Human Embryonic Stem Cells Research Articles

Generation of a DMD loss-of-function mutant human embryonic stem cell lines by CRISPR base editing

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disorder, which is caused mostly by frame-disrupting, out-of-frame variation in the dystrophin (DMD) gene. Loss-of- function mutations are the most common type of mutation in DMD, accounting for approximately 60–90% of all DMD variations. In this study, we used adenine base editing to generate a human embryonic stem cell line with splice-site mutations to mimic exon deletion variants in clinical Duchenne muscular dystrophy patients. This cell line has differentiation potential and a normal karyotypic.

RNA m6A modification regulates L1 retrotransposons in human spermatogonial stem cell differentiation in vitro and in vivo

The maintenance of genome integrity in the germline is crucial for mammalian development. Long interspersed element type 1 (LINE-1, L1) is a mobile genetic element that makes up about 17% of the human genome and poses a threat to genome integrity. N6-methyl-adenosine (m6A) plays an essential role in regulating various biological processes. However, the function of m6A modification in L1 retrotransposons and human germline development remains largely unknown. Here we knocked out the m6A methyltransferase METTL3 or the m6A reader YTHDF2 in human embryonic stem cells (hESCs) and discovered that METTL3 and YTHDF2 are crucial for inducing human spermatogonial stem cells (hSSCs) from hESCs in vitro. The removal of METTL3 or YTHDF2 resulted in increased L1 retrotransposition and reduced the efficiency of SSC differentiation in vitro. Further analysis showed that YTHDF2 recognizes the METTL3-catalyzed m6A modification of L1 retrotransposons and degrades L1 mRNA through autophagy, thereby blocking L1 retrotransposition. Moreover, the study confirmed that m6A modification in human fetal germ cells promotes the degradation of L1 retrotransposon RNA, preventing the insertion of new L1 retrotransposons into the genome. Interestingly, L1 retrotransposon RNA was highly expressed while METTL3 was significantly downregulated in the seminal plasma of azoospermic patients with meiotic arrest compared to males with normal fertility. Additionally, we identified some potentially pathogenic variants in m6A-related genes in azoospermic men with meiotic arrest. In summary, our study suggests that m6A modification serves as a guardian of genome stability during human germline development and provides novel insights into the function and regulatory mechanisms of m6A modification in restricting L1 retrotransposition.

Targeting the glycan epitope type I N-acetyllactosamine enables immunodepletion of human pluripotent stem cells from early differentiated cells.

Cell surface biomarkers are fundamental for specific characterization of human pluripotent stem cells (hPSCs). Importantly, they can be applied for hPSC enrichment and/or purification but also to remove potentially teratoma-forming hPSCs from differentiated populations before clinical application. Several specific markers for hPSCs are glycoconjugates comprising the glycosphingolipid (GSL)-based glycans SSEA-3 and SSEA-4. We applied an analytical approach based on multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection to quantitatively assess the GSL glycome of human embryonic stem cells and human induced pluripotent stem cells as well as during early stages of differentiation into mesoderm, endoderm, and ectoderm. Thereby, we identified the GSL lacto-N-tetraosylceramide (Lc4-Cer, Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Cer), which comprises a terminal type 1 LacNAc (T1LN) structure (Galβ1-3GlcNAc), to be rapidly decreased upon onset of differentiation. Using a specific antibody, we could confirm a decline of T1LN-terminating glycans during the first four days of differentiation by live-cell staining and subsequent flow cytometry. We could further separate T1LN-positive and T1LN-negative cells out of a mixed population of pluripotent and differentiated cells by magnetic activated cell sorting. Notably, not only the T1LN-positive but also the T1LN-negative population was positive for SSEA-3, SSEA-4, and SSEA-5 while expression of nuclear pluripotency markers OCT4 and NANOG was highly reduced in the T1LN-negative population, exclusively. Our findings suggest T1LN as a pluripotent stem cell-specific glycan epitope that is more rapidly down-regulated upon differentiation than SSEA-3, SSEA-4, and SSEA-5.

Conserved mechanisms of self-renewal and pluripotency in mouse and human ESCs regulated by simulated microgravity using a 3D clinostat

Embryonic stem cells (ESCs) exhibit unique attributes of boundless self-renewal and pluripotency, making them invaluable for fundamental investigations and clinical endeavors. Previous examinations of microgravity effects on ESC self-renewal and differentiation have predominantly maintained a descriptive nature, constrained by limited experimental opportunities and techniques. In this investigation, we present compelling evidence derived from murine and human ESCs, demonstrating that simulated microgravity (SMG)-induced stress significantly impacts self-renewal and pluripotency through a previously unidentified conserved mechanism. Specifically, SMG induces the upregulation of heat shock protein genes, subsequently enhancing the expression of core pluripotency factors and activating the Wnt and/or LIF/STAT3 signaling pathways, thereby fostering ESC self-renewal. Notably, heightened Wnt pathway activity, facilitated by Tbx3 upregulation, prompts mesoendodermal differentiation in both murine and human ESCs under SMG conditions. Recognizing potential disparities between terrestrial SMG simulations and authentic microgravity, forthcoming space flight experiments are imperative to validate the impact of reduced gravity on ESC self-renewal and differentiation mechanisms.

HAND factors regulate cardiac lineage commitment and differentiation from human pluripotent stem cells

BackgroundTranscription factors HAND1 and HAND2 (HAND1/2) play significant roles in cardiac organogenesis. Abnormal expression and deficiency of HAND1/2 result in severe cardiac defects. However, the function and mechanism of HAND1/2 in regulating human early cardiac lineage commitment and differentiation are still unclear.MethodsWith NKX2.5eGFP H9 human embryonic stem cells (hESCs), we established single and double knockout cell lines for HAND1 and HAND2, respectively, whose cardiomyocyte differentiation efficiency could be monitored by assessing NKX2.5-eGFP+ cells with flow cytometry. The expression of specific markers for heart fields and cardiomyocyte subtypes was examined by quantitative PCR, western blot and immunofluorescence staining. Microelectrode array and whole-cell patch clamp were performed to determine the electrophysiological characteristics of differentiated cardiomyocytes. The transcriptomic changes of HAND knockout cells were revealed by RNA sequencing. The HAND1/2 target genes were identified and validated experimentally by integrating with HAND1/2 chromatin immunoprecipitation sequencing data.ResultsEither HAND1 or HAND2 knockout did not affect the cardiomyocyte differentiation kinetics, whereas depletion of HAND1/2 resulted in delayed differentiation onset. HAND1 knockout biased cardiac mesoderm toward second heart field progenitors at the expense of first heart field progenitors, leading to increased expression of atrial and outflow tract cardiomyocyte markers, which was further confirmed by the appearance of atrial-like action potentials. By contrast, HAND2 knockout cardiomyocytes had reduced expression of atrial cardiomyocyte markers and displayed ventricular-like action potentials. HAND1/2-deficient hESCs were more inclined to second heart field lineage and its derived cardiomyocytes with atrial-like action potentials than HAND1 single knockout during differentiation. Further mechanistic investigations suggested TBX5 as one of the downstream targets of HAND1/2, whose overexpression partially restored the abnormal cardiomyocyte differentiation in HAND1/2-deficient hESCs.ConclusionsHAND1/2 have specific and redundant roles in cardiac lineage commitment and differentiation. These findings not only reveal the essential function of HAND1/2 in cardiac organogenesis, but also provide important information on the pathogenesis of HAND1/2 deficiency-related congenital heart diseases, which could potentially lead to new therapeutic strategies.

Human primordial germ cell-like cells specified from resetting precursors develop in human hindgut organoids.

Human primordial germ cells (hPGCs), the precursors of eggs and sperm, start their complex development shortly after specification and during their migration to the primitive gonads. Here, we describe protocols for specifying hPGC-like cells (hPGCLCs) from resetting precursors and progressing them with the support of human hindgut organoids. Resetting hPGCLCs (rhPGCLCs) are specified from human embryonic stem cells (hESCs) transitioning from the primed into the naive state of pluripotency. Hindgut organoids are also derived from hESCs after a sequential differentiation into a posterior endoderm/hindgut fate. Both rhPGCLCs and hindgut organoids are combined and co-cultured for 25 d. The entire procedure takes ~1.5 months and can be successfully implemented by a doctoral or graduate student with basic skills and experience in hESC cultures. The co-culture system supports the progression of rhPGCLCs at a developmental timing analogous to that observed in vivo. Compared with previously developed hPGCLC progression protocols, which depend on co-cultures with mouse embryonic gonadal tissue, our co-culture system represents a developmentally relevant model closer to the environment that hPGCs first encounter after specification. Together with the potential for investigations of events during hPGC specification and early development, these protocols provide a practical approach to designing efficient models for in vitro gametogenesis. Notably, the rhPGCLC-hindgut co-culture system can also be adapted to study failings in hPGC migration, which are associated with the etiology of some forms of infertility and germ cell tumors.

Tris(1,3-dichloro-2-propyl) phosphate disrupts cellular metabolism within human embryonic kidney (HEK293) cells

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a widely used, additive flame retardant that migrates from end-use products, leading to ubiquitous exposure of humans around the world. However, little is known about whether TDCIPP disrupts the physiology of human embryonic cells. Therefore, the objective of this study was to determine whether TDCIPP alters cell viability, cellular metabolism, cytosine methylation, and reactive oxygen species (ROS) levels within human embryonic kidney (HEK293) cells. Relative to vehicle controls, TDCIPP (0.015–0.1225 µM) resulted in a concentration-dependent increase in cell viability, a finding that was driven by an increase in relative ATP abundance. Interestingly, TDCIPP (0.061–0.98 µM) increased the rate of glycolysis – an adaptive mechanism consistent with the Warburg effect exhibited by tumorigenic cells. Moreover, relative to vehicle-treated cells, TDCIPP (0.245–15.63 µM) exposure for 48 h (but not 24 h) resulted in a significant, concentration-dependent decrease in ROS in situ, and TDCIPP (0.245 µM) exposure significantly increased carnosine within the histidine metabolism pathway. However, TDCIPP did not affect global 5-methylcytosine (5-mC) methylation (0.015–15.63 µM), cell membrane integrity (0.061–0.98 µM), nor the abundance of mitochondria (0.061–1.95 µM). Overall, our findings with TDCIPP point to a novel mechanism of action that may be relevant to human embryonic stem cells.

Applications of Plant-Made Fibroblast Growth Factor for Human Pluripotent Stem Cells.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) hold great potential in regenerative medicine. These cells can be expanded indefinitely in theory and are able to differentiate into different types of cells for cell therapies, drug screening, and basic biology studies. The reliable and effective propagation of hESCs and hiPSCs is important for their downstream applications. Basic fibroblast growth factor (bFGF) is critical to hESCs and hiPSCs for maintaining their pluripotency. Plant-produced growth factors are safe to use without potential contamination of infectious viruses and are less expensive to produce. In this study, we used rice cell-made basic fibroblast growth factor (RbFGF) to propagate hESCs and hiPSCs for at least eight passages. Both hESCs and hiPSCs cultured with RbFGF not only maintained the morphology but also the specific expression (OCT4, SSEA4, SOX2, and TRA-1-60) of PSCs, similar to those cultured with the commercial Escherichia coli-produced bFGF. Furthermore, both gene chip-based PluriTest and TaqMan hPSC Scorecard pluripotency analysis demonstrated the pluripotent expression profile of the hESCs cultured with RbFGF. In vitro trilineage assays further showed that these hESCs and hiPSCs cultured on RbFGF were capable of giving rise to cell derivatives of ectoderm, mesoderm, and endoderm, further demonstrating their pluripotency. Finally, chromosome stability was also maintained in hESCs cultured with RbFGF as demonstrated by normal karyotypes. This study suggests broad applications for plant-made growth factors in stem cell culture and regenerative medicine.

Stem Cells and Infertility: A Review of Clinical Applications and Legal Frameworks.

Infertility is a condition defined by the failure to establish a clinical pregnancy after 12 months of regular, unprotected sexual intercourse or due to an impairment of a person's capacity to reproduce either as an individual or with their partner. The authors have set out to succinctly investigate, explore, and assess infertility treatments, harnessing the potential of stem cells to effectively and safely treat infertility; in addition, this paper will present the legal and regulatory complexities at the heart of stem cell research, with an overview of the legislative state of affairs in six major European countries. For couples who cannot benefit from assisted reproductive technologies (ART) to treat their infertility, stem-cells-based approaches have been shown to be a highly promising approach. Nonetheless, lingering ethical and immunological uncertainties require more conclusive findings and data before such treatment avenues can become mainstream and be applied on a large scale. The isolation of human embryonic stem cells (ESCs) is ethically controversial, since their collection involves the destruction of human embryonic tissue. Overall, stem cell research has resulted in important new breakthroughs in the treatment of infertility. The effort to untangle the complex web of ethical and legal issues associated with such therapeutic approaches will have to rely on evidence-based, broadly shared standards, guidelines, and best practices to make sure that the procreative rights of patients can be effectively reconciled with the core values at the heart of medical ethics.

Characterization of Novel Human β-glucocerebrosidase Antibodies for Parkinson's Disease Research.

Mutations in GBA1, which encodes the lysosome enzyme β-glucocerebrosidase (also referred to as acid β-glucosidase or GCase), are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Evidence also suggests that loss of GCase activity is implicated in PD without GBA1 mutations. Consequently, therapies targeting GCase are actively being pursued as potential strategies to modify the progression of PD and related synucleinopathies. Despite this significant interest in GCase as a therapeutic target, the lack of well-characterized GCase antibodies continues to impede progress in the development of GCase-targeted therapies. This study aims to independently evaluate human GCase (hGCase) antibodies to provide recommendations for western blot, immunofluorescence, immunoprecipitation, and AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay) assays. Two mouse monoclonal antibodies, hGCase-1/17 and hGCase-1/23, were raised against hGCase using imiglucerase, the recombinant enzyme developed to treat patients, as the antigen. These novel antibodies, alongside commonly used antibodies in the field, underwent evaluation in a variety of assays. The characterization of hGCase-1/17 and hGCase-1/23 using genetic models including GBA1 loss-of-function human neuroglioma H4 line and neurons differentiated from human embryonic stem cells revealed their remarkable specificity and potency in immunofluorescence and immunoprecipitation assays. Furthermore, a hGCase AlphaLISA assay with excellent sensitivity, a broad dynamic range, and suitability for high throughput applications was developed using hGCase-1/17 and hGCase-1/23, which enabled a sandwich assay configuration. The hGCase immunofluorescence, immunoprecipitation, and AlphaLISA assays utilizing hGCase-1/17 and hGCase-1/23 will not only facilitate improved investigations of hGCase biology, but can also serve as tools to assess the distribution and effectiveness of GCase-targeted therapies for PD and related synucleinopathies.

Enhanced innate responses in microglia derived from retinoblastoma patient-specific iPSCs.

RB1 deficiency leads to retinoblastoma (Rb), the most prevalent intraocular malignancy. Tumor-associated macrophages (TAMs) are related to local inflammation disorder, particularly by increasing cytokines and immune escape. Microglia, the unique resident macrophages for retinal homeostasis, are the most important immune cells of Rb. However, whether RB1 deficiency affects microglial function remain unknown. In this study, microglia were successfully differentiated from Rb patient- derived human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), and then we investigated the function of RB1 in microglia by live imaging phagocytosis assay, immunofluorescence, RNA-seq, qRT-PCR, ELISA and retina organoids/microglia co-culturing. RB1 was abundantly expressed in microglia and predominantly located in the nucleus. We then examined the phagocytosis ability and secretion function of iMGs in vitro. We found that RB1 deficiency did not affect the expression of microglia-specific markers or the phagocytic abilities of these cells by live-imaging. Upon LPS stimulation, RB1-deficient microglia displayed enhanced innate immune responses, as evidenced by activated MAPK signaling pathway and elevated expression of IL-6 and TNF-α at both mRNA and protein levels, compared to wildtype microglia. Furthermore, retinal structure disruption was observed when retinal organoids were co-cultured with RB1-deficient microglia, highlighting the potential contribution of microglia to Rb development and potential therapeutic strategies for retinoblastoma.

Notch signalling influences cell fate decisions and HOX gene induction in axial progenitors.

The generation of the post-cranial embryonic body relies on the coordinated production of spinal cord neurectoderm and presomitic mesoderm cells from neuromesodermal progenitors (NMPs). This process is orchestrated by pro-neural and pro-mesodermal transcription factors that are co-expressed in NMPs together with Hox genes, which are essential for axial allocation of NMP derivatives. NMPs reside in a posterior growth region, which is marked by the expression of Wnt, FGF and Notch signalling components. Although the importance of Wnt and FGF in influencing the induction and differentiation of NMPs is well established, the precise role of Notch remains unclear. Here, we show that the Wnt/FGF-driven induction of NMPs from human embryonic stem cells (hESCs) relies on Notch signalling. Using hESC-derived NMPs and chick embryo grafting, we demonstrate that Notch directs a pro-mesodermal character at the expense of neural fate. We show that Notch also contributes to activation of HOX gene expression in human NMPs, partly in a non-cell-autonomous manner. Finally, we provide evidence that Notch exerts its effects via the establishment of a negative-feedback loop with FGF signalling.

ISRIB facilitates the co-culture of human trophoblast stem cells and embryonic stem cells.

The embryo-like structures (embryoids) constructed by aggregating embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) have provided revolutionary tools for studying the intricate interaction between embryonic and extra-embryonic tissues during early embryonic development, which has been achieved in mice. However, due to the opposite dependence on some signalling pathways for in vitro culture of human ESCs (hESCs) and TSCs (hTSCs), particularly WNT and TGFβ signalling pathways, which limits the construction of human post-implantation embryoids by aggregating hESCs and hTSCs. To overcome this challenge, here, by screening 1639 chemicals, we found that an inhibitor of integrated stress response, ISRIB, can replace WNT agonists and TGFβ inhibitors to maintain the stemness and differentiation capacity of hTSCs. Thus, we developed an ISRIB-dependent in vitro culture medium for hTSCs, namely nTSM. Furthermore, we demonstrated that ISRIB could also maintain the hESC stemness. Using a 3D co-culture system (hESCs and hTSCs aggregate, ETA), we demonstrated that a 1:1 mixture of hESC culture medium (ESM) and nTSM improved the cell proliferation and organisation of both hESC- and hTSC-compartments and the lumenogenesis of hESC-compartment in ETAs. Overall, our study provided an ISRIB-dependent system for co-culturing hESCs and hTSCs, which facilitated the construction of human embryoids by aggregating hESCs and hTSCs.

Generation of a gene-edited H9 embryonic stem cell line carrying a DOX-inducible NGN2 expression cassette in the CLYBL locus

The pro-neural transcription factor neurogenin-2 (NGN2) possesses the ability to rapidly and effectively transform stem cells into fully operational neurons. Here we report the successful generation of a modified H9 human embryonic H9 stem cell line containing a doxycycline (DOX) inducible NGN2 expression construct featuring a floxed Blasticidin/mApple selection module in the safe-harbor locus CLYBL. This cell line retains its pluripotent state in the absence of DOX, yet readily transitions into a neuronal state upon DOX introduction.

Epigenetic repression of CHCHD2 enhances survival from single cell dissociation through attenuated Rho A kinase activity

During in vitro culture, human pluripotent stem cells (hPSCs) often acquire survival advantages characterized by decreased susceptibility to mitochondrial cell death, known as “culture adaptation.” This adaptation is associated with genetic and epigenetic abnormalities, including TP53 mutations, copy number variations, trisomy, and methylation changes. Understanding the molecular mechanisms underlying this acquired survival advantage is crucial for safe hPSC-based cell therapies. Through transcriptome and methylome analysis, we discovered that the epigenetic repression of CHCHD2, a mitochondrial protein, is a common occurrence during in vitro culture using enzymatic dissociation. We confirmed this finding through genetic perturbation and reconstitution experiments in normal human embryonic stem cells (hESCs). Loss of CHCHD2 expression conferred resistance to single cell dissociation-induced cell death, a common stress encountered during in vitro culture. Importantly, we found that the downregulation of CHCHD2 significantly attenuates the activity of Rho-associated protein kinase (ROCK), which is responsible for inducing single cell death in hESCs. This suggests that hESCs may survive routine enzyme-based cell dissociation by downregulating CHCHD2 and thereby attenuating ROCK activity. These findings provide insights into the mechanisms by which hPSCs acquire survival advantages and adapt to in vitro culture conditions.

N-acetyltransferase NAT10 controls cell fates via connecting mRNA cytidine acetylation to chromatin signaling.

Cell fate transition involves dynamic changes of gene regulatory network and chromatin landscape, requiring multiple levels of regulation, yet the cross-talk between epitranscriptomic modification and chromatin signaling remains largely unknown. Here, we uncover that suppression of N-acetyltransferase 10 (NAT10), the writer for mRNA N4-acetylcytidine (ac4C) modification, can notably affect human embryonic stem cell (hESC) lineage differentiation and pluripotent reprogramming. With integrative analysis, we identify that NAT10-mediated ac4C modification regulates the protein levels of a subset of its targets, which are strongly enriched for fate-instructive chromatin regulators, and among them, histone chaperone ANP32B is experimentally verified and functionally relevant. Furthermore, NAT10-ac4C-ANP32B axis can modulate the chromatin landscape of their downstream genes (e.g., key regulators of Wnt and TGFβ pathways). Collectively, we show that NAT10 is an essential regulator of cellular plasticity, and its catalyzed mRNA cytidine acetylation represents a critical layer of epitranscriptomic modulation and uncover a previously unrecognized, direct cross-talk between epitranscriptomic modification and chromatin signaling during cell fate transitions.

Autophagy is essential for human myelopoiesis

Emergency myelopoiesis (EM) is essential in immune defense against pathogens for rapid replenishing of mature myeloid cells. During the EM process, a rapid cell-cycle switch from the quiescent hematopoietic stem cells (HSCs) to highly proliferative myeloid progenitors (MPs) is critical. How the rapid proliferation of MPs during EM is regulated remains poorly understood. Here, we reveal that ATG7, a critical autophagy factor, is essential for the rapid proliferation of MPs during human myelopoiesis. Peripheral blood (PB)-mobilized hematopoietic stem/progenitor cells (HSPCs) with ATG7 knockdown or HSPCs derived from ATG7-/- human embryonic stem cells (hESCs) exhibit severe defect in proliferation during fate transition from HSPCs to MPs. Mechanistically, we show that ATG7 deficiency reduces p53 localization in lysosome for a potential autophagy-mediated degradation. Together, we reveal a previously unrecognized role of autophagy to regulate p53 for a rapid proliferation of MPs in human myelopoiesis.

HOXA3 functions as the on-off switch to regulate the development of hESC-derived third pharyngeal pouch endoderm through EPHB2-mediated Wnt pathway.

Normal commitment of the endoderm of the third pharyngeal pouch (3PP) is essential for the development and differentiation of the thymus. The aim of this study was to investigate the role of transcription factor HOXA3 in the development and differentiation of 3PP endoderm (3PPE) from human embryonic stem cells (hESCs). The 3PPE was differentiated from hESC-derived definitive endoderm (DE) by mimicking developmental queues with Activin A, WNT3A, retinoic acid and BMP4. The function of 3PPE was assessed by further differentiating into functional thymic epithelial cells (TECs). The effect of HOXA3 inhibition on cells of 3PPE was subsequently investigated. A highly efficient approach for differentiating 3PPE cells was developed and these cells expressed 3PPE related genes HOXA3, SIX1, PAX9 as well as EpCAM. 3PPE cells had a strong potential to develop into TECs which expressed both cortical TEC markers K8 and CD205, and medullary TEC markers K5 and AIRE, and also promoted the development and maturation of T cells. More importantly, transcription factor HOXA3 not only regulated the differentiation of 3PPE, but also had a crucial role for the proliferation and migration of 3PPE cells. Our further investigation revealed that HOXA3 controlled the commitment and function of 3PPE through the regulation of Wnt signaling pathway by activating EPHB2. Our results demonstrated that HOXA3 functioned as the on-off switch to regulate the development of hESC-derived 3PPE through EPHB2-mediated Wnt pathway, and our findings will provide new insights into studying the development of 3PP and thymic organ in vitro and in vivo.

Evaluation of Pancreatic β-cell Differentiation Efficiency of Human iPSC Lines for Clinical Use

Transplantation of pancreatic β-cells generated from human induced pluripotent stem cells (hiPSCs) has great potential as a root treatment for type 1 diabetes. However, their current level of efficiency to differentiate into β-cells is still not at par for clinical use. Previous research has shown that differentiation efficiency varies among human embryonic stem cells and mouse-induced pluripotent stem cell lines. Therefore, selecting a suitable cell line for efficient induction into desired tissues and organs is crucial. In this study, we have evaluated the efficiency of 15 hiPSC lines available for clinical use to differentiate into pancreatic β-cells. Our investigation has revealed induction efficiency to differ among the hiPSC lines, even when derived from the same donor. Among the hiPSC lines tested, the 16A01 cell line exhibited the highest Insulin expression and low Glucagon expression, suggesting that this cell line is suitable for differentiation into β-cells. Our study has demonstrated the importance of selecting a suitable hiPSC line for effective differentiation into β-cells.

Single-cell joint profiling of multiple epigenetic proteins and gene transcription.

Sculpting the epigenome with a combination of histone modifications and transcription factor occupancy determines gene transcription and cell fate specification. Here, we first develop uCoTarget, utilizing a split-pool barcoding strategy for realizing ultrahigh-throughput single-cell joint profiling of multiple epigenetic proteins. Through extensive optimization for sensitivity and multimodality resolution, we demonstrate that uCoTarget enables simultaneous detection of five histone modifications (H3K27ac, H3K4me3, H3K4me1, H3K36me3, and H3K27me3) in 19,860 single cells. We applied uCoTarget to the in vitro generation of hematopoietic stem/progenitor cells (HSPCs) from human embryonic stem cells, presenting multimodal epigenomic profiles in 26,418 single cells. uCoTarget reveals establishment of pairing of HSPC enhancers (H3K27ac) and promoters (H3K4me3) and RUNX1 engagement priming for H3K27ac activation along the HSPC path. We then develop uCoTargetX, an expansion of uCoTarget to simultaneously measure transcriptome and multiple epigenome targets. Together, our methods enable generalizable, versatile multimodal profiles for reconstructing comprehensive epigenome and transcriptome landscapes and analyzing the regulatory interplay at single-cell level.