Cell-derived Cells Research Articles

The future of personalized cardiovascular medicine demands 3D and 4D printing, stem cells, and artificial intelligence

Cardiovascular diseases remain the most lethal disorders worldwide. Employing preeminent techniques is paramount for addressing this global challenge. Recent advances in lab-on-a-chip technology have the potential to transform cardiovascular medicine by providing new tools for understanding the biological variability that underlies disease and drug response. Coupling improved fabrication techniques and cardiovascular cellular models with artificial intelligence-based tools for design and analysis primes the field to model and explore cardiovascular biology more accurately than ever before. The fabrication of lab-on-a-chip systems has dramatically evolved with the development of additive manufacturing technologies. Not only can 3D printing be used to fabricate intricate microfluidic networks, but now through the incorporation of smart materials, 4D printing can change the shape and properties of devices based on external stimuli. By leveraging advances in induced pluripotent stem cells, lab-on-a-chip devices open new opportunities within personalized cardiovascular medicine. As customizable systems, devices can be fabricated and populated with donor stem cell-derived cardiovascular cells to mimic the cardiovascular system. These capabilities are only going to improve with the increasingly important role of artificial intelligence in lab-on-a-chip centric design, real-time analysis, data gathering, processing and analysis. Here, we address the potential to personalize cardiovascular medicine through the lens of advances in the fabrication of lab-on-a-chip devices, development of induced pluripotent stem cells derived engineered vasculature and heart tissues, and explore how artificial intelligence continues to shape this field.

Cell Replacement Therapy for Brain Repair: Recent Progress and Remaining Challenges for Treating Parkinson's Disease and Cortical Injury.

Neural transplantation represents a promising approach to repairing damaged brain circuitry. Cellular grafts have been shown to promote functional recovery through "bystander effects" and other indirect mechanisms. However, extensive brain lesions may require direct neuronal replacement to achieve meaningful restoration of function. While fetal cortical grafts have been shown to integrate with the host brain and appear to develop appropriate functional attributes, the significant ethical concerns and limited availability of this tissue severely hamper clinical translation. Induced pluripotent stem cell-derived cells and tissues represent a more readily scalable alternative. Significant progress has recently been made in developing protocols for generating a wide range of neural cell types in vitro. Here, we discuss recent progress in neural transplantation approaches for two conditions with distinct design needs: Parkinson's disease and cortical injury. We discuss the current status and future application of injections of dopaminergic cells for the treatment of Parkinson's disease as well as the use of structured grafts such as brain organoids for cortical repair.

Pervasive Positive Selection in Blood in 200,618 Individuals and Novel Drivers of Clonal Hematopoiesis

In the last decade, genomic sequencing of blood samples has revealed that clonal hematopoiesis (CH) is common in elderly subjects and associated with hematological malignancies, cardiovascular disease, and all-cause mortality. Initial estimates identified CH in > 10% of those over the age of 70 when screening for mutations in a known set of canonical CH genes (Bick et al. Nature 2020). However, alternative approaches demonstrate the frequency of CH to be grossly underestimated (Zink et al. Blood 2017). Using single-cell sequencing approaches, dozens of parallel clonal expansions can be found in blood in all individuals by the 7th-8th decade of life, with most expansions lacking known driver mutations (Mitchell et al. Nature 2022). It has been estimated that 70% of driver mutations in blood currently remain unknown (Poon et al. Nat Genet 2021). The UK Biobank (UKBB) provides a large cohort with which to assess gene fitness effects and their associated health effects (Szustakowski et al. Nat Genet 2021). We exploited the idea that a gene can be identified as under positive selection if one finds an enrichment of non-synonymous mutations compared to neutral synonymous mutations within that gene's coding sequence, and comprehensively examined 200,618 UKBB whole blood exomes for the presence of positive selection leading to clonal expansions. We confirmed the discovery of genes under positive selection using two somatic variant calling approaches. We queried the UKBB database to examine the clinical phenotypes and health outcomes associated with these novel drivers of CH. We further validated our findings in more than 10,000 genomes from single cell-derived hematopoietic cells. We identified 17 new genes, including lymphoid-expressed genes, under positive selection in blood from 681 variants (VAF > 0.1) among 660 individuals. Notably, 93% of these individuals did not otherwise harbour mutations in canonical CH genes, representing an 18% increase in the cohort of individuals with large clone CH in UKBB (VAF > .0.1). As will be demonstrated in detail, newly identified genes show distinctive mutation landscapes, contribute to both small and large clones in blood, and increase in frequency and size with age - effectively demonstrating characteristics of known drivers of CH. Moreover, across 10,837 single cell-derived clonal hematopoietic cell colonies from 50 individuals (including both those with healthy hematopoiesis and a range of hematological malignancies), we identified 150 somatic mutations across our set of novel CH drivers. Over two-thirds of these genes also demonstrated evidence of positive selection and a strikingly similar pattern of non-synonymous mutations to that seen in the bulk whole exome sequencing data from UKBB. CH driven by mutations in novel genes was associated with increased hazards for hematological malignancies (including lymphoid malignancies), infections, and death, with poor health outcomes influenced by clone size (Figure). Mutations in novel genes conferred some of the strongest fitness effects among all CH genes, corresponding to an excess HSC division rate of 15-20% per year. We believe the expanded list of genetic drivers of CH now accounts for up to half of the full compendium of mutations capable of driving large clonal expansions in blood. Given their clinical relevance, fitness effects, and prevalence, we propose that these novel genes should now be routinely added to diagnostic gene sequencing panels for blood and included in future studies of CH. Figure. Clinical outcome associations with novel drivers of clonal hematopoiesis (CH). Cox-proportional hazard ratios for a range of poor health outcomes against different CH categories with 95% confidence intervals of log hazard ratios. Novel genes under selection in UKBB are shown in orange; canonical genes under selection in UKBB are shown in blue; other canonical genes associated with CH but not under positive selection in UKBB are shown in green; COPD, chronic obstructive pulmonary disease; MI, myocardial infarction; heme, hematological.

API-192, an Allogeneic CD19/20 CAR-NKT Cell Product Derived from Cord Blood CD34+ HSPCs for the Treatment of B-Cell Malignancies

Autologous chimeric antigen receptor (CAR)-T cell therapies induce durable remissions and improve survival in patients with advanced B cell malignancies. However, challenges such as manufacturing variability, cost, and time to treatment highlight the need for an allogeneic, off-the-shelf approach that preserves the efficacy of autologous CAR-T. Additionally, antigen escape is one of the established mechanisms of resistance to current CAR-T therapies which target a single antigen. Classical natural killer T (NKT) cells express an invariant T cell receptor (iTCR) that is restricted to the conserved CD1d protein, making it a promising cell type for allogeneic therapies as they do not require TCR gene editing to avoid graft-vs-host (GvH) disease. Furthermore, NKT cells mediate dendritic cell cross-priming, target immunosuppressive myeloid cells/macrophages in the tumor microenvironment, and directly kill CD1d + tumor cells through the iTCR. Endogenous NKT cells are an extremely rare population in the blood (<1% of lymphocytes), posing manufacturing challenges. API-192 is a human cord blood CD34 + hematopoietic stem and progenitor cell-derived NKT cell product engineered to express dual CARs targeting CD19 and CD20 and armored with soluble IL-15 for improved expansion and persistence. The Appia Cells Utilized for Allogeneic (ACUA) platform enables the production of large numbers of product NKT cells co-expressing multiple transgenes at high purity (>90% iTCR + 19CAR + 20CAR +) with no purification step or gene editing. Phenotypically, API-192 cells are predominantly CD4 -CD8 - or CD4 -CD8 +, express major activating NK receptors, and harbor intracellular stores of cytotoxic molecules, resembling endogenous NKT cells. API-192 kills Raji and Nalm6 tumor cells in a CAR-specific and dose-dependent manner and can control multiple rounds of tumor challenge in vitro. API-192 secretes pro-inflammatory cytokines and proliferates in response to tumor antigen . In human tumor xenograft models in immunocompromised mice, API-192 robustly expands, suppresses tumor growth, and exhibits prolonged persistence, enabling control of a tumor rechallenge 70 days after initial dosing. API-192 expresses lower levels of MHC I and MHC II molecules relative to conventional CAR-T cells and elicits less IFN-γ release by NK and T cells in mixed lymphocyte reaction (MLR) assays, indicative of a more favorable immunogenic profile. Compared to conventional CAR-T cells, API-192 also secretes minimal levels of IFN-γ in response to co-culture with healthy donor peripheral blood mononuclear cells, consistent with the expression of a non-HLA restricted TCR and low GvH risk. These results demonstrate the feasibility, potency, and potential safety of API-192, warranting further development as a novel allogeneic therapy.

Genome editing for engineering stem cell-derived pancreatic β cells: recent trends and future perspectives

Rapid advancements in biotechnology have enabled the development of new therapeutic modalities for refractory diseases. Among these, stem cell-derived β (SC-β) cells are emerging as next-generation therapeutics for diabetes. However, several challenges must be overcome for these cell-based therapies to enter widespread use. Current SC-β cells exhibit functional immaturity, and transplanted cells face rejection by the host’s immune system. The cells also undergo apoptosis shortly after transplantation due to insufficient oxygen supply. Additionally, the use of stem cell-derived live cells may raise safety concerns, including the risk of teratoma formation. To address these challenges, genome-editing tools, particularly clustered regularly interspaced short palindromic repeats/Cas9 technologies, are being extensively explored owing to their ease of use, modularity, and robustness across diverse cell types. Herein, we provide a comprehensive overview of the various issues in the development of cell-based therapies for diabetes and discuss how genome-editing tools can enhance this emerging therapeutic modality. We also discuss the use of genome editing with SC-β cells for modeling genetic variants in diabetic β cells and correcting pathogenic variants to enable personalized diabetes therapy. Furthermore, we propose future research directions for leveraging genome-editing tools to improve the performance of cell-based therapeutics for diabetes.

Ultrastructural Abnormalities in Induced Pluripotent Stem Cell-Derived Neural Stem Cells and Neurons of Two Cohen Syndrome Patients.

Cohen syndrome is an autosomal recessive disorder caused by VPS13B (COH1) gene mutations. This syndrome is significantly underdiagnosed and is characterized by intellectual disability, microcephaly, autistic symptoms, hypotension, myopia, retinal dystrophy, neutropenia, and obesity. VPS13B regulates intracellular membrane transport and supports the Golgi apparatus structure, which is critical for neuron formation. We generated induced pluripotent stem cells from two patients with pronounced manifestations of Cohen syndrome and differentiated them into neural stem cells and neurons. Using transmission electron microscopy, we documented multiple new ultrastructural changes associated with Cohen syndrome in the neuronal cells. We discovered considerable disturbances in the structure of some organelles: Golgi apparatus fragmentation and swelling, endoplasmic reticulum structural reorganization, mitochondrial defects, and the accumulation of large autophagosomes with undigested contents. These abnormalities underline the ultrastructural similarity of Cohen syndrome to many neurodegenerative diseases. The cell models that we developed based on patient-specific induced pluripotent stem cells can serve to uncover not only neurodegenerative processes, but the causes of intellectual disability in general.

Genome editing for engineering stem cell-derived pancreatic β cells: recent trends and future perspectives

Rapid advancements in biotechnology have enabled the development of new therapeutic modalities for refractory diseases. Among these, stem cell-derived β (SC-β) cells are emerging as next-generation therapeutics for diabetes. However, several challenges must be overcome for these cell-based therapies to enter widespread use. Current SC-β cells exhibit functional immaturity, and transplanted cells face rejection by the host’s immune system. The cells also undergo apoptosis shortly after transplantation due to insufficient oxygen supply. Additionally, the use of stem cell-derived live cells may raise safety concerns, including the risk of teratoma formation. To address these challenges, genome-editing tools, particularly clustered regularly interspaced short palindromic repeats/Cas9 technologies, are being extensively explored owing to their ease of use, modularity, and robustness across diverse cell types. Herein, we provide a comprehensive overview of the various issues in the development of cell-based therapies for diabetes and discuss how genome-editing tools can enhance this emerging therapeutic modality. We also discuss the use of genome editing with SC-β cells for modeling genetic variants in diabetic β cells and correcting pathogenic variants to enable personalized diabetes therapy. Furthermore, we propose future research directions for leveraging genome-editing tools to improve the performance of cell-based therapeutics for diabetes.

Developmental potency of human ES cell-derived mesenchymal stem cells revealed in mouse embryos following blastocyst injection

Mesenchymal stem cells (MSCs) are present in almost all the tissues in the body, critical for their homeostasis and regeneration. However, the stemness of MSCs is mainly an invitro observation, and lacking exclusive markers for endogenous MSCs makes it difficult to study the multipotency of MSCs invivo, especially for human MSCs. To address this hurdle, we injected GFP-tagged human embryonic stem cell (hESC)-derived MSCs (EMSCs) into mouse blastocysts. EMSCs survived well and penetrated both the inner cell mass and trophectoderm, correlating to the higher anti-apoptotic capability of EMSCs than hESCs. Injected EMSCs contributed to skeletal, dermal, and extraembryonic tissues in the resultant chimera and partially rescued skeletal defects in Sox9+/- mouse fetuses. Thus, this study provides evidence for the stemness and developmental capability of human MSCs through chimerization with the mouse blastocyst, serving as a model for studying human mesenchymal and skeletal development.

Development of synthetic modulator enabling long-term propagation and neurogenesis of human embryonic stem cell-derived neural progenitor cells

Neural progenitor cells (NPCs) are essential for in vitro drug screening and cell-based therapies for brain-related disorders, necessitating well-defined and reproducible culture systems. Current strategies employing protein growth factors pose challenges in terms of both reproducibility and cost. In this study, we developed a novel DNA-based modulator to regulate FGFR signaling in NPCs, thereby facilitating the long-term maintenance of stemness and promoting neurogenesis. This DNA-based FGFR-agonist effectively stimulated FGFR1 phosphorylation and activated the downstream ERK signaling pathway in human embryonic stem cell (HESC)-derived NPCs. We replaced the basic fibroblast growth factor (bFGF) in the culture medium with our DNA-based FGFR-agonist to artificially modulate FGFR signaling in NPCs. Utilizing a combination of cell experiments and bioinformatics analyses, we showed that our FGFR-agonist could enhance NPC proliferation, direct migration, and promote neurosphere formation, thus mimicking the functions of bFGF. Notably, transcriptomic analysis indicated that the FGFR-agonist could specifically influence the transcriptional program associated with stemness while maintaining the neuronal differentiation program, closely resembling the effects of bFGF. Furthermore, our culture conditions allowed for the successful propagation of NPCs through over 50 passages while retaining their ability to efficiently differentiate into neurons. Collectively, our approach offers a highly effective method for expanding NPCs, thereby providing new avenues for disease-in-dish research and drug screening aimed at combating neural degeneration.

Scalable and stable human pluripotent stem cell-derived microvascular-like endothelial cells for cardiac regeneration and disease modelling applications

Abstract Introduction The microvasculature is a vital constituent of the adult human vasculature 1. Cardiac tissue engineering, in vitro models of cardiac remodelling and regeneration, and disease models of the microvasculature would benefit from the inclusion of well-characterised, phenotypically stable microvascular-like endothelial cells (ECs) that could be generated at scale without xenogeneic reagents. Primary microvascular ECs have been utilised for such applications however, they are susceptible to passaging-induced senescence². Moreover, alternative solutions including current protocols for the derivation of human pluripotent stem cell-derived ECs (hPSC-ECs) generate cells with a heterogenous vascular identity that often transdifferentiate into non-EC lineages³. Purpose The purpose of this study was to create a 3D, xenogeneic-free differentiation protocol that yielded stable hPSC-derived microvascular-like ECs (hPSC-MVECs) at scale. Methods Endothelial differentiation of hPSCs was conducted in 2D adherent monolayers and within stirred 3D bioreactors for 12 days. Thereafter, CD31pos ECs were isolated via fluorescence-activated cell sorting (FACS) and cultured for a further 14 days. Gene expression analysis of hPSCs undergoing 2D and 3D differentiation was evaluated via RT-qPCR. The day 26 hPSC-ECs were treated with VEGFA (0 – 50 ng/ml) for a further 7 days and evaluated for CD31 expression via high-content image analysis. The angiogenic profiles of 2D-, 3D-, and 3D-hPSC-ECs cultured in high concentration VEGFA (herein, 3D+V), relative to primary Human Cardiac Microvascular Endothelial Cells (HMVEC-Cs) was assessed using the Proteome Profiler Human Angiogenesis Array. Transcriptomic analysis of these EC populations was subsequently conducted via single-cell RNA sequencing (scRNA-seq). Results Complex vascular clusters emerged by day 12 of the 3D differentiation protocol (fig. 1). The emergent 3D hPSC-ECs had a comparable expression of PECAM1 and CDH5 relative to 2D-derived hPSC-ECs however, VEGFA expression was significantly reduced (P<0.01, fig. 2A). VEGFA treatment at 50 ng/ml yielded phenotypically stable hPSC-ECs (3D+V, fig. 2B). Principal component analysis of the angiogenic factors assayed for revealed 3D+V hPSC-ECs clustered with HMVEC-Cs (fig. 2C). scRNA-seq identified a subset of 3D-derived hPSC-ECs expressing genes associated with microvascular vessels (GMFG, PLVAP), capillary ECs (RGCC, GLUL), and angiogenic tip cells (ESM1, HSPG2). Conclusion The scalable and xenogeneic-free 3D protocol outlined within allows for the attainment of hPSC-derived microvascular-like ECs with high angiogenic potential. The absence of Matrigel enhances the translational capacity of the hPSC-MVECs arising from this protocol that could now be derived from patient-specific hPSC lines for evaluation in pre-clinical models of post-myocardial infarction cardiac regeneration or for in vivo models of cardiac remodelling and microvasculature disease.

Induced pluripotent stem cell-derived vascular smooth muscle cells as an in vitro model of coronary spastic angina

Abstract Background Coronary spastic angina (CSA), also known as Prinzmetal’s variant angina, is a form of angina caused by coronary artery spasm. CSA is characterized by enhanced basal tone of coronary artery smooth muscle and hyperreactivity to vasoconstrictor stimuli. The vascular smooth muscle hyperreactivity is likely due to a post-receptorial cellular abnormality in the regulation of myofibril contraction, suggesting the possibility of multiple stimuli acting on various cellular receptors. However, its detailed mechanism is still unclear. To investigate it, various experiments using coronary artery smooth muscle cells obtained from the CSA patients is preferable, but it is impossible to do it clinically and ethically. Purpose The purpose of this study was to establish induced pluripotent stem cell (iPSC)-derived vascular smooth muscle cells (VSMCs) and to characterize this VSMC model for CSA research. Methods Three CSA patients with ST-segment elevation, which is a characteristic of variant angina, were recruited for this study. The isolated skin fibroblasts or peripheral blood mononuclear cells were reprogrammed into iPSCs through electroporation of episomal plasmids encoding the reprogramming genes OCT3/4, SOX2, KLF4, L-MYC, LIN28, and mouse p53DD. We differentiated mesodermal using Glycogen synthase kinase 3 inhibition and Bone morphogenetic protein 4 treatment from iPSCs and resulted in the differentiation of VSMCs by platelet-derived growth factor-BB and Activin A. The differentiation of VSMCs was confirmed by VSMC-specific gene expression in qRT–PCR and fluorescence-activated cell sorting (FACS). We examined biological features and intracellular calcium response to acetylcholine (ACh) in VSMCs obtained from 3 CSA patients and 3 healthy controls. Intracellular free Ca2+ concentration ([Ca2+]i; nM) was measured by fura-2. Results CSA iPSCs were confirmed to have normal karyotypes and pluripotency to differentiate. FACS showed that VSMCs differentiated from iPSCs exhibited smooth muscle cell marker expressions including alpha-smooth muscle actin and SM22-alpha. qRT–PCR showed that VSMCs from iPSCs had expressions of ACTA2 and TAGLN gene similar to the human coronary artery smooth muscle cells. Differentiation induction rate of VSMCs differentiated from iPSCs between CSA and control did not show significant difference. [Ca2+]i at baseline did not differ significantly between them (143 nM in CSA versus 120 nM in control, p=0.63, n=3), but peak increase in [Ca2+]i from baseline after ACh stimulation was significantly increased in CSA group compared with control (372 nM versus 177 nM, p<0.05, n=3). Conclusions iPSC-derived VSMCs obtained from CSA patients showed an enhanced intracellular calcium response to ACh, which is likely responsible for CSA phenotype. Our iPSC-derived VSMC model can be useful for elucidating the mechanism of coronary spasm and developing therapeutic strategy such as drug efficacy.

Massive expansion of functional human iPSC-derived cardiomyocytes by concomitant glycogen synthase kinase-3 beta inhibition and removal of cell-cell contact

Abstract Background Adult mammalian heart has limited capacity for regeneration. Cardiac injuries such as myocardial infarction lead to significant cardiomyocyte (CM) loss and subsequent heart failure with significant morbidity and mortality. Cell-based therapeutic approaches such as injection of stem cell-derived cells or transplantation of engineered cardiac tissue patches have shown promise to re-muscularize the damaged myocardium and enhance cardiac functions. Rationale: Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (< 5-fold). Results Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e. 100 to 250-fold) by glycogen synthase kinase-3β (GSK-3β) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show GSK-3β inhibition suppresses CM maturation while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10- to 25-times greater expansion beyond GSK-3β inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from Yes associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. Conclusion In summary, we uncovered a molecular interplay that enables massive hiPSC-CMs expansion for large-scale drug screening and tissue engineering applications.

Abstract 12562: DYRK1A/PPARG/EGR1 Pathway Elicits Endothelial Dysfunctions and Predispose to Developing Pulmonary Hypertension in Down Syndrome

Introduction: Patients with Down syndrome (DS) have a higher prevalence of pulmonary arterial hypertension (PAH). Pulmonary vascular endothelial dysfunction has been recognized to be one of the predisposing factors to develop PAH, but pathophysiology of pulmonary endothelial cells in DS specific PAH is still unknown. Hypothesis: Endothelial dysfunctions contribute to developing PAH in DS. Methods: Induced pluripotent stem cell-derived vascular endothelial cells (iPSC-ECs) from DS patients were utilized for analyzing cellular and mitochondrial functions. A Cre-loxP mediated isogenic correction line of disomy 21 was established and used as a control. Results: DS-iPSC-ECs showed increased apoptosis, reduced angiogenesis, increased mitochondrial reactive oxygen species, and decreased ATP production, which were all consistent with features of endothelial dysfunctions previously reported in other types of PAH. RNA-seq revealed increased expression of EGR1 in DS-iPSC-ECs, and its inhibition restored cellular and mitochondrial dysfunctions, demonstrated by si EGR1 and pioglitazone treatment. Moreover, we revealed that increased expression of DYRK1a , one of the major pathogenic genes in DS critical region on chromosome 21, led to elevated EGR1 expression thorough PPARG suppression. These experimental results were reinforced by the observations that pulmonary ECs of a DS patient with severe PAH also exhibited increased EGR1. Conclusions: DYRK1A/PPARG/EGR1 pathway in DS is responsible for endothelial dysfunctions and might be a new therapeutic target for DS specific PAH.

Abstract 195: Dose-Dependent Induction of Nitric Oxide Production in Human Induced Pluripotent Stem Cell-Derived Endothelial Cells by Poloxamer 188

Background: Poloxamer 188 (P188), a triblock copolymer, protects against ischemia/reperfusion injury. The precise mechanisms remain incompletely understood. Human induced pluripotent stem cells (hiPSCs) constitute a unique model system, allowing the parallel generation of different cell types from individual patients to study drug-specific effects in human tissue. Here, we aimed to investigate the potential of P188 to induce nitric oxide (NO) production in hiPSC-derived endothelial cells (ECs) and its effect on cell viability during hypoxia-reoxygenation. Methods: hiPSC-ECs were cultured in phenol red-free endothelial Cell Growth Medium-2 and subjected to hypoxia (1% oxygen) at 37°C for 36 hours. At the end of hypoxia, the cell culture medium was supplemented with NO-sensitive fluorescent dye DAF-FM diacetate (1:5000 dilution, final concentration 1 uM, 30 min). Hypoxic medium was removed, and P188 at different concentrations (0.1 nM to 100 nM) was added for 8 hours. DAF fluorescence and cell viability were assessed at the end of reoxygenation. One-Way ANOVA & Dunn's post hoc test was utilized for statistical analysis (* P < 0.05 vs. untreated control group). Results: P188 dose-dependently increased hiPSC-ECs viability (control group: 53±2%, 0.01 nM P188: 59±2%, 100 nM P188: 64±5%). Interestingly, DAF fluorescence increased dose-dependently as well (control group: 182±9%, 0.1 nM P188: 199±11%, 100 nM P188: 264±4%, data expressed as percent of normoxic cells, n=3 / treatment). Conclusion: This suggests that P188 protects hiPSC-ECs with increased NO production, shedding new light on the underlying mechanisms of its cardioprotective effects.

Abstract 15024: Immunomodulatory Effects of Human Embryonic Stem Cell-Derived Epicardial Cells in Myocardial Infarct Healing

Background: Acute myocardial infarction (AMI) causes excessive myocardium damage, including epicardium. Cell therapy by implantation of human pluripotent stem cell (hPSC)-derived cardiovascular cells has been tested in the infarcted hearts of preclinical models and clinical studies. However, whether hPSC-derived epicardial cells (hEPs) can be a therapeutic approach for infarcted hearts remains unclear. AMI induces inflammatory cascades, leading to multifaceted processes of myocardial injury and healing, whereas it is unknown whether hEPs regulate immune responses, especially macrophage plasticity at the acute phase of MI. Aims: to determine whether hEP implantation promotes myocardial infarct healing and modulates inflammatory response. Results: intramyocardial injection of hEPs at the acute phase of MI ameliorated functional worsening (p<0.001 vs. MI control) and scar formation of adult male NOD-SCID mouse hearts (p<0.05 vs. MI control) at day 28 post-MI, concomitantly with enhanced cardiomyocyte survival, angiogenesis, and lymphangiogenesis (p<0.05 vs. MI control). Mechanistically, hEPs suppressed AMI-induced increases of type I interferon (IFN-I) response, infiltration and cytokine-release of inflammatory cells, and promoted polarization of reparative macrophages. These effects were blocked by a IFN-I receptor agonist RO8191 in mouse model of MI. Moreover, hEP abundantly secreted intelectin 1 (ITLN1), which interacted with IFN-β and mimicked the effects of hEP-condition medium in the suppression of IFN-β-stimulated responses in macrophages and promotion of reparative macrophage polarization. However, ITLN1 downregulation in the hEPs canceled beneficial effects of hEPs in anti-inflammation and anti-IFN-I response, and in cardiac repair of infarcted hearts. Conclusions: our resutls demonstrate the reparative effects of hEP implantations in the infarcted hearts and reveal hEPs as an inflammatory modulator in promoting infarct healing via paracrine factor-mediated cell-cell communications. These findings provide new knowledges for the functions and mechanisms of hEPs for cardiac repair and suggest a therapeutic option of hEPs for ischemic myocardial repair.

Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage.

Retinal pigment epithelial(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in individuals with retinal degenerative diseases; however, studies on the stable and secure banking of these therapeutic cells are scarce. Highly variable cell viability and functional recovery ofRPE cells after cryopreservation are the most commonly encountered issues. In the present protocol, we aimed to achieve the best cell recovery rate after thawing by selecting the optimal cell phase for freezing based on the original experimental conditions. Cells were frozen in the exponential phase determined by using the 5-ethynyl-2'-deoxyuridine labeling assay, which improved cell viability and recovery rate after thawing. Stable and functional cells were obtained shortly after thawing, independent of a long differentiation process. The methods described here allow the simple, efficient, and inexpensive preservation and thawing of hESC-derived RPE cells. Although this protocol focuses on RPE cells, this freezing strategy may be applied to many other types of differentiated cells.

Microvesicles facilitate the differentiation of mesenchymal stem cells into pancreatic beta-like cells via miR-181a-5p/150-5p

Transplantation of pancreatic islet cells is a promising strategy for the long-term treatment of type 1 diabetes (T1D). The stem cell-derived beta cells showed great potential as substitute sources of transplanted pancreatic islet cells. However, the current efficiency of stem cell differentiation still cannot match the requirements for clinical transplantation. Here, we report that microvesicles (MVs) from insulin-producing INS-1 cells could induce mesenchymal stem cell (MSC) differentiation into pancreatic beta-like cells. The combination of MVs with small molecules, nicotinamide and insulin-transferrin-selenium (ITS), dramatically improved the efficiency of MSC differentiation. Notably, the function of MVs in MSC differentiation requires their entry into MSCs through giant pinocytosis. The MVs-treated or MVs combined with small molecules-treated MSCs show pancreatic beta-like cell morphology and response to glucose stimulation in insulin secretion. Using high throughput small RNA-sequencing, we found that MVs induced MSC differentiation into the beta-like cells through miR-181a-5p/150-5p. Together, our findings reveal the role of MVs or the MV-enriched miR-181a-5p/150-5p as a class of biocompatible reagents to differentiate MSCs into functional beta-like cells and demonstrate that the combined usage of MVs or miR-181a-5p/150-5p with small molecules can potentially be used in making pancreatic islet cells for future clinical purposes.

Pluripotent stem cell-derived neural progenitor cells can be used to model effects of IL-6 on human neurodevelopment

ABSTRACT Maternal immune activation (MIA) increases the risks for neurodevelopmental disorders in offspring through inflammatory cytokines, including interleukin-6 (IL-6). We therefore aimed to establish a human two-dimensional (2D) in vitro neural model to investigate the effects of IL-6 exposure on neurodevelopment. IL-6 signal transduction requires two receptors: interleukin-6 signal transducer (IL6ST) and interleukin-6 receptor (IL6R). Prenatally, neural cells lack IL6R, and hence cannot elicit cis IL-6 signaling, but IL6R can be provided by microglia in trans. We demonstrate here that an immortalized human neural progenitor cell (NPC) line, ReNCell CX, expresses IL6ST and elicits both cis and trans IL-6 signaling, limiting its use as a model of MIA. In contrast, induced pluripotent stem cell (iPSC)-derived NPCs only activate the IL-6 cascade in trans. Activation of the trans IL-6 cascade did not result in increased proliferation of iPSC-derived NPCs or ReNCell CX, as has been demonstrated in animal models. iPSC-derived NPCs upregulated NR2F1 expression in response to IL-6 signaling in line with analogous experiments in organoids. Thus, iPSC-derived NPCs can be used to model gene expression changes in response to MIA in 2D cultures.

Reconstituted ovaries self-assemble without an ovarian surface epithelium

Three-dimensional (3D) stem cell models of the ovary have the potential to benefit women's reproductive health research. One such model, the reconstituted ovary (rOvary) self-assembles with pluripotent stem cell-derived germ cells creating a 3D ovarian mimic competent to support the differentiation of functional oocytes inside follicles. In this study, we evaluated the cellular composition of the rOvary revealing the capacity to generate multiple follicles surrounded by NR2F2+ stroma cells. However, the rOvary does not develop a surface epithelium, the source of second-wave pre-granulosa cells, or steroidogenic theca. Therefore, the rOvary models represent the self-assembly of activated follicles in a pre-pubertal ovary poised but not yet competent for hormone production.

Functional interrogation of twenty type 2 diabetes-associated genes using isogenic human embryonic stem cell-derived β-like cells

Genetic studies have identified numerous loci associated with type 2 diabetes (T2D), but the functional roles of many loci remain unexplored. Here, we engineered isogenic knockout human embryonic stem cell lines for 20 genes associated with T2D risk. We examined the impacts of each knockout on β cell differentiation, functions, and survival. We generated gene expression and chromatin accessibility profiles on β cells derived from each knockout line. Analyses of T2D-association signals overlapping HNF4A-dependent ATAC peaks identified a likely causal variant at the FAIM2 T2D-association signal. Additionally, the integrative association analyses identified four genes (CP, RNASE1, PCSK1N, and GSTA2) associated with insulin production, and two genes (TAGLN3 and DHRS2) associated with β cell sensitivity to lipotoxicity. Finally, we leveraged deep ATAC-seq read coverage to assess allele-specific imbalance at variants heterozygous in the parental line and identified a single likely functional variant at each of 23 T2D-association signals.