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Stancioiu FA, Bogdan R, Ivanescu B, Dumitrescu R. Autologous cord blood vs individualized supplements in autistic spectrum disorder: CORDUS study results. World J Clin Pediatr 2025; 14:96643. [DOI: 10.5409/wjcp.v14.i1.96643] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/11/2024] [Revised: 10/03/2024] [Accepted: 12/06/2024] [Indexed: 12/20/2024] Open
Abstract
BACKGROUND Cellular therapies have started an important new therapeutic direction in autistic spectrum disorder (ASD), and the ample diversity of ASD pathophysiology and the different types of cell therapies prompt an equally ample effort to employ clinical studies for studying the ASD causes and cell therapies. Stem cells have yielded so far mixed results in clinical trials, and at patient level the results varied from impressive to no improvement. In this context we have administered autologous cord blood (ACB) and a non-placebo, material intervention represented by an individualized combination of supplements (ICS) to ASD children.
AIM To compare the efficacy of ACB vs ICS and find markers correlated with the child's progress in order to better predict ACB efficacy.
METHODS CORDUS clinical study is a crossover study in which both oral ICS and intravenous ACB were sequentially administered to 56 children; ACB was infused as an inpatient procedure. Treatment efficacy was evaluated pre-treatment and post-treatment at 6 months by an independent psychotherapist with Autism Treatment Evaluation Checklist, Quantitative Checklist for Autism in Toddlers and a 16-item comparative table score, after interviewing the children’s parents and therapists. Before and after each intervention participants had a set of blood tests including inflammatory, metabolic and oxidative markers, and the neuronal specific enolase.
RESULTS No serious adverse reactions were noted during and after cord blood or supplement administration. ACB improved evaluation scores in 78% of children with age 3–7-years (n = 28), but was much less effective in kids older than 8 years or with body weight of more than 35 kg (n = 28; only 11% of children improved scores). ICS yielded better results than ACB in 5 cases out of 28, while in 23 kids ACB brought more improvement than ICS (P < 0.05); high initial levels of inflammation and ferritin were associated with no improvement. Ample individual differences were noted in children's progress, and statistically significant improvements were seen after ACB on areas such as verbalization and social interaction, but not on irritability or aggressive behavior.
CONCLUSION ACB has superior efficacy to ICS in ASD; high inflammation, ferritin, age and body weight predict less improvement; more clinical studies are needed for studying ACB efficacy in ASD.
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Affiliation(s)
- Felician A Stancioiu
- Department of Clinical Research, Bio-Forum Foundation, Bucharest 040245, Bucuresti, Romania
| | - Raluca Bogdan
- Department of Pediatrics, Medicover Hospital Bucharest, Bucharest 013982, Bucuresti, Romania
| | | | - Radu Dumitrescu
- Department of Anesthesiology and Intensive Therapy, Medicover Hospital, Bucharest 013982, Bucuresti, Romania
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Diane A, Mu-U-Min RBA, Al-Siddiqi HH. Epigenetic memory as crucial contributing factor in directing the differentiation of human iPSC into pancreatic β-cells in vitro. Cell Tissue Res 2025; 399:267-276. [PMID: 39883142 PMCID: PMC11870940 DOI: 10.1007/s00441-025-03952-8] [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: 10/16/2024] [Accepted: 01/20/2025] [Indexed: 01/31/2025]
Abstract
Impaired insulin secretion contributes to the pathogenesis of type 1 diabetes mellitus through autoimmune destruction of pancreatic β-cells and the pathogenesis of severe forms of type 2 diabetes mellitus through β-cell dedifferentiation and other mechanisms. Replenishment of malfunctioning β-cells via islet transplantation has the potential to induce long-term glycemic control in the body. However, this treatment option cannot widely be implemented in clinical due to healthy islet donor shortage. Emerging β-cell replacement with human-induced pluripotent stem cell (iPSC) provides high remedial therapy hopes. Thus, tremendous progress has been made in developing β-cell differentiation protocols in vitro; however, most of the differentiated iPSC-derived β-cells showed immature phenotypes associated with low efficiency depending on the iPSC lines used, creating a crucial barrier for their clinical implementation. Multiple mechanisms including differences in genetic, cell cycle patterns, and mitochondrial dysfunction underlie the defective differentiation propensity of iPSC into insulin-producing β-cells. Accumulating evidence recently indicated that, following the reprogramming, epigenetic memory inherited from parental cells substantially affects the differentiation capacity of many iPSC lines. Therefore, differences in epigenetic signature are likely to be essential contributing factors influencing the propensity of iPSC differentiation. In this review, we will document the impact of the epigenome on the reprogramming efficacy and differentiation potential of iPSCs and how targeting the epigenetic residual memory could be an additional strategy to improve the differentiation efficiency of existing protocols to generate fully functional hPSC-derived pancreatic β-cells for diabetes therapy and drug screening.
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Affiliation(s)
- Abdoulaye Diane
- Diabetes Research Center, Qatar Biomedical Research Institute (QBRI), Qatar Foundation (QF), Hamad Bin Khalifa University (HBKU), Doha, Qatar.
| | - Razik Bin Abdul Mu-U-Min
- Diabetes Research Center, Qatar Biomedical Research Institute (QBRI), Qatar Foundation (QF), Hamad Bin Khalifa University (HBKU), Doha, Qatar
| | - Heba Hussain Al-Siddiqi
- Diabetes Research Center, Qatar Biomedical Research Institute (QBRI), Qatar Foundation (QF), Hamad Bin Khalifa University (HBKU), Doha, Qatar
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Genova E, Rispoli P, Fengming Y, Kohei J, Bramuzzo M, Bulla R, Lucafò M, Ferraro RM, Decorti G, Stocco G. Time-efficient strategies in human iPS cell-derived pancreatic progenitor differentiation and cryopreservation: advancing towards practical applications. Stem Cell Res Ther 2024; 15:483. [PMID: 39695795 DOI: 10.1186/s13287-024-04068-6] [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: 08/30/2024] [Accepted: 11/20/2024] [Indexed: 12/20/2024] Open
Abstract
BACKGROUND Differentiation of patient-specific induced pluripotent stem cells (iPS) helps researchers to study the individual sensibility to drugs. However, differentiation protocols are time-consuming, and not all tissues have been studied. Few works are available regarding pancreatic exocrine differentiation of iPS cells, and little is known on culturing and cryopreserving these cells. METHODS We differentiated the iPS cells of two pediatric Crohn's disease patients into pancreatic progenitors and exocrine cells, adapting and shortening a protocol for differentiating embryonic stem cells. We analyzed the expression of key genes and proteins of the differentiation process by qPCR and immunofluorescence, respectively. We explored the possibility of keeping differentiated cells in culture and freezing and thawing them to shorten the time needed for the differentiation. We analyzed the cell cycle of undifferentiated and differentiated cells by flow cytometry. RESULTS The analysis of mRNA levels of key pancreatic differentiation genes PDX1 and pancreatic amylase indicate that iPS cells were successfully differentiated into pancreatic exocrine cells with expression of PDX1 (one way ANOVA p < 0.0001), and the two isoforms of amylase (one way ANOVA p < 0.05) significantly higher in exocrine cells in comparison to iPS cells. Differentiation efficiency was also confirmed by immunofluorescence analysis of PDX1 and amylase. We confirmed the possibility of shortening the time necessary for obtaining pancreatic cells without losing differentiation efficiency. Pancreatic progenitors and exocrine cells were maintained in culture and cryopreserved. Interestingly, the stemness marker OCT4 resulted significantly lower after subculturing (OCT4 p < 0.001; one-way ANOVA) and after freezing and thawing procedures (p < 0.05, one-way ANOVA) suggesting a reduction of undifferentiated stem cells leading to a purer population of pancreatic progenitor cells. Also, the stemness marker NANOG resulted lower after passaging, corroborating this result. CONCLUSIONS In this work, we optimized the generation of patient-specific pancreatic differentiated cells and laid the foundation for creating a bank of patient-specific pancreatic lines exploitable for tailored pharmacological assays. TRIAL REGISTRATION The study was approved by the Ethical Committee of the Institute of Maternal and Child Health IRCCS Burlo Garofolo, with approval number 1556 (internal ID RC 44/22).
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Affiliation(s)
- Elena Genova
- Institute for Maternal and Child Health - IRCCS Burlo Garofolo, Trieste, Italy
| | - Paola Rispoli
- Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy
| | - Yue Fengming
- Department of Histology and Embryology, Shinshu University School of Medicine, Matsumoto, Japan
- Institute for Biomedical Sciences, Shinshu University Interdisciplinary Cluster for Cutting Edge Research, Matsumoto, Japan
| | - Johkura Kohei
- Department of Histology and Embryology, Shinshu University School of Medicine, Matsumoto, Japan
| | - Matteo Bramuzzo
- Institute for Maternal and Child Health - IRCCS Burlo Garofolo, Trieste, Italy
| | - Roberta Bulla
- Department of Life Sciences, University of Trieste, Trieste, Italy
| | - Marianna Lucafò
- Department of Life Sciences, University of Trieste, Trieste, Italy
| | - Rosalba Monica Ferraro
- Angelo Nocivelli Institute for Molecular Medicine, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
| | - Giuliana Decorti
- Institute for Maternal and Child Health - IRCCS Burlo Garofolo, Trieste, Italy
- Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy
| | - Gabriele Stocco
- Institute for Maternal and Child Health - IRCCS Burlo Garofolo, Trieste, Italy.
- Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy.
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Modaresi S, Pacelli S, Chakraborty A, Coyle A, Luo W, Singh I, Paul A. Engineering a Microfluidic Platform to Cryopreserve Stem Cells: A DMSO-Free Sustainable Approach. Adv Healthc Mater 2024; 13:e2401264. [PMID: 39152923 PMCID: PMC11582517 DOI: 10.1002/adhm.202401264] [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: 04/05/2024] [Revised: 06/24/2024] [Indexed: 08/19/2024]
Abstract
Human adipose-derived stem cells (hASCs) are cryopreserved traditionally using dimethyl sulfoxide (DMSO) as the cryoprotectant agent. DMSO penetrates cell membranes and prevents cellular damage during cryopreservation. However, DMSO is not inert to cells, inducing cytotoxic effects by causing mitochondrial dysfunction, reduced cell proliferation, and impaired hASCs transplantation. Additionally, large-scale production of DMSO and contamination can adversely impact the environment. A sustainable, green alternative to DMSO is trehalose, a natural disaccharide cryoprotectant agent that does not pose any risk of cytotoxicity. However, the cellular permeability of trehalose is less compared to DMSO. Here, a microfluidic chip is developed for the intracellular delivery of trehalose in hASCs. The chip is designed for mechanoporation, which creates transient pores in cell membranes by mechanical deformation. Mechanoporation allows the sparingly permeable trehalose to be internalized within the cell cytosol. The amount of trehalose delivered intracellularly is quantified and optimized based on cellular compatibility and functionality. Furthermore, whole-transcriptome sequencing confirms that less than 1% of all target genes display at least a twofold change in expression when cells are passed through the chip compared to untreated cells. Overall, the results confirm the feasibility and effectiveness of using this microfluidic chip for DMSO-free cryopreservation of hASCs.
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Affiliation(s)
- Saman Modaresi
- Department of Chemical and Petroleum Engineering, Bioengineering Graduate Program, School of Engineering, The University of Kansas, Lawrence, KS, 66045, USA
| | - Settimio Pacelli
- Department of Biomedical Engineering, Illinois Institute of Technology, Chicago, IL, 60616, USA
| | - Aishik Chakraborty
- Department of Chemical and Biochemical Engineering, The University of Western Ontario, London, ON, N6A 5B9, Canada
- Collaborative Specialization in Musculoskeletal Health Research and Bone and Joint Institute, The University of Western Ontario, London, ON, N6A 5B9, Canada
| | - Ali Coyle
- School of Biomedical Engineering, The University of Western Ontario, London, ON, N6A 5B9, Canada
| | - Wei Luo
- Department of Chemical and Biochemical Engineering, The University of Western Ontario, London, ON, N6A 5B9, Canada
| | - Irtisha Singh
- Department of Cell Biology and Genetics, College of Medicine, Texas A&M University, Bryan, TX, 77807, USA
- Department of Biomedical Engineering, College of Engineering, Texas A&M University, College Station, TX, 77843, USA
- Interdisciplinary Program in Genetics and Genomics, Texas A&M University, College Station, TX, 77840, USA
| | - Arghya Paul
- Department of Chemical and Biochemical Engineering, The University of Western Ontario, London, ON, N6A 5B9, Canada
- Collaborative Specialization in Musculoskeletal Health Research and Bone and Joint Institute, The University of Western Ontario, London, ON, N6A 5B9, Canada
- School of Biomedical Engineering, The University of Western Ontario, London, ON, N6A 5B9, Canada
- Department of Chemistry, The Center for Advanced Materials and Biomaterials Research, The University of Western Ontario, London, ON, N6A 5B9, Canada
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Liu M, Liang L, Yu C, Guo B, Zhang H, Yao F, Zhang H, Li J. Enhancing cell cryopreservation with acidic polyamino acids integrated liquid marbles. Colloids Surf B Biointerfaces 2024; 241:114055. [PMID: 38936034 DOI: 10.1016/j.colsurfb.2024.114055] [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: 01/02/2024] [Revised: 05/16/2024] [Accepted: 06/22/2024] [Indexed: 06/29/2024]
Abstract
Cryopreservation is highly desired for long-term maintenance of the viability of living biosamples, while effective cell cryopreservation still relies heavily on the addition of dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS). However, the intrinsic toxicity of DMSO is still a bottleneck, which could not only cause the clinical side effect but also induce cell genetic variants. In the meantime, the addition of FBS may bring potentially the risk of pathogenic microorganism contamination. The liquid marbles (LMs), a novel biotechnology tool for cell cryopreservation, which not only have a small volume system that facilitated recovery, but the hydrophobic shell also resisted the harm to cells caused by adverse environments. Previous LM-based cell cryopreservation relied heavily on the addition of FBS. In this work, we introduced acidic polyaspartic acid and polyglutamic acid as cryoprotectants to construct LM systems. LMs could burst in an instant to facilitate and achieve ultrarapid recovery process, and the hydrophilic carboxyl groups of the cryoprotectants could form hydrogen bonds with water molecules and further inhibit ice growth/formation to protect cells from cryoinjuries. The L929 cells could be well cryopreserved by acidic polyamino acid-based LMs. This new biotechnology platform is expected to be widely used for cell cryopreservation, which has the potential to propel LMs for the preservation of various functional cells in the future.
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Affiliation(s)
- Min Liu
- Department of Polymer Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Lei Liang
- Department of Polymer Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Chaojie Yu
- Department of Polymer Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Bingyan Guo
- Department of Polymer Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Haitao Zhang
- Department of Polymer Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Fanglian Yao
- Department of Polymer Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China; Frontiers Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300350, China
| | - Hong Zhang
- Department of Polymer Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
| | - Junjie Li
- Department of Polymer Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China; Frontiers Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300350, China.
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Gokaltun A, Asik E, Byrne D, Yarmush ML, Usta OB. Supercooled preservation of cultured primary rat hepatocyte monolayers. Front Bioeng Biotechnol 2024; 12:1429412. [PMID: 39076209 PMCID: PMC11284110 DOI: 10.3389/fbioe.2024.1429412] [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: 05/08/2024] [Accepted: 06/27/2024] [Indexed: 07/31/2024] Open
Abstract
Supercooled preservation (SCP) is a technology that involves cooling a substance below its freezing point without initiating ice crystal formation. It is a promising alternative to prolong the preservation time of cells, tissues, engineered tissue products, and organs compared to the current practices of hypothermic storage. Two-dimensional (2D) engineered tissues are extensively used in in vitro research for drug screening and development and investigation of disease progression. Despite their widespread application, there is a lack of research on the SCP of 2D-engineered tissues. In this study, we presented the effects of SCP at -2 and -6°C on primary rat hepatocyte (PRH) monolayers for the first time and compared cell viability and functionality with cold storage (CS, + 4°C). We preserved PRH monolayers in two different commercially available solutions: Hypothermosol-FRS (HTS-FRS) and the University of Wisconsin (UW) with and without supplements (i.e., polyethylene glycol (PEG) and 3-O-Methyl-Α-D-Glucopyranose (3-OMG)). Our findings revealed that UW with and without supplements were inadequate for the short-term preservation of PRH monolayers for both SCP and CS with high viability, functionality, and monolayer integrity. The combination of supplements (PEG and 3-OMG) in the HTS-FRS solution outperformed the other groups and yielded the highest viability and functional capacity. Notably, PRH monolayers exhibited superior viability and functionality when stored at -2°C through SCP for up to 3 days compared to CS. Overall, our results demonstrated that SCP is a feasible approach to improving the short-term preservation of PRH monolayers and enables readily available 2D-engineered tissues to advance in vitro research. Furthermore, our findings provide insights into preservation outcomes across various biological levels, from cells to tissues and organs, contributing to the advancement of bioengineering and biotechnology.
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Affiliation(s)
- Aslihan Gokaltun
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States
- Shriners Hospitals for Children, Boston, MA, United States
- Department of Chemical and Biological Engineering, Tufts University, Medford, MA, United States
- Department of Chemical Engineering, Hacettepe University, Ankara, Türkiye
| | - Eda Asik
- Shriners Hospitals for Children, Boston, MA, United States
- Department of Bioengineering, Hacettepe University, Ankara, Türkiye
| | - Delaney Byrne
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States
- Shriners Hospitals for Children, Boston, MA, United States
| | - Martin L. Yarmush
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States
- Shriners Hospitals for Children, Boston, MA, United States
- Department of Biomedical Engineering, Rutgers University, Newark, NJ, United States
| | - O. Berk Usta
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States
- Shriners Hospitals for Children, Boston, MA, United States
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Aranda-Anzaldo A, Dent MAR, Segura-Anaya E, Martínez-Gómez A. Protein folding, cellular stress and cancer. PROGRESS IN BIOPHYSICS AND MOLECULAR BIOLOGY 2024; 191:40-57. [PMID: 38969306 DOI: 10.1016/j.pbiomolbio.2024.07.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/12/2024] [Revised: 06/30/2024] [Accepted: 07/02/2024] [Indexed: 07/07/2024]
Abstract
Proteins are acknowledged as the phenotypical manifestation of the genotype, because protein-coding genes carry the information for the strings of amino acids that constitute the proteins. It is widely accepted that protein function depends on the corresponding "native" structure or folding achieved within the cell, and that native protein folding corresponds to the lowest free energy minimum for a given protein. However, protein folding within the cell is a non-deterministic dissipative process that from the same input may produce different outcomes, thus conformational heterogeneity of folded proteins is the rule and not the exception. Local changes in the intracellular environment promote variation in protein folding. Hence protein folding requires "supervision" by a host of chaperones and co-chaperones that help their client proteins to achieve the folding that is most stable according to the local environment. Such environmental influence on protein folding is continuously transduced with the help of the cellular stress responses (CSRs) and this may lead to changes in the rules of engagement between proteins, so that the corresponding protein interactome could be modified by the environment leading to an alternative cellular phenotype. This allows for a phenotypic plasticity useful for adapting to sudden and/or transient environmental changes at the cellular level. Starting from this perspective, hereunder we develop the argument that the presence of sustained cellular stress coupled to efficient CSRs may lead to the selection of an aberrant phenotype as the resulting adaptation of the cellular proteome (and the corresponding interactome) to such stressful conditions, and this can be a common epigenetic pathway to cancer.
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Affiliation(s)
- Armando Aranda-Anzaldo
- Laboratorio de Biología Molecular y Neurociencias, Facultad de Medicina, Universidad Autónoma del Estado de México, Paseo Tollocan y Jesús Carranza s/n, Toluca, 50180, Edo. Méx., Mexico.
| | - Myrna A R Dent
- Laboratorio de Biología Molecular y Neurociencias, Facultad de Medicina, Universidad Autónoma del Estado de México, Paseo Tollocan y Jesús Carranza s/n, Toluca, 50180, Edo. Méx., Mexico
| | - Edith Segura-Anaya
- Laboratorio de Biología Molecular y Neurociencias, Facultad de Medicina, Universidad Autónoma del Estado de México, Paseo Tollocan y Jesús Carranza s/n, Toluca, 50180, Edo. Méx., Mexico
| | - Alejandro Martínez-Gómez
- Laboratorio de Biología Molecular y Neurociencias, Facultad de Medicina, Universidad Autónoma del Estado de México, Paseo Tollocan y Jesús Carranza s/n, Toluca, 50180, Edo. Méx., Mexico
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Lundin BF, Knight GT, Fedorchak NJ, Krucki K, Iyer N, Maher JE, Izban NR, Roberts A, Cicero MR, Robinson JF, Iskandar BJ, Willett R, Ashton RS. RosetteArray ® Platform for Quantitative High-Throughput Screening of Human Neurodevelopmental Risk. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.04.01.587605. [PMID: 38798648 PMCID: PMC11118315 DOI: 10.1101/2024.04.01.587605] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/29/2024]
Abstract
Neural organoids have revolutionized how human neurodevelopmental disorders (NDDs) are studied. Yet, their utility for screening complex NDD etiologies and in drug discovery is limited by a lack of scalable and quantifiable derivation formats. Here, we describe the RosetteArray® platform's ability to be used as an off-the-shelf, 96-well plate assay that standardizes incipient forebrain and spinal cord organoid morphogenesis as micropatterned, 3-D, singularly polarized neural rosette tissues (>9000 per plate). RosetteArrays are seeded from cryopreserved human pluripotent stem cells, cultured over 6-8 days, and immunostained images can be quantified using artificial intelligence-based software. We demonstrate the platform's suitability for screening developmental neurotoxicity and genetic and environmental factors known to cause neural tube defect risk. Given the presence of rosette morphogenesis perturbation in neural organoid models of NDDs and neurodegenerative disorders, the RosetteArray platform could enable quantitative high-throughput screening (qHTS) of human neurodevelopmental risk across regulatory and precision medicine applications.
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Affiliation(s)
- Brady F. Lundin
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53705, USA
- Medical Scientist Training Program, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, WI, 53705 USA
- Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA
| | - Gavin T. Knight
- Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA
- Neurosetta LLC, 330 N. Orchard Street Rm 4140A, Madison, WI 53715 USA
| | | | - Kevin Krucki
- Neurosetta LLC, 330 N. Orchard Street Rm 4140A, Madison, WI 53715 USA
| | - Nisha Iyer
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53705, USA
- Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA
| | - Jack E. Maher
- Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA
| | - Nicholas R. Izban
- Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA
| | - Abilene Roberts
- Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA
| | - Madeline R. Cicero
- Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA
| | - Joshua F. Robinson
- Center of Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Bermans J. Iskandar
- Department of Neurological Surgery, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, WI 53705, USA
| | - Rebecca Willett
- Neurosetta LLC, 330 N. Orchard Street Rm 4140A, Madison, WI 53715 USA
- Departments of Statistics and Computer Science, University of Chicago, Chicago, IL 60637, USA
| | - Randolph S. Ashton
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53705, USA
- Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA
- Neurosetta LLC, 330 N. Orchard Street Rm 4140A, Madison, WI 53715 USA
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Tunçer Çağlayan S, Gurbanov R. Modulation of bacterial membranes and cellular macromolecules by dimethyl sulfoxide: A dose-dependent study providing novel insights. Int J Biol Macromol 2024; 267:131581. [PMID: 38615866 DOI: 10.1016/j.ijbiomac.2024.131581] [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: 11/10/2023] [Revised: 04/10/2024] [Accepted: 04/11/2024] [Indexed: 04/16/2024]
Abstract
Using Escherichia coli as a model, this manuscript delves into the intricate interactions between dimethyl sulfoxide (DMSO) and membranes, cellular macromolecules, and the effects on various aspects of bacterial physiology. Given DMSO's wide-ranging use as a solvent in microbiology, we investigate the impacts of both non-growth inhibitory (1.0 % and 2.5 % v/v) and slightly growth-inhibitory (5.0 % v/v) concentrations of DMSO. The results demonstrate that DMSO causes alterations in bacterial membrane potential, influences the electrochemical characteristics of the cell surface, and exerts substantial effects on the composition and structure of cellular biomolecules. Genome-wide gene expression data from DMSO-treated E. coli was used to further investigate and bolster the results. The findings of this study provide valuable insights into the complex relationship between DMSO and biological systems, with potential implications in drug delivery and cellular manipulation. However, it is essential to exercise caution when utilizing DMSO to enhance the solubility and delivery of bioactive compounds, as even at low concentrations, DMSO exerts non-inert effects on cellular macromolecules and processes.
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Affiliation(s)
- Sinem Tunçer Çağlayan
- Vocational School of Health Services, Department of Medical Services and Techniques, Bilecik Şeyh Edebali University, 11100 Bilecik, Turkey.
| | - Rafig Gurbanov
- Department of Bioengineering, Bilecik Şeyh Edebali University, 11100 Bilecik, Turkey; Central Research Laboratory, Bilecik Şeyh Edebali University, 11100 Bilecik, Turkey
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10
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Agriesti F, Cela O, Capitanio N. "Time Is out of Joint" in Pluripotent Stem Cells: How and Why. Int J Mol Sci 2024; 25:2063. [PMID: 38396740 PMCID: PMC10889767 DOI: 10.3390/ijms25042063] [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: 01/10/2024] [Revised: 02/02/2024] [Accepted: 02/06/2024] [Indexed: 02/25/2024] Open
Abstract
The circadian rhythm is necessary for the homeostasis and health of living organisms. Molecular clocks interconnected by transcription/translation feedback loops exist in most cells of the body. A puzzling exemption to this, otherwise, general biological hallmark is given by the cell physiology of pluripotent stem cells (PSCs) that lack circadian oscillations gradually acquired following their in vivo programmed differentiation. This process can be nicely phenocopied following in vitro commitment and reversed during the reprogramming of somatic cells to induce PSCs. The current understanding of how and why pluripotency is "time-uncoupled" is largely incomplete. A complex picture is emerging where the circadian core clockwork is negatively regulated in PSCs at the post-transcriptional/translational, epigenetic, and other-clock-interaction levels. Moreover, non-canonical functions of circadian core-work components in the balance between pluripotency identity and metabolic-driven cell reprogramming are emerging. This review selects and discusses results of relevant recent investigations providing major insights into this context.
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Affiliation(s)
- Francesca Agriesti
- Department of Clinical and Experimental Medicine, University of Foggia, 71122 Foggia, Italy; (O.C.); (N.C.)
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11
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Ding L, Oh S, Shrestha J, Lam A, Wang Y, Radfar P, Warkiani ME. Scaling up stem cell production: harnessing the potential of microfluidic devices. Biotechnol Adv 2023; 69:108271. [PMID: 37844769 DOI: 10.1016/j.biotechadv.2023.108271] [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: 04/29/2023] [Revised: 10/08/2023] [Accepted: 10/13/2023] [Indexed: 10/18/2023]
Abstract
Stem cells are specialised cells characterised by their unique ability to both self-renew and transform into a wide array of specialised cell types. The widespread interest in stem cells for regenerative medicine and cultivated meat has led to a significant demand for these cells in both research and practical applications. Despite the growing need for stem cell manufacturing, the industry faces significant obstacles, including high costs for equipment and maintenance, complicated operation, and low product quality and yield. Microfluidic technology presents a promising solution to the abovementioned challenges. As an innovative approach for manipulating liquids and cells within microchannels, microfluidics offers a plethora of advantages at an industrial scale. These benefits encompass low setup costs, ease of operation and multiplexing, minimal energy consumption, and the added advantage of being labour-free. This review presents a thorough examination of the prominent microfluidic technologies employed in stem cell research and explores their promising applications in the burgeoning stem cell industry. It thoroughly examines how microfluidics can enhance cell harvesting from tissue samples, facilitate mixing and cryopreservation, streamline microcarrier production, and efficiently conduct cell separation, purification, washing, and final cell formulation post-culture.
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Affiliation(s)
- Lin Ding
- Smart MCs Pty Ltd, Ultimo, Sydney, 2007, Australia.
| | - Steve Oh
- Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, 138668, Singapore
| | - Jesus Shrestha
- School of Biomedical Engineering, University of Technology Sydney, Sydney, NSW 2007, Australia
| | - Alan Lam
- Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, 138668, Singapore
| | - Yaqing Wang
- School of Biomedical Engineering, University of Science and Technology of China, Hefei 230026, China; Suzhou Institute for Advanced Research, University of Science and Technology of China, Suzhou 215123, China
| | - Payar Radfar
- Smart MCs Pty Ltd, Ultimo, Sydney, 2007, Australia
| | - Majid Ebrahimi Warkiani
- School of Biomedical Engineering, University of Technology Sydney, Sydney, NSW 2007, Australia..
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12
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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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13
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Liang S, Zhao J, Baker RK, Tran E, Zhan L, Kieffer TJ. Differentiation of stem cell-derived pancreatic progenitors into insulin-secreting islet clusters in a multiwell-based static 3D culture system. CELL REPORTS METHODS 2023; 3:100466. [PMID: 37323565 PMCID: PMC10261893 DOI: 10.1016/j.crmeth.2023.100466] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/02/2022] [Revised: 11/08/2022] [Accepted: 04/12/2023] [Indexed: 06/17/2023]
Abstract
Orbital shaker-based suspension culture systems have been in widespread use for differentiating human pluripotent stem cell (hPSC)-derived pancreatic progenitors toward islet-like clusters during endocrine induction stages. However, reproducibility between experiments is hampered by variable degrees of cell loss in shaking cultures, which contributes to variable differentiation efficiencies. Here, we describe a 96-well-based static suspension culture method for differentiation of pancreatic progenitors into hPSC-islets. Compared with shaking culture, this static 3D culture system induces similar islet gene expression profiles during differentiation processes but significantly reduces cell loss and improves cell viability of endocrine clusters. This static culture method results in more reproducible and efficient generation of glucose-responsive, insulin-secreting hPSC-islets. The successful differentiation and well-to-well consistency in 96-well plates also provides a proof of principle that the static 3D culture system can serve as a platform for small-scale compound screening experiments as well as facilitating further protocol development.
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Affiliation(s)
- Shenghui Liang
- Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
| | - Jia Zhao
- Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
| | - Robert K. Baker
- Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
| | - Elisa Tran
- Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
| | - Lisa Zhan
- Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
| | - Timothy J. Kieffer
- Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
- Department of Surgery, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
- School of Biomedical Engineering, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
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14
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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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15
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Mattingly Z, Chetty S. Generation of Oligodendrocytes from Human Pluripotent and Embryonic Stem Cells. Methods Mol Biol 2023; 2683:89-101. [PMID: 37300769 DOI: 10.1007/978-1-0716-3287-1_8] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/12/2023]
Abstract
Oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes (OLs) can be generated using human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). By manipulating culture conditions, pluripotent cell types are serially guided through intermediary cell types, developing first into neural progenitor cells (NPCs) then OPCs before maturing as CNS-specific OLs. This procedure is conducted under adherent, feeder-free conditions to derive mature OLs in as few as 28 days.
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Affiliation(s)
- Zoe Mattingly
- Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA
| | - Sundari Chetty
- Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA.
- Department of Psychiatry, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
- Harvard Stem Cell Institute, Cambridge, MA, USA.
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16
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Liu M, Chen C, Yu J, Zhang H, Liang L, Guo B, Qiu Y, Yao F, Zhang H, Li J. The gelatin-based liquid marbles for cell cryopreservation. Mater Today Bio 2022; 17:100477. [DOI: 10.1016/j.mtbio.2022.100477] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2022] [Revised: 10/23/2022] [Accepted: 10/26/2022] [Indexed: 11/06/2022] Open
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17
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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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18
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Cheng H, Han Y, Zhang J, Zhang S, Zhai Y, An X, Li Q, Duan J, Zhang X, Li Z, Tang B, Shen H. Effects of dimethyl sulfoxide (DMSO) on DNA methylation and histone modification in parthenogenetically activated porcine embryos. Reprod Fertil Dev 2022; 34:598-607. [PMID: 35397781 DOI: 10.1071/rd21083] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2021] [Accepted: 12/20/2021] [Indexed: 12/18/2022] Open
Abstract
Epigenetic mechanisms play an important role in oogenesis and early embryo development in mammals. Dimethyl sulfoxide (DMSO) is frequently used as a solvent in biological studies and as a vehicle for drug therapy. Recent studies suggest that DMSO detrimentally affects porcine embryonic development, yet the mechanism of the process in parthenogenetically activated porcine embryos has not been reported. In this study, we found that treatment of embryos with 1.5% DMSO significantly decreased the cleavage and blastocyst rates, total cell number of blastocysts and the anti-apoptotic gene BCL-2 transcription level; however, the percentage of apoptotic cells and the expression levels of the pro-apoptotic gene BAX were not changed. Treatment with DMSO significantly decreased the expression levels of DNMT1 , DNMT3a , DNMT3b , TET1 , TET2 , TET3 , KMT2C , MLL2 and SETD3 in most of the stages of embryonic development and increased 5-mC signals, while the staining intensity for 5-hmC had no change in porcine preimplantation embryos from 2-cell to the blastocyst stages. Meanwhile, DMSO decreased the level of H3K4me3 during the development of parthenogenetically activated porcine embryos. After treatment with DMSO, expression levels of the pluripotency-related genes POU5F1 and NANOG decreased significantly (P <0.01), whereas the imprinted gene H19 did not change (P >0.05). In conclusion, these results suggest that DMSO can affect genome-wide DNA methylation and histone modification by regulating the expression of epigenetic modification enzymes, and DMSO also influences the expression level of pluripotent genes. These dysregulations lead to defects in embryonic development.
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Affiliation(s)
- Hui Cheng
- College of Veterinary Medicine, Jilin University, Changchun, Jilin 130062, China
| | - Yu Han
- College of Veterinary Medicine, Jilin University, Changchun, Jilin 130062, China
| | - Jian Zhang
- College of Veterinary Medicine, Jilin University, Changchun, Jilin 130062, China
| | - Sheng Zhang
- Academy of Translational Medicine, First Hospital, Jilin University, Changchun, Jilin 130021, China
| | - Yanhui Zhai
- College of Veterinary Medicine, Jilin University, Changchun, Jilin 130062, China
| | - Xinglan An
- Academy of Translational Medicine, First Hospital, Jilin University, Changchun, Jilin 130021, China
| | - Qi Li
- Academy of Translational Medicine, First Hospital, Jilin University, Changchun, Jilin 130021, China
| | - Jiahui Duan
- College of Veterinary Medicine, Jilin University, Changchun, Jilin 130062, China
| | - Xueming Zhang
- College of Veterinary Medicine, Jilin University, Changchun, Jilin 130062, China
| | - Ziyi Li
- Academy of Translational Medicine, First Hospital, Jilin University, Changchun, Jilin 130021, China
| | - Bo Tang
- College of Veterinary Medicine, Jilin University, Changchun, Jilin 130062, China
| | - Haiqing Shen
- College of Veterinary Medicine, Jilin University, Changchun, Jilin 130062, China
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Piao Z, Patel M, Park JK, Jeong B. Poly(l-alanine- co-l-lysine)- g-Trehalose as a Biomimetic Cryoprotectant for Stem Cells. Biomacromolecules 2022; 23:1995-2006. [PMID: 35412815 DOI: 10.1021/acs.biomac.1c01701] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Poly(l-alanine-co-l-lysine)-graft-trehalose (PAKT) was synthesized as a natural antifreezing glycopolypeptide (AFGP)-mimicking cryoprotectant for cryopreservation of mesenchymal stem cells (MSCs). FTIR and circular dichroism spectra indicated that the content of the α-helical structure of PAK decreased after conjugation with trehalose. Two protocols were investigated in cryopreservation of MSCs to prove the significance of the intracellularly delivered PAKT. In protocol I, MSCs were cryopreserved at -196 °C for 7 days by a slow-cooling procedure in the presence of both PAKT and free trehalose. In protocol II, MSCs were preincubated at 37 °C in a PAKT solution, followed by cryopreservation at -196 °C in the presence of free trehalose for 7 days by the slow-cooling procedure. Polymer and trehalose concentrations were varied by 0.0-1.0 and 0.0-15.0 wt %, respectively. Cell recovery was significantly improved by protocol II with preincubation of the cells in the PAKT solution. The recovered cells from protocol II exhibited excellent proliferation and maintained multilineage potentials into osteogenic, chondrogenic, and adipogenic differentiation, similar to MSCs recovered from cryopreservation in the traditional 10% dimethyl sulfoxide system. Ice recrystallization inhibition (IRI) activity of the polymers/trehalose contributed to cell recovery; however, intracellularly delivered PEG-PAKT was the major contributor to the enhanced cell recovery in protocol II. Inhibitor studies suggested that macropinocytosis and caveolin-dependent endocytosis are the main mechanisms for the intracellular delivery of PEG-PAKT. 1H NMR and FTIR spectra suggested that the intracellular PEG-PAKTs interact with water and stabilize the cells during cryopreservation.
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Affiliation(s)
- Zhengyu Piao
- Department of Chemistry and Nanoscience, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 03760, Korea
| | - Madhumita Patel
- Department of Chemistry and Nanoscience, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 03760, Korea
| | - Jin Kyung Park
- Department of Chemistry and Nanoscience, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 03760, Korea
| | - Byeongmoon Jeong
- Department of Chemistry and Nanoscience, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 03760, Korea
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20
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Soloperto A, Quaglio D, Baiocco P, Romeo I, Mori M, Ardini M, Presutti C, Sannino I, Ghirga S, Iazzetti A, Ippoliti R, Ruocco G, Botta B, Ghirga F, Di Angelantonio S, Boffi A. Rational design and synthesis of a novel BODIPY-based probe for selective imaging of tau tangles in human iPSC-derived cortical neurons. Sci Rep 2022; 12:5257. [PMID: 35347170 PMCID: PMC8960764 DOI: 10.1038/s41598-022-09016-z] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2021] [Accepted: 03/15/2022] [Indexed: 12/26/2022] Open
Abstract
Numerous studies have shown a strong correlation between the number of neurofibrillary tangles of the tau protein and Alzheimer's disease progression, making the quantitative detection of tau very promising from a clinical point of view. However, the lack of highly reliable fluorescent probes for selective imaging of tau neurofibrillary tangles is a major challenge due to sharing similar β–sheet motifs with homologous Amyloid-β fibrils. In the current work, we describe the rational design and the in silico evaluation of a small-size focused library of fluorescent probes, consisting of a BODIPY core (electron acceptor) featuring highly conjugated systems (electron donor) with a length in the range 13–19 Å at C3. Among the most promising probes in terms of binding mode, theoretical affinity and polarity, BT1 has been synthesized and tested in vitro onto human induced pluripotent stem cells derived neuronal cell cultures. The probe showed excellent photophysical properties and high selectivity allowing in vitro imaging of hyperphosphorylated tau protein filaments with minimal background noise. Our findings offer new insight into the structure-activity relationship of this class of tau selective fluorophores, paving the way for boosting tau tangle detection in patients possibly through retinal spectral scans.
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Affiliation(s)
- Alessandro Soloperto
- Center for Life Nano- & Neuro-Science, Istituto Italiano Di Tecnologia, 00161, Rome, Italy
| | - Deborah Quaglio
- Department of Chemistry and Technology of Drugs, Department of Excellence 2018-2022, Sapienza University of Rome, 00185, Rome, Italy
| | - Paola Baiocco
- Center for Life Nano- & Neuro-Science, Istituto Italiano Di Tecnologia, 00161, Rome, Italy.,Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, 00185, Rome, Italy
| | - Isabella Romeo
- Center for Life Nano- & Neuro-Science, Istituto Italiano Di Tecnologia, 00161, Rome, Italy.,Department of Chemistry and Technology of Drugs, Department of Excellence 2018-2022, Sapienza University of Rome, 00185, Rome, Italy
| | - Mattia Mori
- Department of Biotechnology, Chemistry and Pharmacy, Department of Excellence 2018-2022, University of Siena, 53100, Siena, Italy
| | - Matteo Ardini
- Department of Life, Health, and Environmental Sciences, University of L'Aquila, 67100, L'Aquila, Italy
| | - Caterina Presutti
- Center for Life Nano- & Neuro-Science, Istituto Italiano Di Tecnologia, 00161, Rome, Italy.,Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, 00185, Rome, Italy
| | - Ida Sannino
- Center for Life Nano- & Neuro-Science, Istituto Italiano Di Tecnologia, 00161, Rome, Italy
| | - Silvia Ghirga
- Center for Life Nano- & Neuro-Science, Istituto Italiano Di Tecnologia, 00161, Rome, Italy
| | - Antonia Iazzetti
- Department of Basic Biotechnological Sciences, Intensivological and Perioperative Clinics, Catholic University of Sacred Heart, 00168, Rome, Italy
| | - Rodolfo Ippoliti
- Department of Life, Health, and Environmental Sciences, University of L'Aquila, 67100, L'Aquila, Italy
| | - Giancarlo Ruocco
- Center for Life Nano- & Neuro-Science, Istituto Italiano Di Tecnologia, 00161, Rome, Italy
| | - Bruno Botta
- Department of Chemistry and Technology of Drugs, Department of Excellence 2018-2022, Sapienza University of Rome, 00185, Rome, Italy
| | - Francesca Ghirga
- Department of Chemistry and Technology of Drugs, Department of Excellence 2018-2022, Sapienza University of Rome, 00185, Rome, Italy.
| | - Silvia Di Angelantonio
- Center for Life Nano- & Neuro-Science, Istituto Italiano Di Tecnologia, 00161, Rome, Italy. .,Department of Physiology and Pharmacology, Sapienza University of Rome, 00185, Rome, Italy.
| | - Alberto Boffi
- Center for Life Nano- & Neuro-Science, Istituto Italiano Di Tecnologia, 00161, Rome, Italy.,Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, 00185, Rome, Italy
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21
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Franck S, Couvreu De Deckersberg E, Bubenik JL, Markouli C, Barbé L, Allemeersch J, Hilven P, Duqué G, Swanson MS, Gheldof A, Spits C, Sermon KD. Myotonic dystrophy type 1 embryonic stem cells show decreased myogenic potential, increased CpG methylation at the DMPK locus and RNA mis-splicing. Biol Open 2022; 11:273965. [PMID: 35019138 PMCID: PMC8764412 DOI: 10.1242/bio.058978] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2021] [Accepted: 11/29/2021] [Indexed: 12/12/2022] Open
Abstract
Skeletal muscle tissue is severely affected in myotonic dystrophy type 1 (DM1) patients, characterised by muscle weakness, myotonia and muscle immaturity in the most severe congenital form of the disease. Previously, it was not known at what stage during myogenesis the DM1 phenotype appears. In this study we differentiated healthy and DM1 human embryonic stem cells to myoblasts and myotubes and compared their differentiation potential using a comprehensive multi-omics approach. We found myogenesis in DM1 cells to be abnormal with altered myotube generation compared to healthy cells. We did not find differentially expressed genes between DM1 and non-DM1 cell lines within the same developmental stage. However, during differentiation we observed an aberrant inflammatory response and increased CpG methylation upstream of the CTG repeat at the myoblast level and RNA mis-splicing at the myotube stage. We show that early myogenesis modelled in hESC reiterates the early developmental manifestation of DM1. Summary: Early developmental abnormalities in myotonic dystrophy type 1 are reiterated in vitro in myotubes differentiated from human embryonic stem cells that carry the mutation.
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Affiliation(s)
- Silvie Franck
- Department Reproduction and Genetics, Vrije Universiteit Brussel, Brussels 1090, Belgium
| | | | - Jodi L Bubenik
- Department of Molecular Genetics and Microbiology, Center for NeuroGenetics and the Genetics Institute, University of Florida, College of Medicine, Gainesville, FL 32610, USA
| | - Christina Markouli
- Department Reproduction and Genetics, Vrije Universiteit Brussel, Brussels 1090, Belgium
| | - Lise Barbé
- Center for Systems and Therapeutics, Gladstone Institutes, San Francisco, 94107 CA, United States
| | | | - Pierre Hilven
- Department Reproduction and Genetics, Vrije Universiteit Brussel, Brussels 1090, Belgium
| | - Geoffrey Duqué
- Department Reproduction and Genetics, Vrije Universiteit Brussel, Brussels 1090, Belgium
| | - Maurice S Swanson
- Department of Molecular Genetics and Microbiology, Center for NeuroGenetics and the Genetics Institute, University of Florida, College of Medicine, Gainesville, FL 32610, USA
| | - Alexander Gheldof
- Department Reproduction and Genetics, Vrije Universiteit Brussel, Brussels 1090, Belgium.,Center for Medical Genetics, UZ Brussel, Brussels 1090, Belgium
| | - Claudia Spits
- Department Reproduction and Genetics, Vrije Universiteit Brussel, Brussels 1090, Belgium
| | - Karen D Sermon
- Department Reproduction and Genetics, Vrije Universiteit Brussel, Brussels 1090, Belgium
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22
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Miura Y, Li MY, Revah O, Yoon SJ, Narazaki G, Pașca SP. Engineering brain assembloids to interrogate human neural circuits. Nat Protoc 2022; 17:15-35. [PMID: 34992269 DOI: 10.1038/s41596-021-00632-z] [Citation(s) in RCA: 82] [Impact Index Per Article: 27.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Accepted: 09/10/2021] [Indexed: 12/12/2022]
Abstract
The development of neural circuits involves wiring of neurons locally following their generation and migration, as well as establishing long-distance connections between brain regions. Studying these developmental processes in the human nervous system remains difficult because of limited access to tissue that can be maintained as functional over time in vitro. We have previously developed a method to convert human pluripotent stem cells into brain region-specific organoids that can be fused and integrated to form assembloids and study neuronal migration. In contrast to approaches that mix cell lineages in 2D cultures or engineer microchips, assembloids leverage self-organization to enable complex cell-cell interactions, circuit formation and maturation in long-term cultures. In this protocol, we describe approaches to model long-range neuronal connectivity in human brain assembloids. We present how to generate 3D spheroids resembling specific domains of the nervous system and then how to integrate them physically to allow axonal projections and synaptic assembly. In addition, we describe a series of assays including viral labeling and retrograde tracing, 3D live imaging of axon projection and optogenetics combined with calcium imaging and electrophysiological recordings to probe and manipulate the circuits in assembloids. The assays take 3-4 months to complete and require expertise in stem cell culture, imaging and electrophysiology. We anticipate that these approaches will be useful in deciphering human-specific aspects of neural circuit assembly and in modeling neurodevelopmental disorders with patient-derived cells.
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Affiliation(s)
- Yuki Miura
- Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA.,Stanford Brain Organogenesis, Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA
| | - Min-Yin Li
- Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA.,Stanford Brain Organogenesis, Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA
| | - Omer Revah
- Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA.,Stanford Brain Organogenesis, Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA
| | - Se-Jin Yoon
- Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA.,Stanford Brain Organogenesis, Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA
| | - Genta Narazaki
- Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA
| | - Sergiu P Pașca
- Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA. .,Stanford Brain Organogenesis, Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA.
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23
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Karvonen E, Krohn KJE, Ranki A, Hau A. Generation and Characterization of iPS Cells Derived from APECED Patients for Gene Correction. Front Endocrinol (Lausanne) 2022; 13:794327. [PMID: 35432216 PMCID: PMC9010864 DOI: 10.3389/fendo.2022.794327] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/13/2021] [Accepted: 03/08/2022] [Indexed: 12/20/2022] Open
Abstract
APECED (Autoimmune-Polyendocrinopathy-Candidiasis-Ectodermal-Dystrophy) is a severe and incurable multiorgan autoimmune disease caused by mutations in the AIRE (autoimmune regulator) gene. Without functional AIRE, the development of central and peripheral immune tolerance is severely impaired allowing the accumulation of autoreactive immune cells in the periphery. This leads to multiple endocrine and non-endocrine autoimmune disorders and mucocutaneous candidiasis in APECED patients. Recent studies have suggested that AIRE also has novel functions in stem cells and contributes to the regulatory network of pluripotency. In preparation of therapeutic gene correction, we generated and assessed patient blood cell-derived iPSCs, potentially suitable for cell therapy in APECED. Here, we describe APECED-patient derived iPSCs's properties, expression of AIRE as well as classical stem cell markers by qPCR and immunocytochemistry. We further generated self-aggregated EBs of the iPSCs. We show that APECED patient-derived iPSCs and EBs do not have any major proliferative or apoptotic defects and that they express all the classical pluripotency markers similarly to healthy person iPSCs. The results suggest that the common AIRE R257X truncation mutation does not affect stem cell properties and that APECED iPSCs can be propagated in vitro and used for subsequent gene-correction. This first study on APECED patient-derived iPSCs validates their pluripotency and confirms their ability for differentiation and potential therapeutic use.
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Affiliation(s)
- Eira Karvonen
- Department of Dermatology and Allergology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
- Clinical Research Institute Helsinki University Central Hospital (HUCH), Helsinki, Finland
| | - Kai J. E. Krohn
- Clinical Research Institute Helsinki University Central Hospital (HUCH), Helsinki, Finland
| | - Annamari Ranki
- Department of Dermatology and Allergology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
- Clinical Research Institute Helsinki University Central Hospital (HUCH), Helsinki, Finland
| | - Annika Hau
- Department of Dermatology and Allergology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
- Clinical Research Institute Helsinki University Central Hospital (HUCH), Helsinki, Finland
- *Correspondence: Annika Hau,
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24
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Memon B, Abdelalim EM. OUP accepted manuscript. Stem Cells Transl Med 2022; 11:704-714. [PMID: 35640144 PMCID: PMC9299517 DOI: 10.1093/stcltm/szac030] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2022] [Accepted: 04/09/2022] [Indexed: 11/14/2022] Open
Abstract
Although genome profiling provides important genetic and phenotypic details for applying precision medicine to diabetes, it is imperative to integrate in vitro human cell models, accurately recapitulating the genetic alterations associated with diabetes. The absence of the appropriate preclinical human models and the unavailability of genetically relevant cells substantially limit the progress in developing personalized treatment for diabetes. Human pluripotent stem cells (hPSCs) provide a scalable source for generating diabetes-relevant cells carrying the genetic signatures of the patients. Remarkably, allogenic hPSC-derived pancreatic progenitors and β cells are being used in clinical trials with promising preliminary results. Autologous hiPSC therapy options exist for those with monogenic and type 2 diabetes; however, encapsulation or immunosuppression must be accompanied with in the case of type 1 diabetes. Furthermore, genome-wide association studies-identified candidate variants can be introduced in hPSCs for deciphering the associated molecular defects. The hPSC-based disease models serve as excellent resources for drug development facilitating personalized treatment. Indeed, hPSC-based diabetes models have successfully provided valuable knowledge by modeling different types of diabetes, which are discussed in this review. Herein, we also evaluate their strengths and shortcomings in dissecting the underlying pathogenic molecular mechanisms and discuss strategies for improving hPSC-based disease modeling investigations.
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Affiliation(s)
- Bushra Memon
- College of Health and Life Sciences, Hamad Bin Khalifa University (HBKU), Qatar Foundation (QF), Education City, Doha, Qatar
- Diabetes Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation (QF), Doha, Qatar
| | - Essam M Abdelalim
- Corresponding author: Essam M. Abdelalim, Diabetes Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa, University (HBKU), Qatar Foundation (QF), PO Box 34110, Doha, Qatar. Tel: +974 445 46432; Fax: +974 445 41770;
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25
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Keller A, Krivec N, Markouli C, Spits C. Measuring Early Germ-Layer Specification Bias in Human Pluripotent Stem Cells. Methods Mol Biol 2022; 2429:57-72. [PMID: 35507155 DOI: 10.1007/978-1-0716-1979-7_5] [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: 06/14/2023]
Abstract
Human pluripotent stem cells have a wide variety of potential applications, ranging from clinical translation to in vitro disease modeling. However, there is significant variation in the potential of individual cell lines to differentiate towards each of the three germ layers as a result of (epi)genetic background, culture conditions, and other factors. We describe here in detail a methodology to evaluate this bias using short directed differentiation towards neuroectoderm, mesendoderm, and definitive endoderm in combination with quantification by RT-qPCR and immunofluorescent stains.
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Affiliation(s)
- Alexander Keller
- Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Jette, Belgium
| | - Nuša Krivec
- Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Jette, Belgium
| | - Christina Markouli
- Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Jette, Belgium
| | - Claudia Spits
- Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Jette, Belgium.
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26
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Jiang B, Li W, Stewart S, Ou W, Liu B, Comizzoli P, He X. Sand-mediated ice seeding enables serum-free low-cryoprotectant cryopreservation of human induced pluripotent stem cells. Bioact Mater 2021; 6:4377-4388. [PMID: 33997514 PMCID: PMC8111032 DOI: 10.1016/j.bioactmat.2021.04.025] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2021] [Revised: 04/14/2021] [Accepted: 04/14/2021] [Indexed: 12/23/2022] Open
Abstract
Human induced pluripotent stem cells (hiPSCs) possess tremendous potential for tissue regeneration and banking hiPSCs by cryopreservation for their ready availability is crucial to their widespread use. However, contemporary methods for hiPSC cryopreservation are associated with both limited cell survival and high concentration of toxic cryoprotectants and/or serum. The latter may cause spontaneous differentiation and/or introduce xenogeneic factors, which may compromise the quality of hiPSCs. Here, sand from nature is discovered to be capable of seeding ice above -10 °C, which enables cryopreservation of hiPSCs with no serum, much-reduced cryoprotectant, and high cell survival. Furthermore, the cryopreserved hiPSCs retain high pluripotency and functions judged by their pluripotency marker expression, cell cycle analysis, and capability of differentiation into the three germ layers. This unique sand-mediated cryopreservation method may greatly facilitate the convenient and ready availability of high-quality hiPSCs and probably many other types of cells/tissues for the emerging cell-based translational medicine.
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Affiliation(s)
- Bin Jiang
- Fischell Department of Bioengineering, University of Maryland, College Park, MD, 20742, USA
| | - Weijie Li
- Fischell Department of Bioengineering, University of Maryland, College Park, MD, 20742, USA
- Institute of Biothermal Technology, University of Shanghai for Science and Technology, Shanghai, 200093, China
| | - Samantha Stewart
- Fischell Department of Bioengineering, University of Maryland, College Park, MD, 20742, USA
| | - Wenquan Ou
- Fischell Department of Bioengineering, University of Maryland, College Park, MD, 20742, USA
| | - Baolin Liu
- Institute of Biothermal Technology, University of Shanghai for Science and Technology, Shanghai, 200093, China
| | - Pierre Comizzoli
- Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC, 20008, USA
| | - Xiaoming He
- Fischell Department of Bioengineering, University of Maryland, College Park, MD, 20742, USA
- Robert E. Fischell Institute for Biomedical Devices, University of Maryland, College Park, MD, 20742, USA
- Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD, 21201, USA
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27
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Ota A, Hyon SH, Sumi S, Matsumura K. Gene expression analysis of human induced pluripotent stem cells cryopreserved by vitrification using StemCell Keep. Biochem Biophys Rep 2021; 28:101172. [PMID: 34825070 PMCID: PMC8605251 DOI: 10.1016/j.bbrep.2021.101172] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2021] [Revised: 11/03/2021] [Accepted: 11/10/2021] [Indexed: 01/11/2023] Open
Abstract
In recent years, regenerative medicine research using human somatic and induced pluripotent stem cells has advanced considerably, promoting clinical applications. However, it is essential that these cells are cryopreserved safely and effectively. Most cryopreservation solution agents contain dimethyl sulfoxide (DMSO), which exhibits strong toxicity and can potentially promote cell differentiation. Hence, it is important to explore substitutes for DMSO in cryoprotectant solutions. One such alternative is StemCell Keep (SCK), a DMSO-free solution that has been reported to effectively cryopreserve human induced pluripotent stem cells (hiPS cells). To clarify the effect of cryopreservation agents on cells, DNA microarray analysis is useful, as it can identify a large number of gene expression differences in cryopreserved cells, as well as functional increases in gene groups. In this study, we performed gene expression analysis of SCK-cryopreserved hiPS cells using a DNA microarray gene chip. The hiPS cells vitrified with SCK or DMSO-based vitrification solutions were thawed and cultured on Matrigel under feeder-free conditions, and RNA was extracted for DNA microarray analysis. Genes obtained from DNA microarray data were classified by the keywords of Gene Ontology Biological Process Term, and their relationships were analyzed using DAVID or the GeneMANIA database. SCK-cryopreserved hiPS cells expressed several anti-apoptotic genes, as well as genes related to cell adhesion or proliferation at levels that were nearly equivalent to those of non-frozen hiPS cells. Gene enrichment analysis with selected genes of SCK-cryopreserved hiPS cells whose expression differences were superior to those of DAP-cryopreserved showed strong interactions of negative regulation of apoptotic process, cell adhesion and positive regulation of cell proliferation in DAVID analysis. We demonstrated that SCK successfully maintained the key functions of hiPS cells, including anti-apoptosis, cell adhesion, and cell proliferation, during cryopreservation.
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Affiliation(s)
| | | | - Shoichiro Sumi
- Department of Organ Reconstruction, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
| | - Kazuaki Matsumura
- School of Materials Science, Japan Advanced Institute of Science and Technology, Nomi, Ishikawa, Japan
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28
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Zhu Y, Cheng C, Chen L, Zhang L, Pan H, Hou L, Sun Z, Zhang L, Fu X, Chan KY, Zhang J. Cell cycle heterogeneity directs spontaneous 2C state entry and exit in mouse embryonic stem cells. Stem Cell Reports 2021; 16:2659-2673. [PMID: 34624246 PMCID: PMC8580870 DOI: 10.1016/j.stemcr.2021.09.003] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2021] [Revised: 09/07/2021] [Accepted: 09/08/2021] [Indexed: 12/01/2022] Open
Abstract
Mouse embryonic stem cells (ESCs) show cell-to-cell heterogeneity. A small number of two-cell-like cells (2CLCs) marked by endogenous retrovirus activation emerge spontaneously. The 2CLCs are unstable and they are prone to transiting back to the pluripotent state without extrinsic stimulus. To understand how this bidirectional transition takes place, we performed single-cell RNA sequencing on isolated 2CLCs that underwent 2C-like state exit and re-entry, and revealed a step-by-step transitional process between 2C-like and pluripotent states. Mechanistically, we found that cell cycle played an important role in mediating these transitions by regulating assembly of the nucleolus and peri-nucleolar heterochromatin to influence 2C gene Dux expression. Collectively, our findings provide a roadmap of the 2C-like state entry and exit in ESCs and also a causal role of the cell cycle in promoting these transitions.
The entry to and exit from the 2C-like state showed a step-by-step roadmap Cell cycle participates in mediating dynamic transitions between ESCs and 2CLCs G1/S phase arrest facilitates the Dux locus escape from heterochromatin Nucleolus-heterochromatin remodeling is involved in 2C activation
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Affiliation(s)
- Yuqing Zhu
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China; Zhejiang University-University of Edinburgh Institute, Zhejiang University School of Medicine, Haining, Zhejiang, China
| | - Chen Cheng
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China
| | - Lang Chen
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China
| | - Li Zhang
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China
| | - Hongru Pan
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China
| | - Linxiao Hou
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China
| | - Zhen Sun
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China
| | - Ling Zhang
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China; Zhejiang Laboratory for Systems and Precision Medicine, Zhejiang University Medical Center, Hangzhou, Zhejiang, China
| | - Xudong Fu
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China; Zhejiang Laboratory for Systems and Precision Medicine, Zhejiang University Medical Center, Hangzhou, Zhejiang, China; Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA
| | - Kuan Yoow Chan
- Zhejiang University-University of Edinburgh Institute, Zhejiang University School of Medicine, Haining, Zhejiang, China
| | - Jin Zhang
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China; Zhejiang Laboratory for Systems and Precision Medicine, Zhejiang University Medical Center, Hangzhou, Zhejiang, China; Institute of Hematology, Zhejiang University, Hangzhou, Zhejiang, China; Center of Gene/Cell Engineering and Genome Medicine, Hangzhou, Zhejiang, China.
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29
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Lim CK, Efthymios M, Tan W, Autio MI, Tiang Z, Li PY, Foo RSY. Dimethyl sulfoxide (DMSO) enhances direct cardiac reprogramming by inhibiting the bromodomain of coactivators CBP/p300. J Mol Cell Cardiol 2021; 160:15-26. [PMID: 34146546 DOI: 10.1016/j.yjmcc.2021.06.008] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/27/2020] [Revised: 06/09/2021] [Accepted: 06/13/2021] [Indexed: 12/01/2022]
Abstract
AIMS Direct cardiac reprogramming represents an attractive way to reversing heart damage caused by myocardial infarction because it removes fibroblasts, while also generating new functional cardiomyocytes. Yet, the main hurdle for bringing this technique to the clinic is the lack of efficacy with current reprogramming protocols. Here, we describe our unexpected discovery that DMSO is capable of significantly augmenting direct cardiac reprogramming in vitro. METHODS AND RESULTS Upon induction with cardiac transcription factors- Gata4, Hand2, Mef2c and Tbx5 (GHMT), the treatment of mouse embryonic fibroblasts (MEFs) with 1% DMSO induced ~5 fold increase in Myh6-mCherry+ cells, and significantly upregulated global expression of cardiac genes, including Myh6, Ttn, Nppa, Myh7 and Ryr2. RNA-seq confirmed upregulation of cardiac gene programmes and downregulation of extracellular matrix-related genes. Treatment of TGF-β1, DMSO, or SB431542, and the combination thereof, revealed that DMSO most likely targets a separate but parallel pathway other than TGF-β signalling. Subsequent experiments using small molecule screening revealed that DMSO enhances direct cardiac reprogramming through inhibition of the CBP/p300 bromodomain, and not its acetyltransferase property. CONCLUSION In conclusion, our work points to a direct molecular target of DMSO, which can be used for augmenting GHMT-induced direct cardiac reprogramming and possibly other cell fate conversion processes.
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Affiliation(s)
- Choon Kiat Lim
- Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive, Singapore 117599, Singapore; Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore; NUS Graduate School of Integrative Sciences and Engineering (NGS), National University of Singapore, Singapore 117456, Singapore
| | - Motakis Efthymios
- Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive, Singapore 117599, Singapore; Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore
| | - Wilson Tan
- Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive, Singapore 117599, Singapore; Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore
| | - Matias Ilmari Autio
- Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore
| | - Zenia Tiang
- Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive, Singapore 117599, Singapore; Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore
| | - Peter Yiqing Li
- Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive, Singapore 117599, Singapore
| | - Roger Sik Yin Foo
- Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive, Singapore 117599, Singapore; Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore.
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30
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Cryopreservation Engineering Strategies for Mass Production of Adipose-Derived Stem Cells. BIOTECHNOL BIOPROC E 2021. [DOI: 10.1007/s12257-019-1359-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
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31
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Roos D, de Boer M. Mutations in cis that affect mRNA synthesis, processing and translation. Biochim Biophys Acta Mol Basis Dis 2021; 1867:166166. [PMID: 33971252 DOI: 10.1016/j.bbadis.2021.166166] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2020] [Revised: 05/03/2021] [Accepted: 05/04/2021] [Indexed: 12/17/2022]
Abstract
Genetic mutations that cause hereditary diseases usually affect the composition of the transcribed mRNA and its encoded protein, leading to instability of the mRNA and/or the protein. Sometimes, however, such mutations affect the synthesis, the processing or the translation of the mRNA, with similar disastrous effects. We here present an overview of mRNA synthesis, its posttranscriptional modification and its translation into protein. We then indicate which elements in these processes are known to be affected by pathogenic mutations, but we restrict our review to mutations in cis, in the DNA of the gene that encodes the affected protein. These mutations can be in enhancer or promoter regions of the gene, which act as binding sites for transcription factors involved in pre-mRNA synthesis. We also describe mutations in polyadenylation sequences and in splice site regions, exonic and intronic, involved in intron removal. Finally, we include mutations in the Kozak sequence in mRNA, which is involved in protein synthesis. We provide examples of genetic diseases caused by mutations in these DNA regions and refer to databases to help identify these regions. The over-all knowledge of mRNA synthesis, processing and translation is essential for improvement of the diagnosis of patients with genetic diseases.
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Affiliation(s)
- Dirk Roos
- Sanquin Blood Supply Organization, Dept. of Blood Cell Research, Landsteiner Laboratory, Amsterdam University Medical Centre, location AMC, University of Amsterdam, Amsterdam, the Netherlands.
| | - Martin de Boer
- Sanquin Blood Supply Organization, Dept. of Blood Cell Research, Landsteiner Laboratory, Amsterdam University Medical Centre, location AMC, University of Amsterdam, Amsterdam, the Netherlands
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Huang J, Guo J, Zhou L, Zheng G, Cao J, Li Z, Zhou Z, Lei Q, Brinker CJ, Zhu W. Advanced Nanomaterials-Assisted Cell Cryopreservation: A Mini Review. ACS APPLIED BIO MATERIALS 2021; 4:2996-3014. [PMID: 35014388 DOI: 10.1021/acsabm.1c00105] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Cell cryopreservation is of vital significance both for transporting and storing cells before experimental/clinical use. Cryoprotectants (CPAs) are necessary additives in the preserving medium in cryopreservation, preventing cells from freeze-thaw injuries. Traditional organic solvents have been widely used in cell cryopreservation for decades. Given the obvious damage to cells due to their undesirable cytotoxicity and the burdensome post-thaw washing cycles before use, traditional CPAs are more and more likely to be replaced by modern ones with lower toxicity, less processing, and higher efficiency. As materials science thrives, nanomaterials are emerging to serve as potent vehicles for delivering nontoxic CPAs or inherent CPAs comparable to or even superior to conventional ones. This review will introduce some advanced nanomaterials (e.g., organic/inorganic nanoCPAs, nanodelivery systems) utilized for cell cryopreservation, providing broader insights into this developing field.
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Affiliation(s)
- Junda Huang
- MOE International Joint Research Laboratory on Synthetic Biology and Medicines, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, P. R. China
| | - Jimin Guo
- Center for Micro-Engineered Materials, Department of Chemical and Biological Engineering, The University of New Mexico, Albuquerque, New Mexico 87131, United States.,Department of Internal Medicine, Molecular Medicine, The University of New Mexico, Albuquerque, New Mexico 87131, United States
| | - Liang Zhou
- MOE International Joint Research Laboratory on Synthetic Biology and Medicines, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, P. R. China
| | - Guansheng Zheng
- MOE International Joint Research Laboratory on Synthetic Biology and Medicines, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, P. R. China
| | - Jiangfan Cao
- MOE International Joint Research Laboratory on Synthetic Biology and Medicines, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, P. R. China
| | - Zeyu Li
- MOE International Joint Research Laboratory on Synthetic Biology and Medicines, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, P. R. China
| | - Zhuang Zhou
- MOE International Joint Research Laboratory on Synthetic Biology and Medicines, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, P. R. China
| | - Qi Lei
- MOE International Joint Research Laboratory on Synthetic Biology and Medicines, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, P. R. China
| | - C Jeffrey Brinker
- Center for Micro-Engineered Materials, Department of Chemical and Biological Engineering, The University of New Mexico, Albuquerque, New Mexico 87131, United States
| | - Wei Zhu
- MOE International Joint Research Laboratory on Synthetic Biology and Medicines, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, P. R. China
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Sustained intrinsic WNT and BMP4 activation impairs hESC differentiation to definitive endoderm and drives the cells towards extra-embryonic mesoderm. Sci Rep 2021; 11:8242. [PMID: 33859268 PMCID: PMC8050086 DOI: 10.1038/s41598-021-87547-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2020] [Accepted: 03/31/2021] [Indexed: 12/13/2022] Open
Abstract
We identified a human embryonic stem cell subline that fails to respond to the differentiation cues needed to obtain endoderm derivatives, differentiating instead into extra-embryonic mesoderm. RNA-sequencing analysis showed that the subline has hyperactivation of the WNT and BMP4 signalling. Modulation of these pathways with small molecules confirmed them as the cause of the differentiation impairment. While activation of WNT and BMP4 in control cells resulted in a loss of endoderm differentiation and induction of extra-embryonic mesoderm markers, inhibition of these pathways in the subline restored its ability to differentiate. Karyotyping and exome sequencing analysis did not identify any changes in the genome that could account for the pathway deregulation. These findings add to the increasing evidence that different responses of stem cell lines to differentiation protocols are based on genetic and epigenetic factors, inherent to the line or acquired during cell culture.
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An Efficient Method for the Differentiation of Human iPSC-Derived Endoderm toward Enterocytes and Hepatocytes. Cells 2021; 10:cells10040812. [PMID: 33917333 PMCID: PMC8067398 DOI: 10.3390/cells10040812] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2021] [Revised: 03/31/2021] [Accepted: 04/02/2021] [Indexed: 12/13/2022] Open
Abstract
The endoderm, differentiated from human induced pluripotent stem cells (iPSCs), can differentiate into the small intestine and liver, which are vital for drug absorption and metabolism. The development of human iPSC-derived enterocytes (HiEnts) and hepatocytes (HiHeps) has been reported. However, pharmacokinetic function-deficiency of these cells remains to be elucidated. Here, we aimed to develop an efficient differentiation method to induce endoderm formation from human iPSCs. Cells treated with activin A for 168 h expressed higher levels of endodermal genes than those treated for 72 h. Using activin A (days 0–7), CHIR99021 and PI−103 (days 0–2), and FGF2 (days 3–7), the hiPSC-derived endoderm (HiEnd) showed 97.97% CD−117 and CD−184 double-positive cells. Moreover, HiEnts derived from the human iPSC line Windy had similar or higher expression of small intestine-specific genes than adult human small intestine. Activities of the drug transporter P-glycoprotein and drug-metabolizing enzyme cytochrome P450 (CYP) 3A4/5 were confirmed. Additionally, Windy-derived HiHeps expressed higher levels of hepatocyte- and pharmacokinetics-related genes and proteins and showed higher CYP3A4/5 activity than those derived through the conventional differentiation method. Thus, using this novel method, the differentiated HiEnts and HiHeps with pharmacokinetic functions could be used for drug development.
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Santarella F, O'Brien FJ, Garlick JA, Kearney CJ. The Development of Tissue Engineering Scaffolds Using Matrix from iPS-Reprogrammed Fibroblasts. Methods Mol Biol 2021; 2454:273-283. [PMID: 33755908 DOI: 10.1007/7651_2021_351] [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: 04/03/2023]
Abstract
Tissue engineering solutions have been widely explored for enhanced healing of skin wounds. Diabetic foot ulcers (DFU) are particularly challenging wounds to heal for a variety of reasons, including aberrant ECM, dysregulation of vascularization, and persistent inflammation. Tissue engineering approaches, such as porous collagen-based scaffolds, have shown promise in replacing the current treatments of surgical debridement and topical treatments. Collagen-glycosaminoglycan scaffolds, which are FDA approved for diabetic foot ulcers, can benefit from further functionalization by incorporation of additional signaling factors or extracellular matrix molecules. One option for this is to incorporate matrix from a rejuvenated cell source, as wounds in younger patients heal more quickly. Induced pluripotent stem cells (iPS) are generated from somatic cells and share many functional similarities with embryonic stem cells (ES), while avoiding the ethical concerns. Fibroblasts differentiated from iPS cells have been shown to enrich their ECM with glycosaminoglycan (GAGs), collagen Type III and fibronectin, to have an increased ECM production, and to be pro-angiogenic. Here we describe a technique to grow matrix from post-iPS fibroblasts, and to develop a scaffold from this matrix, in combination with collagen, with the goal of enhancing wound healing. By activating scaffolds with extracellular matrix (ECM) from fibroblasts derived from an iPS source (post-iPSF), the scaffolds are enriched with beneficial elements like GAGs, collagen type III, fibronectin, and VEGF. We believe these scaffolds can enhance skin regeneration and that the techniques can be modified for other tissue engineering applications.
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Affiliation(s)
- Francesco Santarella
- Tissue Engineering Research Group (TERG), Royal College of Surgeons in Ireland (RCSI), Dublin 2, Ireland
| | - Fergal J O'Brien
- Tissue Engineering Research Group (TERG), Royal College of Surgeons in Ireland (RCSI), Dublin 2, Ireland
- Trinity Centre for Biomedical Engineering, The University of Dublin Trinity College (TCD), Dublin 2, Ireland
- Advanced Materials and Bioengineering Research Centre, RCSI & TCD, Dublin 2, Ireland
| | - Jonathan A Garlick
- Department of Diagnostic Sciences, Tufts University School of Dental Medicine, Boston, MA, USA
| | - Cathal J Kearney
- Department of Biomedical Engineering, University of Massachusetts Amherst, Amherst, MA, USA.
- Tissue Engineering Research Group (TERG), Royal College of Surgeons in Ireland (RCSI), Dublin 2, Ireland.
- Advanced Materials and Bioengineering Research Centre, RCSI & TCD, Dublin 2, Ireland.
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Assis RIF, Schmidt AG, Racca F, da Silva RA, Zambuzzi WF, Silvério KG, Nociti FH, Pecorari VG, Wiench M, Andia DC. DNMT1 Inhibitor Restores RUNX2 Expression and Mineralization in Periodontal Ligament Cells. DNA Cell Biol 2021; 40:662-674. [PMID: 33751901 DOI: 10.1089/dna.2020.6239] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Periodontal ligament cells (PDLCs) have well documented osteogenic potential; however, this commitment can be highly heterogenous, limiting their applications in tissue regeneration. In this study, we use PDLC populations characterized by high and low osteogenic potential (h-PDLCs and l-PDLCs, respectively) to identify possible sources of such heterogeneity and to investigate whether the osteogenic differentiation can be enhanced by epigenetic modulation. In h-PDLCs, low basal expression levels of pluripotency markers (NANOG, OCT4), DNA methyltransferases (DNMT1, DNMT3B), and enzymes involved in active DNA demethylation (TET1, TET3) were prerequisite to high osteogenic potential. Furthermore, these genes were downregulated upon early osteogenesis, possibly allowing for the increase in expression of the key osteogenic transcription factors, Runt-related transcription factor 2 (RUNX2) and SP7, and ultimately, mineral nodule formation. l-PDLCs appeared locked in the multipotent state and this was further enhanced upon early osteogenic stimulation, correlating with low RUNX2 expression and impaired mineralization. Further upregulation of DNMTs was also evident, while pretreatment with RG108, the DNMTs' inhibitor, enhanced the osteogenic program in l-PDLCs through downregulation of DNMTs, increased RUNX2 expression and nuclear localization, accelerated expression of osteogenic markers, and increased mineralization. These findings point toward the role of DNMTs and Ten Eleven Translocations (TETs) in osteogenic commitment and support application of epigenetic approaches to modulate biomineralization in PDLCs.
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Affiliation(s)
- Rahyza I F Assis
- Department of Prosthodontics and Periodontics, Piracicaba Dental School, University of Campinas, Piracicaba, Brazil
| | - Arthur G Schmidt
- Health Science Institute, School of Dentistry, Paulista University-UNIP, São Paulo, Brazil
| | - Francesca Racca
- Department of Prosthodontics and Periodontics, Piracicaba Dental School, University of Campinas, Piracicaba, Brazil
| | - Rodrigo A da Silva
- Program in Environmental and Experimental Pathology, Paulista University-UNIP, São Paulo, Brazil
| | - William F Zambuzzi
- Department of Chemistry and Biochemistry, Biosciences Institute, São Paulo State University, Botucatu, Brazil
| | - Karina G Silvério
- Department of Prosthodontics and Periodontics, Piracicaba Dental School, University of Campinas, Piracicaba, Brazil
| | - Francisco H Nociti
- Department of Prosthodontics and Periodontics, Piracicaba Dental School, University of Campinas, Piracicaba, Brazil
| | - Vanessa G Pecorari
- Health Science Institute, School of Dentistry, Paulista University-UNIP, São Paulo, Brazil
| | - Malgorzata Wiench
- Institute of Clinical Sciences, Institute of Cancer and Genomic Sciences, School of Dentistry, University of Birmingham, Birmingham, United Kingdom
| | - Denise C Andia
- Health Science Institute, School of Dentistry, Paulista University-UNIP, São Paulo, Brazil
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Abdelalim EM. Modeling different types of diabetes using human pluripotent stem cells. Cell Mol Life Sci 2021; 78:2459-2483. [PMID: 33242105 PMCID: PMC11072720 DOI: 10.1007/s00018-020-03710-9] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2020] [Revised: 10/19/2020] [Accepted: 11/11/2020] [Indexed: 12/22/2022]
Abstract
Diabetes mellitus (DM) is a metabolic disease characterized by chronic hyperglycemia as a result of progressive loss of pancreatic β cells, which could lead to several debilitating complications. Different paths, triggered by several genetic and environmental factors, lead to the loss of pancreatic β cells and/or function. Understanding these many paths to β cell damage or dysfunction could help in identifying therapeutic approaches specific for each path. Most of our knowledge about diabetes pathophysiology has been obtained from studies on animal models, which do not fully recapitulate human diabetes phenotypes. Currently, human pluripotent stem cell (hPSC) technology is a powerful tool for generating in vitro human models, which could provide key information about the disease pathogenesis and provide cells for personalized therapies. The recent progress in generating functional hPSC-derived β cells in combination with the rapid development in genomic and genome-editing technologies offer multiple options to understand the cellular and molecular mechanisms underlying the development of different types of diabetes. Recently, several in vitro hPSC-based strategies have been used for studying monogenic and polygenic forms of diabetes. This review summarizes the current knowledge about different hPSC-based diabetes models and how these models improved our current understanding of the pathophysiology of distinct forms of diabetes. Also, it highlights the progress in generating functional β cells in vitro, and discusses the current challenges and future perspectives related to the use of the in vitro hPSC-based strategies.
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Affiliation(s)
- Essam M Abdelalim
- Diabetes Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation (QF), PO Box 34110, Doha, Qatar.
- College of Health and Life Sciences, Hamad Bin Khalifa University (HBKU), Qatar Foundation (QF), Education City, Doha, Qatar.
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Brown J, Barry C, Schmitz MT, Argus C, Bolin JM, Schwartz MP, Van Aartsen A, Steill J, Swanson S, Stewart R, Thomson JA, Kendziorski C. Interspecies chimeric conditions affect the developmental rate of human pluripotent stem cells. PLoS Comput Biol 2021; 17:e1008778. [PMID: 33647016 PMCID: PMC7951976 DOI: 10.1371/journal.pcbi.1008778] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2020] [Revised: 03/11/2021] [Accepted: 02/08/2021] [Indexed: 12/17/2022] Open
Abstract
Human pluripotent stem cells hold significant promise for regenerative medicine. However, long differentiation protocols and immature characteristics of stem cell-derived cell types remain challenges to the development of many therapeutic applications. In contrast to the slow differentiation of human stem cells in vitro that mirrors a nine-month gestation period, mouse stem cells develop according to a much faster three-week gestation timeline. Here, we tested if co-differentiation with mouse pluripotent stem cells could accelerate the differentiation speed of human embryonic stem cells. Following a six-week RNA-sequencing time course of neural differentiation, we identified 929 human genes that were upregulated earlier and 535 genes that exhibited earlier peaked expression profiles in chimeric cell cultures than in human cell cultures alone. Genes with accelerated upregulation were significantly enriched in Gene Ontology terms associated with neurogenesis, neuron differentiation and maturation, and synapse signaling. Moreover, chimeric mixed samples correlated with in utero human embryonic samples earlier than human cells alone, and acceleration was dose-dependent on human-mouse co-culture ratios. The altered gene expression patterns and developmental rates described in this report have implications for accelerating human stem cell differentiation and the use of interspecies chimeric embryos in developing human organs for transplantation.
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Affiliation(s)
- Jared Brown
- Department of Statistics, University of Wisconsin-Madison, Wisconsin, United States of America
- * E-mail: (JB); (CK)
| | - Christopher Barry
- Morgridge Institute for Research, Madison, Wisconsin, United States of America
| | - Matthew T. Schmitz
- Morgridge Institute for Research, Madison, Wisconsin, United States of America
| | - Cara Argus
- Morgridge Institute for Research, Madison, Wisconsin, United States of America
| | - Jennifer M. Bolin
- Morgridge Institute for Research, Madison, Wisconsin, United States of America
| | - Michael P. Schwartz
- NSF Center for Sustainable Nanotechnology, Department of Chemistry, University of Wisconsin-Madison, Wisconsin, United States of America
| | - Amy Van Aartsen
- Morgridge Institute for Research, Madison, Wisconsin, United States of America
| | - John Steill
- Morgridge Institute for Research, Madison, Wisconsin, United States of America
| | - Scott Swanson
- Morgridge Institute for Research, Madison, Wisconsin, United States of America
| | - Ron Stewart
- Morgridge Institute for Research, Madison, Wisconsin, United States of America
| | - James A. Thomson
- Morgridge Institute for Research, Madison, Wisconsin, United States of America
- Department of Cell and Regenerative Biology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America
- Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California, United States of America
| | - Christina Kendziorski
- Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Wisconsin, United States of America
- * E-mail: (JB); (CK)
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Huang Y, Su T, Wang C, Dong L, Liu S, Zhu Y, Hao K, Xia Y, Jiang Q, Qin J. Rbbp4 Suppresses Premature Differentiation of Embryonic Stem Cells. Stem Cell Reports 2021; 16:566-581. [PMID: 33606987 PMCID: PMC7940252 DOI: 10.1016/j.stemcr.2021.01.009] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2020] [Revised: 01/16/2021] [Accepted: 01/19/2021] [Indexed: 12/30/2022] Open
Abstract
Polycomb group (PcG) proteins exist in distinct multi-protein complexes and play a central role in silencing developmental genes, yet the underlying mechanisms remain elusive. Here, we show that deficiency of retinoblastoma binding protein 4 (RBBP4), a component of the Polycomb repressive complex 2 (PRC2), in embryonic stem cells (ESCs) leads to spontaneous differentiation into mesendodermal lineages. We further show that Rbbp4 and core PRC2 share an important number of common genomic targets, encoding regulators involved in early germ layer specification. Moreover, we find that Rbbp4 is absolutely essential for genomic targeting of PRC2 to a subset of developmental genes. Interestingly, we demonstrate that Rbbp4 is necessary for sustaining the expression of Oct4 and Sox2 and that the forced co-expression of Oct4 and Sox2 fully rescues the pluripotency of Rbbp4-null ESCs. Therefore, our study indicates that Rbbp4 links maintenance of the pluripotency regulatory network with repression of mesendoderm lineages.
RBBP4 deficiency in ESCs leads to spontaneous differentiation into mesendodermal lineages Rbbp4 binding sites in ESCs substantially overlap with PRC2 binding Rbbp4 is absolutely essential for PRC2 chromatin occupancy Rbbp4 is necessary for sustaining the expression levels of Oct4 and Sox2
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Affiliation(s)
- Yikai Huang
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, School of Medicine, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China
| | - Ting Su
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, School of Medicine, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China
| | - Congcong Wang
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, School of Medicine, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China
| | - Lixia Dong
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, School of Medicine, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China
| | - Shuang Liu
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, School of Medicine, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China
| | - Yaru Zhu
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, School of Medicine, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China
| | - Kunying Hao
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, School of Medicine, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China
| | - Yin Xia
- School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China.
| | - Qing Jiang
- Department of Sports Medicine and Adult Reconstructive Surgery, Nanjing Drum Tower Hospital Affiliated to Medical School of Nanjing University, Nanjing, China.
| | - Jinzhong Qin
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, School of Medicine, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China.
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Vats N, Dubey RC, Sanal MG, Taneja P, Venugopal SK. Glibenclamide, ATP and metformin increases the expression of human bile salt export pump ABCB11. F1000Res 2020; 9:1497. [PMID: 33763207 PMCID: PMC7953918 DOI: 10.12688/f1000research.26632.1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 10/02/2020] [Indexed: 12/30/2022] Open
Abstract
Background: Bile salt export pump (BSEP/ABCB11) is important in the maintenance of the enterohepatic circulation of bile acids and drugs. Drugs such as rifampicin and glibenclamide inhibit BSEP. Progressive familial intrahepatic cholestasis type-2, a lethal pediatric disease, some forms of intrahepatic cholestasis of pregnancy, and drug-induced cholestasis are associated with BSEP dysfunction. Methods: We started with a bioinformatic approach to identify the relationship between ABCB11 and other proteins, microRNAs, and drugs. A microarray data set of the liver samples from ABCB11 knockout mice was analyzed using GEO2R. Differentially expressed gene pathway enrichment analysis was conducted using ClueGo. A protein-protein interaction network was constructed using STRING application in Cytoscape. Networks were analyzed using Cytoscape. CyTargetLinker was used to screen the transcription factors, microRNAs and drugs. Predicted drugs were validated on human liver cell line, HepG2. BSEP expression was quantified by real-time PCR and western blotting. Results:ABCB11 knockout in mice was associated with a predominant upregulation and downregulation of genes associated with cellular component movement and sterol metabolism, respectively. We further identified the hub genes in the network. Genes related to immune activity, cell signaling, and fatty acid metabolism were dysregulated. We further identified drugs (glibenclamide and ATP) and a total of 14 microRNAs targeting the gene. Western blot and real-time PCR analysis confirmed the upregulation of BSEP on the treatment of HepG2 cells with glibenclamide, ATP, and metformin. Conclusions: The differential expression of cell signaling genes and those related to immune activity in ABCB11 KO animals may be secondary to cell injury. We have found glibenclamide, ATP, and metformin upregulates BSEP. The mechanisms involved and the clinical relevance of these findings need to be investigated.
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Affiliation(s)
- Nisha Vats
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi, Delhi, 110070, India
| | - Ravi Chandra Dubey
- Department of Life Sciences, South Asian University, New Delhi, Delhi, 110021, India
| | - Madhusudana Girija Sanal
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi, Delhi, 110070, India
| | - Pankaj Taneja
- Department of Biotechnology, Sharda University, Noida, Uttar Pradesh, 201310, India
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Qiu J, Nordling S, Vasavada HH, Butcher EC, Hirschi KK. Retinoic Acid Promotes Endothelial Cell Cycle Early G1 State to Enable Human Hemogenic Endothelial Cell Specification. Cell Rep 2020; 33:108465. [PMID: 33264627 PMCID: PMC8105879 DOI: 10.1016/j.celrep.2020.108465] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2020] [Revised: 08/27/2020] [Accepted: 11/10/2020] [Indexed: 12/01/2022] Open
Abstract
Development of blood-forming (hemogenic) endothelial cells that give rise to hematopoietic stem and progenitor cells (HSPCs) is critical during embryogenesis to generate the embryonic and postnatal hematopoietic system. We previously demonstrated that the specification of murine hemogenic endothelial cells is promoted by retinoic acid (RA) signaling and requires downstream endothelial cell cycle control. Whether this mechanism is conserved in human hemogenic endothelial cell specification is unknown. Here, we present a protocol to derive primordial endothelial cells from human embryonic stem cells and promote their specification toward hemogenic endothelial cells. Furthermore, we demonstrate that RA treatment significantly increases human hemogenic endothelial cell specification. That is, RA promotes endothelial cell cycle arrest to enable RA-induced instructive signals to upregulate the genes needed for hematopoietic transition. These insights provide guidance for the ex vivo generation of autologous human hemogenic endothelial cells that are needed to produce human HSPCs for regenerative medicine applications.
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Affiliation(s)
- Jingyao Qiu
- Department of Medicine, Yale University School of Medicine, New Haven, CT 06520, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA; Yale Cardiovascular Research Center, Yale University School of Medicine, New Haven, CT 06520, USA; Vascular Biology and Therapeutics Program, Yale University School of Medicine, New Haven, CT 06520, USA; Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Sofia Nordling
- Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Hema H Vasavada
- Yale Cardiovascular Research Center, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Eugene C Butcher
- Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA; Palo Alto Veterans Institute for Research, VA Palo Alto Health Care System, Palo Alto, CA 94304, USA; The Center for Molecular Biology and Medicine, VA Palo Alto Health Care System, Palo Alto, CA 94304, USA
| | - Karen K Hirschi
- Department of Medicine, Yale University School of Medicine, New Haven, CT 06520, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA; Yale Cardiovascular Research Center, Yale University School of Medicine, New Haven, CT 06520, USA; Vascular Biology and Therapeutics Program, Yale University School of Medicine, New Haven, CT 06520, USA; Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT 06520, USA; Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA.
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Insulin/Glucose-Responsive Cells Derived from Induced Pluripotent Stem Cells: Disease Modeling and Treatment of Diabetes. Cells 2020; 9:cells9112465. [PMID: 33198288 PMCID: PMC7696367 DOI: 10.3390/cells9112465] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2020] [Revised: 11/03/2020] [Accepted: 11/09/2020] [Indexed: 12/21/2022] Open
Abstract
Type 2 diabetes, characterized by dysfunction of pancreatic β-cells and insulin resistance in peripheral organs, accounts for more than 90% of all diabetes. Despite current developments of new drugs and strategies to prevent/treat diabetes, there is no ideal therapy targeting all aspects of the disease. Restoration, however, of insulin-producing β-cells, as well as insulin-responsive cells, would be a logical strategy for the treatment of diabetes. In recent years, generation of transplantable cells derived from stem cells in vitro has emerged as an important research area. Pluripotent stem cells, either embryonic or induced, are alternative and feasible sources of insulin-secreting and glucose-responsive cells. This notwithstanding, consistent generation of robust glucose/insulin-responsive cells remains challenging. In this review, we describe basic concepts of the generation of induced pluripotent stem cells and subsequent differentiation of these into pancreatic β-like cells, myotubes, as well as adipocyte- and hepatocyte-like cells. Use of these for modeling of human disease is now feasible, while development of replacement therapies requires continued efforts.
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Kuroda K, Komori T, Ishibashi K, Uto T, Kobayashi I, Kadokawa R, Kato Y, Ninomiya K, Takahashi K, Hirata E. Non-aqueous, zwitterionic solvent as an alternative for dimethyl sulfoxide in the life sciences. Commun Chem 2020; 3:163. [PMID: 36703409 PMCID: PMC9814479 DOI: 10.1038/s42004-020-00409-7] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2020] [Accepted: 10/14/2020] [Indexed: 01/29/2023] Open
Abstract
Dimethyl sulfoxide (DMSO) is widely used as a solvent in the life sciences, however, it is somewhat toxic and affects cell behaviours in a range of ways. Here, we propose a zwitterionic liquid (ZIL), a zwitterion-type ionic liquid containing histidine-like module, as a new alternative to DMSO. ZIL is not cell permeable, less toxic to cells and tissues, and has great potential as a vehicle for various hydrophobic drugs. Notably, ZIL can serve as a solvent for stock solutions of platinating agents, whose anticancer effects are completely abolished by dissolution in DMSO. Furthermore, ZIL possesses suitable affinity to the plasma membrane and acts as a cryoprotectant. Our results suggest that ZIL is a potent, multifunctional and biocompatible solvent that compensates for many shortcomings of DMSO.
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Affiliation(s)
- Kosuke Kuroda
- grid.9707.90000 0001 2308 3329Faculty of Biological Science and Technology, Institute of Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192 Japan
| | - Tetsuo Komori
- grid.9707.90000 0001 2308 3329Faculty of Biological Science and Technology, Institute of Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192 Japan
| | - Kojiro Ishibashi
- grid.9707.90000 0001 2308 3329Division of Tumor Cell Biology and Bioimaging, Cancer Research Institute of Kanazawa University, Kakuma-machi, Kanazawa, 920-1192 Japan
| | - Takuya Uto
- grid.410849.00000 0001 0657 3887Organization for Promotion of Tenure Track, University of Miyazaki, Nishi 1-1 Gakuen-Kibanadai, Miyazaki, 889-2192 Japan
| | - Isao Kobayashi
- grid.9707.90000 0001 2308 3329Faculty of Biological Science and Technology, Institute of Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192 Japan
| | - Riki Kadokawa
- grid.9707.90000 0001 2308 3329Faculty of Biological Science and Technology, Institute of Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192 Japan
| | - Yui Kato
- grid.9707.90000 0001 2308 3329Faculty of Biological Science and Technology, Institute of Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192 Japan
| | - Kazuaki Ninomiya
- grid.9707.90000 0001 2308 3329Institute for Frontier Science Initiative, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192 Japan
| | - Kenji Takahashi
- grid.9707.90000 0001 2308 3329Faculty of Biological Science and Technology, Institute of Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192 Japan
| | - Eishu Hirata
- grid.9707.90000 0001 2308 3329Division of Tumor Cell Biology and Bioimaging, Cancer Research Institute of Kanazawa University, Kakuma-machi, Kanazawa, 920-1192 Japan ,grid.9707.90000 0001 2308 3329Nano Life Science Institute of Kanazawa University, Kakuma-machi, Kanazawa, 920-1192 Japan
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Assetta B, Tang C, Bian J, O'Rourke R, Connolly K, Brickler T, Chetty S, Huang YWA. Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions. J Vis Exp 2020. [PMID: 33226027 DOI: 10.3791/61778] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
In Alzheimer's disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.
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Affiliation(s)
- Benedetta Assetta
- Department of Molecular Biology, Cell Biology and Biochemistry, Brown University
| | - Changyong Tang
- Department of Molecular Biology, Cell Biology and Biochemistry, Brown University; Department of Neurology, The Third Affiliated Hospital of Sun Yat-Sen University
| | - Jing Bian
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine
| | - Ryan O'Rourke
- Department of Molecular Biology, Cell Biology and Biochemistry, Brown University
| | - Kevin Connolly
- Department of Molecular Biology, Cell Biology and Biochemistry, Brown University
| | - Thomas Brickler
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine
| | - Sundari Chetty
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine
| | - Yu-Wen Alvin Huang
- Department of Molecular Biology, Cell Biology and Biochemistry, Brown University; Department of Neurology, Warren Alpert Medical School of Brown University; Center for Translational Neuroscience, Robert J. and Nancy D. Carney Institute for Brain Science and Brown Institute for Translational Science, Brown University;
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Wei F, Zhao L, Jing Y. Mechanisms underlying dimethyl sulfoxide-induced cellular migration in human normal hepatic cells. ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY 2020; 80:103489. [PMID: 32911099 DOI: 10.1016/j.etap.2020.103489] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/26/2019] [Revised: 08/03/2020] [Accepted: 09/04/2020] [Indexed: 06/11/2023]
Abstract
Numerous studies have reported that low-dose dimethyl sulfoxide (DMSO, <1.5%, v/v) can interfere with various cellular processes, such as cell proliferation, differentiation, apoptosis, and cycle. By contrast, minimal information is available about the effect of low-dose DMSO on cell migration. Here, we report the effect of DMSO (0.0005%-0.5%, v/v) on cellular migration in human normal hepatic L02 cells. We used the Cell Counting Kit-8 assay to measure cell viability, scratch wound healing assay to observe cellular migration, flow cytometry to analyze cell cycle and death pattern, reverse transcription quantitative polymerase chain reaction to evaluate mRNA expression, and Western blot to detect protein levels. After treatment with 0.0005% (v/v) DMSO, more cells entered S phase arrest, the MMP1/TIMP1 ratio increased, and HSP27 expression was elevated. After treatment with 0.1% (v/v) DMSO, more cells entered G0/G1 phase arrest, the MMP2/TIMP2 ratio increased, the p-p38 and p-Smad3 signaling pathways were activated, and neuropilin-1 expression was elevated. These results showed that cells migrate when their MMP1/TIMP1 and MMP2/TIMP2 ratios are imbalanced. Such migration is modulated by the p38/HSP27 signaling pathway and TGF-β/Smad3 dependent signaling pathway.
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Affiliation(s)
- Fengmei Wei
- Department of Physiology and Psychology, School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu Province 730000, PR China
| | - Long Zhao
- Department of Orthopaedics, Lanzhou University First Affiliated Hospital, Lanzhou, Gansu Province 730000, PR China
| | - Yuhong Jing
- Institute of Anatomy and Histology & Embryology, Neuroscience, School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu, 730000, PR China; Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Lanzhou University, Lanzhou, Gansu, 730000, PR China.
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46
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Stewart S, Arminan A, He X. NANOPARTICLE-MEDIATED DELIVERY OF CRYOPROTECTANTS FOR CRYOPRESERVATION. CRYO LETTERS 2020; 41:308-316. [PMID: 33814648 PMCID: PMC8015346] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Subscribe] [Scholar Register] [Indexed: 06/12/2023]
Abstract
Nanotechnology research has continued to garner interest and is investigated across a number of fields and industries, ranging from water treatment to clinical and biomedical applications. In biomedical research, for example, polymeric nanoparticles can be leveraged for controlled delivery of drugs and chemical compounds into cells. In cryobiological applications, polymeric nanoparticles can be utilized to deliver cryoprotectants (CPAs) and other protective agents, particularly those impermeable to the cell membrane, into cells to study their effects on cells during cooling down and warming back and at low temperatures. This perspective will discuss how polymeric nanoparticles have been used in cryobiology, with particular focus on how delivery systems have been specifically developed for low temperature applications and the potential for these systems going forward.
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Affiliation(s)
- Samantha Stewart
- Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, United States
| | - Alyssa Arminan
- Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, United States
| | - Xiaoming He
- Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, United States
- Robert E Fischell Institute for Biomedical Devices, University of Maryland, College Park, MD 20742, United States
- Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD 21202, United States
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Effects of DMSO on the Pluripotency of Cultured Mouse Embryonic Stem Cells (mESCs). Stem Cells Int 2020; 2020:8835353. [PMID: 33123203 PMCID: PMC7584961 DOI: 10.1155/2020/8835353] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2020] [Revised: 09/24/2020] [Accepted: 09/30/2020] [Indexed: 01/07/2023] Open
Abstract
DMSO is a commonly used solvent in biological studies, as it is an amphipathic molecule soluble in both aqueous and organic media. For that reason, it is the vehicle of choice for several water-insoluble substances used in research. At the molecular and cellular level, DMSO is a hydrogen-bound disrupter, an intercellular electrical uncoupler, and a cryoprotectant, among other properties. Importantly, DMSO often has overlooked side effects. In stem cell research, the literature is scarce, but there are reports on the effect of DMSO in human embryoid body differentiation and on human pluripotent stem cell priming towards differentiation, via modulation of cell cycle. However, in mouse embryonic stem cell (mESC) culture, there is almost no available information. Taking into consideration the almost ubiquitous use of DMSO in experiments involving mESCs, we aimed to understand the effect of very low doses of DMSO (0.0001%-0.2%), usually used to introduce pharmacological inhibitors/modulators, in mESCs cultured in two different media (2i and FBS-based media). Our results show that in the E14Tg2a mESC line used in this study, even the smallest concentration of DMSO had minor effects on the total number of cells in serum-cultured mESCs. However, these effects could not be explained by alterations in cell cycle or apoptosis. Furthermore, DMSO did not affect pluripotency or differentiation potential. All things considered, and although control experiments should be carried out in each cell line that is used, it is reasonable to conclude that DMSO at the concentrations used here has a minimal effect on this particular mESC line.
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48
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Yi JK, Park S, Ha JJ, Kim DH, Huang H, Park SJ, Lee MH, Ryoo ZY, Kim SH, Kim MO. Effects of Dimethyl Sulfoxide on the Pluripotency and Differentiation Capacity of Mouse Embryonic Stem Cells. Cell Reprogram 2020; 22:244-253. [DOI: 10.1089/cell.2020.0006] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Affiliation(s)
- Jun-Koo Yi
- Department of Embryo Transfer Research, Gyeongbuk Livestock Research Institute, Yeongju, Korea
| | - Song Park
- Core Protein Resources Center, DGIST, Daegu, Republic of Korea
| | - Jae-Jung Ha
- Department of Embryo Transfer Research, Gyeongbuk Livestock Research Institute, Yeongju, Korea
| | - Dae-Hyun Kim
- Department of Embryo Transfer Research, Gyeongbuk Livestock Research Institute, Yeongju, Korea
| | - Hai Huang
- Department of Animal Science and Biotechnology, Kyungpook National University, Sangju, Korea
| | - Si-Jun Park
- Department of Life Sciences and Biotechnology, School of Life Sciences and Biotechnology, Kyungpook National University, Daegu, Korea
| | - Mee-Hyun Lee
- College of Korean Medicine, Dongshin University, Naju, Jeollanamdo, Korea
- China-US (Henan) Hormel Cancer Institute, No. 127 Dongming Road, Zhengzhou, Henan, China
| | - Zae-Young Ryoo
- Department of Life Sciences and Biotechnology, School of Life Sciences and Biotechnology, Kyungpook National University, Daegu, Korea
| | - Sung-Hyun Kim
- Life Medicine Analysis Korea Polytechnics Institute, Nonsan, Korea
| | - Myoung-Ok Kim
- Department of Animal Science and Biotechnology, Kyungpook National University, Sangju, Korea
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Cha B, Kim J, Bello A, Lee G, Kim D, Kim BJ, Arai Y, Choi B, Park H, Lee S. Efficient Isolation and Enrichment of Mesenchymal Stem Cells from Human Embryonic Stem Cells by Utilizing the Interaction between Integrin α5 β1 and Fibronectin. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2020; 7:2001365. [PMID: 32995130 PMCID: PMC7507081 DOI: 10.1002/advs.202001365] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Revised: 06/02/2020] [Indexed: 05/09/2023]
Abstract
Human pluripotent stem cells (hPSCs) are a potent source of clinically relevant mesenchymal stem cells (MSCs) that confer functional and structural benefits in cell therapy and tissue regeneration. Obtaining sufficient numbers of MSCs in a short period of time and enhancing the differentiation potential of MSCs can be offered the potential to improve the regenerative activity of MSCs therapy. In addition, the underlying processes in the isolation and derivation of MSCs from hPSCs are still poorly understood and controlled. To overcome these clinical needs, an efficient and simplified technique on the isolation of MSCs from spontaneously differentiated human embryonic stem cells (hESCs) via integrin α5β1 (fibronectin (FN) receptor)-to-FN interactions (hESC-FN-MSCs) is successfully developed. It is demonstrated that hESC-FN-MSCs exhibit a typical MSC surface phenotype, cellular morphology, with the whole transcriptome similar to conventional adult MSCs; but show higher proliferative capacity, more efficient trilineage differentiation, enhanced cytokine secretion, and attenuated cellular senescence. In addition, the therapeutic potential and regenerative capacity of the isolated hESC-FN-MSCs are confirmed by in vitro and in vivo multilineage differentiation. This novel method will be useful in the generation of abundant amounts of clinically relevant MSCs for stem cell therapeutics and regenerative medicine.
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Affiliation(s)
- Byung‐Hyun Cha
- Division of Cardio‐Thoracic SurgeryDepartment of SurgeryCollege of MedicineUniversity of ArizonaTucsonAZ85724USA
| | - Jin‐Su Kim
- CellenGene R&D CenterOpen Innovation BuildingSeoul02455Republic of Korea
- Department of Biomedical ScienceCHA UniversityCHA BiocomplexSeongnam‐siGyeonggi‐do13488Republic of Korea
| | - Alvin Bello
- Department of Integrative EngineeringChung‐Ang UniversitySeoul06974Republic of Korea
| | - Geun‐Hui Lee
- Department of Biomedical ScienceCHA UniversityCHA BiocomplexSeongnam‐siGyeonggi‐do13488Republic of Korea
| | - Do‐Hyun Kim
- Department of Medical BiotechnologyDongguk University32 Dongguk‐ro, Ilsandong‐guGoyangGyeonggi10326Republic of Korea
| | - Byoung Ju Kim
- Department of Medical BiotechnologyDongguk University32 Dongguk‐ro, Ilsandong‐guGoyangGyeonggi10326Republic of Korea
| | - Yoshie Arai
- Department of Medical BiotechnologyDongguk University32 Dongguk‐ro, Ilsandong‐guGoyangGyeonggi10326Republic of Korea
| | - Bogyu Choi
- Department of Biomedical ScienceCHA UniversityCHA BiocomplexSeongnam‐siGyeonggi‐do13488Republic of Korea
| | - Hansoo Park
- Department of Integrative EngineeringChung‐Ang UniversitySeoul06974Republic of Korea
| | - Soo‐Hong Lee
- Department of Medical BiotechnologyDongguk University32 Dongguk‐ro, Ilsandong‐guGoyangGyeonggi10326Republic of Korea
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50
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Santarella F, Sridharan R, Marinkovic M, Do Amaral RJFC, Cavanagh B, Smith A, Kashpur O, Gerami‐Naini B, Garlick JA, O'Brien FJ, Kearney CJ. Scaffolds Functionalized with Matrix from Induced Pluripotent Stem Cell Fibroblasts for Diabetic Wound Healing. Adv Healthc Mater 2020; 9:e2000307. [PMID: 32597577 DOI: 10.1002/adhm.202000307] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2020] [Revised: 06/12/2020] [Indexed: 12/15/2022]
Abstract
Diabetic foot ulcers (DFUs) are chronic wounds, with 20% of cases resulting in amputation, despite intervention. A recently approved tissue engineering product-a cell-free collagen-glycosaminoglycan (GAG) scaffold-demonstrates 50% success, motivating its functionalization with extracellular matrix (ECM). Induced pluripotent stem cell (iPSC) technology reprograms somatic cells into an embryonic-like state. Recent findings describe how iPSCs-derived fibroblasts ("post-iPSF") are proangiogenic, produce more ECM than their somatic precursors ("pre-iPSF"), and their ECM has characteristics of foetal ECM (a wound regeneration advantage, as fetuses heal scar-free). ECM production is 45% higher from post-iPSF and has favorable components (e.g., Collagen I and III, and fibronectin). Herein, a freeze-dried scaffold using ECM grown by post-iPSF cells (Post-iPSF Coll) is developed and tested vs precursors ECM-activated scaffolds (Pre-iPSF Coll). When seeded with healthy or DFU fibroblasts, both ECM-derived scaffolds have more diverse ECM and more robust immune responses to cues. Post-iPSF-Coll had higher GAG, higher cell content, higher Vascular Endothelial Growth Factor (VEGF) in DFUs, and higher Interleukin-1-receptor antagonist (IL-1ra) vs. pre-iPSF Coll. This work constitutes the first step in exploiting ECM from iPSF for tissue engineering scaffolds.
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Affiliation(s)
- Francesco Santarella
- Royal College of Surgeons in Ireland 123 St Stephen's Green, Saint Peter's Dublin D02 YN77 Ireland
| | - Rukmani Sridharan
- Royal College of Surgeons in Ireland 123 St Stephen's Green, Saint Peter's Dublin D02 YN77 Ireland
| | - Milica Marinkovic
- Royal College of Surgeons in Ireland 123 St Stephen's Green, Saint Peter's Dublin D02 YN77 Ireland
| | - Ronaldo Jose Farias Correa Do Amaral
- Royal College of Surgeons in Ireland 123 St Stephen's Green, Saint Peter's Dublin D02 YN77 Ireland
- Biomedical Sciences, National University of Ireland Galway Newcastle Road Galway H91 W2TY Ireland
| | - Brenton Cavanagh
- Royal College of Surgeons in Ireland 123 St Stephen's Green, Saint Peter's Dublin D02 YN77 Ireland
| | - Avi Smith
- Department of Diagnostic SciencesTufts University School of Dental Medicine Boston MA 02111 USA
| | - Olga Kashpur
- Department of Diagnostic SciencesTufts University School of Dental Medicine Boston MA 02111 USA
| | - Behzad Gerami‐Naini
- Department of Diagnostic SciencesTufts University School of Dental Medicine Boston MA 02111 USA
| | - Jonathan A. Garlick
- Department of Diagnostic SciencesTufts University School of Dental Medicine Boston MA 02111 USA
| | - Fergal J. O'Brien
- Royal College of Surgeons in Ireland 123 St Stephen's Green, Saint Peter's Dublin D02 YN77 Ireland
- The University of Dublin Trinity College, College Street Dublin Dublin 2, D02 R590 Ireland
- Advanced Materials and Bioengineering Research Centre (AMBER)RCSI and TCD Dublin D02 HP52 Ireland
| | - Cathal J. Kearney
- Royal College of Surgeons in Ireland 123 St Stephen's Green, Saint Peter's Dublin D02 YN77 Ireland
- The University of Dublin Trinity College, College Street Dublin Dublin 2, D02 R590 Ireland
- Advanced Materials and Bioengineering Research Centre (AMBER)RCSI and TCD Dublin D02 HP52 Ireland
- Department of Biomedical EngineeringUniversity of Massachusetts Amherst Amherst MA 01003‐9292 USA
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