Multiple hPSC Lines Research Articles

Abstract We016: Generative Artificial Intelligence for hPSC-derived Cardiac Organoid Florescence Generation

Background: Human pluripotent stem cell (hPSC)-derived cardiac organoids (COs) are extensively applied in modeling heart development and disease. COs are daily monitored by bright-field microscopic imaging for tracking organoid differentiation, morphology, and size, however, it does not provide important information on cell distribution and network in COs. Then, fluorescence microscopic imaging is required with extra steps of experiments when multiple hPSC lines are required. Recently, generative artificial intelligence (AI) has been increasingly applied for image colorization, which extends the possibility of colorizing COs with fluorescence from phase/contrast images. While the phase/contrast image is routinely applied and conveniently used in every biomedical lab daily, is it possible to generate accurate and useful fluorescence information or image colorization from the phase/contrast images of cardiac organoids under the same differentiation protocol is crucial for a broader characterization and analysis of hPSC-derived cardiac organoids and further applications? Hypothesis: We hypothesize that generative AI will generate accurate fluorescence images from phase/contrast images of COs. Approach: 1300 images from COs (differentiated from one reporter hPSC line) were used as the training dataset for the Pix2Pix Conditional Generative Adversarial Networks (GANs) model ( Fig.1a ) with the Convolution Block Attention Module (CBAM) in the U-Net generator ( Fig.1b ). Three models were designed: U-Net (Model 1), U-Net generator with CBAM (Model 2), and U-Net with CBAM and Generator Iteration (Model 3). Three different metrics were included: Peak Signal-to-Noise Ratio (PSNR), Structural Similarity Index (SSIM), and Weighted Patch Histogram to evaluate the prediction outcomes. Results: The predicted fluorescence images from phase/contrast images of different COs are very similar to the ground truths ( Fig.1c ). The similarities evaluated by all three metrics ( Fig.1d ) have the scores of PSNR over 30, SSIM over 0.92, and a Weighted Patch Histogram score over 0.75, which are the most accurate and similar to the ground truth. Conclusions: A generative AI model was established to colorize phase images of COs using cGANs and CBAM, which capture intricate fluorescence details to support the biomedical research of COs with enhanced interpretability and analysis of cardiac organoid morphology and structure in biomedical research and applications.

MAGIK: A rapid and efficient method to create lineage-specific reporters in human pluripotent stem cells

Precise insertion of fluorescent proteins into lineage-specific genes in human pluripotent stem cells (hPSCs) presents challenges due to low knockin efficiency and difficulties in isolating targeted cells. To overcome these hurdles, we present the modified mRNA (ModRNA)-based Activation for Gene Insertion and Knockin (MAGIK) method. MAGIK operates in two steps: first, it uses a Cas9-2A-p53DD modRNA with a mini-donor plasmid (without a drug selection cassette) to significantly enhance efficiency. Second, a deactivated Cas9 activator modRNA and a 'dead' guide RNA are used to temporarily activate the targeted gene, allowing for live cell sorting of targeted cells. Consequently, MAGIK eliminates the need for drug selection cassettes or labor-intensive single-cell colony screening, expediting precise gene editing. We showed MAGIK can be utilized to insert fluorescent proteins into various genes, including SOX17, NKX6.1, NKX2.5, and PDX1, across multiple hPSC lines. This underscores its robust performance and offers a promising solution for achieving knockin in hPSCs within a significantly shortened time frame.

Aggregation of cryopreserved mid-hindgut endoderm for more reliable and reproducible hPSC-derived small intestinal organoid generation.

SummaryA major technical limitation hindering the widespread adoption of human pluripotent stem cell (hPSC)-derived gastrointestinal (GI) organoid technologies is the need for de novo hPSC differentiation and dependence on spontaneous morphogenesis to produce detached spheroids. Here, we report a method for simple, reproducible, and scalable production of small intestinal organoids (HIOs) based on the aggregation of cryopreservable hPSC-derived mid-hindgut endoderm (MHE) monolayers. MHE aggregation eliminates variability in spontaneous spheroid production and generates HIOs that are comparable to those arising spontaneously. With a minor modification to the protocol, MHE can be cryopreserved, thawed, and aggregated, facilitating HIO production without de novo hPSC differentiation. Finally, aggregation can also be used to generate antral stomach organoids and colonic organoids. This improved method removes significant barriers to the implementation and successful use of hPSC-derived GI organoid technologies and provides a framework for improved dissemination and increased scalability of GI organoid production.

The transcriptional regulator ZNF398 mediates pluripotency and epithelial character downstream of TGF-beta in human PSCs

Human pluripotent stem cells (hPSCs) have the capacity to give rise to all differentiated cells of the adult. TGF-beta is used routinely for expansion of conventional hPSCs as flat epithelial colonies expressing the transcription factors POU5F1/OCT4, NANOG, SOX2. Here we report a global analysis of the transcriptional programme controlled by TGF-beta followed by an unbiased gain-of-function screening in multiple hPSC lines to identify factors mediating TGF-beta activity. We identify a quartet of transcriptional regulators promoting hPSC self-renewal including ZNF398, a human-specific mediator of pluripotency and epithelial character in hPSCs. Mechanistically, ZNF398 binds active promoters and enhancers together with SMAD3 and the histone acetyltransferase EP300, enabling transcription of TGF-beta targets. In the context of somatic cell reprogramming, inhibition of ZNF398 abolishes activation of pluripotency and epithelial genes and colony formation. Our findings have clear implications for the generation of bona fide hPSCs for regenerative medicine.

Scalable Generation of Mesenchymal Stem Cells and Adipocytes from Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) can provide unlimited supply for mesenchymal stem cells (MSCs) and adipocytes that can be used for therapeutic applications. Here we developed a simple and highly efficient all-trans-retinoic acid (RA)-based method for generating an off-the-shelf and scalable number of human pluripotent stem cell (hPSC)-derived MSCs with enhanced adipogenic potential. We showed that short exposure of multiple hPSC lines (hESCs/hiPSCs) to 10 μM RA dramatically enhances embryoid body (EB) formation through regulation of genes activating signaling pathways associated with cell proliferation, survival and adhesion, among others. Disruption of cell adhesion induced the subsequent differentiation of the highly expanded RA-derived EB-forming cells into a pure population of multipotent MSCs (up to 1542-fold increase in comparison to RA-untreated counterparts). Interestingly, the RA-derived MSCs displayed enhanced differentiation potential into adipocytes. Thus, these findings present a novel RA-based approach for providing an unlimited source of MSCs and adipocytes that can be used for regenerative medicine, drug screening and disease modeling applications.

BIG-TREE: Base-Edited Isogenic hPSC Line Generation Using a Transient Reporter for Editing Enrichment.

SummaryCurrent CRISPR-targeted single-nucleotide modifications and subsequent isogenic cell line generation in human pluripotent stem cells (hPSCs) require the introduction of deleterious double-stranded DNA breaks followed by inefficient homology-directed repair (HDR). Here, we utilize Cas9 deaminase base-editing technologies to co-target genomic loci and an episomal reporter to enable single-nucleotide genomic changes in hPSCs without HDR. Together, this method entitled base-edited isogenic hPSC line generation using a transient reporter for editing enrichment (BIG-TREE) allows for single-nucleotide editing efficiencies of >80% across multiple hPSC lines. In addition, we show that BIG-TREE allows for efficient generation of loss-of-function hPSC lines via introduction of premature stop codons. Finally, we use BIG-TREE to achieve efficient multiplex editing of hPSCs at several independent loci. This easily adoptable method will allow for the precise and efficient base editing of hPSCs for use in developmental biology, disease modeling, drug screening, and cell-based therapies.

A Reproducible and Simple Method to Generate Red Blood Cells from Human Pluripotent Stem Cells

Erythroid cells generated from human pluripotent stem cells (hPSCs) can potentially offer an unlimited and safe supply of red blood cells (RBCs) for transfusion. Human PSC-derived erythroid cells at various stages of differentiation can also be used to model blood diseases, test new drug candidates, and develop cellular and genetic therapies. Although several protocols for deriving RBCs from hPSCs have been described, these are typically complex, involving multiple culture steps that may include co-culture with feeder cells, and exhibit large variability in erythroid cell yields between hPSC lines and replicate experiments. We have developed a straightforward, serum-free and feeder-free culture method to generate erythroid cells from hPSCs with high yields and high purity. The method has been validated on multiple human embryonic stem (ES) cell lines (H1, H7, H9) and induced pluripotent stem (iPS) cell lines (WLS-1C, STiPS-F016, STiPS-B004). The protocol involves two steps: hematopoietic specification of hPSCs, followed by differentiation of hPSC-derived hematopoietic stem and progenitor cells (HSPCs) into erythroid cells. Lineage specification and differentiation is driven by only three supplements that combine cytokines and other factors to support optimal differentiation efficiency and cell yield across cell lines. In the first step, small hPSC aggregates routinely maintained in feeder-free maintenance medium, are plated onto matrigel-coated microwells, and specification to mesoderm and subsequent hematoendothelial differentiation is induced by addition of successive expansion supplements. This phase promotes extensive hematopoietic progenitor cell generation, with a single hPSC producing on average 142 HSPCs (range: 50 - 360, n = 3 experiments) by day 10 across all six ES and iPS cell lines tested. The average frequency of cells expressing CD43, an embryonic pan-hematopoietic marker, is 92% (range: 85 - 95%), and the frequency of CD34+ cells ranges between 24-55%. In the second, erythroid differentiation step, hPSC-derived HSPCs expand on average 300-fold (range: 80 - 1000) within 10 - 14 days, and the average frequency of GlyA+ cells is 75% (range: 70 - 85%). Cumulatively, this results in the generation of on average 30,000 GlyA+ cells (range: 10,000 - 80,000) per initial hPSC after 20 - 24 days. Further maturation in 7-day cultures containing EPO and human serum resulted in a > 90% pure population of GlyA+ erythroid cells. Notably, no cell loss was observed during the maturation phase, resulting in an average yield of 50,000 GlyA+ cells (range: 10,000 - 220,000) per single initial hPSC on day 31. Differentiated cells were characterized by orthochromatic normoblast morphology and decreased CD71 expression, consistent with erythroid maturation. Erythroid cells generated in this differentiation culture system expressed a mix of 'primitive' and 'definitive' hemoglobin types, but with adult and fetal hemoglobin being expressed at higher levels than embryonic hemoglobin. The observed enucleation rates of hPSC-derived erythroid cells are consistent with current reports and are subject to further optimization. In summary, we have developed a two-step, serum- and feeder-free erythroid differentiation method to generate large numbers of erythroid cells from multiple hPSC lines. This culture system provides a simple, standardized and reproducible platform to generate RBCs from hPSCs with high yields and efficiency for basic and translational research. Disclosures Walasek: STEMCELL Technologies, Inc: Employment. Chau:STEMCELL Technologies, Inc: Employment. Barborini:STEMCELL Technologies, Inc: Employment. Richardson:STEMCELL Technologies, Inc: Employment. Szilvassy:STEMCELL Technologies, Inc: Employment. Louis:STEMCELL Technologies, Inc: Employment. Thomas:STEMCELL Technologies, Inc: Employment. Eaves:STEMCELL Technologies, Inc: Employment. Wognum:STEMCELL Technologies, Inc: Employment.

Engineering human pluripotent stem cells into a functional skeletal muscle tissue

The generation of functional skeletal muscle tissues from human pluripotent stem cells (hPSCs) has not been reported. Here, we derive induced myogenic progenitor cells (iMPCs) via transient overexpression of Pax7 in paraxial mesoderm cells differentiated from hPSCs. In 2D culture, iMPCs readily differentiate into spontaneously contracting multinucleated myotubes and a pool of satellite-like cells endogenously expressing Pax7. Under optimized 3D culture conditions, iMPCs derived from multiple hPSC lines reproducibly form functional skeletal muscle tissues (iSKM bundles) containing aligned multi-nucleated myotubes that exhibit positive force–frequency relationship and robust calcium transients in response to electrical or acetylcholine stimulation. During 1-month culture, the iSKM bundles undergo increased structural and molecular maturation, hypertrophy, and force generation. When implanted into dorsal window chamber or hindlimb muscle in immunocompromised mice, the iSKM bundles survive, progressively vascularize, and maintain functionality. iSKM bundles hold promise as a microphysiological platform for human muscle disease modeling and drug development.

Top-Down Inhibition of BMP Signaling Enables Robust Induction of hPSCs Into Neural Crest in Fully Defined, Xeno-free Conditions.

SummaryDefects in neural crest development have been implicated in many human disorders, but information about human neural crest formation mostly depends on extrapolation from model organisms. Human pluripotent stem cells (hPSCs) can be differentiated into in vitro counterparts of the neural crest, and some of the signals known to induce neural crest formation in vivo are required during this process. However, the protocols in current use tend to produce variable results, and there is no consensus as to the precise signals required for optimal neural crest differentiation. Using a fully defined culture system, we have now found that the efficient differentiation of hPSCs to neural crest depends on precise levels of BMP signaling, which are vulnerable to fluctuations in endogenous BMP production. We present a method that controls for this phenomenon and could be applied to other systems where endogenous signaling can also affect the outcome of differentiation protocols.

A Modular Platform for Differentiation of Human PSCs into All Major Ectodermal Lineages

Directing the fate of human pluripotent stem cells (hPSCs) into different lineages requires variable starting conditions and components with undefined activities, introducing inconsistencies that confound reproducibility and assessment of specific perturbations. Here we introduce a simple, modular protocol for deriving the four main ectodermal lineages from hPSCs. By precisely varying FGF, BMP, WNT, and TGFβ pathway activity in a minimal, chemically defined medium, we show parallel, robust, and reproducible derivation of neuroectoderm, neural crest (NC), cranial placode (CP), and non-neural ectoderm in multiple hPSC lines, on different substrates independently of cell density. We highlight the utility ofthis system by interrogating the role of TFAP2 transcription factors in ectodermal differentiation, revealing the importance of TFAP2A in NC and CPspecification, and performing a small-molecule screen that identified compounds that further enhance CP differentiation. This platform provides a simple stage for systematic derivation of the entire range of ectodermal cell types.

ROCKII inhibition promotes the maturation of human pancreatic beta-like cells

Diabetes is linked to loss of pancreatic beta-cells. Pluripotent stem cells offer a valuable source of human beta-cells for basic studies of their biology and translational applications. However, the signalling pathways that regulate beta-cell development and functional maturation are not fully understood. Here we report a high content chemical screen, revealing that H1152, a ROCK inhibitor, promotes the robust generation of insulin-expressing cells from multiple hPSC lines. The insulin expressing cells obtained after H1152 treatment show increased expression of mature beta cell markers and improved glucose stimulated insulin secretion. Moreover, the H1152-treated beta-like cells show enhanced glucose stimulated insulin secretion and increased capacity to maintain glucose homeostasis after transplantation. Conditional gene knockdown reveals that inhibition of ROCKII promotes the generation and maturation of glucose-responding cells. This study provides a strategy to promote human beta-cell maturation and identifies an unexpected role for the ROCKII pathway in the development and maturation of beta-like cells.

Directed differentiation and long-term maintenance of epicardial cells derived from human pluripotent stem cells under fully defined conditions.

Here, we describe how to efficiently direct human pluripotent stem cells (hPSCs) differentiation into self-renewing epicardial cells in a completely defined, xeno-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate differentiation-stage-specific application of Gsk3 inhibitor, Wnt inhibitor, and Gsk3 inhibitor (GiWiGi) is sufficient to produce cells expressing epicardial markers and exhibiting epicardial phenotypes with a high yield and purity from multiple hPSC lines in 16 d. Characterization of differentiated cells is performed via flow cytometry and immunostaining to assess quantitative expression and localization of epicardial cell-specific proteins. In vitro differentiation into fibroblasts and smooth muscle cells (SMCs) is also described. In addition, culture in the presence of transforming growth factor (TGF)-β inhibitors allows long-term expansion of hPSC-derived epicardial cells (for at least 25 population doublings). Functional human epicardial cells differentiated via this protocol may constitute a potential cell source for heart disease modeling, drug screening, and cell-based therapeutic applications.

Dynamic culture yields engineered myocardium with near-adult functional output.

Engineered cardiac tissues hold promise for cell therapy and drug development, but exhibit inadequate function and maturity. In this study, we sought to significantly improve the function and maturation of rat and human engineered cardiac tissues. We developed dynamic, free-floating culture conditions for engineering "cardiobundles", 3-dimensional cylindrical tissues made from neonatal rat cardiomyocytes or human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) embedded in fibrin-based hydrogel. Compared to static culture, 2-week dynamic culture of neonatal rat cardiobundles significantly increased expression of sarcomeric proteins, cardiomyocyte size (∼2.1-fold), contractile force (∼3.5-fold), and conduction velocity of action potentials (∼1.4-fold). The average contractile force per cross-sectional area (59.7mN/mm2) and conduction velocity (52.5cm/s) matched or approached those of adult rat myocardium, respectively. The inferior function of statically cultured cardiobundles was rescued by transfer to dynamic conditions, which was accompanied by an increase in mTORC1 activity and decline in AMPK phosphorylation and was blocked by rapamycin. Furthermore, dynamic culture effects did not stimulate ERK1/2 pathway and were insensitive to blockers of mechanosensitive channels, suggesting increased nutrient availability rather than mechanical stimulation as the upstream activator of mTORC1. Direct comparison with phenylephrine treatment confirmed that dynamic culture promoted physiological cardiomyocyte growth rather than pathological hypertrophy. Optimized dynamic culture conditions also augmented function of human cardiobundles made reproducibly from cardiomyocytes derived from multiple hPSC lines, resulting in significantly increased contraction force (∼2.5-fold) and conduction velocity (∼1.4-fold). The average specific force of 23.2mN/mm2 and conduction velocity of 25.8cm/s approached the functional metrics of adult human myocardium. In conclusion, we have developed a versatile methodology for engineering cardiac tissues with a near-adult functional output without the need for exogenous electrical or mechanical stimulation, and have identified mTOR signaling as an important mechanism for advancing tissue maturation and function invitro.

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-β, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing > 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Directed differentiation of basal forebrain cholinergic neurons from human pluripotent stem cells

BackgroundBasal forebrain cholinergic neurons (BFCNs) play critical roles in learning, memory and cognition. Dysfunction or degeneration of BFCNs may connect to neuropathology, such as Alzheimer’s disease, Down’s syndrome and dementia. Generation of functional BFCNs may contribute to the studies of cell-based therapy and pathogenesis that is related to learning and memory deficits. New methodHere we describe a detail method for robust generation of BFCNs from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). In this method, BFCN progenitors are patterned from hESC or hiPSC-derived primitive neuroepithelial cells, with the treatment of sonic hedgehog (SHH) or combination with its agonist Purmorphamine, and by co-culturing with human astrocytes. ResultsAt day 20, ∼90% hPSC-derived progenitors expressed NKX2.1, which is a transcriptional marker for MGE. Moreover, around 40% of NKX2.1+ cells co-expressed OLIG2 and ∼15% of NKX2.1+ cells co-expressed ISLET1, which are ventral markers. At day 35, ∼40% neurons robustly express ChAT, most of which are co-labeled with NKX2.1, ISLET1 and FOXG1, indicating the basal forebrain-like identity. At day 45, these neurons express mature neuronal markers MAP2, Synapsin, and VAChT. Comparison with existing method(s)In this method, undefined conditions including genetic modification or cell-sorting are avoided. As a choice, feeder free conditions are used to avoid ingredients of animal origin. Moreover, Purmorphamine can be substituted for SHH to induce ventral progenitors effectively and economically. ConclusionWe provide an efficient method to generate BFCNs from multiple hPSC lines, which offers the potential application for disease modeling and pharmacological studies.

Isolation and cultivation of naive-like human pluripotent stem cells based on HERVH expression.

The ability to derive and stably maintain ground-state human pluripotent stem cells (hPSCs) that resemble the cells seen in vivo in the inner cell mass has the potential to be an invaluable tool for researchers developing stem cell-based therapies. To date, derivation of human naive-like pluripotent stem cell lines has been limited to a small number of lineages, and their long-term culturing remains problematic. We describe a protocol for genetic and phenotypic tagging, selecting and maintaining naive-like hPSCs. We tag hPSCs by GFP, expressed by the long terminal repeat (LTR7) of HERVH endogenous retrovirus. This simple and efficient protocol has been reproduced with multiple hPSC lines, including embryonic and induced pluripotent stem cells, and it takes ∼6 weeks. By using the reporter, homogeneous hPSC cultures can be derived, characterized and maintained for the long term by repeated re-sorting and re-plating steps. The HERVH-expressing cells have a similar, but nonidentical, expression pattern to other naive-like cells, suggesting that alternative pluripotent states might exist.

Highly Synchronized Expression of Lineage‐Specific Genes during In Vitro Hepatic Differentiation of Human Pluripotent Stem Cell Lines

Human pluripotent stem cells- (hPSCs-) derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, and CYP3A4) which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models.

A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells.

Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-β and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays. Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC-CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end-stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC-CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (∼90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC-CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high-throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease.

Increased culture density is linked to decelerated proliferation, prolonged G1 phase, and enhanced propensity for differentiation of self-renewing human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) display a very short G1 phase and rapid proliferation kinetics. Regulation of the cell cycle, which is linked to pluripotency and differentiation, is dependent on the stem cell environment, particularly on culture density. This link has been so far empirical and central to disparities in the growth rates and fractions of self-renewing hPSCs residing in different cycle phases. In this study, hPSC cycle progression in conjunction with proliferation and differentiation were comprehensively investigated for different culture densities. Cell proliferation decelerated significantly at densities beyond 50×10(4) cells/cm(2). Correspondingly, the G1 fraction increased from 25% up to 60% at densities greater than 40×10(4) cells/cm(2) while still hPSC pluripotency marker expression was maintained. In parallel, expression of the cycle inhibitor CDKN1A (p21) was increased, while that of p27 and p53 did not change significantly. After 4 days of culture in an unconditioned medium, greater heterogeneity was noted in the differentiation outcomes and was limited by reducing the density variation. A quantitative model was constructed for self-renewing and differentiating hPSC ensembles to gain a better understanding of the link between culture density, cycle progression, and stem cell state. Results for multiple hPSC lines and medium types corroborated experimental findings. Media commonly used for maintenance of self-renewing hPSCs exhibited the slowest kinetics of induction of differentiation (kdiff), while BMP4 supplementation led to 14-fold higher kdiff values. Spontaneous differentiation in a growth factor-free medium exhibited the largest variation in outcomes at different densities. In conjunction with the quantitative framework, our findings will facilitate rationalizing the selection of cultivation conditions for the generation of stem cell therapeutics.

Abstract 15972: Fibronectin is Essential for Mesendoderm Induction During Cardiac Differentiation of Human Pluripotent Stem Cells

Extracellular matrix (ECM) plays important roles in development, and we have previously demonstrated a matrix sandwich protocol for efficient cardiac differentiation of human pluripotent stem cells (hPSCs). The purpose of the present study was to identify the key ECM proteins required to initiate cardiogenic differentiation of hPSCs and define their mechanism of action. We tested purified ECM proteins including human laminin (LN111, LN521), fibronectin (FN) and collagen IV in the matrix sandwich protocol, and found FN promoted efficient cardiac differentiation. To determine if FN is essential for the early stages of cardiogenesis we tested three FN blockers: FUD peptide that binds the N-terminal 70-kDa fragment of FN, anti-human FN antibody clone10 which recognizes FN domain III2, and anti-human FN antibody clone 3E3 that binds on the cell attachment domain. All three blockers showed concentration-dependent inhibition of cardiac differentiation of hPSCs when added at day 0 for 24 hours, measured by flow cytometry for cTnT+ cells in the Matrigel/Matrigel (bottom/overlay) and FN/FN sandwich culture as well as in the small molecule GiWi protocol. Furthermore, clone10 showed significant inhibition of cardiac differentiation even in the absence of adding exogenous FN due to the robust endogenous expression of FN by the hPSCs. Gene expression analysis with qRT-PCR demonstrated that blocking FN by clone10 caused a significant suppression of gene expression related to: 1) epithelial to mesenchymal transition, e.g. SNAIL1, SNAIL2, GSC, VIM, CDH2 and FN1; 2) Mesendoderm e.g. T, MIXL1, SOX17 and precardiac mesoderm of MESP1; 3) Cardiac transcription factors ISL1 and NKX2.5. To investigate the cellular signaling activated by FN, we tested a blocking antibody (clone P5D2) to the major FN receptor, integrin β1, and found that P5D2 produced a concentration-dependent inhibition of cardiogenesis in multiple hPSC lines. Furthermore, the integrin-linked kinase (ILK) inhibitor, Cpd22, inhibited cardiac differentiation in a concentration-dependent fashion as well when added on day 0. We conclude that FN, acting via integrin β1-activated ILK signaling, is essential to mesendoderm formation enabling cardiogenesis of hPSCs.