)
- Lin Zhang
- Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China
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Nijiati N, Wubuli D, Li X, Zhou Z, Julaiti M, Huang P, Hu B. The Construction of Stem Cell-Induced Hepatocyte Model and Its Application in Evaluation of Developmental Hepatotoxicity of Environmental Pollutants. Stem Cells Dev 2024; 33:575-585. [PMID: 39109950 DOI: 10.1089/scd.2024.0117] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/24/2024] Open
Abstract
Stem cells, with their ability to self-renew and differentiate into various cell types, are a unique and valuable resource for medical research and toxicological studies. The liver is the most crucial metabolic organ in the human body and serves as the primary site for the accumulation of environmental pollutants. Enrichment with environmental pollutants can disrupt the early developmental processes of the liver and have a significant impact on liver function. The liver comprises a complex array of cell types, and different environmental pollutants have varying effects on these cells. Currently, there is a lack of well-established research models that can effectively demonstrate the mechanisms by which environmental pollutants affect human liver development. The emergence of liver cells and organoids derived from stem cells offers a promising tool for investigating the impact of environmental pollutants on human health. Therefore, this study systematically reviewed the developmental processes of different types of liver cells and provided an overview of studies on the developmental toxicity of various environmental pollutants using stem cell models.
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Affiliation(s)
- Nadire Nijiati
- State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Xinjiang Key Laboratory of Molecular Biology for Endemic Diseases, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi, China
| | - Dilixiati Wubuli
- Department of Physiology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi, China
| | - Xiaobing Li
- The Third Clinical Medicine College of Xinjiang Medical University, Urumqi, China
| | - Zidong Zhou
- The Third Clinical Medicine College of Xinjiang Medical University, Urumqi, China
| | - Mulati Julaiti
- State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Xinjiang Key Laboratory of Molecular Biology for Endemic Diseases, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi, China
| | - Pengfei Huang
- State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Xinjiang Key Laboratory of Molecular Biology for Endemic Diseases, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi, China
| | - Bowen Hu
- State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Xinjiang Key Laboratory of Molecular Biology for Endemic Diseases, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi, China
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5
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Intoh A, Watanabe-Susaki K, Kato T, Kiritani H, Kurisaki A. EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells. Stem Cells Transl Med 2024; 13:763-775. [PMID: 38811016 PMCID: PMC11328934 DOI: 10.1093/stcltm/szae036] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2023] [Accepted: 04/14/2024] [Indexed: 05/31/2024] Open
Abstract
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) possess the intrinsic ability to differentiate into diverse cellular lineages, marking them as potent instruments in regenerative medicine. Nonetheless, the proclivity of these stem cells to generate teratomas post-transplantation presents a formidable obstacle to their therapeutic utility. In previous studies, we identified an array of cell surface proteins specifically expressed in the pluripotent state, as revealed through proteomic analysis. Here we focused on EPHA2, a protein found to be abundantly present on the surface of undifferentiated mouse ESCs and is diminished upon differentiation. Knock-down of Epha2 led to the spontaneous differentiation of mouse ESCs, underscoring a pivotal role of EPHA2 in maintaining an undifferentiated cell state. Further investigations revealed a strong correlation between EPHA2 and OCT4 expression during the differentiation of both mouse and human PSCs. Notably, removing EPHA2+ cells from mouse ESC-derived hepatic lineage reduced tumor formation after transplanting them into immune-deficient mice. Similarly, in human iPSCs, a larger proportion of EPHA2+ cells correlated with higher OCT4 expression, reflecting the pattern observed in mouse ESCs. Conclusively, EPHA2 emerges as a potential marker for selecting undifferentiated stem cells, providing a valuable method to decrease tumorigenesis risks after stem-cell transplantation in regenerative treatments.
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Affiliation(s)
- Atsushi Intoh
- Division of Biological Science, Nara Institute of Science and Technology, Nara, 630-0192, Japan
- Organ Development Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, 305-8560, Japan
| | - Kanako Watanabe-Susaki
- Organ Development Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, 305-8560, Japan
| | - Taku Kato
- Division of Biological Science, Nara Institute of Science and Technology, Nara, 630-0192, Japan
| | - Hibiki Kiritani
- Division of Biological Science, Nara Institute of Science and Technology, Nara, 630-0192, Japan
| | - Akira Kurisaki
- Division of Biological Science, Nara Institute of Science and Technology, Nara, 630-0192, Japan
- Organ Development Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, 305-8560, Japan
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Son B, Park S, Cho S, Kim JA, Baek SH, Yoo KH, Han D, Joo J, Park HH, Park TH. Improved Neural Inductivity of Size-Controlled 3D Human Embryonic Stem Cells Using Magnetic Nanoparticles. Biomater Res 2024; 28:0011. [PMID: 38500782 PMCID: PMC10944702 DOI: 10.34133/bmr.0011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2023] [Accepted: 02/12/2024] [Indexed: 03/20/2024] Open
Abstract
Background: To improve the efficiency of neural development from human embryonic stem cells, human embryoid body (hEB) generation is vital through 3-dimensional formation. However, conventional approaches still have limitations: long-term cultivation and laborious steps for lineage determination. Methods: In this study, we controlled the size of hEBs for ectodermal lineage specification using cell-penetrating magnetic nanoparticles (MNPs), which resulted in reduced time required for initial neural induction. The magnetized cells were applied to concentrated magnetic force for magnet-derived multicellular organization. The uniformly sized hEBs were differentiated in neural induction medium (NIM) and suspended condition. This neurally induced MNP-hEBs were compared with other groups. Results: As a result, the uniformly sized MNP-hEBs in NIM showed significantly improved neural inductivity through morphological analysis and expression of neural markers. Signaling pathways of the accelerated neural induction were detected via expression of representative proteins; Wnt signaling, dopaminergic neuronal pathway, intercellular communications, and mechanotransduction. Consequently, we could shorten the time necessary for early neurogenesis, thereby enhancing the neural induction efficiency. Conclusion: Overall, this study suggests not only the importance of size regulation of hEBs at initial differentiation stage but also the efficacy of MNP-based neural induction method and stimulations for enhanced neural tissue regeneration.
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Affiliation(s)
- Boram Son
- School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
- Department of Bioengineering, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea
| | - Sora Park
- School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
| | - Sungwoo Cho
- School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
| | - Jeong Ah Kim
- Center for Scientific Instrumentation, Korea Basic Science Institute, Cheongju, Chungbuk 28119, Republic of Korea
| | - Seung-Ho Baek
- Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), Ulsan 44429, Korea
| | - Ki Hyun Yoo
- SIMPLE Planet Inc., 48 Achasan-ro 17-gil, Seongdong-gu, Seoul 04799, Korea
| | - Dongoh Han
- SIMPLE Planet Inc., 48 Achasan-ro 17-gil, Seongdong-gu, Seoul 04799, Korea
| | - Jinmyoung Joo
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea
| | - Hee Ho Park
- Department of Bioengineering, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul 04763, Republic of Korea
- Research Institute for Convergence of Basic Science, Hanyang University, Seoul 04763, Republic of Korea
| | - Tai Hyun Park
- School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
- Department of Nutritional Science and Food Management, Ewha Womans University, Seodaemun-gu, Seoul 03760, Republic of Korea
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7
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Baert L, Rudy S, Pellisson M, Doll T, Rocchetti R, Kaiser M, Mäser P, Müller M. Induced pluripotent stem cell-derived human macrophages as an infection model for Leishmania donovani. PLoS Negl Trop Dis 2024; 18:e0011559. [PMID: 38166146 PMCID: PMC10786377 DOI: 10.1371/journal.pntd.0011559] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2023] [Revised: 01/12/2024] [Accepted: 12/19/2023] [Indexed: 01/04/2024] Open
Abstract
The parasite Leishmania donovani is one of the species causing visceral leishmaniasis in humans, a deadly infection claiming up to 40,000 lives each year. The current drugs for leishmaniasis treatment have severe drawbacks and there is an urgent need to find new anti-leishmanial compounds. However, the search for drug candidates is complicated by the intracellular lifestyle of Leishmania. Here, we investigate the use of human induced pluripotent stem cell (iPS)-derived macrophages (iMACs) as host cells for L. donovani. iMACs obtained through embryoid body differentiation were infected with L. donovani promastigotes, and high-content imaging techniques were used to optimize the iMACs seeding density and multiplicity of infection, allowing us to reach infection rates up to 70% five days after infection. IC50 values obtained for miltefosine and amphotericin B using the infected iMACs or mouse peritoneal macrophages as host cells were comparable and in agreement with the literature, showing the potential of iMACs as an infection model for drug screening.
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Affiliation(s)
- Lore Baert
- Swiss Tropical and Public Health Institute (SwissTPH), Basel, Switzerland
- University of Basel, Basel, Switzerland
| | - Serena Rudy
- Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland
| | - Mélanie Pellisson
- Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland
| | - Thierry Doll
- Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland
| | - Romina Rocchetti
- Swiss Tropical and Public Health Institute (SwissTPH), Basel, Switzerland
- University of Basel, Basel, Switzerland
| | - Marcel Kaiser
- Swiss Tropical and Public Health Institute (SwissTPH), Basel, Switzerland
- University of Basel, Basel, Switzerland
| | - Pascal Mäser
- Swiss Tropical and Public Health Institute (SwissTPH), Basel, Switzerland
- University of Basel, Basel, Switzerland
| | - Matthias Müller
- Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland
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8
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da Silva Nunes Barreto R, da Silva Júnior LN, Henrique Doná Rodrigues Almeida G, de Oliveira Horvath-Pereira B, da Silva TS, Garcia JM, Smith LC, Carreira ACO, Miglino MA. Placental scaffolds as a potential biological platform for embryonic stem cells differentiation into hepatic-like cells lineage: A pilot study. Tissue Cell 2023; 84:102181. [PMID: 37515966 DOI: 10.1016/j.tice.2023.102181] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2023] [Revised: 07/20/2023] [Accepted: 07/21/2023] [Indexed: 07/31/2023]
Abstract
Hepatic microenvironment plays an essential role in liver regeneration, providing the necessary conditions for cell proliferation, differentiation and tissue rearrangement. One of the key factors for hepatic tissue reconstruction is the extracellular matrix (ECM), which through collagenous and non-collagenous proteins provide a three-dimensional structure that confers support for cell adhesion and assists on their survival and maintenance. In this scenario, placental ECM may be eligible for hepatic tissue reconstruction, once these scaffolds hold the major components required for cell support. Therefore, this preliminary study aimed to access the possibility of mouse embryonic stem cells differentiation into hepatocyte-like cells on placental scaffolds in a three-dimensional dynamic system using a Rotary Cell Culture System. Following a four-phase differentiation protocol that simulates liver embryonic development events, the preliminary results showed that a significant quantity of cells adhered and interacted with the scaffold through outer and inner surfaces. Positive immunolabelling for alpha fetus protein and CK7 suggest presence of hepatoblast phenotype cells, and CK18 and Albumin positive immunolabelling suggest the presence of hepatocyte-like phenotype cells, demonstrating the presence of a heterogeneous population into the recellularized scaffolds. Periodic Acid Schiff-Diastase staining confirmed the presence of glycogen storage, indicating that differentiate cells acquired a hepatic-like phenotype. In conclusion, these preliminary results suggested that mouse placental scaffolds might be used as a biological platform for stem cells differentiation into hepatic-like cells and their establishment, which may be a promissing biomaterial for hepatic tissue reconstruction.
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Affiliation(s)
| | | | | | | | - Thamires Santos da Silva
- Department of Surgery, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, SP, Brazil
| | - Joaquim Mansano Garcia
- Department of Preventive Veterinary Medicine and Animal Reproduction, Faculty of Agricultural and Veterinary Sciences, State University of São Paulo, Jaboticabal, SP, Brazil
| | - Lawrence Charles Smith
- Centre de Recherche en Reproduction et Fertilité, University of Montreal, Montreal, QC, Canada
| | - Ana Claudia Oliveira Carreira
- Department of Surgery, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, SP, Brazil; Centre of Human and Natural Sciences, Federal University of ABC, Santo André, SP, Brazil
| | - Maria Angelica Miglino
- Department of Surgery, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, SP, Brazil.
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Song Y, Lu Z, Shu W, Xiang Z, Wang Z, Wei X, Xu X. Arouse potential stemness: Intrinsic and acquired stem cell therapeutic strategies for advanced liver diseases. CELL INSIGHT 2023; 2:100115. [PMID: 37719773 PMCID: PMC10502372 DOI: 10.1016/j.cellin.2023.100115] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/08/2023] [Revised: 08/10/2023] [Accepted: 08/10/2023] [Indexed: 09/19/2023]
Abstract
Liver diseases are a major health issue, and prolonged liver injury always progresses. Advanced liver disorders impair liver regeneration. Millions of patients die yearly worldwide, even with the available treatments of liver transplantation and artificial liver support system. With its abundant cell resources and significant differentiative potential, stem cell therapy is a viable treatment for various disorders and offers hope to patients waiting for orthotopic liver transplantation. Considering such plight, stem cell therapeutic strategies deliver hope to the patients. Moreover, we conclude intrinsic and acquired perspectives based on stem cell sources. The properties and therapeutic uses of these stem cells' specific types or sources were then reviewed. Owing to the recent investigations of the above cells, a safe and effective therapy will emerge for advanced liver diseases soon.
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Affiliation(s)
- Yisu Song
- Department of Hepatobiliary and Pancreatic Surgery Affiliated Hangzhou First People’s Hospital Zhejiang University School of Medicine Hangzhou, Zhejiang, 310006, China
- Zhejiang University School of Medicine, Hangzhou, China
- Key Laboratory of Integrated Oncology and Intelligent Medicine of Zhejiang Province, Hangzhou, 310006, China
| | - Zhengyang Lu
- Department of Hepatobiliary and Pancreatic Surgery Affiliated Hangzhou First People’s Hospital Zhejiang University School of Medicine Hangzhou, Zhejiang, 310006, China
- Key Laboratory of Integrated Oncology and Intelligent Medicine of Zhejiang Province, Hangzhou, 310006, China
- Zhejiang Chinese Medical University, Hangzhou, 310053, PR China
| | - Wenzhi Shu
- Department of Hepatobiliary and Pancreatic Surgery Affiliated Hangzhou First People’s Hospital Zhejiang University School of Medicine Hangzhou, Zhejiang, 310006, China
- Key Laboratory of Integrated Oncology and Intelligent Medicine of Zhejiang Province, Hangzhou, 310006, China
| | - Ze Xiang
- Zhejiang University School of Medicine, Hangzhou, China
| | - Zhengxin Wang
- Department of General Surgery, Huashan Hospital, Fudan University Shanghai, 200040, China
| | - Xuyong Wei
- Department of Hepatobiliary and Pancreatic Surgery Affiliated Hangzhou First People’s Hospital Zhejiang University School of Medicine Hangzhou, Zhejiang, 310006, China
- Key Laboratory of Integrated Oncology and Intelligent Medicine of Zhejiang Province, Hangzhou, 310006, China
| | - Xiao Xu
- Zhejiang University School of Medicine, Hangzhou, China
- Key Laboratory of Integrated Oncology and Intelligent Medicine of Zhejiang Province, Hangzhou, 310006, China
- Institute of Organ Transplantation, Zhejiang University, Hangzhou, 310003, China
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10
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Fattahi P, de Hoyos-Vega JM, Choi JH, Duffy CD, Gonzalez-Suarez AM, Ishida Y, Nguyen KM, Gwon K, Peterson QP, Saito T, Stybayeva G, Revzin A. Guiding Hepatic Differentiation of Pluripotent Stem Cells Using 3D Microfluidic Co-Cultures with Human Hepatocytes. Cells 2023; 12:1982. [PMID: 37566061 PMCID: PMC10417547 DOI: 10.3390/cells12151982] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2023] [Revised: 07/21/2023] [Accepted: 07/26/2023] [Indexed: 08/12/2023] Open
Abstract
Human pluripotent stem cells (hPSCs) are capable of unlimited proliferation and can undergo differentiation to give rise to cells and tissues of the three primary germ layers. While directing lineage selection of hPSCs has been an active area of research, improving the efficiency of differentiation remains an important objective. In this study, we describe a two-compartment microfluidic device for co-cultivation of adult human hepatocytes and stem cells. Both cell types were cultured in a 3D or spheroid format. Adult hepatocytes remained highly functional in the microfluidic device over the course of 4 weeks and served as a source of instructive paracrine cues to drive hepatic differentiation of stem cells cultured in the neighboring compartment. The differentiation of stem cells was more pronounced in microfluidic co-cultures compared to a standard hepatic differentiation protocol. In addition to improving stem cell differentiation outcomes, the microfluidic co-culture system described here may be used for parsing signals and mechanisms controlling hepatic cell fate.
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Affiliation(s)
- Pouria Fattahi
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA; (P.F.); (J.M.d.H.-V.); (J.H.C.); (C.D.D.); (A.M.G.-S.); (K.M.N.); (K.G.); (Q.P.P.); (G.S.)
- Department of Biomedical Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Jose M. de Hoyos-Vega
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA; (P.F.); (J.M.d.H.-V.); (J.H.C.); (C.D.D.); (A.M.G.-S.); (K.M.N.); (K.G.); (Q.P.P.); (G.S.)
| | - Jong Hoon Choi
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA; (P.F.); (J.M.d.H.-V.); (J.H.C.); (C.D.D.); (A.M.G.-S.); (K.M.N.); (K.G.); (Q.P.P.); (G.S.)
| | - Caden D. Duffy
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA; (P.F.); (J.M.d.H.-V.); (J.H.C.); (C.D.D.); (A.M.G.-S.); (K.M.N.); (K.G.); (Q.P.P.); (G.S.)
| | - Alan M. Gonzalez-Suarez
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA; (P.F.); (J.M.d.H.-V.); (J.H.C.); (C.D.D.); (A.M.G.-S.); (K.M.N.); (K.G.); (Q.P.P.); (G.S.)
| | - Yuji Ishida
- Department of Medicine, Division of Gastrointestinal and Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; (Y.I.); (T.S.)
- Research and Development Unit, PhoenixBio Co., Ltd., Higashi-Hiroshima 739-0046, Japan
| | - Kianna M. Nguyen
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA; (P.F.); (J.M.d.H.-V.); (J.H.C.); (C.D.D.); (A.M.G.-S.); (K.M.N.); (K.G.); (Q.P.P.); (G.S.)
| | - Kihak Gwon
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA; (P.F.); (J.M.d.H.-V.); (J.H.C.); (C.D.D.); (A.M.G.-S.); (K.M.N.); (K.G.); (Q.P.P.); (G.S.)
| | - Quinn P. Peterson
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA; (P.F.); (J.M.d.H.-V.); (J.H.C.); (C.D.D.); (A.M.G.-S.); (K.M.N.); (K.G.); (Q.P.P.); (G.S.)
| | - Takeshi Saito
- Department of Medicine, Division of Gastrointestinal and Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; (Y.I.); (T.S.)
| | - Gulnaz Stybayeva
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA; (P.F.); (J.M.d.H.-V.); (J.H.C.); (C.D.D.); (A.M.G.-S.); (K.M.N.); (K.G.); (Q.P.P.); (G.S.)
| | - Alexander Revzin
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA; (P.F.); (J.M.d.H.-V.); (J.H.C.); (C.D.D.); (A.M.G.-S.); (K.M.N.); (K.G.); (Q.P.P.); (G.S.)
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11
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Ite K, Toyoda M, Akiyama S, Enosawa S, Yoshioka S, Yukitake T, Yamazaki-Inoue M, Tatsumi K, Akutsu H, Nishina H, Kimura T, Otani N, Nakazawa A, Fukuda A, Kasahara M, Umezawa A. Stem cell challenges and opportunities. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2023; 199:379-395. [PMID: 37678981 DOI: 10.1016/bs.pmbts.2023.03.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/09/2023]
Abstract
Hepatocyte-like cells (HLCs) generated from human pluripotent stem cells (PSCs) exhibit hepatocytic properties in vitro; however, their engraftment and functionality in vivo remain unsatisfactory. Despite optimization of differentiation protocols, HLCs did not engraft in a mouse model of liver injury. In contrast, organ-derived hepatocytes reproducibly formed colonies in the liver injury mouse model. As an extension of the phenomenon observed in hematopoietic stem cells giving rise to colonies within the spleen, commonly referred to as "colony-forming units in spleen (CFU-s)", we hypothesize that "colony-forming units in liver (CFU-L)" serves as a reliable indicator of stemness, engraftment, and functionality of hepatocytes. The uniform expression of the randomly inactivated gene in a single colony, as reported by Sugahara et al. 2022, suggests that the colonies generated by isolated hepatocytes likely originate from a single cell. We, therefore, propose that CFU-L can be used to quantify the number of "hepatocytes that engraft and proliferate in vivo" as a quantitative assay for stem cells that utilize colony-forming ability, similar to that observed in hematopoietic stem cells.
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Affiliation(s)
- Kenta Ite
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan; Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
| | - Masashi Toyoda
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan; Research team for Aging Science (Vascular Medicine), Tokyo Metropolitan Institute for Geriatrics and Gerontology, Tokyo, Japan
| | - Saeko Akiyama
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan; Department of Advanced Pediatric Medicine (National Center for Child Health and Development), Tohoku University School of Medicine, Miyagi, Japan
| | - Shin Enosawa
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
| | - Saeko Yoshioka
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
| | - Takaaki Yukitake
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan; Department of Applied Biological Science, Tokyo University of Science, Tokyo, Japan
| | - Mayu Yamazaki-Inoue
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
| | - Kuniko Tatsumi
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
| | - Hidenori Akutsu
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
| | - Hiroshi Nishina
- Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
| | - Toru Kimura
- Department of BioSciences, Kitasato University School of Science, Kanagawa, Japan
| | - Naoko Otani
- Department of Applied Biological Science, Tokyo University of Science, Tokyo, Japan
| | - Atsuko Nakazawa
- Department of Pathology, National Center for Child Health and Development Hospital, Tokyo, Japan
| | - Akinari Fukuda
- Department of Pathology, National Center for Child Health and Development Hospital, Tokyo, Japan
| | - Mureo Kasahara
- Department of Pathology, National Center for Child Health and Development Hospital, Tokyo, Japan
| | - Akihiro Umezawa
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan; Department of Advanced Pediatric Medicine (National Center for Child Health and Development), Tohoku University School of Medicine, Miyagi, Japan.
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12
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Ietto G, Iori V, Gritti M, Inversini D, Costantino A, Izunza Barba S, Jiang ZG, Carcano G, Dalla Gasperina D, Pettinato G. Multicellular Liver Organoids: Generation and Importance of Diverse Specialized Cellular Components. Cells 2023; 12:1429. [PMID: 37408262 PMCID: PMC10217024 DOI: 10.3390/cells12101429] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2023] [Revised: 05/11/2023] [Accepted: 05/17/2023] [Indexed: 07/07/2023] Open
Abstract
Over 40,000 patients in the United States are estimated to suffer from end-stage liver disease and acute hepatic failure, for which liver transplantation is the only available therapy. Human primary hepatocytes (HPH) have not been employed as a therapeutic tool due to the difficulty in growing and expanding them in vitro, their sensitivity to cold temperatures, and tendency to dedifferentiate following two-dimensional culture. The differentiation of human-induced pluripotent stem cells (hiPSCs) into liver organoids (LO) has emerged as a potential alternative to orthotropic liver transplantation (OLT). However, several factors limit the efficiency of liver differentiation from hiPSCs, including a low proportion of differentiated cells capable of reaching a mature phenotype, the poor reproducibility of existing differentiation protocols, and insufficient long-term viability in vitro and in vivo. This review will analyze various methodologies being developed to improve hepatic differentiation from hiPSCs into liver organoids, paying particular attention to the use of endothelial cells as supportive cells for their further maturation. Here, we demonstrate why differentiated liver organoids can be used as a research tool for drug testing and disease modeling, or employed as a bridge for liver transplantation following liver failure.
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Affiliation(s)
- Giuseppe Ietto
- General, Emergency and Transplant Surgery Department, ASST-Sette Laghi, 21100 Varese, Italy
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
| | - Valentina Iori
- General, Emergency and Transplant Surgery Department, ASST-Sette Laghi, 21100 Varese, Italy
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
| | - Mattia Gritti
- Department of General Surgery, Humanitas Clinical and Research Center, Rozzano, 20089 Milan, Italy
| | - Davide Inversini
- General, Emergency and Transplant Surgery Department, ASST-Sette Laghi, 21100 Varese, Italy
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
| | - Angelita Costantino
- Department of Drug and Health Sciences, University of Catania, 95124 Catania, Italy;
| | - Sofia Izunza Barba
- Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
| | - Z. Gordon Jiang
- Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
| | - Giulio Carcano
- General, Emergency and Transplant Surgery Department, ASST-Sette Laghi, 21100 Varese, Italy
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
| | - Daniela Dalla Gasperina
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
- Department of Infectious Diseases, ASST-Sette Laghi, 21100 Varese, Italy
| | - Giuseppe Pettinato
- Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
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13
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A dataset of definitive endoderm and hepatocyte differentiations from human induced pluripotent stem cells. Sci Data 2023; 10:93. [PMID: 36788249 PMCID: PMC9929325 DOI: 10.1038/s41597-023-02001-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2022] [Accepted: 02/03/2023] [Indexed: 02/16/2023] Open
Abstract
Hepatocytes are a major parenchymal cell type in the liver and play an essential role in liver function. Hepatocyte-like cells can be differentiated in vitro from induced pluripotent stem cells (iPSCs) via definitive endoderm (DE)-like cells and hepatoblast-like cells. Here, we explored the in vitro differentiation time-course of hepatocyte-like cells. We performed methylome and transcriptome analyses for hepatocyte-like cell differentiation. We also analyzed DE-like cell differentiation by methylome, transcriptome, chromatin accessibility, and GATA6 binding profiles, using finer time-course samples. In this manuscript, we provide a detailed description of the dataset and the technical validations. Our data may be valuable for the analysis of the molecular mechanisms underlying hepatocyte and DE differentiations.
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14
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Liu S, Wang J, Chen S, Han Z, Wu H, Chen H, Duan Y. C/EBPβ Coupled with E2F2 Promoted the Proliferation of hESC-Derived Hepatocytes through Direct Binding to the Promoter Regions of Cell-Cycle-Related Genes. Cells 2023; 12:cells12030497. [PMID: 36766839 PMCID: PMC9914899 DOI: 10.3390/cells12030497] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2022] [Revised: 01/09/2023] [Accepted: 01/31/2023] [Indexed: 02/05/2023] Open
Abstract
Human embryonic stem cells (hESCs) hold the potential to solve the problem of the shortage of functional hepatocytes in clinical applications and drug development. However, a large number of usable hepatocytes derived from hESCs cannot be effectively obtained due to the limited proliferation capacity. In this study, we found that enhancement of liver transcription factor C/EBPβ during hepatic differentiation could not only significantly promote the expression of hepatic genes, such as albumin, alpha fetoprotein, and alpha-1 antitrypsin, but also dramatically reinforce proliferation-related phenotypes, including increasing the expression of proliferative genes, such as CDC25C, CDC45L, and PCNA, and the activation of cell cycle and DNA replication pathways. In addition, the analysis of CUT&Tag sequencing further revealed that C/EBPβ is directly bound to the promoter region of proliferating genes to promote cell proliferation; this interaction between C/EBPβ and DNA sequences of the promoters was verified by luciferase assay. On the contrary, the knockdown of C/EBPβ could significantly inhibit the expression of the aforementioned proliferative genes. RNA transcriptome analysis and GSEA enrichment indicated that the E2F family was enriched, and the expression of E2F2 was changed with the overexpression or knockdown of C/EBPβ. Moreover, the results of CUT&Tag sequencing showed that C/EBPβ also directly bound the promoter of E2F2, regulating E2F2 expression. Interestingly, Co-IP analysis exhibited a direct binding between C/EBPβ and E2F2 proteins, and this interaction between these two proteins was also verified in the LO2 cell line, a hepatic progenitor cell line. Thus, our results demonstrated that C/EBPβ first initiated E2F2 expression and then coupled with E2F2 to regulate the expression of proliferative genes in hepatocytes during the differentiation of hESCs. Therefore, our findings open a new avenue to provide an in vitro efficient approach to generate proliferative hepatocytes to potentially meet the demands for use in cell-based therapeutics as well as for pharmaceutical and toxicological studies.
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Affiliation(s)
- Shoupei Liu
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, Guangzhou 510006, China
| | - Jue Wang
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, Guangzhou 510006, China
| | - Sen Chen
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, Guangzhou 510006, China
| | - Zonglin Han
- School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China
| | - Haibin Wu
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, Guangzhou 510006, China
| | - Honglin Chen
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, Guangzhou 510006, China
- Correspondence: (H.C.); (Y.D.)
| | - Yuyou Duan
- Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, Guangzhou 510006, China
- Correspondence: (H.C.); (Y.D.)
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15
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Yuan Y, Cotton K, Samarasekera D, Khetani SR. Engineered Platforms for Maturing Pluripotent Stem Cell-Derived Liver Cells for Disease Modeling. Cell Mol Gastroenterol Hepatol 2023; 15:1147-1160. [PMID: 36738860 PMCID: PMC10034210 DOI: 10.1016/j.jcmgh.2023.01.013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/08/2022] [Revised: 01/25/2023] [Accepted: 01/26/2023] [Indexed: 02/06/2023]
Abstract
Several liver diseases (eg, hepatitis B/C viruses, alcoholic/nonalcoholic fatty liver, malaria, monogenic diseases, and drug-induced liver injury) significantly impact global mortality and morbidity. Species-specific differences in liver functions limit the use of animals to fully elucidate/predict human outcomes; therefore, in vitro human liver models are used for basic and translational research to complement animal studies. However, primary human liver cells are in short supply and display donor-to-donor variability in viability/quality. In contrast, human hepatocyte-like cells (HLCs) differentiated from induced pluripotent stem cells and embryonic stem cells are a near infinite cell resource that retains the patient/donor's genetic background; however, conventional protocols yield immature phenotypes. HLC maturation can be significantly improved using advanced techniques, such as protein micropatterning to precisely control cell-cell interactions, controlled sized spheroids, organoids with multiple cell types and layers, 3-dimensional bioprinting to spatially control cell populations, microfluidic devices for automated nutrient exchange and to induce liver zonation via soluble factor gradients, and synthetic biology to genetically modify the HLCs to accelerate and enhance maturation. Here, we present design features and characterization for representative advanced HLC maturation platforms and then discuss HLC use for modeling various liver diseases. Lastly, we discuss desirable advances to move this field forward. We anticipate that with continued advances in this space, pluripotent stem cell-derived liver models will provide human-relevant data much earlier in preclinical drug development and reduce animal usage, help elucidate liver disease mechanisms for the discovery of efficacious and safe therapeutics, and be useful as cell-based therapies for patients suffering from end-stage liver failure.
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Affiliation(s)
- Yang Yuan
- Department of Biomedical Engineering, University of Illinois at Chicago, Chicago, Illinois
| | - Kristen Cotton
- Department of Biomedical Engineering, University of Illinois at Chicago, Chicago, Illinois
| | - Dinithi Samarasekera
- Department of Biomedical Engineering, University of Illinois at Chicago, Chicago, Illinois
| | - Salman R Khetani
- Department of Biomedical Engineering, University of Illinois at Chicago, Chicago, Illinois.
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16
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Madrigal P, Deng S, Feng Y, Militi S, Goh KJ, Nibhani R, Grandy R, Osnato A, Ortmann D, Brown S, Pauklin S. Epigenetic and transcriptional regulations prime cell fate before division during human pluripotent stem cell differentiation. Nat Commun 2023; 14:405. [PMID: 36697417 PMCID: PMC9876972 DOI: 10.1038/s41467-023-36116-9] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2022] [Accepted: 01/17/2023] [Indexed: 01/26/2023] Open
Abstract
Stem cells undergo cellular division during their differentiation to produce daughter cells with a new cellular identity. However, the epigenetic events and molecular mechanisms occurring between consecutive cell divisions have been insufficiently studied due to technical limitations. Here, using the FUCCI reporter we developed a cell-cycle synchronised human pluripotent stem cell (hPSC) differentiation system for uncovering epigenome and transcriptome dynamics during the first two divisions leading to definitive endoderm. We observed that transcription of key differentiation markers occurs before cell division, while chromatin accessibility analyses revealed the early inhibition of alternative cell fates. We found that Activator protein-1 members controlled by p38/MAPK signalling are necessary for inducing endoderm while blocking cell fate shifting toward mesoderm, and that enhancers are rapidly established and decommissioned between different cell divisions. Our study has practical biomedical utility for producing hPSC-derived patient-specific cell types since p38/MAPK induction increased the differentiation efficiency of insulin-producing pancreatic beta-cells.
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Affiliation(s)
- Pedro Madrigal
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, CB10 1SA, UK
- Wellcome - MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, CB2 0SZ, UK
- European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, CB10 1SD, UK
| | - Siwei Deng
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Headington, Oxford, OX3 7LD, UK
| | - Yuliang Feng
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Headington, Oxford, OX3 7LD, UK
| | - Stefania Militi
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Headington, Oxford, OX3 7LD, UK
| | - Kim Jee Goh
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
- The Francis Crick Institute, London, NW1 1AT, UK
| | - Reshma Nibhani
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Headington, Oxford, OX3 7LD, UK
| | - Rodrigo Grandy
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
| | - Anna Osnato
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
| | - Daniel Ortmann
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
| | - Stephanie Brown
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
| | - Siim Pauklin
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Headington, Oxford, OX3 7LD, UK.
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17
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Ouchi R, Koike H. Modeling human liver organ development and diseases with pluripotent stem cell-derived organoids. Front Cell Dev Biol 2023; 11:1133534. [PMID: 36875751 PMCID: PMC9974642 DOI: 10.3389/fcell.2023.1133534] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2022] [Accepted: 02/06/2023] [Indexed: 02/17/2023] Open
Abstract
The discoveries of human pluripotent stem cells (PSCs) including embryonic stem cells and induced pluripotent stem cells (iPSCs) has led to dramatic advances in our understanding of basic human developmental and cell biology and has also been applied to research aimed at drug discovery and development of disease treatments. Research using human PSCs has been largely dominated by studies using two-dimensional cultures. In the past decade, however, ex vivo tissue "organoids," which have a complex and functional three-dimensional structure similar to human organs, have been created from PSCs and are now being used in various fields. Organoids created from PSCs are composed of multiple cell types and are valuable models with which it is better to reproduce the complex structures of living organs and study organogenesis through niche reproduction and pathological modeling through cell-cell interactions. Organoids derived from iPSCs, which inherit the genetic background of the donor, are helpful for disease modeling, elucidation of pathophysiology, and drug screening. Moreover, it is anticipated that iPSC-derived organoids will contribute significantly to regenerative medicine by providing treatment alternatives to organ transplantation with which the risk of immune rejection is low. This review summarizes how PSC-derived organoids are used in developmental biology, disease modeling, drug discovery, and regenerative medicine. Highlighted is the liver, an organ that play crucial roles in metabolic regulation and is composed of diverse cell types.
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Affiliation(s)
- Rie Ouchi
- Institute of Research, Tokyo Medical and Dental University (TMDU), Tokyo, Japan
| | - Hiroyuki Koike
- Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan
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18
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Inui J, Ueyama-Toba Y, Mitani S, Mizuguchi H. Development of a method of passaging and freezing human iPS cell-derived hepatocytes to improve their functions. PLoS One 2023; 18:e0285783. [PMID: 37200286 DOI: 10.1371/journal.pone.0285783] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2022] [Accepted: 04/29/2023] [Indexed: 05/20/2023] Open
Abstract
Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells (HLCs) are expected to replace primary human hepatocytes as a new source of functional hepatocytes in various medical applications. However, the hepatic functions of HLCs are still low and it takes a long time to differentiate them from human iPS cells. Furthermore, HLCs have very low proliferative capacity and are difficult to be passaged due to loss of hepatic functions after reseeding. To overcome these problems, we attempted to develop a technology to dissociate, cryopreserve, and reseed HLCs in this study. By adding epithelial-mesenchymal transition inhibitors and optimizing the cell dissociation time, we have developed a method for passaging HLCs without loss of their functions. After passage, HLCs showed a hepatocyte-like polygonal cell morphology and expressed major hepatocyte marker proteins such as albumin and cytochrome P450 3A4 (CYP3A4). In addition, the HLCs had low-density lipoprotein uptake and glycogen storage capacity. The HLCs also showed higher CYP3A4 activity and increased gene expression levels of major hepatocyte markers after passage compared to before passage. Finally, they maintained their functions even after their cryopreservation and re-culture. By applying this technology, it will be possible to provide ready-to-use availability of cryopreserved HLCs for drug discovery research.
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Affiliation(s)
- Jumpei Inui
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
| | - Yukiko Ueyama-Toba
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
- Laboratory of Functional Organoid for Drug Discovery, National Institute of Biomedical Innovation, Health and Nutrition, Osaka, Japan
- Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Osaka, Japan
| | - Seiji Mitani
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
| | - Hiroyuki Mizuguchi
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
- Laboratory of Functional Organoid for Drug Discovery, National Institute of Biomedical Innovation, Health and Nutrition, Osaka, Japan
- Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Osaka, Japan
- Global Center for Medical Engineering and Informatics, Osaka University, Osaka, Japan
- Center for Infectious Disease Education and Research (CiDER), Osaka University, Osaka, Japan
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19
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Shi Y, Deng J, Sang X, Wang Y, He F, Chen X, Xu A, Wu F. Generation of Hepatocytes and Nonparenchymal Cell Codifferentiation System from Human-Induced Pluripotent Stem Cells. Stem Cells Int 2022; 2022:3222427. [PMID: 36467281 PMCID: PMC9709383 DOI: 10.1155/2022/3222427] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2022] [Revised: 09/22/2022] [Accepted: 10/13/2022] [Indexed: 02/05/2024] Open
Abstract
To date, hepatocytes derived from human-induced pluripotent stem cells (hiPSC) provide a potentially unlimited resource for clinical application and drug development. However, most hiPSC-derived hepatocyte-like cells initiated differentiation from highly purified definitive endoderm, which are insufficient to accurately replicate the complex regulation of signals among multiple cells and tissues during liver organogenesis, thereby displaying an immature phenotypic and short survival time in vitro. Here, we described a protocol to achieve codifferentiation of endoderm-derived hepatocytes and mesoderm-derived nonparenchymal cells by the inclusion of BMP4 into hepatic differentiation medium, which has a beneficial effect on the hepatocyte maturation and lifespan in vitro. Our codifferentiation system suggests the important role of nonparenchymal cells in liver organogenesis. Hopefully, these hepatocytes described here provide a promising approach in the therapy of liver diseases.
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Affiliation(s)
- Ying Shi
- State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, College of Life Sciences, Sun Yat-sen University, Guangzhou, China
| | - Jiali Deng
- State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, College of Life Sciences, Sun Yat-sen University, Guangzhou, China
| | - Xiaopu Sang
- School of Life Sciences, Beijing University of Chinese Medicine, Beijing, China
| | - Yihang Wang
- State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, College of Life Sciences, Sun Yat-sen University, Guangzhou, China
| | - Fei He
- Department of Central Laboratory, Shenzhen Hospital, Beijing University of Chinese Medicine, Shenzhen, China
| | - Xiaoni Chen
- Department of Central Laboratory, Shenzhen Hospital, Beijing University of Chinese Medicine, Shenzhen, China
| | - Anlong Xu
- State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, College of Life Sciences, Sun Yat-sen University, Guangzhou, China
- School of Life Sciences, Beijing University of Chinese Medicine, Beijing, China
| | - Fenfang Wu
- State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, College of Life Sciences, Sun Yat-sen University, Guangzhou, China
- Department of Central Laboratory, Shenzhen Hospital, Beijing University of Chinese Medicine, Shenzhen, China
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20
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Wang SX, Yan JS, Chan YS. Advancements in MAFLD Modeling with Human Cell and Organoid Models. Int J Mol Sci 2022; 23:11850. [PMID: 36233151 PMCID: PMC9569457 DOI: 10.3390/ijms231911850] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2022] [Revised: 09/27/2022] [Accepted: 09/28/2022] [Indexed: 11/17/2022] Open
Abstract
Metabolic (dysfunction) associated fatty liver disease (MAFLD) is one of the most prevalent liver diseases and has no approved therapeutics. The high failure rates witnessed in late-phase MAFLD drug trials reflect the complexity of the disease, and how the disease develops and progresses remains to be fully understood. In vitro, human disease models play a pivotal role in mechanistic studies to unravel novel disease drivers and in drug testing studies to evaluate human-specific responses. This review focuses on MAFLD disease modeling using human cell and organoid models. The spectrum of patient-derived primary cells and immortalized cell lines employed to model various liver parenchymal and non-parenchymal cell types essential for MAFLD development and progression is discussed. Diverse forms of cell culture platforms utilized to recapitulate tissue-level pathophysiology in different stages of the disease are also reviewed.
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Affiliation(s)
- Shi-Xiang Wang
- Guangzhou Laboratory, No. 9 Xing Dao Huan Bei Road, Guangzhou International Bio Island, Guangzhou 510005, China
| | - Ji-Song Yan
- Guangzhou Laboratory, No. 9 Xing Dao Huan Bei Road, Guangzhou International Bio Island, Guangzhou 510005, China
- School of Life Sciences, Yunnan University, Kunming 650500, China
| | - Yun-Shen Chan
- Guangzhou Laboratory, No. 9 Xing Dao Huan Bei Road, Guangzhou International Bio Island, Guangzhou 510005, China
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21
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Wesley BT, Ross ADB, Muraro D, Miao Z, Saxton S, Tomaz RA, Morell CM, Ridley K, Zacharis ED, Petrus-Reurer S, Kraiczy J, Mahbubani KT, Brown S, Garcia-Bernardo J, Alsinet C, Gaffney D, Horsfall D, Tysoe OC, Botting RA, Stephenson E, Popescu DM, MacParland S, Bader G, McGilvray ID, Ortmann D, Sampaziotis F, Saeb-Parsy K, Haniffa M, Stevens KR, Zilbauer M, Teichmann SA, Vallier L. Single-cell atlas of human liver development reveals pathways directing hepatic cell fates. Nat Cell Biol 2022; 24:1487-1498. [PMID: 36109670 PMCID: PMC7617064 DOI: 10.1038/s41556-022-00989-7] [Citation(s) in RCA: 57] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2020] [Accepted: 07/29/2022] [Indexed: 12/14/2022]
Abstract
The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances have been hampered by the lack of knowledge concerning human hepatic development. Here, we addressed this limitation by describing the developmental trajectories of different cell types that make up the human liver at single-cell resolution. These transcriptomic analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing essential roles during organogenesis. We utilized this information to derive bipotential hepatoblast organoids and then exploited this model system to validate the importance of signalling pathways in hepatocyte and cholangiocyte specification. Further insights into hepatic maturation also enabled the identification of stage-specific transcription factors to improve the functionality of hepatocyte-like cells generated from human pluripotent stem cells. Thus, our study establishes a platform to investigate the basic mechanisms directing human liver development and to produce cell types for clinical applications.
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Affiliation(s)
- Brandon T Wesley
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | - Alexander D B Ross
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
- Department of Paediatrics, University of Cambridge, Cambridge, UK
| | - Daniele Muraro
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
- Wellcome Sanger Institute, Hinxton, UK
| | - Zhichao Miao
- Wellcome Sanger Institute, Hinxton, UK
- European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, Cambridge, UK
| | - Sarah Saxton
- Departments of Bioengineering and Pathology, University of Washington, Seattle, WA, USA
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA
| | - Rute A Tomaz
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | - Carola M Morell
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | - Katherine Ridley
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Paediatrics, University of Cambridge, Cambridge, UK
| | - Ekaterini D Zacharis
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | - Sandra Petrus-Reurer
- Department of Surgery, University of Cambridge, Cambridge, UK
- NIHR Cambridge Biomedical Research Centre, Cambridge, UK
| | - Judith Kraiczy
- Department of Paediatrics, University of Cambridge, Cambridge, UK
| | | | - Stephanie Brown
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | | | | | | | - Dave Horsfall
- Digital Institute, Newcastle University, Newcastle upon Tyne, UK
| | - Olivia C Tysoe
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | - Rachel A Botting
- Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK
| | - Emily Stephenson
- Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK
| | | | | | - Gary Bader
- University of Toronto, Toronto, Ontario, Canada
| | - Ian D McGilvray
- Multi-Organ Transplant Program, Toronto General Hospital Research Institute, Toronto, Ontario, Canada
| | - Daniel Ortmann
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | - Fotios Sampaziotis
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | - Kourosh Saeb-Parsy
- Department of Surgery, University of Cambridge, Cambridge, UK
- NIHR Cambridge Biomedical Research Centre, Cambridge, UK
| | - Muzlifah Haniffa
- Wellcome Sanger Institute, Hinxton, UK
- Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK
- Department of Dermatology and NIHR Newcastle Biomedical Research Centre, Newcastle Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK
| | - Kelly R Stevens
- Departments of Bioengineering and Pathology, University of Washington, Seattle, WA, USA
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA
| | - Matthias Zilbauer
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Paediatrics, University of Cambridge, Cambridge, UK
| | - Sarah A Teichmann
- Wellcome Sanger Institute, Hinxton, UK
- Theory of Condensed Matter Group, Cavendish Laboratory, University of Cambridge, Cambridge, UK
| | - Ludovic Vallier
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK.
- Department of Surgery, University of Cambridge, Cambridge, UK.
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22
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Tomaz RA, Zacharis ED, Bachinger F, Wurmser A, Yamamoto D, Petrus-Reurer S, Morell CM, Dziedzicka D, Wesley BT, Geti I, Segeritz CP, de Brito MC, Chhatriwala M, Ortmann D, Saeb-Parsy K, Vallier L. Generation of functional hepatocytes by forward programming with nuclear receptors. eLife 2022; 11:71591. [PMID: 35959725 PMCID: PMC9374437 DOI: 10.7554/elife.71591] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2021] [Accepted: 07/25/2022] [Indexed: 11/13/2022] Open
Abstract
Production of large quantities of hepatocytes remains a major challenge for a number of clinical applications in the biomedical field. Directed differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells (HLCs) provides an advantageous solution and a number of protocols have been developed for this purpose. However, these methods usually follow different steps of liver development in vitro, which is time consuming and requires complex culture conditions. In addition, HLCs lack the full repertoire of functionalities characterising primary hepatocytes. Here, we explore the interest of forward programming to generate hepatocytes from hPSCs and to bypass these limitations. This approach relies on the overexpression of three hepatocyte nuclear factors (HNF1A, HNF6, and FOXA3) in combination with different nuclear receptors expressed in the adult liver using the OPTi-OX platform. Forward programming allows for the rapid production of hepatocytes (FoP-Heps) with functional characteristics using a simplified process. We also uncovered that the overexpression of nuclear receptors such as RORc can enhance specific functionalities of FoP-Heps thereby validating its role in lipid/glucose metabolism. Together, our results show that forward programming could offer a versatile alternative to direct differentiation for generating hepatocytes in vitro.
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Affiliation(s)
- Rute A Tomaz
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.,Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom
| | - Ekaterini D Zacharis
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.,Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom
| | - Fabian Bachinger
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.,Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom
| | - Annabelle Wurmser
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.,Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom
| | - Daniel Yamamoto
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.,Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom
| | - Sandra Petrus-Reurer
- Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom
| | - Carola M Morell
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.,Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom
| | - Dominika Dziedzicka
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.,Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom
| | - Brandon T Wesley
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom
| | - Imbisaat Geti
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.,Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom
| | - Charis-Patricia Segeritz
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.,Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom
| | - Miguel C de Brito
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.,Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom
| | - Mariya Chhatriwala
- Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom
| | - Daniel Ortmann
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.,Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom
| | - Kourosh Saeb-Parsy
- Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom
| | - Ludovic Vallier
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.,Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom.,Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom
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23
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Eiro N, Fraile M, González-Jubete A, González LO, Vizoso FJ. Mesenchymal (Stem) Stromal Cells Based as New Therapeutic Alternative in Inflammatory Bowel Disease: Basic Mechanisms, Experimental and Clinical Evidence, and Challenges. Int J Mol Sci 2022; 23:ijms23168905. [PMID: 36012170 PMCID: PMC9408403 DOI: 10.3390/ijms23168905] [Citation(s) in RCA: 30] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2022] [Revised: 07/26/2022] [Accepted: 07/26/2022] [Indexed: 12/13/2022] Open
Abstract
Inflammatory bowel diseases (IBD) are an example of chronic diseases affecting 40% of the population, which involved tissue damage and an inflammatory process not satisfactorily controlled with current therapies. Data suggest that mesenchymal stem cells (MSC) may be a therapeutic option for these processes, and especially for IBD, due to their multifactorial approaches such as anti-inflammatory, anti-oxidative stress, anti-apoptotic, anti-fibrotic, regenerative, angiogenic, anti-tumor, or anti-microbial. However, MSC therapy is associated with important limitations as safety issues, handling difficulties for therapeutic purposes, and high economic cost. MSC-derived secretome products (conditioned medium or extracellular vesicles) are therefore a therapeutic option in IBD as they exhibit similar effects to their parent cells and avoid the issues of cell therapy. In this review, we proposed further studies to choose the ideal tissue source of MSC to treat IBD, the implementation of new standardized production strategies, quality controls and the integration of other technologies, such as hydrogels, which may improve the therapeutic effects of derived-MSC secretome products in IBD.
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Affiliation(s)
- Noemi Eiro
- Research Unit, Fundación Hospital de Jove, Av. de Eduardo Castro, 161, 33290 Gijón, Spain
- Correspondence: (N.E.); (F.J.V.); Tel.: +34-98-5320050 (ext. 84216) (N.E.); Fax: +34-98-531570 (N.E.)
| | - Maria Fraile
- Research Unit, Fundación Hospital de Jove, Av. de Eduardo Castro, 161, 33290 Gijón, Spain
| | | | - Luis O. González
- Department of Anatomical Pathology, Fundación Hospital de Jove, Av. de Eduardo Castro, 161, 33290 Gijón, Spain
| | - Francisco J. Vizoso
- Research Unit, Fundación Hospital de Jove, Av. de Eduardo Castro, 161, 33290 Gijón, Spain
- Department of Surgery, Fundación Hospital de Jove, Av. de Eduardo Castro, 161, 33290 Gijón, Spain
- Correspondence: (N.E.); (F.J.V.); Tel.: +34-98-5320050 (ext. 84216) (N.E.); Fax: +34-98-531570 (N.E.)
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24
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Dubois-Pot-Schneider H, Aninat C, Kattler K, Fekir K, Jarnouen K, Cerec V, Glaise D, Salhab A, Gasparoni G, Takashi K, Ishida S, Walter J, Corlu A. Transcriptional and Epigenetic Consequences of DMSO Treatment on HepaRG Cells. Cells 2022; 11:cells11152298. [PMID: 35892596 PMCID: PMC9331440 DOI: 10.3390/cells11152298] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2022] [Revised: 07/21/2022] [Accepted: 07/22/2022] [Indexed: 11/16/2022] Open
Abstract
Dimethyl sulfoxide (DMSO) is used to sustain or favor hepatocyte differentiation in vitro. Thus, DMSO is used in the differentiation protocol of the HepaRG cells that present the closest drug-metabolizing enzyme activities to primary human hepatocytes in culture. The aim of our study is to clarify its influence on liver-specific gene expression. For that purpose, we performed a large-scale analysis (gene expression and histone modification) to determine the global role of DMSO exposure during the differentiation process of the HepaRG cells. The addition of DMSO drives the upregulation of genes mainly regulated by PXR and PPARα whereas genes not affected by this addition are regulated by HNF1α, HNF4α, and PPARα. DMSO-differentiated-HepaRG cells show a differential expression for genes regulated by histone acetylation, while differentiated-HepaRG cells without DMSO show gene signatures associated with histone deacetylases. In addition, we observed an interplay between cytoskeleton organization and EMC remodeling with hepatocyte maturation.
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Affiliation(s)
- Hélène Dubois-Pot-Schneider
- INSERM, Université de Rennes, INRAE, Institut NuMeCan (Nutrition, Metabolisms and Cancer), F-35000 Rennes, France; (C.A.); (K.F.); (K.J.); (V.C.); (D.G.); (A.C.)
- Correspondence: ; Tel.: +33-372746115
| | - Caroline Aninat
- INSERM, Université de Rennes, INRAE, Institut NuMeCan (Nutrition, Metabolisms and Cancer), F-35000 Rennes, France; (C.A.); (K.F.); (K.J.); (V.C.); (D.G.); (A.C.)
| | - Kathrin Kattler
- Department of Genetics, University of Saarland (UdS), 66123 Saarbrücken, Germany; (K.K.); (A.S.); (G.G.); (J.W.)
| | - Karim Fekir
- INSERM, Université de Rennes, INRAE, Institut NuMeCan (Nutrition, Metabolisms and Cancer), F-35000 Rennes, France; (C.A.); (K.F.); (K.J.); (V.C.); (D.G.); (A.C.)
| | - Kathleen Jarnouen
- INSERM, Université de Rennes, INRAE, Institut NuMeCan (Nutrition, Metabolisms and Cancer), F-35000 Rennes, France; (C.A.); (K.F.); (K.J.); (V.C.); (D.G.); (A.C.)
| | - Virginie Cerec
- INSERM, Université de Rennes, INRAE, Institut NuMeCan (Nutrition, Metabolisms and Cancer), F-35000 Rennes, France; (C.A.); (K.F.); (K.J.); (V.C.); (D.G.); (A.C.)
| | - Denise Glaise
- INSERM, Université de Rennes, INRAE, Institut NuMeCan (Nutrition, Metabolisms and Cancer), F-35000 Rennes, France; (C.A.); (K.F.); (K.J.); (V.C.); (D.G.); (A.C.)
| | - Abdulrahman Salhab
- Department of Genetics, University of Saarland (UdS), 66123 Saarbrücken, Germany; (K.K.); (A.S.); (G.G.); (J.W.)
| | - Gilles Gasparoni
- Department of Genetics, University of Saarland (UdS), 66123 Saarbrücken, Germany; (K.K.); (A.S.); (G.G.); (J.W.)
| | - Kubo Takashi
- Division of Pharmacology, National Institute of Health Sciences, Kawasaki-ku, Kawasaki 2109501, Japan; (K.T.); (S.I.)
| | - Seiichi Ishida
- Division of Pharmacology, National Institute of Health Sciences, Kawasaki-ku, Kawasaki 2109501, Japan; (K.T.); (S.I.)
| | - Jörn Walter
- Department of Genetics, University of Saarland (UdS), 66123 Saarbrücken, Germany; (K.K.); (A.S.); (G.G.); (J.W.)
| | - Anne Corlu
- INSERM, Université de Rennes, INRAE, Institut NuMeCan (Nutrition, Metabolisms and Cancer), F-35000 Rennes, France; (C.A.); (K.F.); (K.J.); (V.C.); (D.G.); (A.C.)
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25
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Clinical Application of Induced Hepatocyte-like Cells Produced from Mesenchymal Stromal Cells: A Literature Review. Cells 2022; 11:cells11131998. [PMID: 35805080 PMCID: PMC9265349 DOI: 10.3390/cells11131998] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2022] [Revised: 06/15/2022] [Accepted: 06/16/2022] [Indexed: 11/17/2022] Open
Abstract
Liver disease is a leading cause of mortality worldwide, resulting in 1.3 million deaths annually. The vast majority of liver disease is caused by metabolic disease (i.e., NASH) and alcohol-induced hepatitis, and to a lesser extent by acute and chronic viral infection. Furthermore, multiple insults to the liver is becoming common due to the prevalence of metabolic and alcohol-related liver diseases. Despite this rising prevalence of liver disease, there are few treatment options: there are treatments for viral hepatitis C and there is vaccination for hepatitis B. Aside from the management of metabolic syndrome, no direct liver therapy has shown clinical efficacy for metabolic liver disease, there is very little for acute alcohol-induced liver disease, and liver transplantation remains the only effective treatment for late-stage liver disease. Traditional pharmacologic interventions have failed to appreciably impact the pathophysiology of alcohol-related liver disease or end-stage liver disease. The difficulties associated with developing liver-specific therapies result from three factors that are common to late-stage liver disease arising from any cause: hepatocyte injury, inflammation, and aberrant tissue healing. Hepatocyte injury results in tissue damage with inflammation, which sensitizes the liver to additional hepatocyte injury and stimulates hepatic stellate cells and aberrant tissue healing responses. In the setting of chronic liver insults, there is progressive scarring, the loss of hepatocyte function, and hemodynamic dysregulation. Regenerative strategies using hepatocyte-like cells that are manufactured from mesenchymal stromal cells may be able to correct this pathophysiology through multiple mechanisms of action. Preclinical studies support their effectiveness and recent clinical studies suggest that cell replacement therapy can be safe and effective in patients with liver disease for whom there is no other option.
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26
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Lee J, Mun SJ, Shin Y, Lee S, Son MJ. Advances in liver organoids: model systems for liver disease. Arch Pharm Res 2022; 45:390-400. [DOI: 10.1007/s12272-022-01390-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2022] [Accepted: 05/26/2022] [Indexed: 12/24/2022]
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27
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Hurrell T, Naidoo J, Scholefield J. Hepatic Models in Precision Medicine: An African Perspective on Pharmacovigilance. Front Genet 2022; 13:864725. [PMID: 35495161 PMCID: PMC9046844 DOI: 10.3389/fgene.2022.864725] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2022] [Accepted: 03/29/2022] [Indexed: 01/02/2023] Open
Abstract
Pharmaceuticals are indispensable to healthcare as the burgeoning global population is challenged by diseases. The African continent harbors unparalleled genetic diversity, yet remains largely underrepresented in pharmaceutical research and development, which has serious implications for pharmaceuticals approved for use within the African population. Adverse drug reactions (ADRs) are often underpinned by unique variations in genes encoding the enzymes responsible for their uptake, metabolism, and clearance. As an example, individuals of African descent (14-34%) harbor an exclusive genetic variant in the gene encoding a liver metabolizing enzyme (CYP2D6) which reduces the efficacy of the breast cancer chemotherapeutic Tamoxifen. However, CYP2D6 genotyping is not required prior to dispensing Tamoxifen in sub-Saharan Africa. Pharmacogenomics is fundamental to precision medicine and the absence of its implementation suggests that Africa has, to date, been largely excluded from the global narrative around stratified healthcare. Models which could address this need, include primary human hepatocytes, immortalized hepatic cell lines, and induced pluripotent stem cell (iPSC) derived hepatocyte-like cells. Of these, iPSCs, are promising as a functional in vitro model for the empirical evaluation of drug metabolism. The scale with which pharmaceutically relevant African genetic variants can be stratified, the expediency with which these platforms can be established, and their subsequent sustainability suggest that they will have an important role to play in the democratization of stratified healthcare in Africa. Here we discuss the requirement for African hepatic models, and their implications for the future of pharmacovigilance on the African continent.
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Affiliation(s)
- Tracey Hurrell
- Bioengineering and Integrated Genomics Group, Next Generation Health Cluster, Council for Scientific and Industrial Research, Pretoria, South Africa
| | - Jerolen Naidoo
- Bioengineering and Integrated Genomics Group, Next Generation Health Cluster, Council for Scientific and Industrial Research, Pretoria, South Africa
| | - Janine Scholefield
- Bioengineering and Integrated Genomics Group, Next Generation Health Cluster, Council for Scientific and Industrial Research, Pretoria, South Africa
- Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
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28
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Luce E, Steichen C, Allouche M, Messina A, Heslan JM, Lambert T, Weber A, Nguyen TH, Christophe O, Dubart-Kupperschmitt A. In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model. Hepatology 2022; 75:866-880. [PMID: 34687060 PMCID: PMC9299628 DOI: 10.1002/hep.32211] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/20/2021] [Revised: 08/13/2021] [Accepted: 10/13/2021] [Indexed: 12/08/2022]
Abstract
BACKGROUND AND AIMS Pluripotent stem cell-derived hepatocytes differentiated in monolayer culture are known to have more fetal than adult hepatocyte characteristics. If numerous studies tend to show that this immature phenotype might not necessarily be an obstacle to their use in transplantation, other applications such as drug screening, toxicological studies, or bioartificial livers are reliant on hepatocyte functionality and require full differentiation of hepatocytes. New technologies have been used to improve the differentiation process in recent years, usually evaluated by measuring the albumin production and CYP450 activity. Here we used the complex production and most importantly the activity of the coagulation factor IX (FIX) produced by mature hepatocytes to assess the differentiation of hemophilia B (HB) patient's induced pluripotent stem cells (iPSCs) in both monolayer culture and organoids. APPROACH AND RESULTS Indeed, HB is an X-linked monogenic disease due to an impaired activity of FIX synthesized by hepatocytes in the liver. We have developed an in vitro model of HB hepatocytes using iPSCs generated from fibroblasts of a severe HB patient. We used CRISPR/Cas9 technology to target the genomic insertion of a coagulation factor 9 minigene bearing the Padua mutation to enhance FIX activity. Noncorrected and corrected iPSCs were differentiated into hepatocytes under both two-dimensional and three-dimensional differentiation protocols and deciphered the production of active FIX in vitro. Finally, we assessed the therapeutic efficacy of this approach in vivo using a mouse model of HB. CONCLUSIONS Functional FIX, whose post-translational modifications only occur in fully mature hepatocytes, was only produced in corrected iPSCs differentiated in organoids. Immunohistochemistry analyses of mouse livers indicated a good cell engraftment, and the FIX activity detected in the plasma of transplanted animals confirmed rescue of the bleeding phenotype.
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Affiliation(s)
- Eléanor Luce
- INSERM Université Paris-SaclayUnité Mixte de Recherche 1193VillejuifFrance.,Féderation Hospitalo-Universitaire Hépatinov, Hôpital Paul BrousseVillejuifFrance
| | - Clara Steichen
- INSERM Université Paris-SaclayUnité Mixte de Recherche 1193VillejuifFrance.,Féderation Hospitalo-Universitaire Hépatinov, Hôpital Paul BrousseVillejuifFrance
| | - Mickaël Allouche
- INSERM Université Paris-SaclayUnité Mixte de Recherche 1193VillejuifFrance.,Féderation Hospitalo-Universitaire Hépatinov, Hôpital Paul BrousseVillejuifFrance
| | - Antonietta Messina
- INSERM Université Paris-SaclayUnité Mixte de Recherche 1193VillejuifFrance.,Féderation Hospitalo-Universitaire Hépatinov, Hôpital Paul BrousseVillejuifFrance
| | | | - Thierry Lambert
- Centre de Référence pour le Traitement des HémophilesHôpital de BicêtreFrance
| | - Anne Weber
- INSERM Université Paris-SaclayUnité Mixte de Recherche 1193VillejuifFrance.,Féderation Hospitalo-Universitaire Hépatinov, Hôpital Paul BrousseVillejuifFrance
| | - Tuan Huy Nguyen
- INSERM Unité Mixte de Recherche 1064CHU Hôtel DieuNantesFrance
| | - Olivier Christophe
- INSERM Unité Mixte de Recherche 1176Hôpital de BicêtreKremlin-BicêtreFrance
| | - Anne Dubart-Kupperschmitt
- INSERM Université Paris-SaclayUnité Mixte de Recherche 1193VillejuifFrance.,Féderation Hospitalo-Universitaire Hépatinov, Hôpital Paul BrousseVillejuifFrance
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29
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Imamura S, Yoshimoto K, Terada S, Takamuro K, Kamei KI. In vitro culture at 39 °C during hepatic maturation of human ES cells facilitates hepatocyte-like cell functions. Sci Rep 2022; 12:5155. [PMID: 35338220 PMCID: PMC8956733 DOI: 10.1038/s41598-022-09119-7] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2022] [Accepted: 03/14/2022] [Indexed: 11/12/2022] Open
Abstract
Hepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) offer an alternative to primary hepatocytes commonly used for drug screenings and toxicological tests. However, these cells do not have hepatic functions comparable to those of hepatocytes in vivo due to insufficient hepatic differentiation. Here we showed that the hepatic functions of hPSC-HLCs were facilitated by applying physiological liver temperatures during hepatic differentiation. We identified the optimal temperature by treating HLCs derived from H9 human embryonic stem cells (hESC-HLCs) at 39 °C; the 42 °C treatment caused significantly greater cell death than the 39 °C treatment. We confirmed the improvement of hepatic functions, such as albumin secretion, cytochrome P450 3A activity, and collagen production, without severe cell damage. In combination with existing hepatic differentiation protocols, the method proposed here may further improve hepatic functions for hPSCs and lead to the realization of drug discovery efforts and drug toxicological tests.
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Affiliation(s)
- Satoshi Imamura
- Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Ushinomiya-cho, Sakyo-ku, Kyoto, 606-8501, Japan
| | - Koki Yoshimoto
- Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Ushinomiya-cho, Sakyo-ku, Kyoto, 606-8501, Japan.,Department of Biosystems Science, Institute for Frontier Life and Medical Sciences, Kyoto University, Shogoin-Kawara-cho, Sakyo-ku, Kyoto, 606-8397, Japan.,Laboratory of Cellular and Molecular Biomechanics, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8397, Japan
| | - Shiho Terada
- Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Ushinomiya-cho, Sakyo-ku, Kyoto, 606-8501, Japan
| | - Kaho Takamuro
- Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Ushinomiya-cho, Sakyo-ku, Kyoto, 606-8501, Japan
| | - Ken-Ichiro Kamei
- Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Ushinomiya-cho, Sakyo-ku, Kyoto, 606-8501, Japan. .,Wuya College of Innovation, Shenyang Pharmaceutical University, Liaoning, 110016, People's Republic of China. .,Department of Pharmaceutics, Shenyang Pharmaceutical University, Liaoning, 110016, People's Republic of China.
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Raggi C, Selleri S, M'Callum MA, Paganelli M. Generation of Complex Syngeneic Liver Organoids from Induced Pluripotent Stem Cells to Model Human Liver Pathophysiology. Curr Protoc 2022; 2:e389. [PMID: 35263041 DOI: 10.1002/cpz1.389] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
The study of human liver pathophysiology has been hampered for decades by the lack of easily accessible, robust, and representative in vitro models. The discovery of induced pluripotent stem cells (iPSCs)-which can be generated from patients' somatic cells, engineered to harbor specific mutations, and differentiated into hepatocyte-like cells-opened the way to more meaningful modeling of liver development and disease. Nevertheless, representative modeling of many complex liver conditions requires the recreation of the interplay between hepatocytes and nonparenchymal liver cells. Here we describe protocols we developed to generate and characterize complex human liver organoids composed of iPSC-derived hepatic, endothelial, and mesenchymal cells. With all cell types derived from the same iPSC population, such organoids reproduce the liver niche, allowing for the study of liver development and the modeling of complex inflammatory and fibrotic conditions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Differentiation of human iPSCs into hepatic progenitor cells (hepatoblasts) Basic Protocol 2: Differentiation of human iPSCs into endothelial progenitor cells Support Protocol 1: Characterization of iPSC-derived endothelial progenitor cells Basic Protocol 3: Differentiation of human iPSCs into mesenchymal progenitor cells Support Protocol 2: Characterization of iPSC-derived mesenchymal progenitor cells Basic Protocol 4: Generation of complex syngeneic liver organoids.
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Affiliation(s)
- Claudia Raggi
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine Research Centre, Montreal, Canada
- Morphocell Technologies, Inc., Montreal, Canada
| | - Silvia Selleri
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine Research Centre, Montreal, Canada
| | - Marie-Agnes M'Callum
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine Research Centre, Montreal, Canada
| | - Massimiliano Paganelli
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine Research Centre, Montreal, Canada
- Pediatric Hepatology, CHU Sainte-Justine, Montreal, Canada
- Department of Pediatrics, Faculty of Medicine, University of Montréal, Montreal, Canada
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31
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Graffmann N, Scherer B, Adjaye J. In vitro differentiation of pluripotent stem cells into hepatocyte like cells - basic principles and current progress. Stem Cell Res 2022; 61:102763. [DOI: 10.1016/j.scr.2022.102763] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/17/2021] [Revised: 03/08/2022] [Accepted: 03/22/2022] [Indexed: 12/11/2022] Open
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Dai Q, Jiang W, Huang F, Song F, Zhang J, Zhao H. Recent Advances in Liver Engineering With Decellularized Scaffold. Front Bioeng Biotechnol 2022; 10:831477. [PMID: 35223793 PMCID: PMC8866951 DOI: 10.3389/fbioe.2022.831477] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2021] [Accepted: 01/24/2022] [Indexed: 12/02/2022] Open
Abstract
Liver transplantation is currently the only effective treatment for patients with end-stage liver disease; however, donor liver scarcity is a notable concern. As a result, extensive endeavors have been made to diversify the source of donor livers. For example, the use of a decellularized scaffold in liver engineering has gained considerable attention in recent years. The decellularized scaffold preserves the original orchestral structure and bioactive chemicals of the liver, and has the potential to create a de novo liver that is fit for transplantation after recellularization. The structure of the liver and hepatic extracellular matrix, decellularization, recellularization, and recent developments are discussed in this review. Additionally, the criteria for assessment and major obstacles in using a decellularized scaffold are covered in detail.
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Affiliation(s)
- Qingqing Dai
- Department of Hepatopancreatobiliary Surgery and Organ Transplantation Center, Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, China
- Department of Internal Medicine IV (Gastroenterology, Hepatology, and Infectious Diseases), Jena University Hospital, Jena, Germany
| | - Wei Jiang
- Department of Burns, The First Affiliated Hospital of Anhui Medical University, Hefei, China
| | - Fan Huang
- Department of Hepatopancreatobiliary Surgery and Organ Transplantation Center, Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, China
| | - Fei Song
- Department of Urology, Jena University Hospital, Jena, Germany
| | - Jiqian Zhang
- Department of Anesthesiology, The First Affiliated Hospital of Anhui Medical University, Hefei, China
- *Correspondence: Jiqian Zhang, ; Hongchuan Zhao,
| | - Hongchuan Zhao
- Department of Hepatopancreatobiliary Surgery and Organ Transplantation Center, Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, China
- *Correspondence: Jiqian Zhang, ; Hongchuan Zhao,
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Raggi C, M'Callum MA, Pham QT, Gaub P, Selleri S, Baratang NV, Mangahas CL, Cagnone G, Reversade B, Joyal JS, Paganelli M. Leveraging interacting signaling pathways to robustly improve the quality and yield of human pluripotent stem cell-derived hepatoblasts and hepatocytes. Stem Cell Reports 2022; 17:584-598. [PMID: 35120625 PMCID: PMC9039749 DOI: 10.1016/j.stemcr.2022.01.003] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2020] [Revised: 01/04/2022] [Accepted: 01/05/2022] [Indexed: 12/24/2022] Open
Abstract
Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs) have shown great potential as an alternative to primary human hepatocytes (PHHs) for in vitro modeling. Several differentiation protocols have been described to direct PSCs toward the hepatic fate. Here, by leveraging recent knowledge of the signaling pathways involved in liver development, we describe a robust, scalable protocol that allowed us to consistently generate high-quality bipotent human hepatoblasts and HLCs from both embryonic stem cells and induced PSC (iPSCs). Although not yet fully mature, such HLCs were more similar to adult PHHs than were cells obtained with previously described protocols, showing good potential as a physiologically representative alternative to PHHs for in vitro modeling. PSC-derived hepatoblasts effectively generated with this protocol could differentiate into mature hepatocytes and cholangiocytes within syngeneic liver organoids, thus opening the way for representative human 3D in vitro modeling of liver development and pathophysiology.
We generated human hepatoblasts and hepatocyte-like cells (HLCs) from pluripotent stem cells Timed action on Wnt/β-catenin and TGFβ pathways improved maturity and yield of HLCs Hepatoblasts matured into hepatocytes and bile ducts within complex liver organoids The protocol is robust and showed potential for scalability and drug testing
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Affiliation(s)
- Claudia Raggi
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine, Montreal, QC, Canada; Morphocell Technologies Inc., Montreal, QC, Canada
| | - Marie-Agnès M'Callum
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine, Montreal, QC, Canada
| | - Quang Toan Pham
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine, Montreal, QC, Canada
| | - Perrine Gaub
- CHU Sainte-Justine Research Center, Montreal, QC, Canada; Morphocell Technologies Inc., Montreal, QC, Canada
| | - Silvia Selleri
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine, Montreal, QC, Canada
| | | | - Chenicka Lyn Mangahas
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine, Montreal, QC, Canada
| | - Gaël Cagnone
- CHU Sainte-Justine Research Center, Montreal, QC, Canada
| | - Bruno Reversade
- Institute of Molecular and Cell Biology and Institute of Medical Biology, A(∗)STAR, Singapore, Singapore
| | - Jean-Sébastien Joyal
- CHU Sainte-Justine Research Center, Montreal, QC, Canada; Department of Pediatrics, Université de Montréal, Montreal, QC, Canada
| | - Massimiliano Paganelli
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine, Montreal, QC, Canada; Department of Pediatrics, Université de Montréal, Montreal, QC, Canada; Morphocell Technologies Inc., Montreal, QC, Canada; Pediatric Hepatology, CHU Sainte-Justine, Montreal, QC, Canada.
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Ramos MJ, Bandiera L, Menolascina F, Fallowfield JA. In vitro models for non-alcoholic fatty liver disease: Emerging platforms and their applications. iScience 2022; 25:103549. [PMID: 34977507 PMCID: PMC8689151 DOI: 10.1016/j.isci.2021.103549] [Citation(s) in RCA: 48] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Non-alcoholic fatty liver disease (NAFLD) represents a global healthcare challenge, affecting 1 in 4 adults, and death rates are predicted to rise inexorably. The progressive form of NAFLD, non-alcoholic steatohepatitis (NASH), can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. However, no medical treatments are licensed for NAFLD-NASH. Identifying efficacious therapies has been hindered by the complexity of disease pathogenesis, a paucity of predictive preclinical models and inadequate validation of pharmacological targets in humans. The development of clinically relevant in vitro models of the disease will pave the way to overcome these challenges. Currently, the combined application of emerging technologies (e.g., organ-on-a-chip/microphysiological systems) and control engineering approaches promises to unravel NAFLD biology and deliver tractable treatment candidates. In this review, we will describe advances in preclinical models for NAFLD-NASH, the recent introduction of novel technologies in this space, and their importance for drug discovery endeavors in the future.
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Affiliation(s)
- Maria Jimenez Ramos
- Centre for Inflammation Research, The University of Edinburgh, The Queen's Medical Research Institute, Edinburgh EH16 4TJ, UK
| | - Lucia Bandiera
- Institute for Bioengineering, The University of Edinburgh, Edinburgh EH9 3BF, UK.,Synthsys - Centre for Synthetic and Systems Biology, The University of Edinburgh, Edinburgh EH9 3BF, UK
| | - Filippo Menolascina
- Institute for Bioengineering, The University of Edinburgh, Edinburgh EH9 3BF, UK.,Synthsys - Centre for Synthetic and Systems Biology, The University of Edinburgh, Edinburgh EH9 3BF, UK
| | - Jonathan Andrew Fallowfield
- Centre for Inflammation Research, The University of Edinburgh, The Queen's Medical Research Institute, Edinburgh EH16 4TJ, UK
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Ramos MJ, Bandiera L, Menolascina F, Fallowfield JA. In vitro models for non-alcoholic fatty liver disease: Emerging platforms and their applications. iScience 2022; 25:103549. [PMID: 34977507 DOI: 10.1016/j.isci] [Citation(s) in RCA: 35] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/23/2023] Open
Abstract
Non-alcoholic fatty liver disease (NAFLD) represents a global healthcare challenge, affecting 1 in 4 adults, and death rates are predicted to rise inexorably. The progressive form of NAFLD, non-alcoholic steatohepatitis (NASH), can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. However, no medical treatments are licensed for NAFLD-NASH. Identifying efficacious therapies has been hindered by the complexity of disease pathogenesis, a paucity of predictive preclinical models and inadequate validation of pharmacological targets in humans. The development of clinically relevant in vitro models of the disease will pave the way to overcome these challenges. Currently, the combined application of emerging technologies (e.g., organ-on-a-chip/microphysiological systems) and control engineering approaches promises to unravel NAFLD biology and deliver tractable treatment candidates. In this review, we will describe advances in preclinical models for NAFLD-NASH, the recent introduction of novel technologies in this space, and their importance for drug discovery endeavors in the future.
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Affiliation(s)
- Maria Jimenez Ramos
- Centre for Inflammation Research, The University of Edinburgh, The Queen's Medical Research Institute, Edinburgh EH16 4TJ, UK
| | - Lucia Bandiera
- Institute for Bioengineering, The University of Edinburgh, Edinburgh EH9 3BF, UK
- Synthsys - Centre for Synthetic and Systems Biology, The University of Edinburgh, Edinburgh EH9 3BF, UK
| | - Filippo Menolascina
- Institute for Bioengineering, The University of Edinburgh, Edinburgh EH9 3BF, UK
- Synthsys - Centre for Synthetic and Systems Biology, The University of Edinburgh, Edinburgh EH9 3BF, UK
| | - Jonathan Andrew Fallowfield
- Centre for Inflammation Research, The University of Edinburgh, The Queen's Medical Research Institute, Edinburgh EH16 4TJ, UK
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Bushweller L, Zhao Y, Zhang F, Wu X. Generation of Human Pluripotent Stem Cell-Derived Polarized Hepatocytes. Curr Protoc 2022; 2:e345. [PMID: 35007406 PMCID: PMC9175647 DOI: 10.1002/cpz1.345] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) are valuable tools to study liver biology. HLCs, however, lack certain key in vivo characteristics relevant to their physiological function. One such characteristic is cellular polarity, which is critical to hepatocyte counter-current flow systems involving canalicular bile secretion and sinusoidal secretion of large quantities of serum proteins into blood. Model systems using non-polarized hepatocytes, therefore, cannot recapitulate this physiological function of hepatocytes. Here, we describe a stepwise protocol to generate hPSC-derived polarized HLCs (pol-HLCs), which feature clearly defined basolateral and apical membranes separated by tight junctions. Pol-HLCs not only display many hepatic functions but are also capable of directional cargo secretion, mimicking the counter-current flow systems. We describe protocols for stem cell culture maintenance and for differentiating hPSCs into pol-HLCs. In addition, we describe protocols to assay the pol-HLCs for basic hepatic functions and polarized hepatic characteristics. Once successfully differentiated, these pol-HLCs can be used as an in vitro model system to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism. The establishment of hepatic polarity from non-polarized hPSCs also provides a useful tool to study the development and maintenance of hepatic polarity. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Maintenance of hPSCs Basic Protocol 2: Differentiation of hPSCs to pol-HLCs Basic Protocol 3: Assaying pol-HLCs for basic hepatic functions Support Protocol 1: Assessment of pol-HLC monolayer tightness Support Protocol 2: Assessment of pol-HLC polarity.
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Affiliation(s)
- Leila Bushweller
- Infection Biology Program and Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio,Cleveland Clinic College of Medicine at Case Western Reserve University, Cleveland, Ohio
| | - Yuanyuan Zhao
- Infection Biology Program and Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio
| | - Fan Zhang
- Infection Biology Program and Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio
| | - Xianfang Wu
- Infection Biology Program and Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio,Corresponding author:
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Loh CH, van Genesen S, Perino M, Bark MR, Veenstra GJC. Loss of PRC2 subunits primes lineage choice during exit of pluripotency. Nat Commun 2021; 12:6985. [PMID: 34848733 PMCID: PMC8632979 DOI: 10.1038/s41467-021-27314-4] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2020] [Accepted: 11/09/2021] [Indexed: 12/11/2022] Open
Abstract
Polycomb Repressive Complex 2 (PRC2) is crucial for the coordinated expression of genes during early embryonic development, catalyzing histone H3 lysine 27 trimethylation. Two distinct PRC2 complexes, PRC2.1 and PRC2.2, contain respectively MTF2 and JARID2 in embryonic stem cells (ESCs). In this study, we explored their roles in lineage specification and commitment, using single-cell transcriptomics and mouse embryoid bodies derived from Mtf2 and Jarid2 null ESCs. We observe that the loss of Mtf2 results in enhanced and faster differentiation towards cell fates from all germ layers, while the Jarid2 null cells are predominantly directed towards early differentiating precursors, with reduced efficiency towards mesendodermal lineages. These effects are caused by derepression of developmental regulators that are poised for activation in pluripotent cells and gain H3K4me3 at their promoters in the absence of PRC2 repression. Upon lineage commitment, the differentiation trajectories are relatively similar to those of wild-type cells. Together, our results uncover a major role for MTF2-containing PRC2.1 in balancing poised lineage-specific gene activation, whereas the contribution of JARID2-containing PRC2 is more selective in nature compared to MTF2. These data explain how PRC2 imposes thresholds for lineage choice during the exit of pluripotency.
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Affiliation(s)
- Chet H. Loh
- grid.5590.90000000122931605Department of Molecular Developmental Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University, Nijmegen, The Netherlands
| | - Siebe van Genesen
- grid.5590.90000000122931605Department of Molecular Developmental Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University, Nijmegen, The Netherlands
| | - Matteo Perino
- grid.5590.90000000122931605Department of Molecular Developmental Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University, Nijmegen, The Netherlands ,grid.4709.a0000 0004 0495 846XPresent Address: Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
| | - Magnus R. Bark
- grid.5590.90000000122931605Department of Molecular Developmental Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University, Nijmegen, The Netherlands
| | - Gert Jan C. Veenstra
- grid.5590.90000000122931605Department of Molecular Developmental Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University, Nijmegen, The Netherlands
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Ma C, Zhang L, He T, Cao H, Ren X, Ma C, Yang J, Huang R, Pan G. Single cell Raman spectroscopy to identify different stages of proliferating human hepatocytes for cell therapy. Stem Cell Res Ther 2021; 12:555. [PMID: 34717753 PMCID: PMC8556950 DOI: 10.1186/s13287-021-02619-9] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2021] [Accepted: 07/23/2021] [Indexed: 12/11/2022] Open
Abstract
Background Cell therapy provides hope for treatment of advanced liver failure. Proliferating human hepatocytes (ProliHHs) were derived from primary human hepatocytes (PHH) and as potential alternative for cell therapy in liver diseases. Due to the continuous decline of mature hepatic genes and increase of progenitor like genes during ProliHHs expanding, it is challenge to monitor the critical changes of the whole process. Raman microspectroscopy is a noninvasive, label free analytical technique with high sensitivity capacity. In this study, we evaluated the potential and feasibility to identify ProliHHs from PHH with Raman spectroscopy. Methods Raman spectra were collected at least 600 single spectrum for PHH and ProliHHs at different stages (Passage 1 to Passage 4). Linear discriminant analysis and a two-layer machine learning model were used to analyze the Raman spectroscopy data. Significant differences in Raman bands were validated by the associated conventional kits. Results Linear discriminant analysis successfully classified ProliHHs at different stages and PHH. A two-layer machine learning model was established and the overall accuracy was at 84.6%. Significant differences in Raman bands have been found within different ProliHHs cell groups, especially changes at 1003 cm−1, 1206 cm−1 and 1440 cm−1. These changes were linked with reactive oxygen species, hydroxyproline and triglyceride levels in ProliHHs, and the hypothesis were consistent with the corresponding assay results. Conclusions In brief, Raman spectroscopy was successfully employed to identify different stages of ProliHHs during dedifferentiation process. The approach can simultaneously trace multiple changes of cellular components from somatic cells to progenitor cells. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-021-02619-9.
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Affiliation(s)
- Chen Ma
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China.,University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Ludi Zhang
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Science, Beijing, China
| | - Ting He
- School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, China
| | - Huiying Cao
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China.,University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Xiongzhao Ren
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, China
| | - Chenhui Ma
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China.,University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Jiale Yang
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China.,University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Ruimin Huang
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China. .,University of Chinese Academy of Sciences, Beijing, 100049, China.
| | - Guoyu Pan
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China. .,University of Chinese Academy of Sciences, Beijing, 100049, China.
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Ebrahim N, Badr OAM, Yousef MM, Hassouna A, Sabry D, Farid AS, Mostafa O, Saihati HAA, Seleem Y, Abd El Aziz E, Khalil AH, Nawar A, Shoulah AA, Aljasir M, Mohamed AZ, El-Sherbiny M, Elsherbiny NM, Eladl MA, Forsyth NR, Salim RF. Functional Recellularization of Acellular Rat Liver Scaffold by Induced Pluripotent Stem Cells: Molecular Evidence for Wnt/B-Catenin Upregulation. Cells 2021; 10:cells10112819. [PMID: 34831042 PMCID: PMC8616374 DOI: 10.3390/cells10112819] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Revised: 10/14/2021] [Accepted: 10/15/2021] [Indexed: 01/08/2023] Open
Abstract
BACKGROUND Liver transplantation remains the only viable therapy for liver failure but has a severely restricted utility. Here, we aimed to decellularize rat livers to form acellular 3D bio-scaffolds suitable for seeding with induced pluripotent cells (iPSCs) as a tool to investigate the role of Wnt/β-catenin signaling in liver development and generation. METHODS Dissected rat livers were randomly divided into three groups: I (control); II (decellularized scaffolds) and III (recellularized scaffolds). Liver decellularization was established via an adapted perfusion procedure and assessed through the measurement of extracellular matrix (ECM) proteins and DNA content. Liver recellularization was assessed through histological examination and measurement of transcript levels of Wnt/β-catenin pathway, hepatogenesis, liver-specific microRNAs and growth factors essential for liver development. Adult rat liver decellularization was confirmed by the maintenance of ECM proteins and persistence of growth factors essential for liver regeneration. RESULTS iPSCs seeded rat decellularized livers displayed upregulated transcript expression of Wnt/β-catenin pathway-related, growth factors, and liver specification genes. Further, recellularized livers displayed restored liver-specific functions including albumin secretion and urea synthesis. CONCLUSION This establishes proof-of-principle for the generation of three-dimensional liver organ scaffolds as grafts and functional re-establishment.
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Affiliation(s)
- Nesrine Ebrahim
- Department of Histology and Cell Biology, Faculty of Medicine, Benha University, Banha 13511, Egypt; (N.E.); (M.M.Y.); (O.M.)
- Stem Cell Unit, Faculty of Medicine, Benha University, Banha 13511, Egypt
| | - Omnia A. M. Badr
- Department of Genetics and Genetic Engineering, Faculty of Agriculture, Benha University, Banha 13511, Egypt;
| | - Mohamed M. Yousef
- Department of Histology and Cell Biology, Faculty of Medicine, Benha University, Banha 13511, Egypt; (N.E.); (M.M.Y.); (O.M.)
| | - Amira Hassouna
- School of Public Health and Interdisciplinary Studies, Faculty of Health and Environmental Sciences, AUT University, Auckland 1010, New Zealand;
| | - Dina Sabry
- Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Cairo University, Cairo 12613, Egypt;
- Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Bader University in Cairo, Cairo 11562, Egypt
| | - Ayman Samir Farid
- Department of Clinical Pathology, Faculty of Veterinary Medicine, Benha University, Banha 13511, Egypt;
| | - Ola Mostafa
- Department of Histology and Cell Biology, Faculty of Medicine, Benha University, Banha 13511, Egypt; (N.E.); (M.M.Y.); (O.M.)
| | - Hajir A. Al Saihati
- Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, University of Hafr Albatin, Hafar Al Batin 39524, Saudi Arabia;
| | - Yasmin Seleem
- Department of Clinical Pharmacology, Faculty of Medicine, Benha University, Banha 13511, Egypt; (Y.S.); (E.A.E.A.)
| | - Eman Abd El Aziz
- Department of Clinical Pharmacology, Faculty of Medicine, Benha University, Banha 13511, Egypt; (Y.S.); (E.A.E.A.)
| | - Ahmed Hassan Khalil
- Department of Surgery & Radiology, Faculty of Veterinary Medicine, Benha University, Banha 13511, Egypt;
| | - Ahmed Nawar
- Department of General Surgery, Faculty of Medicine, Benha University, Banha 13511, Egypt; (A.N.); (A.A.S.)
| | - Ahmed A. Shoulah
- Department of General Surgery, Faculty of Medicine, Benha University, Banha 13511, Egypt; (A.N.); (A.A.S.)
| | - Mohammad Aljasir
- Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 52571, Saudi Arabia;
| | - Amira Zaki Mohamed
- Department of Microbiology, Faculty of Science, Tanta University, Tanta 31527, Egypt;
| | - Mohamed El-Sherbiny
- Department of Basic Medical Sciences, College of Medicine, AlMaarefa University, Riyadh 71666, Saudi Arabia;
- Department of Anatomy, Mansoura Faculty of Medicine, Mansoura University, Mansoura 35516, Egypt
| | - Nehal M. Elsherbiny
- Department of Biochemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Tabuk, Tabuk 47512, Saudi Arabia
- Correspondence: or (N.M.E.); (M.A.E.); (R.F.S.)
| | - Mohamed Ahmed Eladl
- Department of Basic Medical Sciences, College of Medicine, University of Sharjah, Sharjah 27272, United Arab Emirates
- Correspondence: or (N.M.E.); (M.A.E.); (R.F.S.)
| | - Nicholas Robert Forsyth
- Guy Hilton Research Laboratories, School of Pharmacy and Bioengineering, Faculty of Medicine and Health Sciences, Keele University, Newcastle ST5 5BG, UK;
| | - Rabab F. Salim
- Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Benha University, Banha 13511, Egypt
- Correspondence: or (N.M.E.); (M.A.E.); (R.F.S.)
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Tang S, Bai L, Duan Z, Zheng S. Stem cells treatment for wilson disease. Curr Stem Cell Res Ther 2021; 17:712-719. [PMID: 34615454 DOI: 10.2174/1574888x16666211006111556] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2021] [Revised: 07/21/2021] [Accepted: 07/27/2021] [Indexed: 11/22/2022]
Abstract
Wilson disease (WD) is a copper excretion disorder, mainly caused by mutations in the ATP7B gene. Pharmacological therapies and liver transplantation are currently the main treatment methods for WD, but they face problems such as drug treatment compliance, adverse reactions, and shortage of liver donors. Stem cell therapy of WD may correct abnormal copper metabolism permanently, which is the focus of current research. In this review, we summarized the latest research on stem cells treatment for WD, as well as current challenges and future expectations.
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Affiliation(s)
- Shan Tang
- The First Unit, Department of Hepatology, Beijing YouAn Hospital, Capital Medical University, Beijing. China
| | - Li Bai
- The Fourth Unit, Department of Hepatology, Beijing YouAn Hospital, Capital Medical University, Beijing. China
| | - Zhongping Duan
- The Fourth Unit, Department of Hepatology, Beijing YouAn Hospital, Capital Medical University, Beijing. China
| | - Sujun Zheng
- The First Unit, Department of Hepatology, Beijing YouAn Hospital, Capital Medical University, Beijing. China
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41
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Chang M, Bogacheva MS, Lou YR. Challenges for the Applications of Human Pluripotent Stem Cell-Derived Liver Organoids. Front Cell Dev Biol 2021; 9:748576. [PMID: 34660606 PMCID: PMC8517247 DOI: 10.3389/fcell.2021.748576] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2021] [Accepted: 09/08/2021] [Indexed: 12/14/2022] Open
Abstract
The current organoid culture systems allow pluripotent and adult stem cells to self-organize to form three-dimensional (3D) structures that provide a faithful recapitulation of the architecture and function of in vivo organs. In particular, human pluripotent stem cell-derived liver organoids (PSC-LOs) can be used in regenerative medicine and preclinical applications, such as disease modeling and drug discovery. New bioengineering tools, such as microfluidics, biomaterial scaffolds, and 3D bioprinting, are combined with organoid technologies to increase the efficiency of hepatic differentiation and enhance the functional maturity of human PSC-LOs by precise control of cellular microenvironment. Long-term stabilization of hepatocellular functions of in vitro liver organoids requires the combination of hepatic endodermal, endothelial, and mesenchymal cells. To improve the biological function and scalability of human PSC-LOs, bioengineering methods have been used to identify diverse and zonal hepatocyte populations in liver organoids for capturing heterogeneous pathologies. Therefore, constructing engineered liver organoids generated from human PSCs will be an extremely versatile tool in in vitro disease models and regenerative medicine in future. In this review, we aim to discuss the recent advances in bioengineering technologies in liver organoid culture systems that provide a timely and necessary study to model disease pathology and support drug discovery in vitro and to generate cell therapy products for transplantation.
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Affiliation(s)
- Mingyang Chang
- Department of Clinical Pharmacy and Drug Administration, School of Pharmacy, Fudan University, Shanghai, China
| | - Mariia S. Bogacheva
- Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland
| | - Yan-Ru Lou
- Department of Clinical Pharmacy and Drug Administration, School of Pharmacy, Fudan University, Shanghai, China
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42
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Xu Q. Human Three-Dimensional Hepatic Models: Cell Type Variety and Corresponding Applications. Front Bioeng Biotechnol 2021; 9:730008. [PMID: 34631680 PMCID: PMC8497968 DOI: 10.3389/fbioe.2021.730008] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2021] [Accepted: 08/30/2021] [Indexed: 12/23/2022] Open
Abstract
Owing to retained hepatic phenotypes and functions, human three-dimensional (3D) hepatic models established with diverse hepatic cell types are thought to recoup the gaps in drug development and disease modeling limited by a conventional two-dimensional (2D) cell culture system and species-specific variability in drug metabolizing enzymes and transporters. Primary human hepatocytes, human hepatic cancer cell lines, and human stem cell-derived hepatocyte-like cells are three main hepatic cell types used in current models and exhibit divergent hepatic phenotypes. Primary human hepatocytes derived from healthy hepatic parenchyma resemble in vivo-like genetic and metabolic profiling. Human hepatic cancer cell lines are unlimitedly reproducible and tumorigenic. Stem cell-derived hepatocyte-like cells derived from patients are promising to retain the donor's genetic background. It has been suggested in some studies that unique properties of cell types endue them with benefits in different research fields of in vitro 3D modeling paradigm. For instance, the primary human hepatocyte was thought to be the gold standard for hepatotoxicity study, and stem cell-derived hepatocyte-like cells have taken a main role in personalized medicine and regenerative medicine. However, the comprehensive review focuses on the hepatic cell type variety, and corresponding applications in 3D models are sparse. Therefore, this review summarizes the characteristics of different cell types and discusses opportunities of different cell types in drug development, liver disease modeling, and liver transplantation.
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Affiliation(s)
- Qianqian Xu
- School of Chinese Medicine, and Centre for Cancer and Inflammation Research, Hong Kong Baptist University, Hong Kong, China
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43
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Ferrari E, Rasponi M. Liver-Heart on chip models for drug safety. APL Bioeng 2021; 5:031505. [PMID: 34286172 PMCID: PMC8282347 DOI: 10.1063/5.0048986] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2021] [Accepted: 06/01/2021] [Indexed: 12/14/2022] Open
Abstract
Current pre-clinical models to evaluate drug safety during the drug development process (DDP) mainly rely on traditional two-dimensional cell cultures, considered too simplistic and often ineffective, or animal experimentations, which are costly, time-consuming, and not truly representative of human responses. Their clinical translation thus remains limited, eventually causing attrition and leading to high rates of failure during clinical trials. These drawbacks can be overcome by the recently developed Organs-on-Chip (OoC) technology. OoC are sophisticated in vitro systems capable of recapitulating pivotal architecture and functionalities of human organs. OoC are receiving increasing attention from the stakeholders of the DDP, particularly concerning drug screening and safety applications. When a drug is administered in the human body, it is metabolized by the liver and the resulting compound may cause unpredicted toxicity on off-target organs such as the heart. In this sense, several liver and heart models have been widely adopted to assess the toxicity of new or recalled drugs. Recent advances in OoC technology are making available platforms encompassing multiple organs fluidically connected to efficiently assess and predict the systemic effects of compounds. Such Multi-Organs-on-Chip (MOoC) platforms represent a disruptive solution to study drug-related effects, which results particularly useful to predict liver metabolism on off-target organs to ultimately improve drug safety testing in the pre-clinical phases of the DDP. In this review, we focus on recently developed liver and heart on chip systems for drug toxicity testing. In addition, MOoC platforms encompassing connected liver and heart tissues have been further reviewed and discussed.
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Affiliation(s)
- Erika Ferrari
- Department of Electronics, Information and Bioengineering, Politecnico di Milano, 20133 Milano, Italy
| | - Marco Rasponi
- Department of Electronics, Information and Bioengineering, Politecnico di Milano, 20133 Milano, Italy
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44
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Osnato A, Brown S, Krueger C, Andrews S, Collier AJ, Nakanoh S, Quiroga Londoño M, Wesley BT, Muraro D, Brumm AS, Niakan KK, Vallier L, Ortmann D, Rugg-Gunn PJ. TGFβ signalling is required to maintain pluripotency of human naïve pluripotent stem cells. eLife 2021; 10:e67259. [PMID: 34463252 PMCID: PMC8410071 DOI: 10.7554/elife.67259] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2021] [Accepted: 07/26/2021] [Indexed: 12/30/2022] Open
Abstract
The signalling pathways that maintain primed human pluripotent stem cells (hPSCs) have been well characterised, revealing a critical role for TGFβ/Activin/Nodal signalling. In contrast, the signalling requirements of naive human pluripotency have not been fully established. Here, we demonstrate that TGFβ signalling is required to maintain naive hPSCs. The downstream effector proteins - SMAD2/3 - bind common sites in naive and primed hPSCs, including shared pluripotency genes. In naive hPSCs, SMAD2/3 additionally bind to active regulatory regions near to naive pluripotency genes. Inhibiting TGFβ signalling in naive hPSCs causes the downregulation of SMAD2/3-target genes and pluripotency exit. Single-cell analyses reveal that naive and primed hPSCs follow different transcriptional trajectories after inhibition of TGFβ signalling. Primed hPSCs differentiate into neuroectoderm cells, whereas naive hPSCs transition into trophectoderm. These results establish that there is a continuum for TGFβ pathway function in human pluripotency spanning a developmental window from naive to primed states.
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Affiliation(s)
- Anna Osnato
- Wellcome–MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of CambridgeCambridgeUnited Kingdom
- Department of Surgery, University of CambridgeCambridgeUnited Kingdom
| | - Stephanie Brown
- Wellcome–MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of CambridgeCambridgeUnited Kingdom
- Department of Surgery, University of CambridgeCambridgeUnited Kingdom
| | - Christel Krueger
- Bioinformatics Group, The Babraham InstituteCambridgeUnited Kingdom
| | - Simon Andrews
- Bioinformatics Group, The Babraham InstituteCambridgeUnited Kingdom
| | - Amanda J Collier
- Epigenetics Programme, The Babraham InstituteCambridgeUnited Kingdom
| | - Shota Nakanoh
- Wellcome–MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of CambridgeCambridgeUnited Kingdom
- Department of Surgery, University of CambridgeCambridgeUnited Kingdom
- Division of Embryology, National Institute for Basic BiologyOkazakiJapan
| | - Mariana Quiroga Londoño
- Wellcome–MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of CambridgeCambridgeUnited Kingdom
- Department of Surgery, University of CambridgeCambridgeUnited Kingdom
| | - Brandon T Wesley
- Wellcome–MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of CambridgeCambridgeUnited Kingdom
- Department of Surgery, University of CambridgeCambridgeUnited Kingdom
| | - Daniele Muraro
- Wellcome–MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of CambridgeCambridgeUnited Kingdom
- Department of Surgery, University of CambridgeCambridgeUnited Kingdom
- Wellcome Sanger Institute, HinxtonCambridgeUnited Kingdom
| | - A Sophie Brumm
- Human Embryo and Stem Cell Laboratory, The Francis Crick InstituteLondonUnited Kingdom
| | - Kathy K Niakan
- Human Embryo and Stem Cell Laboratory, The Francis Crick InstituteLondonUnited Kingdom
- Centre for Trophoblast Research, University of CambridgeCambridgeUnited Kingdom
| | - Ludovic Vallier
- Wellcome–MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of CambridgeCambridgeUnited Kingdom
- Department of Surgery, University of CambridgeCambridgeUnited Kingdom
| | - Daniel Ortmann
- Wellcome–MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of CambridgeCambridgeUnited Kingdom
- Department of Surgery, University of CambridgeCambridgeUnited Kingdom
| | - Peter J Rugg-Gunn
- Wellcome–MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of CambridgeCambridgeUnited Kingdom
- Epigenetics Programme, The Babraham InstituteCambridgeUnited Kingdom
- Centre for Trophoblast Research, University of CambridgeCambridgeUnited Kingdom
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45
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Luo S, Ai Y, Xiao S, Wang B, Wang Y. Functional hit 1 (FH1)-based rapid and efficient generation of functional hepatocytes from human mesenchymal stem cells: a novel strategy for hepatic differentiation. ANNALS OF TRANSLATIONAL MEDICINE 2021; 9:1087. [PMID: 34422999 PMCID: PMC8339809 DOI: 10.21037/atm-21-2829] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/19/2021] [Accepted: 06/25/2021] [Indexed: 12/19/2022]
Abstract
Background Because the liver is central to the physiology of the body, primary hepatocytes are widely used in liver pathology and physiological research, such as liver drug screening, bioartificial liver support system, and cell therapy for liver diseases. However, the source of primary hepatocytes is limited. We describe a novel non-transgenic protocol that facilitates the rapid generation of hepatocyte-like cells from human umbilical cord-derived mesenchymal stem cells (hUC-MSCs), providing a new source of functional hepatocytes. Methods In this study, we used hUC-MSCs and human induced pluripotent cells (iPSCs) derived mesenchymal stem cells (iMSCs) to investigate the new induction strategy. Passage 3 MSCs were induced into hepatocyte-like cells using small-molecule compounds combined with cell factors in vitro. Functional hit 1 (FH1), a promising small molecule compound was achieved to replace HGF in the hepatocyte maturation stage to induce the hepatocyte-like cells differentiation. Results We rapidly induced hUC-MSCs and human iMSCs into hepatocyte-like cells within 10 days in vitro, and the cells were morphologically similarly to both hepatocytes derived from the hepatocyte growth factor (HGF)-based method and the primary hepatocytes. They expressed mature hepatocyte special genes and achieved functions such as glycogen storage, albumin expression, urea secretion, cytochrome P450 activity, Low-density lipoprotein (LDL) uptake, and indocyanine green (ICG) uptake. Conclusions We successfully established a small-molecule protocol without using HGF to differentiate MSCs into hepatocyte-like cells, which provides a rapid and cost-effective platform for in vitro studies of liver disease.
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Affiliation(s)
- Sang Luo
- State Key Laboratory of Virology, School of Life Sciences, Wuhan University, Wuhan, China
| | - Yang Ai
- State Key Laboratory of Virology, School of Life Sciences, Wuhan University, Wuhan, China
| | - Shuai Xiao
- State Key Laboratory of Virology, School of Life Sciences, Wuhan University, Wuhan, China
| | - Ben Wang
- State Key Laboratory of Virology, School of Life Sciences, Wuhan University, Wuhan, China
| | - Yefu Wang
- State Key Laboratory of Virology, School of Life Sciences, Wuhan University, Wuhan, China
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46
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Polidoro MA, Ferrari E, Marzorati S, Lleo A, Rasponi M. Experimental liver models: From cell culture techniques to microfluidic organs-on-chip. Liver Int 2021; 41:1744-1761. [PMID: 33966344 DOI: 10.1111/liv.14942] [Citation(s) in RCA: 39] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/31/2021] [Revised: 05/02/2021] [Accepted: 05/03/2021] [Indexed: 12/12/2022]
Abstract
The liver is one of the most studied organs of the human body owing to its central role in xenobiotic and drug metabolism. In recent decades, extensive research has aimed at developing in vitro liver models able to mimic liver functions to study pathophysiological clues in high-throughput and reproducible environments. Two-dimensional (2D) models have been widely used in screening potential toxic compounds but have failed to accurately reproduce the three-dimensionality (3D) of the liver milieu. To overcome these limitations, improved 3D culture techniques have been developed to recapitulate the hepatic native microenvironment. These models focus on reproducing the liver architecture, representing both parenchymal and nonparenchymal cells, as well as cell interactions. More recently, Liver-on-Chip (LoC) models have been developed with the aim of providing physiological fluid flow and thus achieving essential hepatic functions. Given their unprecedented ability to recapitulate critical features of the liver cellular environments, LoC have been extensively adopted in pathophysiological modelling and currently represent a promising tool for tissue engineering and drug screening applications. In this review, we discuss the evolution of experimental liver models, from the ancient 2D hepatocyte models, widely used for liver toxicity screening, to 3D and LoC culture strategies adopted for mirroring a more physiological microenvironment for the study of liver diseases.
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Affiliation(s)
- Michela Anna Polidoro
- Hepatobiliary Immunopathology Laboratory, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy
| | - Erika Ferrari
- Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milan, Italy
| | - Simona Marzorati
- Hepatobiliary Immunopathology Laboratory, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy
| | - Ana Lleo
- Department of Biomedical Sciences, Humanitas University, Pieve Emanuele, Milan, Italy.,Division of Internal Medicine and Hepatology, Department of Gastroenterology, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy
| | - Marco Rasponi
- Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milan, Italy
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47
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Luce E, Messina A, Duclos-Vallée JC, Dubart-Kupperschmitt A. Advanced Techniques and Awaited Clinical Applications for Human Pluripotent Stem Cell Differentiation into Hepatocytes. Hepatology 2021; 74:1101-1116. [PMID: 33420753 PMCID: PMC8457237 DOI: 10.1002/hep.31705] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/13/2020] [Revised: 11/16/2020] [Accepted: 12/19/2020] [Indexed: 12/22/2022]
Abstract
Liver transplantation is currently the only curative treatment for several liver diseases such as acute liver failure, end-stage liver disorders, primary liver cancers, and certain genetic conditions. Unfortunately, despite improvements to transplantation techniques, including live donor transplantation, the number of organs available remains insufficient to meet patient needs. Hepatocyte transplantation has enabled some encouraging results as an alternative to organ transplantation, but primary hepatocytes are little available and cannot be amplified using traditional two-dimensional culture systems. Indeed, although recent studies have tended to show that three-dimensional culture enables long-term hepatocyte culture, it is still agreed that, like most adult primary cell types, hepatocytes remain refractory to in vitro expansion. Because of their exceptional properties, human pluripotent stem cells (hPSCs) can be amplified indefinitely and differentiated into any cell type, including liver cells. While many teams have worked on hepatocyte differentiation, there has been a consensus that cells obtained after hPSC differentiation have more fetal than adult hepatocyte characteristics. New technologies have been used to improve the differentiation process in recent years. This review discusses the technical improvements made to hepatocyte differentiation protocols and the clinical approaches developed to date and anticipated in the near future.
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Affiliation(s)
- Eléanor Luce
- INSERMUniversité Paris-SaclayUnité Mixte de Recherche (UMR_S) 1193VillejuifFrance.,Fédération Hospitalo-Universitaire HépatinovHôpital Paul-BrousseVillejuifFrance
| | - Antonietta Messina
- INSERMUniversité Paris-SaclayUnité Mixte de Recherche (UMR_S) 1193VillejuifFrance.,Fédération Hospitalo-Universitaire HépatinovHôpital Paul-BrousseVillejuifFrance
| | - Jean-Charles Duclos-Vallée
- INSERMUniversité Paris-SaclayUnité Mixte de Recherche (UMR_S) 1193VillejuifFrance.,Fédération Hospitalo-Universitaire HépatinovHôpital Paul-BrousseVillejuifFrance
| | - Anne Dubart-Kupperschmitt
- INSERMUniversité Paris-SaclayUnité Mixte de Recherche (UMR_S) 1193VillejuifFrance.,Fédération Hospitalo-Universitaire HépatinovHôpital Paul-BrousseVillejuifFrance
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48
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Pasqua M, Di Gesù R, Chinnici CM, Conaldi PG, Francipane MG. Generation of Hepatobiliary Cell Lineages from Human Induced Pluripotent Stem Cells: Applications in Disease Modeling and Drug Screening. Int J Mol Sci 2021; 22:8227. [PMID: 34360991 PMCID: PMC8348238 DOI: 10.3390/ijms22158227] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2021] [Revised: 07/28/2021] [Accepted: 07/28/2021] [Indexed: 12/13/2022] Open
Abstract
The possibility to reproduce key tissue functions in vitro from induced pluripotent stem cells (iPSCs) is offering an incredible opportunity to gain better insight into biological mechanisms underlying development and disease, and a tool for the rapid screening of drug candidates. This review attempts to summarize recent strategies for specification of iPSCs towards hepatobiliary lineages -hepatocytes and cholangiocytes-and their use as platforms for disease modeling and drug testing. The application of different tissue-engineering methods to promote accurate and reliable readouts is discussed. Space is given to open questions, including to what extent these novel systems can be informative. Potential pathways for improvement are finally suggested.
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Affiliation(s)
- Mattia Pasqua
- Fondazione Ri.MED, 90133 Palermo, Italy; (M.P.); (R.D.G.); (C.M.C.)
| | - Roberto Di Gesù
- Fondazione Ri.MED, 90133 Palermo, Italy; (M.P.); (R.D.G.); (C.M.C.)
| | - Cinzia Maria Chinnici
- Fondazione Ri.MED, 90133 Palermo, Italy; (M.P.); (R.D.G.); (C.M.C.)
- Dipartimento della Ricerca, IRCCS ISMETT, 90127 Palermo, Italy;
| | | | - Maria Giovanna Francipane
- Fondazione Ri.MED, 90133 Palermo, Italy; (M.P.); (R.D.G.); (C.M.C.)
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA
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49
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Luo C, Lü D, Zheng L, Zhang F, Zhang X, Lü S, Zhang C, Jia X, Shu X, Li P, Li Z, Long M. Hepatic differentiation of human embryonic stem cells by coupling substrate stiffness and microtopography. Biomater Sci 2021; 9:3776-3790. [PMID: 33876166 DOI: 10.1039/d1bm00174d] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Mechanical or physical cues are associated with the growth and differentiation of embryonic stem cells (ESCs). While the substrate stiffness or topography independently affects the differentiation of ESCs, their cooperative regulation on lineage-specific differentiation remains largely unknown. Here, four topographical configurations on stiff or soft polyacrylamide hydrogel were combined to direct hepatic differentiation of human H1 cells via a four-stage protocol, and the coupled impacts of stiffness and topography were quantified at distinct stages. Data indicated that the substrate stiffness is dominant in stemness maintenance on stiff gel and hepatic differentiation on soft gel while substrate topography assists the differentiation of hepatocyte-like cells in positive correlation with the circularity of H1 clones initially formed on the substrate. The differentiated cells exhibited liver-specific functions such as maintaining the capacities of CYP450 metabolism, glycogen synthesis, ICG engulfment, and repairing liver injury in CCl4-treated mice. These results implied that the coupling of substrate stiffness and topography, combined with the biochemical signals, is favorable to improve the efficiency and functionality of hepatic differentiation of human ESCs.
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Affiliation(s)
- Chunhua Luo
- Center for Biomechanics and Bioengineering, Key Laboratory of Microgravity (National Microgravity Laboratory) and Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, China.
| | - Dongyuan Lü
- Center for Biomechanics and Bioengineering, Key Laboratory of Microgravity (National Microgravity Laboratory) and Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, China. and School of Engineering Science, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Lu Zheng
- Center for Biomechanics and Bioengineering, Key Laboratory of Microgravity (National Microgravity Laboratory) and Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, China. and School of Engineering Science, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Fan Zhang
- Center for Biomechanics and Bioengineering, Key Laboratory of Microgravity (National Microgravity Laboratory) and Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, China. and School of Engineering Science, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Xiao Zhang
- Center for Biomechanics and Bioengineering, Key Laboratory of Microgravity (National Microgravity Laboratory) and Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, China. and School of Engineering Science, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Shouqin Lü
- Center for Biomechanics and Bioengineering, Key Laboratory of Microgravity (National Microgravity Laboratory) and Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, China. and School of Engineering Science, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Chen Zhang
- Center for Biomechanics and Bioengineering, Key Laboratory of Microgravity (National Microgravity Laboratory) and Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, China.
| | - Xiaohua Jia
- CAS Key Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of Sciences, Beijing 100190, China
| | - Xinyu Shu
- Center for Biomechanics and Bioengineering, Key Laboratory of Microgravity (National Microgravity Laboratory) and Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, China. and School of Engineering Science, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Peiwen Li
- Center for Biomechanics and Bioengineering, Key Laboratory of Microgravity (National Microgravity Laboratory) and Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, China.
| | - Zhan Li
- Center for Biomechanics and Bioengineering, Key Laboratory of Microgravity (National Microgravity Laboratory) and Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, China.
| | - Mian Long
- Center for Biomechanics and Bioengineering, Key Laboratory of Microgravity (National Microgravity Laboratory) and Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, China. and School of Engineering Science, University of Chinese Academy of Sciences, Beijing 100049, China
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50
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Dannemann M, Gallego Romero I. Harnessing pluripotent stem cells as models to decipher human evolution. FEBS J 2021; 289:2992-3010. [PMID: 33876573 DOI: 10.1111/febs.15885] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2021] [Revised: 03/18/2021] [Accepted: 04/16/2021] [Indexed: 12/13/2022]
Abstract
The study of human evolution, long constrained by a lack of experimental model systems, has been transformed by the emergence of the induced pluripotent stem cell (iPSC) field. iPSCs can be readily established from noninvasive tissue sources, from both humans and other primates; they can be maintained in the laboratory indefinitely, and they can be differentiated into other tissue types. These qualities mean that iPSCs are rapidly becoming established as viable and powerful model systems with which it is possible to address questions in human evolution that were until now logistically and ethically intractable, especially in the quest to understand humans' place among the great apes, and the genetic basis of human uniqueness. In this review, we discuss the key lessons and takeaways of this nascent field; from the types of research, iPSCs make possible to lingering challenges and likely future directions. We provide a comprehensive overview of how the seemingly unlikely combination of iPSCs and explicit evolutionary frameworks is transforming what is possible in our understanding of humanity's past and present.
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Affiliation(s)
| | - Irene Gallego Romero
- Institute of Genomics, University of Tartu, Estonia.,Melbourne Integrative Genomics, The University of Melbourne, Parkville, Australia.,School of BioSciences, The University of Melbourne, Parkville, Australia.,The Centre for Stem Cell Systems, The University of Melbourne, Parkville, Australia
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