Biomaterials play an important role in medicine from contact lenses to joint replacements. High-throughput screening coupled with machine learning has identified synthetic polymers that prevent bacterial biofilm formation, prevent fungal cell attachment, control immune cell attachment and phenotype, or direct stem cell fate. In-vitro preadsorption of proteins from culture medium plays a pivotal role in controlling cell response. However, there is a paucity of studies on the screening of protein adsorption into material libraries. Here, we show how quantitative analysis of protein adsorption on a 208-member polymer microarray can be achieved using liquid extraction surface analysis, combined with an adaptation of the droplet microarray (DMA) approach and tandem mass spectrometry (LESA-MS/MS) for protein identification. This study uses a fully defined cell culture medium containing only four proteins (Essential 8) to demonstrate the feasibility of the analysis approach. Our findings show that we can generate quantitative and predictive machine learning models of protein adsorption that elucidate key polymer features that describe the relationship between surface chemistry and protein adsorption. This information is of use for the rational design of new materials with bespoke protein attachment properties for biomaterials, medical devices, or in vitro compound screening.

Two human induced pluripotent stem cell (hiPSC) lines, FLENIi002-A and FLENIi003-A, were generated from peripheral blood mononuclear cells (PBMCs) using the lentiviral-hSTEMCCA-loxP vector. FLENIi002-A was derived from a 52-year-old Down syndrome patient with Alzheimer's disease and amyloid-beta brain accumulation. FLENIi003-A was derived from a cognitively unimpaired 51-year-old Down syndrome patient exhibiting no brain amyloid-beta deposition. Both lines retained the trisomy 21 genotype, and their pluripotency and differentiation potential were confirmed.

The process of a single-celled zygote developing into a complex multicellular organism is precisely regulated at spatial and temporal levels in vivo. However, understanding the mechanisms underlying development, particularly in humans, has been constrained by technical and ethical limitations associated with studying natural embryos. Harnessing the intrinsic ability of embryonic stem cells (ESCs) to self-organize when induced and assembled, researchers have established several embryo models as alternative approaches to studying early development in vitro. Recent studies have revealed the critical role of extraembryonic cells in early development; and many groups have created more sophisticated and precise ESC-derived embryo models by incorporating extraembryonic stem cell lines, such as trophoblast stem cells (TSCs), extraembryonic mesoderm cells (EXMCs), extraembryonic endoderm cells (XENs, in rodents), and hypoblast stem cells (in primates). Here, we summarize the characteristics of existing mouse and human embryonic and extraembryonic stem cells and review recent advancements in developing mouse and human embryo models.

Maintaining an optimal mitochondrial distribution is critical to ensure an adequate supply of energy and metabolites to support important cellular functions. How cells balance dynamic mitochondrial processes to achieve homeostasis is incompletely understood. Here, we show that ARMC1 partitioning between distinct mitochondrial protein complexes is a key determinant of mitochondrial distribution. In one complex, the mitochondrial trafficking adaptor MIRO recruits ARMC1, which mediates the assembly of a mitochondrial fission regulator (MTFR). MTFR stability depends on ARMC1, and MIRO-MTFR complexes specifically antagonize retrograde mitochondrial movement. In another complex, DNAJC11 facilitates ARMC1 release from mitochondria. Disrupting MIRO-MTFR assembly fails to rescue aberrant mitochondrial distributions clustered in the perinuclear area observed with ARMC1 deletion, while disrupting ARMC1 interaction with DNAJC11 leads to excessive mitochondrially localized ARMC1 and distinct mitochondrial defects. Thus, the abundance and trafficking impact of MIRO-MTFR complexes require ARMC1, whose mito-cytoplasmic shuttling balanced by DNAJC11 tunes steady-state mitochondrial distributions.

Human induced pluripotent stem cells (hiPSCs) hold the potential to generate any human tissue for transplantation in regenerative therapies. These complex cell therapies require billions of cells, which is challenging to acquire in planar adherent cultures. Transitioning hiPSCs to 3D suspension culture on microcarrier materials, often bead-shaped, improves the total surface area accessible to cells, thereby enabling culture scale-up. However, bead-shaped microcarriers do not have the optimal shape configuration, because it is the lowest surface-to-volume ratio of all geometrical shapes, and it also induces uncontrolled cell clumping. Application of synthetic, microfibrous rafts as a replacement for bead-shaped microcarriers potentially solves these issues. Here, microfibrous rafts are engineered by first screening a supramolecular biomaterial library composed of bisurea (BU)-peptide conjugate additives for its ability to induce hiPSC adhesion and maintenance of its pluripotent state, followed by electrospinning the screening-hit into raft-like structures. The resulting rafts contain cylinder-like microfibers, which have a higher surface-to-volume ratio compared to conventional bead-shaped microcarriers, and the flat configuration of the rafts prevents clumping.

Insulin is an important component of stem cell cultures; however, its role in the proliferation of avian primordial germ cells (PGCs) is unknown. The proliferation of PGCs in cultures varies and the growth factors and signaling pathways necessary to induce the proliferation of PGCs in chickens are unknown. Therefore, we conducted the present study to investigate the effect of insulin on the survival and proliferation of PGCs. In this study, we observed that under this culture system, PGCs proliferate in the presence of insulin, but do not proliferate in the absence of insulin. Furthermore, in insulin-lacking media, the expression of pluripotency genes, including LIN28, NANOG, POUV, and SOX2, was markedly decreased. Similarly, the expression of cell adhesion proteins ZO-1, Occludin, and JAM-A was significantly reduced. Elevated levels of ROS, GSSG, and MDA reduced the redox capacity of the cells and induced apoptosis. Subsequent transcriptome analyses revealed that insulin is one of the key factors in the proliferation of chicken PGCs through the regulation of downstream genes by PI3K/AKT, ECM-receptor interaction, Wnt, and P53 signaling, and that these downstream genes may be important for PGCs' proliferation and survival.

Arrhythmogenic cardiomyopathy (ACM) is one of the leading causes of sudden cardiac death in children, young adults, and athletes and is characterized by the fibro-fatty replacement of the myocardium, predominantly of the right ventricle. Sixty percent of patients with ACM have a known genetic cause, but for the remainder, the pathogenesis is unknown. This lack of mechanistic understanding has slowed the development of disease-modifying therapies, and children with ACM have a high degree of morbidity and mortality.

Induced pluripotent stem cells (iPSCs) from 3 family members were differentiated into cardiac myocytes (CMs). Calcium imaging was conducted by labeling calcium with CAL-520 and confocal imaging to capture calcium sparks after iPSC-CMs were electrically paced. A cardiac-specific, inducible knockout mouse (Tax1bp3-/-) was made and intracardiac electrophysiology studies conducted to observe arrhythmia inducibility following pacing.

We identified a kindred with multiple members affected by ACM cosegregating with biallelic variants in the gene TAX1BP3, which encodes the protein TAX1BP3 (Tax1-binding protein 3). iPSC-CMs derived from this kindred demonstrated increased intracellular lipid droplets, induction of TRPV4 (transient receptor potential vanilloid type 4) expression, and inducible TRPV4 current. This was associated with depletion of the intracellular sarcoplasmic reticulum Ca2+ store and increased RyR2 (ryanodine receptor 2)-mediated store Ca2+ leak and delayed afterdepolarizations, a known mechanism of Ca2+-mediated arrhythmogenesis. Similarly, Tax1bp3 cardiac-specific knockout mice had increased Ca2+ leak and were predisposed to ventricular arrhythmias compared with wild-type mice. Ca2+ leak in both the iPSC-CMs and mouse ventricular myocytes was rescued by small molecule TRPV4 inhibition. This strategy also effectively reduced Ca2+ leak in a PKP2 (plakophilin 2) p.His773AlafsX8 iPSC-CM model of ACM.

We conclude that TAX1BP3 is associated with rare autosomal recessive ACM through TRPV4-mediated Ca2+ leak from RyR2. Further, TRPV4 current inhibition has the potential to be a new therapeutic target for ACM.

Pooled processing, in which cells from multiple sources are cultured or captured together, is an increasingly popular strategy for droplet-based single cell sequencing studies. This design allows efficient scaling of experiments, isolation of cell-intrinsic differences, and mitigation of batch effects. We present CellBouncer, a computational toolkit for demultiplexing and analyzing single-cell sequencing data from pooled experiments. We demonstrate that CellBouncer can separate and quantify multi-species and multi-individual cell mixtures, identify unknown mitochondrial haplotypes in cells, assign treatments from lipid-conjugated barcodes or CRISPR sgRNAs, and infer pool composition, outperforming existing methods. We also introduce methods to quantify ambient RNA contamination per cell, infer individual donors' contributions to the ambient RNA pool, and determine a consensus doublet rate harmonized across data types. Applying these tools to tetraploid composite cells, we identify a competitive advantage of human over chimpanzee mitochondria across 10 cell fusion lines and provide evidence for inter-mitochondrial incompatibility and mito-nuclear incompatibility between species.

Blood vessels permeate all organs and execute myriad roles in health and disease. Here, we present a protocol to efficiently generate human artery and vein endothelial cells (ECs) from pluripotent stem cells within 3-4 days of differentiation. We delineate how to seed human pluripotent stem cells and sequentially differentiate them into primitive streak, lateral mesoderm, and either artery or vein ECs. We differentiate stem cells in defined, serum-free culture media in monolayers, without feeder cells or genetic manipulations. For complete details on the use and execution of this protocol, please refer to Ang et al. 1.

Hematopoietic stem cells (HSCs) generate blood and immune cells. Here, we present a protocol to differentiate human pluripotent stem cells (hPSCs) into hematopoietic progenitors that express the signature HSC transcription factors HLF, HOXA5, HOXA7, HOXA9, and HOXA10. hPSCs are dissociated, seeded, and then sequentially differentiated into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and hematopoietic progenitors through the sequential addition of defined, serum-free media. This 10-day protocol enables the manufacturing of blood and immune cells in monolayer cultures. For complete details on the use and execution of this protocol, please refer to Fowler and Zheng et al.1.

The rising number of cats as pets and the growing interest in animal welfare have led to an increased need for the latest treatments in feline veterinary medicine. Among these, veterinary regenerative medicine using pluripotent stem cells is gaining significant attention. However, there have been no reports on establishing feline embryonic stem cell (ESC) lines that possess the pluripotent potential and the ability to differentiate into three germ layers.

In this study, we isolated three inner cell masses from feline in vitro-derived blastocysts and subcultured them in a chemically defined medium (StemFit AK02N). We assessed the expression of undifferentiated markers, the ability to differentiate into the three germ layers, and the karyotype structure.

We established three feline ESC lines. Feline ESCs exhibited positive staining for alkaline phosphatase. RT-qPCR analysis revealed that these cells express undifferentiated marker genes in vitro. Immunostaining and flow cytometry analysis demonstrated that feline ESCs express undifferentiated marker proteins in vitro. In the KSR/FBS medium with or without Activin A, feline ESCs differentiated into all three germ layers (ectoderm, endoderm, and mesoderm), expressing specific marker genes and proteins for each germ layer, as evidenced by RT-qPCR, immunostaining, and flow cytometry. Furthermore, we confirmed that feline ESCs formed teratomas comprising all three germ layers in mouse testes, demonstrating de novo pluripotency in vivo. We also verified that the feline ESCs maintained a normal karyotype.

We successfully established three feline ESC lines, each possessing pluripotent potential and capable of differentiating into all three germ layers, derived from the inner cell masses of blastocysts produced in vitro.

The faithful production of primordial germ cells (PGCs) in vitro opens a wide range of novel applications in reproductive biology and medicine. However, the reproducibility of PGCs culture conditions across different laboratories or breeds remains a challenge. Therefore, it is necessary to research the molecular dynamics that lead to the gradual establishment of cultured PGCs lines network. Here, the results of single-cell RNA-seq indicated that the cell cycle drove cellular heterogeneity. The active populations engaged in PGC self-renewal and the characteristics of the aging cell fate have been identified. The active self-renewal populations presented a rising expression of DNA repair genes, couple with a high proportion of cells in G1/S phase and a low frequency of cells in G2 phase. Notably, Hippo, FoxO, AMPK and MAPK pathways are active within these populations. The combination of six activator or inhibitors, targeting these active pathways, resulted in a significantly higher proliferation rate of PGCs and an increased number of cells entering the G1 and S phases. Importantly, they greatly reduced the establishment time to a minimum of 26 days and increased the efficiency of male PGC line establishment to 59 % in FS medium. Our results provided several new insights into the PGCs.

Our knowledge of testis development and function mainly comes from research using mammalian model organisms, primarily the mouse. However, there are integral differences between men and other mammalian species regarding cellular composition and expression profiles during fetal and post-natal testis development and in the mature testis. Therefore, to specifically learn more about human testis development and function, there is a need to use human testis tissue for research. Human testicular tissues that have been donated for research have allowed extensive molecular and cytological assessments, as well as single-cell transcriptome and epigenome analyses. These tissues have also been used for the development of cell technologies and in vitro models that aim to improve infertility treatments and diagnostics. Biopsied material taken from patients and designated for research is usually very small in size and is unsuitable for comprehensive studies. On the other hand, research using whole testes obtained from deceased, deidentified donors has become a valuable resource to assess conservation between humans and other organisms and identify human-specific phenomena. This review discusses the acquisition of donated deidentified human testes and their use for basic science research.

Tibetan adaptation to high-altitude hypoxia remains a classic example of Darwinian selection in humans. Amongst Tibetan populations, alleles in the EPAS1 gene - whose protein product, HIF-2α, is a central regulator of the hypoxia response - have repeatedly been shown to carry some of the strongest signals of positive selection in humans. However, selective sweep signals alone may only account for some of the phenotypes that differentiate high-altitude adapted populations from closely related lowlanders. Therefore, there is a pressing need to functionally probe adaptive alleles and their impact at both the locus-specific and genome-wide levels and across cell types to uncover the full range of beneficial traits. To this end, we established a library of induced pluripotent stem cells (iPSCs) derived from Tibetan and Han Chinese individuals, a robust model system allowing precise exploration of allelic effects on transcriptional responses, and we differentiated them into vascular endothelium. Using this system, we focus first on a hypoxia-dependent enhancer (ENH5) that contributes to the regulation of EPAS1 to investigate its locus-specific effects in endothelium. Then, to cast a wider net, we harness the same experimental system to compare the transcriptome of Tibetan and Han Chinese cells in hypoxia and find evidence that angiogenesis, energy metabolism and immune pathways differ between these two populations with different histories of long-term residence at high altitude. Coupled with evidence of polygenic adaptations targeting the same pathways, these results suggests that the observed transcriptional differences between the two populations were shaped by natural selection.

In adults, the limbal stem cells (LSC) reside in the limbal region of the eye, at the junction of the cornea and the sclera where they renew the outer epithelial layer of the cornea assuring transparency. LSC deficiencies (LSCD) due to disease or injury account for one of the major causes of blindness. Among current treatments for LSCD, cornea transparency can be restored by providing new LSC to the damaged eye and induced pluripotent stem cells (iPSC) holds great promise as a new advanced cell source. A synthetic mRNA-based protocol to produce human iPSC from bone marrow mesenchymal stem cells has been defined. The results demonstrate a standardizable method that can be easily adaptable for clinical-grade production standards, produce high-purity LSC-like cells in a relatively rapid timeframe of 12 days, and can be successfully seeded on amniotic membrane or a biodegradable fibrin gel for transplantation. In vivo data demonstrated it is feasible to transplant the iPSC-LSC fibrin patch. In conclusion, an efficient method has been developed to produce patient-specific LSC and seed them on a scaffold fibrin gel for future treatment of LSC-deficiency disease.

Lysosomal storage diseases (LSDs) comprise ~50 monogenic disorders marked by the buildup of cellular material in lysosomes, yet systematic global molecular phenotyping of proteins and lipids is lacking. We present a nanoflow-based multiomic single-shot technology (nMOST) workflow that quantifies HeLa cell proteomes and lipidomes from over two dozen LSD mutants. Global cross-correlation analysis between lipids and proteins identified autophagy defects, notably the accumulation of ferritinophagy substrates and receptors, especially in NPC1-/- and NPC2-/- mutants, where lysosomes accumulate cholesterol. Autophagic and endocytic cargo delivery failures correlated with elevated lysophosphatidylcholine species and multilamellar structures visualized by cryo-electron tomography. Loss of mitochondrial cristae, MICOS complex components, and OXPHOS components rich in iron-sulfur cluster proteins in NPC2-/- cells was largely alleviated when iron was provided through the transferrin system. This study reveals how lysosomal dysfunction affects mitochondrial homeostasis and underscores nMOST as a valuable discovery tool for identifying molecular phenotypes across LSDs.

The cultivation and differentiation of human embryonic stem cells (hESCs) into organoids are crucial for advancing of new drug development and personalized cell therapies. Despite establishing of chemically defined hESC culture media over the past decade, these media's reliance on growth factors, which are costly and prone to degradation, poses a challenge for sustained and stable cell culture. Here, we introduce an hESC culture system(E6Bs) that facilitates the long-term, genetically stable expansion of hESCs, enabling cells to consistently sustain high levels of pluripotency markers, including NANOG, SOX2, TRA-1-60, and SSEA4, across extended periods. Moreover, organoids derived from hESCs using this medium were successfully established and expanded for at least one month, exhibiting differentiation into cortical organoids, GABAergic precursor organoids and heart-forming organoids. This innovative system offers a robust tool for preserving hESC homeostasis and modeling the nervous system in vitro.

The genetic variants of LMNA cause an array of diseases that often affect the heart. LMNA-related cardiomyopathy exhibits high-penetrance and early-onset phenotypes that lead to late-stage heart failure or lethal arrhythmia. As a subtype of dilated cardiomyopathy and arrhythmogenic cardiomyopathy, LMNA-related cardiac dysfunction is resistant to existing cardiac therapeutic strategies, leaving a major unmet clinical need in cardiomyopathy management.

Here we comprehensively summarize current knowledge about the genetic basis, disease models and pathological mechanisms of LMNA-related cardiomyopathy. Recent translational studies were highlighted to indicate new therapeutic modalities such as gene supplementation, gene silencing and genome editing therapy, which offer potential opportunities to overcome the difficulties in the development of specific drugs for this disease.

LMNA-related cardiomyopathy involves many diverse disease mechanisms that preclude small-molecule drugs that target only a small fraction of the mechanisms. Agreeing to this notion, the first-in-human clinical trial for this disease recently reported futility. By contrast, gene therapy offers the new hope to directly intervene LMNA variants and demonstrates a tremendous potential for breakthrough therapy for this disease. Concepts in this review are also applicable to studies of other genetic diseases that lack effective therapeutics.

Interest in animal cell-based meat (ACBM) as an environmentally conscious replacement for livestock production has been increasing; however, a life cycle assessment (LCA) for the existing production methods of ACBM has not been conducted. Currently, ACBM products are being produced at a small scale, but ACBM companies are intending to scale-up production. Updated findings from recent technoeconomic assessments (TEAs) of ACBM were utilized to perform an LCA of near-term ACBM production. A scenario analysis was conducted utilizing the metabolic requirements examined in the TEAs of ACBM, and a purification factor was utilized to account for growth medium component processing. The results indicate that the environmental impact of near-term ACBM production has the potential to be significantly higher than beef if a highly refined growth medium is utilized for ACBM production. This study highlights the need to develop a sustainable animal cell growth medium that is optimized for high-density animal cell proliferation for ACBM to generate positive economic and environmental benefits.

Since their discovery, human induced pluripotent stem cells (hiPSCs) have been instrumental in biomedical research, particularly in the fields of disease modelling, drug screening and regenerative therapies. Their use has significantly increased over recent years driven by the ability of hiPSCs to provide differentiated cell models without requiring embryonic stem cells. Furthermore, the transition from integrating to non-integrating reprogramming methodologies has contributed to the increase in utilisation. This shift minimises the risk of genomic alterations, enhancing the safety and reliability of hiPSCs. However, the factors that contribute to reprogramming success are still not well understood. In this study, we conducted a comparative analysis of the most prevalent non-integrating reprogramming methods across a range of starting source materials to assess their impact on reprogramming success rates. We found that while source material does not significantly impact success rates, the Sendai virus reprogramming method yields significantly higher success rates relative to the episomal reprogramming method. Our findings offer important insights from a biobanking perspective, for which long-term reliability, integrity and reproducibility of hiPSCs are crucial.

Definitive endoderm (DE) derived from human pluripotent stem cells (hPSCs) holds great promise for cell-based therapies and drug discovery. However, current DE differentiation methods required undefined components and/or expensive recombinant proteins, limiting their scalable manufacture and clinical use. Homogeneous DE differentiation in defined and recombinant protein-free conditions remains a major challenge. Here, by systematic optimization and high-throughput screening, we report a chemically defined, small-molecule-based defined system that contains only four components (4C), enabling highly efficient and cost-effective DE specification of hPSCs in the absence of recombinant proteins. 4C-induced DE can differentiate into functional hepatocytes, lung epithelium, and pancreatic β cells in vitro and multiple DE derivatives in vivo. Genomic accessibility analysis reveals that 4C reconfigures chromatin architecture to allow key DE transcription factor binding while identifying TEAD3 as a novel key regulator of the process. This system may facilitate mass production of DE derivatives for drug discovery, disease modeling, and cell therapy.

This paper describes a detailed protocol for human pluripotent stem cells (hPSCs) cultivation as matrix-free cell-only aggregates in defined and xeno-free culture medium in stirred tank bioreactors (STBRs). Starting with a frozen stock pre-expanded on conventional culture dishes (2D), the ultimate process is performed in 150 mL culture scale in stirred tank bioreactors (3D) and is designed to produce up to 500 million pluripotent hPSC within 7 days. The culture strategy includes perfusion-based cell feeding facilitating process control, automation, and higher cell yields. Ultimately, this detailed protocol describes an important step for generating a defined starting cell population for directed lineage differentiation and subsequently fueling human cell-based assays and regenerative medicine approaches.

Within the glomerulus, podocytes are highly specialized visceral epithelial cells, that are part of the glomerular filtration barrier. Human podocyte cell culture is rather challenging for primary or immortalized cells, due to the non-proliferative state of the cells. In addition, de-differentiation is often observed rapidly. Hence, iPSC-derived podocytes offer an exciting alternative to culture podocyte-like cells from different donors over a prolonged time. Here we report an improved protocol of a simple and rapid one-step differentiation that drives iPSC into podocyte-like cells in 10 days and offers the possibility to transfer the podocyte-like cells into another plate format once.

With no effective treatments for functional recovery after injury, spinal cord injury (SCI) remains one of the unresolved healthcare challenges. Human induced pluripotent stem cell (hiPSC) transplantation is a versatile patient-specific regenerative approach for functional recovery after SCI. Injectable electroconductive hydrogel (ECH) can further enhance the cell transplantation efficacy through a minimally invasive manner as well as recapitulate the native bioelectrical microenvironment of neural tissue. Given these considerations, we report a novel ECH prepared through self-assembly facilitated in situ gelation of natural silk fibroin (SF) derived from mulberry Bombyx mori silk and electrically conductive PEDOT:PSS. PEDOT:PSS was pre-stabilized to prevent the potential delamination of its hydrophilic PSS chain under aqueous environment using 3% (v/v) (3-glycidyloxypropyl)trimethoxysilane (GoPS) and 3% (w/v) poly(ethylene glycol)diglycidyl ether (PeGDE). The resultant ECH formulations are easily injectable with standard hand force with flow point below 100 Pa and good shear-thinning properties. The ECH formulations with unmodified and GoPS-modified PEDOT:PSS, that is, SF/PEDOT and SF/PEDOTGoP maintain comparable elastic modulus to spinal cord (~10-60 kPa) under physiological condition, indicating their flexibility. The GoPS-modified ECHs also display improved structural recoverability (~70%-90%) as compared to the unmodified versions of the ECHs (~30%-80%), as indicated by the three interval time thixotropy (3ITT) test. Additionally, these ECHs possess electrical conductivity in the range of ~0.2-1.2 S/m comparable to spinal cord (1-10 S/m), indicating their ability to mimic native bioelectrical environment. Approximately 80% or more cell survival was observed when hiPSC-derived cortical neurons and astrocytes were encapsulated within these ECHs. These ECHs support the maturation of cortical neurons when embedded for 7 days, fostering the development of a complex, interconnected network of long axonal processes and promoting synaptogenesis. These results underline the potential of silk ECHs in cell transplantation therapy for spinal cord regeneration.

Mutations in genes coding sarcomere components are the major causes of human inherited cardiomyopathy. Genome editing is widely applied to genetic modification of human pluripotent stem cells (hPSCs) before hPSCs were differentiated into cardiomyocytes to model cardiomyopathy. Whether genetic mutations influence the early hPSC differentiation process or solely the terminally differentiated cardiomyocytes during cardiac pathogenesis remains challenging to distinguish. To solve this problem, here we harnessed chemically modified mRNA (modRNA) and synthetic single-guide RNA to develop an efficient genome editing approach in hPSC-derived cardiomyocytes (hPSC-CMs). We showed that modRNA-based CRISPR/Cas9 mutagenesis of TNNT2, the coding gene for cardiac troponin T, results in sarcomere disassembly and contractile dysfunction in hPSC-CMs. These structural and functional phenotypes were associated with profound downregulation of oxidative phosphorylation genes and upregulation of cardiac stress markers NPPA and NPPB. These data confirmed that sarcomeres regulate gene expression in hPSC-CMs and highlighted the RNA technology as a powerful tool to achieve stage-specific genome editing during hPSC differentiation.

Extracellular vesicles (EVs) are nanovesicles released from different cell types from biofluids such as blood, urine, and cerebrospinal fluid. They vary in size and biomarkers, and their biogenesis pathways allow them to be divided into three major types: exosomes, micro-vesicles, and apoptotic bodies. EVs have been studied in the context of diagnosis and therapeutic intervention of various pathological conditions such as cancer, neurodegenerative diseases, and pulmonary diseases. However, the production of EV-based therapeutics can be affected by the source, heterogeneity, or disease, raising questions about the manufacturing and validation of EVs of clinical grade and their scope regarding good manufacturing practice (GMP) in the industry. To address this, we have discussed the state-of-the-art requirements for EV production that must occur in a GMP-compliant environment with a reliable and traceable source. Additionally, EVs' homogeneity and the therapeutics' purity and stability must be analyzed and validated. Quality control measures must also be established to ensure the safety and efficacy of EVs. In conclusion, these considerations must be weighed carefully when manufacturing and validating EVs of clinical grade to ensure their safety and efficacy for therapeutic use.

Astrocytes are the most abundant type of glial cell in the central nervous system and they play pivotal roles in both normal health and disease. Their dysfunction is detrimental to many brain related pathologies. Under pathological conditions, such as Alzheimer's disease, astrocytes adopt an activated reactive phenotype which can contribute to disease progression. A prominent risk factor for many neurodegenerative diseases is neuroinflammation which is the purview of glial cells, such as astrocytes and microglia. Human in vitro models have the potential to reveal relevant disease specific mechanisms, through the study of individual cell types such as astrocytes or the addition of specific factors, such as those secreted by microglia. The aim of this study was to generate human cortical astrocytes, in order to assess their protein and gene expression, examine their reactivity profile in response to exposure to the microglial secreted factors IL-1α, TNFα and C1q and assess their functionality in terms of calcium signalling and metabolism. The successfully differentiated and stimulated reactive astrocytes display increased IL-6, RANTES and GM-CSF secretion, and increased expression of genes associated with reactivity including, IL-6, ICAM1, LCN2, C3 and SERPINA3. Functional assessment of these reactive astrocytes showed a delayed and sustained calcium response to ATP and a concomitant decrease in the expression of connexin-43. Furthermore, it was demonstrated these astrocytes had an increased glycolytic capacity with no effect on oxidative phosphorylation. These findings not only increase our understanding of astrocyte reactivity but also provides a functional platform for drug discovery.

The discovery of mouse embryonic stem cells in 1981 transformed research in mammalian developmental biology and functional genomics. The subsequent generation of human pluripotent stem cells (PSCs) and the development of molecular reprogramming have opened unheralded avenues for drug discovery and cell replacement therapy. Here, I review the history of PSCs from the perspective that long-term self-renewal is a product of the in vitro signaling environment, rather than an intrinsic feature of embryos. I discuss the relationship between pluripotent states captured in vitro to stages of epiblast in the embryo and suggest key considerations for evaluation of PSCs. A remaining fundamental challenge is to determine whether naïve pluripotency can be propagated from the broad range of mammals by exploiting common principles in gene regulatory architecture.

Cell lineage specification is tightly associated with profound morphological changes in the developing human embryo, particularly during gastrulation. The interplay between mechanical forces and biochemical signals is poorly understood.

Here, we dissect the effects of biochemical cues and physical confinement on a 3D in vitro model based on spheroids formed from human induced pluripotent stem cells (hiPSCs).

First, we compare self-renewing versus differentiating media conditions in free-floating cultures and observe the emergence of tri-germ layers. In these unconfined conditions, BMP4 exposure induces polarised expression of SOX17 in conjunction with spheroid elongation. We then physically confine spheroids using PEG-peptide hydrogels and observe dramatically reduced SOX17 expression, albeit rescued if gels that soften over time are used instead.

Our study combines high-content imaging, synthetic hydrogels, and hiPSCs-derived models of early development to define the drivers that cause changes in the shape and the emergence of germ layers.

Type 1 diabetes, an autoimmune disorder leading to the destruction of pancreatic β-cells, requires lifelong insulin therapy. Islet transplantation offers a promising solution but faces challenges such as limited availability and the need for immunosuppression. Induced pluripotent stem cells (iPSCs) provide a potential alternative source of functional β-cells and have the capability for large-scale production. However, current differentiation protocols, predominantly conducted in hybrid or 2D settings, lack scalability and optimal conditions for suspension culture.

We examined a range of bioreactor scaleup process parameters and quality target product profiles that might affect the differentiation process. This investigation was conducted using an optimized High Dimensional Design of Experiments (HD-DoE) protocol designed for scalability and implemented in 0.5L (PBS-0.5 Mini) vertical wheel bioreactors.

A three stage suspension manufacturing process is developed, transitioning from adherent to suspension culture, with TB2 media supporting iPSC growth during scaling. Stage-wise optimization approaches and extended differentiation times are used to enhance marker expression and maturation of iPSC-derived islet-like clusters. Continuous bioreactor runs were used to study nutrient and growth limitations and impact on differentiation. The continuous bioreactors were compared to a Control media change bioreactor showing metabolic shifts and a more β-cell-like differentiation profile. Cryopreserved aggregates harvested from the runs were recovered and showed maintenance of viability and insulin secretion capacity post-recovery, indicating their potential for storage and future transplantation therapies.

This study demonstrated that stage time increase and limited media replenishing with lactate accumulation can increase the differentiation capacity of insulin producing cells cultured in a large-scale suspension environment.

Genome-wide association studies implicate common genetic variations in the LRP1 (low-density lipoprotein receptor-related protein 1 gene) locus at risk for multiple vascular diseases and traits. However, the underlying biological mechanisms are unknown.

Fine mapping analyses included Bayesian colocalization to identify the most likely causal variant. Human induced pluripotent stem cells were genome-edited using CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein 9) to delete or modify candidate enhancer regions and generate LRP1 knockout cell lines. Cells were differentiated into smooth muscle cells through a mesodermal lineage. Transcription regulation was assessed using luciferase reporter assay, transcription factor knockdown, and chromatin immunoprecipitation. Phenotype changes in cells were conducted using cellular assays, bulk RNA sequencing, and mass spectrometry.

Multitrait colocalization analyses pointed at rs11172113 as the most likely causal variant in LRP1 for fibromuscular dysplasia, migraine, pulse pressure, and spontaneous coronary artery dissection. We found the rs11172113-T allele to associate with higher LRP1 expression. Genomic deletion in induced pluripotent stem cell-derived smooth muscle cells supported rs11172113 to locate in an enhancer region regulating LRP1 expression. We found transcription factors MECP2 (methyl CpG binding protein 2) and SNAIL (Zinc Finger Protein SNAI1) to repress LRP1 expression through an allele-specific mechanism, involving SNAIL interaction with disease risk allele. LRP1 knockout decreased induced pluripotent stem cell-derived smooth muscle cell proliferation and migration. Differentially expressed genes were enriched for collagen-containing extracellular matrix and connective tissue development. LRP1 knockout and deletion of rs11172113 enhancer showed potentiated canonical TGF-β (transforming growth factor beta) signaling through enhanced phosphorylation of SMAD2/3 (Mothers against decapentaplegic homolog 2/3). Analyses of the protein content of decellularized extracts indicated partial extracellular matrix remodeling involving enhanced secretion of CYR61 (cystein rich angiogenic protein 61), a known LRP1 ligand involved in vascular integrity and TIMP3 (Metalloproteinase inhibitor 3), implicated in extracellular matrix maintenance and also known to interact with LRP1.

Our findings support allele-specific LRP1 expression repression by the endothelial-to-mesenchymal transition regulator SNAIL. We propose decreased LRP1 expression in smooth muscle cells to remodel the extracellular matrix enhanced by TGF-β as a potential mechanism of this pleiotropic locus for vascular diseases.

Approval of induced pluripotent stem cells (iPSCs) for the manufacture of cell therapies to support clinical trials is now becoming realized after 20 years of research and development. In 2022 the International Society for Cell and Gene Therapy (ISCT) established a Working Group on Emerging Regenerative Medicine Technologies, an area in which iPSCs-derived technologies are expected to play a key role. In this article, the Working Group surveys the steps that an end user should consider when generating iPSCs that are stable, well-characterised, pluripotent, and suitable for making differentiated cell types for allogeneic or autologous cell therapies. The objective is to provide the reader with a holistic view of how to achieve high-quality iPSCs from selection of the starting material through to cell banking. Key considerations include: (i) intellectual property licenses; (ii) selection of the raw materials and cell sources for creating iPSC intermediates and master cell banks; (iii) regulatory considerations for reprogramming methods; (iv) options for expansion in 2D vs. 3D cultures; and (v) available technologies and equipment for harvesting, washing, concentration, filling, cryopreservation, and storage. Some key process limitations are highlighted to help drive further improvement and innovation, and includes recommendations to close and automate current open and manual processes.

Human pluripotent stem cells (hPSCs) are an important tool in the field of regenerative medicine due to their ability to differentiate towards all tissues of the adult organism. An important task in the study of hPSCs is to understand the factors that influence the maintenance of pluripotent and clonal characteristics of colonies represented by their morphological phenotype. Such factors include the ability of colonies to migrate during growth. In this work, we measured and analyzed the migration trajectories of hPSC colonies obtained from bright-field images of three cell lines, including induced hPSC lines AD3 and HPCASRi002-A (CaSR) and human embryonic stem cell line H9. To represent the pluripotent status, the colonies were visually phenotyped into two classes having a "good" or "bad" morphological phenotype. As for the migration characteristics, we calculated the colony speed and distance traveled (mobility measures), meandering index (motion persistence measures), outreach ratio (trajectory tortuosity characteristic), as well as the velocity autocorrelation function. The analysis revealed that the discrimination of phenotypes by the migration characteristics depended on both the cell line and growth environment. In particular, when the mTESR1/Matrigel culture environment was used, "good" AD3 colonies demonstrated a higher average migration speed than the "bad" ones. The reverse relationship between average speeds of "good" and "bad" colonies was found for the H9 line. The CaSR cell line did not show significant differences in the migration speed between the "good" and "bad" phenotypes. We investigated the type of motion exhibited by the colonies by applying two diffusion models to the mean squared displacement dynamics, one model corresponding to normal and the other to anomalous diffusion. The type of diffusion and diffusion parameter values resulting from the model fitting to data demonstrated a similar cell line, environment, and phenotype dependency. Colonies mainly showed a superdiffusive behavior for the mTESR1/Matrigel culture conditions, characterized by longer migration steps compared to the normal random walk. The specific properties of migration features and the patterns of their variation demonstrated in our work can be useful for the development and/or improvement of automated solutions for quality control of hPSCs.

Heart organoid (HO) technology has successfully overcome the limitations of two-dimensional (2D) disease modeling and drug testing, thereby emerging as a valuable tool in drug discovery for assessing toxicity and efficacy. However, its ability to distinguish drug responses among individuals remain unclear, which is crucial for developing predictive models. We addressed this gap by comparing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with human induced pluripotent stem cell-derived heart organoids (hiPSC-HOs) in the context of doxorubicin-induced cardiotoxicity (DIC). For this study, we utilized hiPSCs generated from breast cancer patients who had previously been treated with doxorubicin. By comparing groups with and without DIC, we examined various parameters, including cell viability, mRNA expression, protein expression and electrophysiological variations. The results of our analysis revealed significant differences between these groups, providing insights into hiPSC-HOs as a potential platform for testing differences in drug responses among patients.

One of the obstacles to autologous chondrocyte implantation (ACI) is obtaining a large quantity of chondrocytes without depletion of their properties. The conditioned medium (CM) from different subpopulations of stem cells (mesenchymal stromal cells (MSC) or induced pluripotent stem cells (iPSC)) could be a gamechanger. MSCs' potential is related to the donor's health and age, which could be omitted when, as a source, iPSCs are used. There is a lack of data regarding their use in the chondrocyte culture expansion. Thus, we wanted to verify whether iPSC-CM could be beneficial for the cell culture of primary chondrocyte cells.

We added the iPSC-CMs from GPCCi001-A and ND 41658*H cells to the culture of primary chondrocyte cell lines isolated from OA patients (n = 6) for other two passages. The composition of the CM was evaluated using Luminex technology. Then, we analysed the senescence, proliferation rate and using flow cytometry: viability, distribution of cell cycle phases, production of reactive oxygen species (ROS) and double-strand breaks. The cartilage-related markers were evaluated using Western blot and immunofluorescence. Additionally, a three-dimensional cell culture was used to determine the potential to form cartilage particles.

iPSC-CM increased proliferation and diminished cell ROS production and senescence. CM influenced the cartilage-related protein expression and promoted the growth of cartilage particles. The cell exposed to CM did not lose the ECM proteins, suggesting the chondroprotective effect for prolonged culture time.

Our preliminary results suggest a beneficial effect on maintaining chondrocyte biology during in vitro expansion.

Pluripotent stem cells have remarkable self-renewal capacity: the ability to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into almost any cell type in the body. To investigate the interplay between these two aspects of self-renewal, we perform four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSCs and the dissolution of primed pluripotent identity during early differentiation. These screens distinguish genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further identify a core set of genes controlling both stem cell fitness and pluripotent identity, including a network of chromatin factors. Here, unbiased screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide a valuable resource for exploring pluripotent stem cell identity versus cell fitness, and offer a framework for categorizing gene function.

The emerging field of synthetic morphogenesis implements synthetic biology tools to investigate the minimal cellular processes sufficient for orchestrating key developmental events. As the field continues to grow, there is a need for new tools that enable scientists to uncover nuances in the molecular mechanisms driving cell fate patterning that emerge during morphogenesis. Here, we present a platform that combines cell engineering with biomaterial design to potentiate artificial signaling in pluripotent stem cells (PSCs). This platform, referred to as PSC-MATRIX, extends the use of programmable biomaterials to PSCs competent to activate morphogen production through orthogonal signaling, giving rise to the opportunity to probe developmental events by initiating morphogenetic programs in a spatially constrained manner through non-native signaling channels. We show that the PSC-MATRIX platform enables temporal and spatial control of transgene expression in response to bulk, soluble inputs in synthetic Notch (synNotch)-engineered human PSCs for an extended culture of up to 11 days. Furthermore, we used PSC-MATRIX to regulate multiple differentiation events via material-mediated artificial signaling in engineered PSCs using the orthogonal ligand green fluorescent protein, highlighting the potential of this platform for probing and guiding fate acquisition. Overall, this platform offers a synthetic approach to interrogate the molecular mechanisms driving PSC differentiation that could be applied to a variety of differentiation protocols.