hiPSC-derived Cells Research Articles

Generation of hiPSC-Derived Skeletal Muscle Cells: Exploiting the Potential of Skeletal Muscle-Derived hiPSCs.

Cell therapies for muscle wasting disorders are on the verge of becoming a realistic clinical perspective. Muscle precursor cells derived from human induced pluripotent stem cells (hiPSCs) represent the key to unrestricted cell numbers indispensable for the treatment of generalized muscle wasting such as cachexia or intensive care unit (ICU)-acquired weakness. We asked how the cell of origin influences efficacy and molecular properties of hiPSC-derived muscle progenitor cells. We generated hiPSCs from primary muscle stem cells and from peripheral blood mononuclear cells (PBMCs) of the same donors (n = 4) and compared their molecular profiles, myogenic differentiation potential, and ability to generate new muscle fibers in vivo. We show that reprogramming into hiPSCs from primary muscle stem cells was faster and 35 times more efficient than from blood cells. Global transcriptome comparison revealed significant differences, but differentiation into induced myogenic cells using a directed transgene-free approach could be achieved with muscle- and PBMC-derived hiPSCs, and both cell types generated new muscle fibers in vivo. Differences in myogenic differentiation efficiency were identified with hiPSCs generated from individual donors. The generation of muscle-stem-cell-derived hiPSCs is a fast and economic method to obtain unrestricted cell numbers for cell-based therapies in muscle wasting disorders, and in this aspect are superior to blood-derived hiPSCs.

Proficiency of Extracellular Vesicles From hiPSC-Derived Neural Stem Cells in Modulating Proinflammatory Human Microglia: Role of Pentraxin-3 and miRNA-21-5p.

Extracellular vesicles (EVs) shed by human-induced pluripotent stem cell (hiPSC)-derived neural stem cells (hNSC-EVs) have shown potent antiinflammatory properties in a mouse macrophage assay and a mouse model of acute neuroinflammation. They can also quickly permeate the entire brain after intranasal administration, making them attractive as an autologous or allogeneic off-the-shelf product for treating neurodegenerative diseases. However, their ability to modulate activated human microglia and specific proteins and miRNAs mediating antiinflammatory effects of hNSC-EVs are unknown. We investigated the proficiency of hNSC-EVs to modulate activated human microglia and probed the role of the protein pentraxin 3 (PTX3) and the miRNA miR-21-5p within hNSC-EVs in mediating the antiinflammatory effects. Mature microglia generated from hiPSCs (iMicroglia) expressed multiple microglia-specific markers. They responded to lipopolysaccharide (LPS) or interferon-gamma challenge by upregulating tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) mRNA expression and protein release. iMicroglia also exhibited proficiency to phagocytose amyloid-beta (Aβ). The addition of hNSC-EVs decreased TNF-α and IL-1β mRNA expression and the release of TNF-α and IL-1β by LPS-stimulated iMicroglia (proinflammatory human Microglia). However, the antiinflammatory activity of hNSC-EVs on LPS-stimulated microglia was considerably diminished when the PTX3 or miR-21-5p concentration was reduced in EVs. The results demonstrate that hNSC-EVs are proficient for modulating the proinflammatory human microglia into non-inflammatory phenotypes, implying their utility to treat neuroinflammation in neurodegenerative diseases. Furthermore, the role of PTX3 and miR-21-5p in the antiinflammatory activity of hNSC-EVs provides a new avenue for improving the antiinflammatory effects of hNSC-EVs through PTX3 and/or miR-21-5p overexpression.

Generation of Human iPSC-Derived Astrocytes with a mature star-shaped phenotype for CNS modeling

The generation of astrocytes from human induced pluripotent stem cells has been hampered by either prolonged differentiation—spanning over two months—or by shorter protocols that generate immature astrocytes, devoid of salient mature astrocytic traits pivotal for central nervous system (CNS) modeling. We directed stable hiPSC-derived neuroepithelial stem cells to human iPSC-derived Astrocytes (hiAstrocytes) with a high percentage of star-shaped cells by orchestrating an astrocytic-tuned culturing environment in 28 days. We employed RT-qPCR and ICC to validate the astrocytic commitment of the neuroepithelial stem cells. To evaluate the inflammatory phenotype, we challenged the hiAstrocytes with the pro-inflammatory cytokine IL-1β (interleukin 1 beta) and quantitatively assessed the secretion profile of astrocyte-associated cytokines and the expression of intercellular adhesion molecule 1 (ICAM-1). Finally, we quantitatively assessed the capacity of hiAstrocytes to synthesize and export the antioxidant glutathione. In under 28 days, the generated cells express canonical and mature astrocytic markers, denoted by the expression of GFAP, AQP4 and ALDH1L1. In addition, the notion of a mature phenotype is reinforced by the expression of both astrocytic glutamate transporters EAAT1 and EAAT2. Thus, hiAstrocytes have a mature phenotype that encompasses traits critical in CNS modeling, including glutathione synthesis and secretion, upregulation of ICAM-1 and a cytokine secretion profile on a par with human fetal astrocytes. This protocol generates a multifaceted astrocytic model suitable for in vitro CNS disease modeling and personalized medicine.Graphical abstract

Human iPSC-Derived Renal Cells Change Their Immunogenic Properties during Maturation: Implications for Regenerative Therapies.

The success of human induced pluripotent stem cell (hiPSC)-based therapy critically depends on understanding and controlling the immunological effects of the hiPSC-derived transplant. While hiPSC-derived cells used for cell therapy are often immature with post-grafting maturation, immunological properties may change, with adverse effects on graft tolerance and control. In the present study, the allogeneic and autologous cellular immunity of hiPSC-derived progenitor and terminally differentiated cells were investigated in vitro. In contrast to allogeneic primary cells, hiPSC-derived early renal progenitors and mature renal epithelial cells are both tolerated not only by autologous but also by allogeneic T cells. These immune-privileged properties result from active immunomodulation and low immune visibility, which decrease during the process of cell maturation. However, autologous and allogeneic natural killer (NK) cell responses are not suppressed by hiPSC-derived renal cells and effectively change NK cell activation status. These findings clearly show a dynamic stage-specific dependency of autologous and allogeneic T and NK cell responses, with consequences for effective cell therapies. The study suggests that hiPSC-derived early progenitors may provide advantageous immune-suppressive properties when applied in cell therapy. The data furthermore indicate a need to suppress NK cell activation in allogeneic as well as autologous settings.

Decellularized Organ-Derived Scaffold Is a Promising Carrier for Human Induced Pluripotent Stem Cells-Derived Hepatocytes.

Human induced pluripotent stem cells (hiPSCs) are a promising cell source for elucidating disease pathology and therapy. The mass supply of hiPSC-derived cells is technically feasible. Carriers that can contain a large number of hiPSC-derived cells and evaluate their functions in vivo-like environments will become increasingly important for understanding disease pathogenesis or treating end-stage organ failure. hiPSC-derived hepatocyte-like cells (hiPSC-HLCs; 5 × 108) were seeded into decellularized organ-derived scaffolds under circumfusion culture. The scaffolds were implanted into immunodeficient microminiature pigs to examine their applicability in vivo. The seeded hiPSC-HLCs demonstrated increased albumin secretion and up-regulated cytochrome P450 activities compared with those in standard two-dimensional culture conditions. Moreover, they showed long-term survival accompanied by neovascularization in vivo. The decellularized organ-derived scaffold is a promising carrier for hiPSC-derived cells for ex vivo and in vivo use and is an essential platform for regenerative medicine and research.

CDH18 is a fetal epicardial biomarker regulating differentiation towards vascular smooth muscle cells

The epicardium is a mesothelial layer covering the myocardium serving as a progenitor source during cardiac development. The epicardium reactivates upon cardiac injury supporting cardiac repair and regeneration. Fine-tuned balanced signaling regulates cell plasticity and cell-fate decisions of epicardial-derived cells (EPCDs) via epicardial-to-mesenchymal transition (EMT). However, powerful tools to investigate epicardial function, including markers with pivotal roles in developmental signaling, are still lacking. Here, we recapitulated epicardiogenesis using human induced pluripotent stem cells (hiPSCs) and identified type II classical cadherin CDH18 as a biomarker defining lineage specification in human active epicardium. The loss of CDH18 led to the onset of EMT and specific differentiation towards cardiac smooth muscle cells. Furthermore, GATA4 regulated epicardial CDH18 expression. These results highlight the importance of tracing CDH18 expression in hiPSC-derived epicardial cells, providing a model for investigating epicardial function in human development and disease and enabling new possibilities for regenerative medicine.

Spatial Environment Affects HNF4A Mutation-Specific Proteome Signatures and Cellular Morphology in hiPSC-Derived β-Like Cells.

Studies of monogenic diabetes are particularly useful because we can gain insight into the molecular events of pancreatic β-cell failure. Maturity-onset diabetes of the young 1 (MODY1) is a form of monogenic diabetes caused by a mutation in the HNF4A gene. Human-induced pluripotent stem cells (hiPSCs) provide an excellent tool for disease modeling by subsequently directing differentiation toward desired pancreatic islet cells, but cellular phenotypes in terminally differentiated cells are notoriously difficult to detect. Re-creating a spatial (three-dimensional [3D]) environment may facilitate phenotype detection. We studied MODY1 by using hiPSC-derived pancreatic β-like patient and isogenic control cell lines in two different 3D contexts. Using size-adjusted cell aggregates and alginate capsules, we show that the 3D context is critical to facilitating the detection of mutation-specific phenotypes. In 3D cell aggregates, we identified irregular cell clusters and lower levels of structural proteins by proteome analysis, whereas in 3D alginate capsules, we identified altered levels of glycolytic proteins in the glucose sensing apparatus by proteome analysis. Our study provides novel knowledge on normal and abnormal function of HNF4A, paving the way for translational studies of new drug targets that can be used in precision diabetes medicine in MODY.

Detection and Functional Evaluation of the P2X7 Receptor in hiPSC Derived Neurons and Microglia-Like Cells.

A large body of evidence suggests the involvement of the ATP-gated purinergic receptor P2X7 (P2X7R) in neurodegenerative diseases, including Alzheimer’s disease. While it is well-described to be present and functional on microglia cells contributing to inflammatory responses, some reports suggest a neuronal expression of the receptor as well. Here, we present experimental results showing P2X7 receptors to be expressed on human hiPSC-derived microglia-like cells, hiPSC-derived neuronal progenitors and hiPSC-derived matured neuronal cells. By applying cell surface protein detection assays, we show that P2X7R is not localized on the cell membrane, despite being detected in neuronal cells and thus may not be available for directly mediating neurotoxicity. On hiPSC-derived microglia-like cells, a clear membranous expression was detected. Additionally, we have not observed differences in P2X7R functions between control and familial Alzheimer’s disease patient-derived neuronal cells. Functional assays employing a P2X7R antagonist JNJ 47965567 confirm these findings by showing P2X7R-dependent modulation of microglia-like cells viability upon treatment with P2X7R agonists ATP and BzATP, while the same effect was absent from neuronal cells. Since the majority of P2X7R research was done on rodent models, our work on human hiPSC-derived cells presents a valuable contribution to the field, extending the work on animal models to the human cellular system and toward clinical translation.

HiPSC-Derived Hepatocyte-like Cells Can Be Used as a Model for Transcriptomics-Based Study of Chemical Toxicity.

Traditional toxicity risk assessment approaches have until recently focussed mainly on histochemical readouts for cell death. Modern toxicology methods attempt to deduce a mechanistic understanding of pathways involved in the development of toxicity, by using transcriptomics and other big data-driven methods such as high-content screening. Here, we used a recently described optimised method to differentiate human induced pluripotent stem cells (hiPSCs) to hepatocyte-like cells (HLCs), to assess their potential to classify hepatotoxic and non-hepatotoxic chemicals and their use in mechanistic toxicity studies. The iPSC-HLCs could accurately classify chemicals causing acute hepatocellular injury, and the transcriptomics data on treated HLCs obtained by TempO-Seq technology linked the cytotoxicity to cellular stress pathways, including oxidative stress and unfolded protein response (UPR). Induction of these stress pathways in response to amiodarone, diclofenac, and ibuprofen, was demonstrated to be concentration and time dependent. The transcriptomics data on diclofenac-treated HLCs were found to be more sensitive in detecting differentially expressed genes in response to treatment, as compared to existing datasets of other diclofenac-treated in vitro hepatocyte models. Hence iPSC-HLCs generated by transcription factor overexpression and in metabolically optimised medium appear suitable for chemical toxicity detection as well as mechanistic toxicity studies.

Differentiation of Human iPS Cells Into Sensory Neurons Exhibits Developmental Stage-Specific Cryopreservation Challenges.

Differentiation of human induced pluripotent stem cells (hiPSCs) generates cell phenotypes valuable for cell therapy and personalized medicine. Successful translation of these hiPSC-derived therapeutic products will rely upon effective cryopreservation at multiple stages of the manufacturing cycle. From the perspective of cryobiology, we attempted to understand how the challenge of cryopreservation evolves between cell phenotypes along an hiPSC-to-sensory neuron differentiation trajectory. Cells were cultivated at three different stages to represent intermediate, differentiated, and matured cell products. All cell stages remained ≥90% viable in a dimethyl sulfoxide (DMSO)-free formulation but suffered ≥50% loss in DMSO before freezing. Raman spectroscopy revealed higher sensitivity to undercooling in hiPSC-derived neuronal cells with lower membrane fluidity and higher sensitivity to suboptimal cooling rates in stem cell developmental stages with larger cell bodies. Highly viable and functional sensory neurons were obtained following DMSO-free cryopreservation. Our study also demonstrated that dissociating adherent cultures plays an important role in the ability of cells to survive and function after cryopreservation.

Evaluation for Retinal Therapy for RPE65 Variation Assessed in hiPSC Retinal Pigment Epithelial Cells.

Human induced pluripotent stem cells (hiPSCs) generated from patients and the derivative retinal cells enable the investigation of pathological and novel variants in relevant cell populations. Biallelic pathogenic variants in RPE65 cause early-onset severe retinal dystrophy (EOSRD) or Leber congenital amaurosis (LCA). Increasingly, regulatory-approved in vivo RPE65 retinal gene replacement therapy is available for patients with these clinical features, but only if they have biallelic pathological variants and sufficient viable retinal cells. In our cohort of patients, we identified siblings with early-onset severe retinal degeneration where genomic studies revealed compound heterozygous variants in RPE65, one a known pathogenic missense variant and the other a novel synonymous variant of uncertain significance. The synonymous variant was suspected to affect RNA splicing. Since RPE65 is very poorly expressed in all tissues except the retinal pigment epithelium (RPE), we generated hiPSC-derived RPE cells from the parental carrier of the synonymous variant. Sequencing of RNA obtained from hiPSC-RPE cells demonstrated heterozygous skipping of RPE65 exon 2 and the introduction of a premature stop codon in the mRNA. Minigene studies confirmed the splicing aberration. Results from this study led to reclassification of the synonymous variant to a pathogenic variant, providing the affected patients with access to RPE65 gene replacement therapy.

Development of human iPSC-derived quiescent hepatic stellate cell-like cells for drug discovery and invitro disease modeling.

SummaryHepatic stellate cells (HSCs) play a central role in the progression of liver fibrosis by producing extracellular matrices. The development of drugs to suppress liver fibrosis has been hampered by the lack of human quiescent HSCs (qHSCs) and an appropriate in vitro model that faithfully recapitulates HSC activation. In the present study, we developed a culture system to generate qHSC-like cells from human-induced pluripotent stem cells (hiPSCs) that can be converted into activated HSCs in culture. To monitor the activation process, a red fluorescent protein (RFP) gene was inserted in hiPSCs downstream of the activation marker gene actin alpha 2 smooth muscle (ACTA2). Using qHSC-like cells derived from RFP reporter iPSCs, we screened a repurposing chemical library and identified therapeutic candidates that prevent liver fibrosis. Hence, hiPSC-derived qHSC-like cells will be a useful tool to study the mechanism of HSC activation and to identify therapeutic agents.

HiPSC-Derived Schwann Cells Influence Myogenic Differentiation in Neuromuscular Cocultures.

Motoneurons, skeletal muscle fibers, and Schwann cells form synapses, termed neuromuscular junctions (NMJs). These control voluntary body movement and are affected in numerous neuromuscular diseases. Therefore, a variety of NMJ in vitro models have been explored to enable mechanistic and pharmacological studies. So far, selective integration of Schwann cells in these models has been hampered, due to technical limitations. Here we present robust protocols for derivation of Schwann cells from human induced pluripotent stem cells (hiPSC) and their coculture with hiPSC-derived motoneurons and C2C12 muscle cells. Upon differentiation with tuned BMP signaling, Schwann cells expressed marker proteins, S100b, Gap43, vimentin, and myelin protein zero. Furthermore, they displayed typical spindle-shaped morphologies with long processes, which often aligned with motoneuron axons. Inclusion of Schwann cells in coculture experiments with hiPSC-derived motoneurons and C2C12 myoblasts enhanced myotube growth and affected size and number of acetylcholine receptor plaques on myotubes. Altogether, these data argue for the availability of a consistent differentiation protocol for Schwann cells and their amenability for functional integration into neuromuscular in vitro models, fostering future studies of neuromuscular mechanisms and disease.

Abstract 12247: Mitochondrial Metabolic Transcriptome Profiles During Cardiomyocyte Differentiation From Mouse and Human Pluripotent Stem Cells

Introduction: Simultaneous increase of myofibrils and mitochondria is a key process of cardiomyocyte differentiation from pluripotent stem cells (PSCs). Specifically, development of mitochondrial oxidative energy metabolism in cardiomyocytes is essential to providing the beating function. Although previous studies reported that mitochondrial oxidative metabolism have some correlation with the differentiation of cardiomyocytes, the mechanism by which mitochondrial oxidative metabolism is regulated and the link between cardiomyogenesis and mitochondrial function are still poorly understood. Methods: We performed transcriptome analysis on cells at specific stages of cardiomyocyte differentiation from mouse embryonic stem cells (mESCs) and human induced PSCs (hiPSCs). We selected highly upregulated mitochondrial metabolic genes at cardiac lineage commitment and time-dependent manner during cardiomyocyte differentiation and identified the protein-protein interaction network connecting between mitochondrial metabolic and cardiac developmental genes. Results: We found several mitochondrial metabolic regulatory genes at cardiac lineage commitment (Cck, Bdnf, Fabp4, Cebpa, Cdkn2a in mESC-derived cells and CCK, NOS3 in hiPSC-derived cells) and time-dependent manner during cardiomyocyte differentiation (Eno3, Pgam2, Cox6a2, Fabp3 in mESC-derived cells and PGAM2, SLC25A4 in hiPSC-derived cells). Notably, mitochondrial metabolic proteins are highly interacted with cardiac developmental proteins time-dependent manner during cardiomyocyte differentiation rather than cardiac lineage commitment. Furthermore, mitochondrial metabolic proteins are mainly interacted with cardiac muscle contractile proteins rather than cardiac transcription factors in cardiomyocyte. Conclusions: Mitochondrial metabolism is critical at cardiac maturation rather than cardiac lineage commitment.

Abstract 10410: Human Stem Cell-Derived Endothelial Cells Suppress Automaticity of Stem Cell-Derived Cardiomyocytes

Clinical translation of stem cell therapies for heart disease is limited by a risk of potentially life-threatening ventricular arrhythmias seen following cardiomyocyte delivery in large animal models. Enhancing cardiomyocyte maturation may reduce this arrhythmogenic risk by reducing automaticity of delivered cardiomyocytes. We tested whether human induced pluripotent stem cell (hiPSC)-derived endothelial cells can enhance maturation and suppress automaticity of iPSC-derived cardiomyocytes in vitro. We found that co-culture of hiPSC-derived endothelial cells with hiPSC-derived cardiomyocytes significantly increased protein expression of cardiac troponin T, cardiac troponin I, Kir2.1, connexin 43, and CD36. In addition, using a stretchable mesh nanoelectronics device, we found that hiPSC-derived endothelial cells accelerated electrical maturation of hiPSC-derived cardiomyocytes and progressively suppressed cardiomyocyte automaticity in vitro (Figure). Using single cell RNA-seq, we identified a subpopulation of hiPSC-derived cardiomyocytes that is eliminated upon co-culture with hiPSC-derived endothelial cells. Further work will investigate whether this subpopulation of cardiomyocytes is responsible for automaticity of cardiomyocyte cultures. Figure. Induced pluripotent stem cell (iPSC)-derived cardiomyocytes alone (CM only) or iPSC-derived cardiomyocytes co-cultured with iPSC-derived endothelial cells (CM+EC) were seeded onto a stretchable mesh nanoelectronics device. Unstimulated voltage tracings at day 30 of cardiomyocyte differentiation show cardiomyocytes with a slower beating rate and more narrow action potential when co-cultured with iPSC-derived endothelial cells compared to iPSC-derived cardiomyocytes alone.

Mitophagy mediates metabolic reprogramming of induced pluripotent stem cells undergoing endothelial differentiation

Pluripotent stem cells are known to shift their mitochondrial metabolism upon differentiation, but the mechanisms underlying such metabolic rewiring are not fully understood. We hypothesized that during differentiation of human induced pluripotent stem cells (hiPSCs), mitochondria undergo mitophagy and are then replenished by the biogenesis of new mitochondria adapted to the metabolic needs of the differentiated cell. To evaluate mitophagy during iPSC differentiation, we performed live cell imaging of mitochondria and lysosomes in hiPSCs differentiating into vascular endothelial cells using confocal microscopy. We observed a burst of mitophagy during the initial phases of hiPSC differentiation into the endothelial lineage, followed by subsequent mitochondrial biogenesis as assessed by the mitochondrial biogenesis biosensor MitoTimer. Furthermore, hiPSCs undergoing differentiation showed greater mitochondrial oxidation of fatty acids and an increase in ATP levels as assessed by an ATP biosensor. We also found that during mitophagy, the mitochondrial phosphatase PGAM5 is cleaved in hiPSC-derived endothelial progenitor cells and in turn activates β-catenin-mediated transcription of the transcriptional coactivator PGC-1α, which upregulates mitochondrial biogenesis. These data suggest that mitophagy itself initiates the increase in mitochondrial biogenesis and oxidative metabolism through transcriptional changes during endothelial cell differentiation. In summary, these findings reveal a mitophagy-mediated mechanism for metabolic rewiring and maturation of differentiating cells via the β-catenin signaling pathway. We propose that such mitochondrial-nuclear cross talk during hiPSC differentiation could be leveraged to enhance the metabolic maturation of differentiated cells.

P53 Is a Crucial Regulator of Hemogenic Endothelial Cell Differentiation from Human Induced Pluripotent Stem Cells

De novo generation of hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (hiPSCs) could provide a virtually unlimited supply of autologous HSCs for clinical transplantation, and offer various approaches that enable gene therapy, drug discovery, disease modeling, and in vitro modeling of human hematopoietic development. However, the derivation of long-term self-renewing HSCs from hiPSCs in culture remains elusive. The tumor suppressor protein p53 plays important roles in normal and malignant hematopoiesis, and Trp53-deficient mice exhibit increased number of HSCs. Although activation of p53 is known to promote differentiation of hPSCs and hPSCs recurrently acquire TP53 dominant negative mutations, its role in hematopoietic differentiation of hiPSCs has not been explored.To differentiate hiPSCs into hematopoietic stem and progenitor cells (HSPCs), we used embryoid body (EB) formation method to first differentiate hiPSCs into hemogenic endothelial (HE) cells that express the CD34 highCD144 +CD73 -CD184 -CD43 -CD235a - cell-surface markers. HE cells were then transferred onto a Matrigel-coated plate to undergo endothelial-to-hematopoietic transition (EHT) to generate HSPCs that express the CD34 midCD45 mid cell-surface markers. Developed HSPCs were functionally evaluated by colony forming assay.We observed that the expression of CDKN1A, a p53 target gene, was upregulated in hiPSC-derived EBs and HSPCs over the course of differentiation. To investigate the role of p53 in the generation of HSPCs from hiPSCs, we genetically deleted TP53 in hiPSCs followed by hematopoietic differentiation. While TP53 deletion increased the growth of EBs, it resulted in severe impairment of differentiation into HE cells and overall production of HSPCs that can form colonies. During HE differentiation from hiPSCs, TP53-deficient EBs showed significant reduction of endothelial-lineage gene expression, such as ETV2, CDH5, and PECAM1, as well as expression of RUNX1, a master transcription factor required for HE specification. These results indicate the indispensable role of p53 in HE differentiation from hiPSCs.We then examined the effect of p53 activation on HE differentiation from hiPSCs by pharmacological activation of p53 in hiPSC-derived cells. Transient activation of p53 by Nutlin-3, a small molecule that inhibits the p53-HDM2 interaction and protects p53 from proteasomal degradation, only during HE differentiation but not during EHT significantly promoted HSPC generation as compared to the vehicle treated control.Our findings shed light on the importance of selecting hiPSC lines that retain normal p53 activity for HE differentiation, and provide an approach to promote hematopoietic differentiation of hiPSCs by transiently activating p53 during HE differentiation. DisclosuresKanaujiya: Synthego: Other: Scientific Advisory; eGenesis: Other: Scientific Advisory.

Human Induced Pluripotent Stem Cell-Derived Vascular Cells: Recent Progress and Future Directions.

Human induced pluripotent stem cells (hiPSCs) hold great promise for cardiovascular regeneration following ischemic injury. Considerable effort has been made toward the development and optimization of methods to differentiate hiPSCs into vascular cells, such as endothelial and smooth muscle cells (ECs and SMCs). In particular, hiPSC-derived ECs have shown robust potential for promoting neovascularization in animal models of cardiovascular diseases, potentially achieving significant and sustained therapeutic benefits. However, the use of hiPSC-derived SMCs that possess high therapeutic relevance is a relatively new area of investigation, still in the earlier investigational stages. In this review, we first discuss different methodologies to derive vascular cells from hiPSCs with a particular emphasis on the role of key developmental signals. Furthermore, we propose a standardized framework for assessing and defining the EC and SMC identity that might be suitable for inducing tissue repair and regeneration. We then highlight the regenerative effects of hiPSC-derived vascular cells on animal models of myocardial infarction and hindlimb ischemia. Finally, we address several obstacles that need to be overcome to fully implement the use of hiPSC-derived vascular cells for clinical application.

Long-term selective stimulation of transplanted neural stem/progenitor cells for spinal cord injury improves locomotor function.

In cell transplantation therapy for spinal cord injury (SCI), grafted human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NS/PCs) mainly differentiate into neurons, forming synapses in a process similar to neurodevelopment. In the developing nervous system, the activity of immature neurons has an important role in constructing and maintaining new synapses. Thus, we investigate how enhancing the activity of transplanted hiPSC-NS/PCs affects both the transplanted cells themselves and the host tissue. We find that chemogenetic stimulation of hiPSC-derived neural cells enhances cell activity and neuron-to-neuron interactions invitro. In a rodent model of SCI, consecutive and selective chemogenetic stimulation of transplanted hiPSC-NS/PCs also enhances the expression of synapse-related genes and proteins in surrounding host tissues and prevents atrophy of the injured spinal cord, thereby improving locomotor function. These findings provide a strategy for enhancing activity within the graft to improve the efficacy of cell transplantation therapy for SCI.

Transplantation of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium in a Swine Model of Geographic Atrophy.

Background: The aim of this study was to test the feasibility and safety of subretinal transplantation of human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells into the healthy margins and within areas of degenerative retina in a swine model of geographic atrophy (GA). Methods: Well-delimited selective outer retinal damage was induced by subretinal injection of NaIO3 into one eye in minipigs (n = 10). Thirty days later, a suspension of hiPSC-derived RPE cells expressing green fluorescent protein was injected into the subretinal space, into the healthy margins, and within areas of degenerative retina. In vivo follow-up was performed by multimodal imaging. Post-mortem retinas were analyzed by immunohistochemistry and histology. Results: In vitro differentiated hiPSC-RPE cells showed a typical epithelial morphology, expressed RPE-related genes, and had phagocytic ability. Engrafted hiPSC-RPE cells were detected in 60% of the eyes, forming mature epithelium in healthy retina extending towards the border of the atrophy. Histological analysis revealed RPE interaction with host photoreceptors in the healthy retina. Engrafted cells in the atrophic zone were found in a patchy distribution but failed to form an epithelial-like layer. Conclusions: These results might support the use of hiPSC-RPE cells to treat atrophic GA by providing a housekeeping function to aid the overwhelmed remnant RPE, which might improve its survival and therefore slow down the progression of GA.