Pluripotent State Research Articles

DNA methylation shapes the Polycomb landscape during the exit from naive pluripotency.

In mammals, 5-methylcytosine (5mC) and Polycomb repressive complex 2 (PRC2)-deposited histone 3 lysine 27 trimethylation (H3K27me3) are generally mutually exclusive at CpG-rich regions. As mouse embryonic stem cells exit the naive pluripotent state, there is massive gain of 5mC concomitantly with restriction of broad H3K27me3 to 5mC-free, CpG-rich regions. To formally assess how 5mC shapes the H3K27me3 landscape, we profiled the epigenome of naive and differentiated cells in the presence and absence of the DNA methylation machinery. Surprisingly, we found that 5mC accumulation is not required to restrict most H3K27me3 domains. Instead, this 5mC-independent H3K27me3 restriction is mediated by aberrant expression of the PRC2 antagonist Ezhip (encoding EZH inhibitory protein). At the subset of regions where 5mC appears to genuinely supplant H3K27me3, we identified 163 candidate genes that appeared to require 5mC deposition and/or H3K27me3 depletion for their activation in differentiated cells. Using site-directed epigenome editing to directly modulate 5mC levels, we demonstrated that 5mC deposition is sufficient to antagonize H3K27me3 deposition and confer gene activation at individual candidates. Altogether, we systematically measured the antagonistic interplay between 5mC and H3K27me3 in a system that recapitulates early embryonic dynamics. Our results suggest that H3K27me3 restraint depends on 5mC, both directly and indirectly. Our study also implies a noncanonical role of 5mC in gene activation, which may be important not only for normal development but also for cancer progression, as oncogenic cells frequently exhibit dynamic replacement of 5mC for H3K27me3 and vice versa.

A novel investigation of NANOG and POU5F1 associations in the pluripotent characterization of ES-like and epiblast cells

The transcription factors NANOG and POU5F1 (OCT4) play crucial roles in maintaining pluripotency in embryonic stem (ES) cells. While their functions have been well-studied, the specific interactions between NANOG and POU5F1 and their combined effects on pluripotency in ES-like and Epiblast cells remain less understood. Understanding these associations is vital for refining pluripotent stem cell characterization and advancing regenerative medicine. In this matter, we investigated the associations between NANOG and POU5F1 in maintaining pluripotency in ES-like and Epiblast cells and how these interactions contribute to the distinct pluripotent states of these cells. In the present paper, we examined the pattern of NANOG expression by the immunocytochemical method in embryonic stem-like (ES-like) cells and compared it with its expression pattern in embryonic stem cells (ESCs). Similarly, we examined the expression pattern of POU5F1 in ES-like cells, ESCs, and epiblast cells and compared the expression pattern of these two genes with each other. On the other hand, using Fluidigm Biomark system analysis, we compared the amount of NANOG mRNA in these three cell lines and differentiated and undifferentiated Spermatogonial stem cells in several passages. Microscopic observations indicated the cytoplasmic expression of NANOG in the considered cells; moreover, they showed a similar expression pattern of NANOG with POU5F1 in the experimented cells. It has also been suggested that the more limited the cell’s pluripotency, the lower the expression of these two genes. However, the decrease in NANOG expression is less than that of POU5F1. Fluidigm real-time RT-PCR analysis also confirmed these results. During the experimental process, protein-protein (PPI) network analysis shows a significant association of NANOG with other stem cell proteins, such as POU5F1. Our findings reveal distinct yet overlapping roles of NANOG and POU5F1 in maintaining pluripotency in ES-like and Epiblast cells. The differential binding patterns and functional interactions between these factors underscore the complexity of pluripotency regulation in different stem cell states. This study provides new insights into the molecular mechanisms governing pluripotency and highlights potential targets for enhancing stem cell-based therapies.

7704 Generating pituitary cells from iPSCs of patients with child onset congenital hypopituitarism harboring PROP1 pathogenic allelic variants

Abstract Disclosure: J.M. Marques: None. F. Fattahi: None. L.R. Carvalho: None. Introduction: Reduced or absent levels of pituitary hormones due to genetic or organic causes is known as combined pituitary hormone deficiency (CPHD) and PROP1 is the most frequent cause of CPHD, but more than 85% of the cases are without molecular diagnosis. Novel Techniques such as induced pluripotent stem cells (iPSC), can be used to generate patient’s specific pituitary cells for disease modeling. Aim: To generate iPSC from CO-CH patients’ peripheral blood mononuclear cells (PBMC) and differentiate them into pituitary cells. Methods: Peripheral blood was collected from 2 siblings (HC1 and HC2) harboring compound heterozygous PROP1 allelic variants and their parents (HC3 and HC4). PBMCs were isolated and expanded with MNC medium prior to nucleoporation and transfection with plasmids containing pluripotency genes (OCT4 and SOX2; cMYC and KLF). After 48h, transfected cells were transferred to Geltrex coated plates and fed with E8, NaB and βFGF until pluripotent colonies were formed. iPSCs were differentiated into cranial placode cells using pituitary placode conditioned medium (PPCM) until day 15. For pituitary cell maturation, pituitary conditioned medium (PPCM without SB431542) was used until day 30. qPCR and flow cytometry using the Kit BD Stemflow - Human Pluripotent Stem Cell Transcription Factor Analysis (BD Biosciences) for stem cells and the eBioscience™ Foxp3 / Transcription Factor Fixation/Permeabilization (Thermo Fisher Scientific) for the placode and pituitary markers were used. Pituitary hormonal levels from cell supernatant were measured at the Laboratory of Hormones and Molecular Genetics - HCFMUSP. Results: we generated iPSCs from patient and control blood cells. iPSCs were more than 90% positive for the pluripotency markers SOX2, NANOG, OCT3/4. Pituitary cells were generated with positivity for the placode marker SIX1 (58%) and pituitary markers, as LHX3 (56%) and PROP1 (59%), at day 15. Control cell lineages expressed higher levels of pituitary hormones at days 15 and 30 at RNA and protein levels while patient cells failed to induce hormone-producing cells properly. Pituitary hormonal release was impaired in patients pituitary cells. Conclusion: CO-CH patients’ PBMC were dedifferentiated to a pluripotent state, being able to generate pituitary hormone producing cells. Patient cells were able to mimic human phenotype. These cells can be used, in the future, to study CO-CH basis in patients specific background, to develop pituitary disease modeling as well as test possible new treatments. Presentation: 6/3/2024

Characterization of a chromatin-associated TCF7L1 complex in human embryonic stem cells.

Human embryonic stem cells (hESCs) resemble the pluripotent epiblast cells found in the early postimplantation human embryo and represent the "primed" state of pluripotency. One factor that helps primed pluripotent cells retain pluripotency and prepare genes for differentiation is the transcription factor TCF7L1, a member of a small family of proteins known as T cell factors/Lymphoid enhancer factors (TCF/LEF) that act as downstream components of the WNT signaling pathway. Transcriptional output of the WNT pathway is regulated, in part, by the activity of TCF/LEFs in conjunction with another component of the WNT pathway, β-CATENIN. Because TCF7L1 plays an important role in regulating pluripotency, we began to characterize the protein complex associated with TCF7L1 when bound to chromatin in hESCs using rapid immunoprecipitation of endogenous proteins (RIME). Data are available via ProteomeXchange with identifierPXD047582. These data identify known and new partners of TCF7L1 on chromatin and provide novel insights into how TCF7L1 and pluripotency itself might be regulated.

A suboptimal OCT4-SOX2 binding site facilitates the naïve-state specific function of a Klf4 enhancer.

Enhancers have critical functions in the precise, spatiotemporal control of transcription during development. It is thought that enhancer grammar, or the characteristics and arrangements of transcription factor binding sites, underlie the specific functions of developmental enhancers. In this study, we sought to identify grammatical constraints that direct enhancer activity in the naïve state of pluripotency, focusing on the enhancers for the naïve-state specific gene, Klf4. Using a combination of biochemical tests, reporter assays, and endogenous mutations in mouse embryonic stem cells, we have studied the binding sites for the transcription factors OCT4 and SOX2. We have found that the three Klf4 enhancers contain suboptimal OCT4-SOX2 composite binding sites. Substitution with a high-affinity OCT4-SOX2 binding site in Klf4 enhancer E2 rescued enhancer function and Klf4 expression upon loss of the ESRRB and STAT3 binding sites. We also observed that the low-affinity of the OCT4-SOX2 binding site is crucial to drive the naïve-state specific activities of Klf4 enhancer E2. Altogether, our work suggests that the affinity of OCT4-SOX2 binding sites could facilitate enhancer functions in specific states of pluripotency.

Evaluating Strategies to Assess the Differentiation Potential of Human Pluripotent Stem Cells: A Review, Analysis and Call for Innovation.

Pluripotent stem cells have the ability to differentiate into all cells and tissues within the human body, and as a result they are attractive resources for use in basic research, drug discovery and regenerative medicine. In order to successfully achieve this application, starting cell sources ideally require in-depth characterisation to confirm their pluripotent status and their ability to differentiate into tissues representative of the three developmental germ layers. Many different methods to assess potency are employed, each having its own distinct advantages and limitations. Some aspects of this characterisation process are not always well standardised, particularly techniques used to assess pluripotency as a function. In this article, we consider the methods used to establish cellular pluripotency and subsequently analyse characterisation data for over 1590 human pluripotent cell lines from publicly available repositories in the UK and USA. In particular, we focus on the teratoma xenograft assay, its use and protocols, demonstrating the level of variation and the frequency with which it is used. Finally, we reflect on the implications of the findings, and suggest in vitro alternatives using modern innovative technology as a way forward.

Arp2/3 complex activity enables nuclear YAP for naïve pluripotency of human embryonic stem cells.

Our understanding of the transitions of human embryonic stem cells between distinct stages of pluripotency relies predominantly on regulation by transcriptional and epigenetic programs with limited insight on the role of established morphological changes. We report remodeling of the actin cytoskeleton of human embryonic stem cells (hESCs) as they transition from primed to naïve pluripotency which includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. Activity of the Arp2/3 complex is required for the actin ring, to establish uniform cell mechanics within naïve colonies, promote nuclear translocation of the Hippo pathway effectors YAP and TAZ, and effective transition to naïve pluripotency. RNA-sequencing analysis confirms that Arp2/3 complex activity regulates Hippo signaling in hESCs, and impaired naïve pluripotency with inhibited Arp2/3 complex activity is rescued by expressing a constitutively active, nuclear-localized YAP-S127A. Moreover, expression of YAP-S127A partially restores the actin filament fence with Arp2/3 complex inhibition, suggesting that actin filament remodeling is both upstream and downstream of YAP activity. These new findings on the cell biology of hESCs reveal a mechanism for cytoskeletal dynamics coordinating cell mechanics to regulate gene expression and facilitate transitions between pluripotency states.

Modeling X chromosome inactivation using t5iLA naive human pluripotent stem cells

BackgroundX chromosome inactivation (XCI) is a critical epigenetic event for dosage compensation of X-linked genes in female mammals, ensuring developmental stability. A robust in vitro model is required for mimicking XCI during the early stages of embryonic development. This methodology article introduces an advanced framework for the in-depth study of XCI using human pluripotent stem cells (hPSCs). By focusing on the transition between naive and primed pluripotent states, we highlight the role of long non-coding RNA X-inactive specific transcript (XIST) and epigenetic alterations in mediating XCI.ResultsOur methodology enables the distinction between naive and primed hESCs based on XIST expression and the activity of X-linked reporters, facilitating the investigation of XCI initiation and maintenance. Through detailed experimental procedures, we demonstrate the utility of our hESC lines in modeling the process of human XCI, including the establishment of conditions for random XCI induction and the analysis of X chromosome reactivation.MethodsThe study outlines a comprehensive approach for characterizing the X chromosome status in hPSCs, employing dual fluorescent reporter hESC lines. These reporter lines enable real-time tracking of XCI dynamics through differentiation processes. We detailed protocols for the induction of X chromosome reactivation and inactivation, as well as the X status characterization methods including cultivation of hESCs, flow cytometric analysis, RNA fluorescence in situ hybridization (FISH), and transcriptome sequencing, providing a step-by-step guide for researchers to investigate XCI mechanisms in vitro.ConclusionsThis article provides a detailed, reproducible methodology for studying XCI mechanisms in vitro, employing hPSCs as a model system. It presents a significant advance in our ability to investigate XCI, offering potential applications in developmental biology, disease modeling, and regenerative medicine. By facilitating the study of XCI dynamics, this methodological framework paves the way for deeper understanding and manipulation of this fundamental biological process.

FoxO transcription factors actuate the formative pluripotency specific gene expression programme

Naïve pluripotency is sustained by a self-reinforcing gene regulatory network (GRN) comprising core and naïve pluripotency-specific transcription factors (TFs). Upon exiting naïve pluripotency, embryonic stem cells (ESCs) transition through a formative post-implantation-like pluripotent state, where they acquire competence for lineage choice. However, the mechanisms underlying disengagement from the naïve GRN and initiation of the formative GRN are unclear. Here, we demonstrate that phosphorylated AKT acts as a gatekeeper that prevents nuclear localisation of FoxO TFs in naïve ESCs. PTEN-mediated reduction of AKT activity upon exit from naïve pluripotency allows nuclear entry of FoxO TFs, enforcing a cell fate transition by binding and activating formative pluripotency-specific enhancers. Indeed, FoxO TFs are necessary and sufficient for the activation of the formative pluripotency-specific GRN. Our work uncovers a pivotal role for FoxO TFs in establishing formative post-implantation pluripotency, a critical early embryonic cell fate transition.

Induction of Pluripotent Stem Cells from Muscle Cells of Large Yellow Croaker (Larimichthys Crocea) Via Electrotransfection.

Induced pluripotent stem cells (iPSCs) are a new type of pluripotent cells reprogrammed from somatic cells back into an embryonic-like pluripotent state of stem cells to study development, disease and potential gene therapies. The induction and regulation mechanisms of iPSCs in fish are still unclear. By using the transfection technique, we investigated the crucial function of the OSKMNL factor co-expression for somatic reprogramming in the muscle cell line of large yellow croaker (Larimichthys crocea) (LYCMs) and successfully established a stable iPSCs line (Lc-OSNL-iPSCs). Stable culturing of iPSCs with high alkaline phosphatase activity and a stable karyotype was achieved. The qRT-PCR and immunofluorescence labeling results revealed that Lc-OSNL-iPSCs displayed a high expression level of pluripotent marker genes such as Nanog, Oct4, and Sox2. There were significant differences between Lc-OSNL-iPSCs, Lc-OSKMNL-iPSCs, and LYCMs, and the expression of several genes in maintaining cell pluripotency was up-regulated when the pluripotency signal pathway of stem cells was activated. The technical system for inducing iPSCs of Larimichthys crocea was constructed in this study. This system can serve as a basic model to understand germ cell differentiation mechanism, gender control, genetics, and breeding of large yellow croaker and a platform for studying iPSCs in fish. Interestingly, the acquired iPSCs serves as a useful material for the directional induction of muscle stem cells, thereby establishing the groundwork for obtaining "artificial fish" in the future.

Direct Cardiac Reprogramming in the Age of Computational Biology.

Heart disease continues to be one of the most fatal conditions worldwide. This is in part due to the maladaptive remodeling process by which ischemic cardiac tissue is replaced with a fibrotic scar. Direct cardiac reprogramming presents a unique solution for restoring injured cardiac tissue through the direct conversion of fibroblasts into induced cardiomyocytes, bypassing the transition through a pluripotent state. Since its inception in 2010, direct cardiac reprogramming using the transcription factors Gata4, Mef2c, and Tbx5 has revolutionized the field of cardiac regenerative medicine. Just over a decade later, the field has rapidly evolved through the expansion of identified molecular and genetic factors that can be used to optimize reprogramming efficiency. The integration of computational tools into the study of direct cardiac reprogramming has been critical to this progress. Advancements in transcriptomics, epigenetics, proteomics, genome editing, and machine learning have not only enhanced our understanding of the underlying mechanisms driving this cell fate transition, but have also driven innovations that push direct cardiac reprogramming closer to clinical application. This review article explores how these computational advancements have impacted and continue to shape the field of direct cardiac reprogramming.

The signaling cascade of induction and maintenance of ES cell diapause.

Nutrient deficiency during pregnancy in numerous animal species can induce the state of embryonic diapause. Diapause is characterized by changes in protein and gene expression that minimize the organism's reliance on external energy sources and ensure survival. Remarkably, the systematic changes associated with diapause appear to spare the gene expression program that supports embryonic cells' maintenance in the pluripotent state. The phenomenon of the differentiation "freeze" during diapause can be reproduced in vitro. Mimicking nutrient deficiency by pharmacological inhibition of mTOR induces the diapause-like state in ES cells without affecting ES cell pluripotency. We discovered a connection between mTOR signaling and the chromatin-bound bromodomain and extra-terminal (BET) transcriptional regulator BRD4, showing a key role of BET-protein in the induction of diapause-like state in ES cells. mTOR inhibition rapidly and negatively impacts BRD4 binding to chromatin, which is associated with changes in gene expression that can contribute to diapause. Conversely, pharmacological inhibition of BET-protein circumvents the diapause dependence on mTOR inhibition and causes the diapause-like state. BET-repressed diapause-like ES cells retain the undifferentiated pluripotent state, which is associated with upregulation of a functionally linked group of genes encoding negative regulators of MAP kinase (MAPK) signaling and inactivation of MAP kinase. The transcriptional switch-off of MAP kinase following chronic BET inhibition imitates the transcriptional de-repression of MAP kinase negative regulators in response to mTOR inhibition. Mechanistically, suppression of mTOR or BET-protein leads to a profound decline in Capicua transcriptional repressor (CIC) at promoters of key negative regulators of MAP kinase. The discovered mTOR-BRD4 axis in the induction of diapause and the rapid transcriptional shut-off of differentiation program is likely to play a major role in the maintenance of embryonic diapause in vivo, as well as in controlling of the undifferentiated state of various types of stem cells during diapause-like metabolic dormancy.

Mesenchymal stem cells from adipose tissue prone to lose their stemness associated markers in obesity related stress conditions

Obesity is a health problem characterized by large expansion of adipose tissue. During this expansion, genotoxic stressors can be accumulated and negatively affect the mesenchymal stem cells (MSCs) of adipose tissue. Due to the oxidative stress generated by these genotoxic stressors, senescence phenotype might be observed in adipose tissue MSCs. Senescent MSCs lose their proliferations and differentiation properties and secrete senescence-associated molecules to their niche thus triggering senescence for the rest of the tissue. Accumulation of senescent cells in adipose tissue results in decreased tissue regeneration and functional impairment not only in the close vicinity but also in the other tissues. Here we hypothesized that declined tissue regeneration might be associated with loss of stemness markers in MSCs population. We analyzed the expression of several stemness-associated genes of in vitro cultured MSCs originated from adipose tissue of high-fat diet and normal diet mice models. Since the heterogenous MSCs population covers a small percentage of the pluripotent stem cells, which have roles in proliferation and tissue regeneration, we measured the percentage of these cells via TRA-1-60 pluripotent state antigen. Additionally, by conducting a shotgun proteomic approach using LC-MS/MS, whole cell proteome of the adipose tissue MSCs of high-fat diet and normal diet mice were analyzed and identified proteins were evaluated via gene ontology and PPI network analysis. MSCs of obese mice showed senescent phenotype and altered cell cycle distribution due to a hostile environment with oxidative stress in adipose tissue where they reside. Additionally, the number of pluripotent markers expressing cells declined in the MSC population of the high-fat diet mice. Gene expression analysis evidenced the loss of stemness with a decrease in the expression of stemness-associated genes. Of the proteomic comparison of the normal and the high-fat diet group, MSCs revealed that stemness-associated molecules were decreased while inflammation and senescence-associated phenotypes emerged in obese mice MSCs. Our results showed us that the MSCs of adipose tissue may lose their stemness properties due to obesity-associated stress conditions.

Propagating pluripotency - The conundrum of self-renewal.

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

High-throughput analysis of topographical cues for the expansion of murine pluripotent stem cells

The expansion of pluripotent stem cells (PSCs)in vitroremains a critical barrier to their use in tissue engineering and regenerative medicine. Biochemical methods for PSC expansion are known to produce heterogeneous cell populations with varying states of pluripotency and are cost-intensive, hindering their clinical translation. Engineering biomaterials to physically control PSC fate offers an alternative approach. Surface or substrate topography is a promising design parameter for engineering biomaterials. Topographical cues have been shown to elicit profound effects on stem cell differentiation and proliferation. Previous reports have shown isotropic substrate topographies to be promising in expanding PSCs. However, the optimal feature to promote PSC proliferation and the pluripotent state has not yet been determined. In this work, the MultiARChitecture (MARC) plate is developed to conduct a high-throughput analysis of topographical cues in a 96-well plate format. The MARC plate is a reproducible and customizable platform for the analysis of multiple topographical patterns and features and is compatible with both microscopic assays and molecular biology techniques. The MARC plate is used to evaluate the expression of pluripotency markersOct4, Nanog, andSox2and the differentiation markerLmnAas well as the proliferation of murine embryonic stem (mES) cells. Our systematic analyses identified three topographical patterns that maintain pluripotency in mES cells after multiple passages: 1µm pillars (1µm spacing, square arrangement), 2µm wells (c-c (x, y) = 4, 4µm), and 5µm pillars (c-c (x, y) = 7.5, 7.5µm). This study represents a step towards developing a biomaterial platform for controlled murine PSC expansion.

EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) possess the intrinsic ability to differentiate into diverse cellular lineages, marking them as potent instruments in regenerative medicine. Nonetheless, the proclivity of these stem cells to generate teratomas post-transplantation presents a formidable obstacle to their therapeutic utility. In previous studies, we identified an array of cell surface proteins specifically expressed in the pluripotent state, as revealed through proteomic analysis. Here we focused on EPHA2, a protein found to be abundantly present on the surface of undifferentiated mouse ESCs and is diminished upon differentiation. Knock-down of Epha2 led to the spontaneous differentiation of mouse ESCs, underscoring a pivotal role of EPHA2 in maintaining an undifferentiated cell state. Further investigations revealed a strong correlation between EPHA2 and OCT4 expression during the differentiation of both mouse and human PSCs. Notably, removing EPHA2+ cells from mouse ESC-derived hepatic lineage reduced tumor formation after transplanting them into immune-deficient mice. Similarly, in human iPSCs, a larger proportion of EPHA2+ cells correlated with higher OCT4 expression, reflecting the pattern observed in mouse ESCs. Conclusively, EPHA2 emerges as a potential marker for selecting undifferentiated stem cells, providing a valuable method to decrease tumorigenesis risks after stem-cell transplantation in regenerative treatments.

Evidence of Immunoproteasome Expression Onset in the Formative State of Pluripotency in Mouse Cells.

Embryonic stem cells (ESCs) are remarkable for the high activity level of ubiquitin-proteasome system-the molecular machinery of protein degradation in the cell. Various forms of the proteasome complexes comprising different subunits and interacting regulators are responsible for the substrate selectivity and degradation. Immunoproteasomes are amongst these forms which play an important role in antigen presentation; however, a body of recent evidence suggests their functions in pluripotent stem cells. Previous studies have established three consecutive phases of pluripotency, featured by epiblast cells and their cultured counterparts: naïve, formative, and primed phase. In this work, we report that immunoproteasomes and their chaperone co-regulators are suppressed in the naïve state but are readily upregulated in the formative phase of the pluripotency continuum, featured by epiblast-like cells (EpiLCs). Our data lay ground for the further investigation of the biological functions of immunoproteasome in the regulation of proteostasis during early mammalian development.

Studying Pathogenetic Contribution of a Variant of Unknown Significance, p.M659I (c.1977G > A) in MYH7, to the Development of Hypertrophic Cardiomyopathy Using CRISPR/Cas9-Engineered Isogenic Induced Pluripotent Stem Cells.

Hypertrophic cardiomyopathy (HCM) is a cardiovascular pathology that is caused by variants in genes encoding sarcomere-associated proteins. However, the clinical significance of numerous variants in HCM-associated genes is still unknown. CRISPR/Cas9 is a tool of nucleotide sequence editing that allows for the unraveling of different biological tasks. In this study, introducing a mutation with CRISPR/Cas9 into induced pluripotent stem cells (iPSCs) of a healthy donor and the directed differentiation of the isogenic iPSC lines into cardiomyocytes were used to assess the pathogenicity of a variant of unknown significance, p.M659I (c.1977G > A) in MYH7, which was found previously in an HCM patient. Using two single-stranded donor oligonucleotides with and without the p.M659I (c.1977G > A) mutation, together with CRISPR/Cas9, an iPSC line heterozygous at the p.M659I (c.1977G > A) variant in MYH7 was generated. No CRISPR/Cas9 off-target activity was observed. The iPSC line with the introduced p.M659I (c.1977G > A) mutation in MYH7 retained its pluripotent state and normal karyotype. Compared to the isogenic control, cardiomyocytes derived from the iPSCs with the introduced p.M659I (c.1977G > A) mutation in MYH7 recapitulated known HCM features: enlarged size, elevated diastolic calcium level, changes in the expression of HCM-related genes, and disrupted energy metabolism. These findings indicate the pathogenicity of the variant.

Abstract We001: Doxorubicin-Induced hiPSC Hormetic Model

Doxorubicin (Dox), an anticancer anthracycline drug, is an important tool to understand how cells develop resistance to anthracyclines. Hormesis is a biological phenomenon where low doses of drugs can have beneficial effects on cell proliferation and differentiation. hiPSCs are becoming the model of choice for Dox studies yet the low dose hormetic effect of Dox on hiPSC proliferation and differentiation is not well understood. We assess the hypothesis that low-dose Dox preconditioning of hiPSCs enhances pluripotency gene expression while maintaining pluripotency state. To determine the low-dose effect (50 nM), we treated the JHU001 hiPSC line with Dox (hiPSC-D50) for two hours and a vehicle control without Dox (hiPSC-D0). Both hiPSC-D50 and hiPSC-D0 were maintained in E8 flex medium culture until reaching 70% confluency and were passaged five times and frozen for experimental studies. hiPSC-D50 and -D0 were thawed at passage 5 and cultured on coverslips for pluripotent gene expression (Oct-4, Nanog, and SSEA-4) analysis by immunocytochemistry. The cells were imaged using epifluorescence Nikon Eclipse Ti2 microscopy and the images were analyzed using NIS-Elements BR software. Phase-contrast microscopy shows significant cell death in hiPSC-D50 24 hours post-treatment compared to hiPSC-D0. However, 72 hours post-Dox-treatment, several colonies with pluripotent morphology were observed in hiPSC-D50 that were similar to hiPSC-D0. While some differentiated cell morphologies were observed in hiPSC-D50, these were significantly reduced after five passages. Immunofluorescent staining of Oct-4 and Nanog showed nuclear localization whereas SSEA-4 was localized on the cell surface, in both hiPSC-D0 and hiPSC-D50 cells at passage five. However, immunocytochemistry analysis of pluripotent markers revealed significantly higher expression of Oct-4, Nanog and SSEA-4 in hiPSC-D50 when compared to hiPSC-D0 (1.3-fold,1.9-fold and 2.9-fold increase with p-values: 0.00057, 0.018, and 0.0000026, respectively; n= 5) suggesting a more robust pluripotent state and self-renewal capacity. Interestingly, higher SSEA-4 expression might also be associated with resistance to Dox. Our data suggest that low-dose Dox preconditioning enhances pluripotency state. This Dox-induced hormesis model of hiPSCs is a unique platform to help understand how Dox upregulate pluripotency markers and the potential link between SSEA-4 and drug resistance.

Generation and characterization of a human iPSC line expressing EGFP-tagged CDH1, KSCBi002-A-3

E-cadherin, a transmembrane protein, is essential for maintaining the integrity and structure of human pluripotent stem cells (hPSCs) by facilitating strong cell–cell adhesion and communication, which is crucial for their colony formation and pluripotency. Here, we used the CRISPR/Cas9 system to introduce the enhanced green fluorescent protein (EGFP)-tagged CDH1 into the AAVS1 locus, a safe harbour site, of human induced pluripotent stem cells (hiPSCs). The engineered cell line, KSCBi002-A-3, expressed functional CDH1-EGFP fusion protein, exhibited normal cell morphology, maintained a normal karyotype, and retained pluripotent state.