Pluripotent Human Embryonic Stem Cells Research Articles

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.

Identification of m6A methylation-related genes in cerebral ischaemia‒reperfusion of Breviscapus therapy based on bioinformatics methods

BackgroundCerebral ischaemia‒reperfusion (I/R) frequently causes late-onset neuronal damage. Breviscapine promotes autophagy in microvascular endothelial cells in I/R and can inhibit oxidative damage and apoptosis. However, the mediation mechanism of breviscapine on neuronal cell death is unclear.MethodsFirst, transcriptome sequencing was performed on three groups of mice: the neuronal normal group (Control group), the oxygen-glucose deprivation/ reoxygenation group (OGD/R group) and the breviscapine administration group (Therapy group). Differentially expressed genes (DEGs) between the OGD/R and control groups and between the Therapy and OGD/R groups were obtained by the limma package. N6-methyladenosine (m6A) methylation-related DEGs were selected by Pearson correlation analysis. Then, prediction and confirmation of drug targets were performed by Swiss Target Prediction and UniProt Knowledgebase (UniProtKB) database, and key genes were obtained by Pearson correlation analysis between m6A-related DEGs and drug target genes. Next, gene set enrichment analysis (GSEA) and Ingenuity pathway analysis (IPA) were used to obtain the pathways of key genes. Finally, a circRNA-miRNA‒mRNA network was constructed based on the mRNAs, circRNAs and miRNAs.ResultsA total of 2250 DEGs between the OGD/R and control groups and 757 DEGs between the Therapy and OGD/R groups were selected by differential analysis. A total of 7 m6A-related DEGs, including Arl4d, Gm10653, Gm1113, Kcns3, Olfml2a, Stk26 and Tfcp2l1, were obtained by Pearson correlation analysis. Four key genes (Tfcp2l1, Kcns3, Olfml2a and Arl4d) were acquired, and GSEA showed that these key genes significantly participated in DNA repair, e2f targets and the g2m checkpoint. IPA revealed that Tfcp2l1 played a significant role in human embryonic stem cell pluripotency. The circRNA-miRNA‒mRNA network showed that mmu_circ_0001258 regulated Tfcp2l1 by mmu-miR-301b-3p.ConclusionsIn conclusion, four key genes, Tfcp2l1, Kcns3, Olfml2a and Arl4d, significantly associated with the treatment of OGD/R by breviscapine were identified, which provides a theoretical basis for clinical trials.

Establishment of Cerebral Organoid‐like Structures from Dental Pulp Stem Cells (DPSCs) to Model Neurodegenerative Diseases.

AbstractBackground3D‐cell cultures are closer to in vivo conditions than the monolayers. There are different 3D‐cell culture models, the most relevant is the Cerebral Organoids (CO) model (Lancaster et al. 2014), it can show complex processes and proved to show molecular and structural changes when modeling Alzheimer´s disease (AD), and other diseases. One of the main limitations of using this model is the requirement for Induced Pluripotent Stem Cells (iPSC) or Human Embryonic Stem Cells (hESC) for the CO generation; because iPSC could undergo epigenetic changes during the process of reprogramming; moreover, hESC are controversial because of their ethical implications. We proposed to use DPSCs instead of iPSC or hESC to generate organoid‐like structures due to their capability to differentiate into the neuronal lineage, lack of pluripotency, and its easy access.MethodThe DPSCs were obtained by dissociating the dental pulp of a healthy 46 years old female with a colagenase‐dispase mix at 37°C, isolated and cultured. The cells phenotype was characterized by Western‐Blot, RT‐PCR and immunofluorescence. We cultured the DPSC into low‐adherence wells for 6 days in a DMEM/F12 medium + thiazovivin 50 mM, bFGF 4ng/mL, BFS 20%, Glutamax® 200x, MEM‐NEAA 1:100, 2‐mercaptoethanol 100 µm and murine LIF 10 µg/mL until cell aggregation into embryoid‐like structures. After that, neural induction was carried out.ResultWe successfully characterized DPSC phenotype by detecting pluripotential markers OCT4, Actin, NanoG, mesenchymal markers CD105, CD73, and the neuronal marker β‐III Tubulin which is always present in the DPSCs lineage, as well as the lack of hematopoietic markers CD14, CD34, and CD45. We obtained Cerebral Organoids‐like structures from de DPSCs after 35 days of the differentiation protocol and detected NeuN, MAP2, β‐III Tubulin, and tau, all neural markers that claim a neuronal phenotype.ConclusionOur results suggest a potential use of this approach to the CO generation using DPSCs instead of iPSC or hESC with the benefits of a shorter time and a less expensive protocol likewise, DPSCs could resemble the molecular aging changes. Further studies are needed to validate the use of this CO to model neurodegenerative diseases such as AD.

Pluripotent stem cell-derived committed cardiac progenitors remuscularize damaged ischemic hearts and improve their function in pigs

Ischemic heart disease, which is often associated with irreversibly damaged heart muscle, is a major global health burden. Here, we report the potential of stem cell-derived committed cardiac progenitors (CCPs) have in regenerative cardiology. Human pluripotent embryonic stem cells were differentiated to CCPs on a laminin 521 + 221 matrix, characterized with bulk and single-cell RNA sequencing, and transplanted into infarcted pig hearts. CCPs differentiated for eleven days expressed a set of genes showing higher expression than cells differentiated for seven days. Functional heart studies revealed significant improvement in left ventricular ejection fraction at four and twelve weeks following transplantation. We also observed significant improvements in ventricular wall thickness and a reduction in infarction size after CCP transplantation (p-value < 0.05). Immunohistology analyses revealed in vivo maturation of the CCPs into cardiomyocytes (CM). We observed temporary episodes of ventricular tachyarrhythmia (VT) in four pigs and persistent VT in one pig, but the remaining five pigs exhibited normal sinus rhythm. Importantly, all pigs survived without the formation of any tumors or VT-related abnormalities. We conclude that pluripotent stem cell-derived CCPs constitute a promising possibility for myocardial infarction treatment and that they may positively impact regenerative cardiology.

Transcriptome profiles associated with human periodontal ligament differentiation

ObjectivesTissue differentiation is regulated by transcription factors. This study aimed to identify candidate transcription factors that induce periodontal ligament (PDL) cell differentiation in human pluripotent stem cells (hPSCs). MethodsHuman PDL tissues were scraped from the root surfaces of extracted teeth for orthodontic treatment and cultured using the explant culture method. We used RNA-seq to generate gene expression profiles of third-passage PDL cells and compared them with those of undifferentiated human induced pluripotent stem cells (hiPSCs) and human embryonic stem cell (hESC)-derived neural crest (NC) cells (publicly available data). ResultsPrimary cultured PDL cells exhibited a spindle-shaped fibroblast-like appearance and the gene expression of several PDL cell-specific markers. The gene expression profiles of PDL cells were relatively similar to those of hESC-derived NC cells but not those of undifferentiated hiPSCs. Thirty-seven transcription factors were identified as upregulated genes in PDL cells. Pathway analysis showed that differentially expressed genes were enriched in several functional groups and pathways, including the SMAD 2/3 nuclear pathway. ConclusionsWe identified 37 upregulated transcription genes in primary cultured PDL cells compared with hESC-derived NC cells. Regulating these genes and the SMAD signaling pathway may be promising ways to induce PDL cells from hPSC-derived NC cells.

Dysregulated cholesterol metabolism, aberrant excitability and altered cell cycle of astrocytes in fragile X syndrome.

Fragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. While neuronal contribution to FXS has been extensively studied in both animal and human-based models of FXS, the roles of astrocytes, a type of glial cells in the brain, are largely unknown. Here, we generated a human-based FXS model via differentiation of astrocytes from human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) and characterized their development, function, and proteomic profiles. We identified shortened cell cycle, enhanced Ca2+ signaling, impaired sterol biosynthesis, and pervasive alterations in the proteome of FXS astrocytes. Our work identified astrocytic impairments that could contribute to the pathogenesis of FXS and highlight astrocytes as a novel therapeutic target for FXS treatment.

Generation of Oligodendrocytes from Human Pluripotent and Embryonic Stem Cells.

Oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes (OLs) can be generated using human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). By manipulating culture conditions, pluripotent cell types are serially guided through intermediary cell types, developing first into neural progenitor cells (NPCs) then OPCs before maturing as CNS-specific OLs. This procedure is conducted under adherent, feeder-free conditions to derive mature OLs in as few as 28days.

Cyclosporin A Promotes Invasion and Migration of Extravillous Trophoblast Cells Derived from Human-Induced Pluripotent Stem Cells and Human Embryonic Stem Cells.

Extravillous trophoblast (EVT) cells play an essential role in the maternal-fetal interaction. Although abnormal development and function of EVT cells, including impaired migration and invasion capability, are believed to be etiologically linked to severe pregnancy disorders including pre-eclampsia, the associated molecular mechanisms are not clear due to the lack of an appropriate cell model in vitro. Cyclosporin A (CsA) is a macrolide immunosuppressant and also used in clinic to improve pregnancy outcomes. However, whether CsA has any effects on the function of EVT cells has not been well investigated. In this study, we induced differentiation of human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) into EVT cells (hiPSC-EVT and hESC-EVT cells, respectively) by Y27632, neuregulin-1 (NRG1), A83-01, and matrigel, and collected these derived EVT cells by flow cytometry for sorting cells positive for double human leukocyte antigen-G (HLA-G) and Cytokeratin7 (KRT7), both of which are EVT markers. We then investigated the effects of CsA on the invasion and migration of these derived EVT cells. We found that the hiPSC-EVT and hESC-EVT cells expressed high levels of the EVT markers such as KRT7, integrin alpha 5 (ITGA5), and HLA-G but low levels of OCT4, a stem cell marker, and that CsA significantly promoted the invasion and migration of hiPSC-EVT and hESC-EVT cells compared with HTR-8/SVneo cells. These results represent a possible cell model for studying the function of EVT cells and mechanism of pregnancy-related disorders associated with EVT. In addition, CsA may be used to treat pregnancy complications in clinic associated with deficient EVT function.

Functional assessment of donated human embryos for the generation of pluripotent embryonic stem cell lines

Research questionCan discarded embryos at blastocyst stage, donated to research because of genetic abnormalities and poor morphological quality, become a reliable source of human embryonic stem cell (HESC) lines? DesignThis study was consecutively conducted with 23 discarded embryos that were donated to research between February 2020 and April 2021. All embryos, except one, were morphologically evaluated and underwent trophectoderm biopsy for preimplantation genetic testing using next-generation sequencing (NGS), and then vitrified. After warming, the embryos were placed in appropriate culture conditions for the generation of HESCs, which was functionally assessed with immunofluorescence and flow cytometry for pluripotency capacity and spontaneous in-vitro differentiation. Cytogenetic assessment of the HESC was conducted with multiplex ligation-dependent probe amplification, and micro array comparative genomic hybridization. ResultsFrom the 23 embryos initially included, 17 survived warming, and 16 of them presented viability. Overall, the embryos presented poor morphological quality after warming. Only the previously untested embryo was capable of generating a new HESC line. Further characterization of this line revealed fully functional, euploid HESCs with preserved pluripotency, becoming a useful resource for research into human development and therapeutic investigation. ConclusionsNone of the donated blastocysts with poor morphological quality in association with genetic abnormalities detected by NGS had the capacity for further in-vitro expansion to originate pluripotent HESC lines. This finding seems to provide extra support to genetic counselling on the suitability of this type of embryo for clinical use.

Human Embryonic Stem Cells as a Model for Hematopoietic Stem Cell Differentiation and Viral Infection.

Pluripotent human embryonic stem cell (hESC) lines are a valuable in vitro tool to differentiate specific cell lineages, including cells from all three germ layers, i.e., neuronal cells, myocytes, and hematopoietic cells, including progenitors (described here), lymphoid cells, and myeloid cells. However, dramatically different cell subtypes and functional properties of specific cells can arise depending on the differentiation technique used. We previously optimized hematopoietic stem cell differentiation from two different NIH-approved hESC lines to generate CD34+ hematopoietic progenitor cells (HPCs). Infection of these HPCs with a common herpesvirus (human cytomegalovirus) results in maintenance of viral latency, capability of viral reactivation, recapitulation of viral mutant phenotypes, and virus-induced myelosuppression of hematopoietic differentiation. However, different HPC subpopulations support different viral latency and reactivation phenotypes, and different hESC-to-HPC differentiation methods alter the ratio of stem cell subsets. In addition, differences in differentiation methods are dependent on both protocol/reagents and user techniques. Here, we report a simplified and optimized method to generate large numbers of CD34+ HPCs with consistent phenotypes and demonstrate a comparison of several common methods that can be used to control the ratio of available HPC subpopulations. A key aspect of this approach is that we achieve consistency in differentiation across users in different laboratories and, importantly, among newly trained individuals. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Maintenance of human embryonic stem cells (hESCs) Basic Protocol 2: Differentiation of hESCs to hematopoietic progenitor cells (HPCs) Basic Protocol 3: Downstream functional differentiation of hESC-derived HPCs to mature lineages Support Protocol 1: Freezing and testing frozen batches of hESCs Support Protocol 2: Counting hESCs Support Protocol 3: Phenotyping by flow cytometry.

GFP Tagged VSELs Help Delineate Novel Stem Cells Biology in Multiple Adult Tissues.

Various types of stem cells are being researched upon to exploit their potential for regenerative medicine including pluripotent human embryonic stem (hES) cells derived from spare human embryos, induced pluripotent stem (iPS) cells by reprogramming somatic cells to a pluripotent state and multipotent mesenchymal stem/stromal cells (MSCs) obtained in vitro from multiple tissues. More than 50 independent groups have reported another novel population of pluripotent stem cells in adult tissues termed very small embryonic-like stem cells (VSELs). VSELs are developmentally linked to primordial germ cells, which rather than giving rise to the germ cells and later ceasing to exist, survive throughout life in multiple organs along with tissue-specific adult stem cells better described as lineage-restricted, tissue-committed progenitors with limited plasticity. VSELs survive total body irradiation in bone marrow, oncotherapy in the gonads, bilateral ovariectomy in the uterus and partial pancreatectomy in the pancreas of mice and participate in the regeneration of multiple organs under normal physiological conditions. VSELs and tissue-specific progenitor cells work together in a subtle manner, maintain life-long tissue homeostasis and their dysfunction leads to various pathologies including cancer. However, due to their quiescent state, VSELs have invariably eluded lineage-tracing studies reported so far. Present article reviews novel insights into VSELs biology and how VSELs enriched from GFP (green fluorescent protein) mice have enabled to delineate their role in various biological processes in vivo. VSELs biology needs to be understood in-depth as this alone will help evolve the field of regenerative medicine and win the war against cancer.

Untangling the Web of Protein Kinase C Mediated Regulation of Naïve Vs Primed State of Pluripotency

Introduction‐Pluripotency of stem cells is associated with the dynamicity of the embryo’s developmental stage from which they are derived. Stem cells derived from the pre‐implantation embryo, called embryonic stem cells (ESCs), are naïve in nature, whereas post‐implantation embryo‐derived stem cells, termed epiblast stem cells (EpiSCs), represent the primed state of pluripotency. Maintaining these stem cells in a naïve state is an essential arena of stem cell research since naïve ESCs are capable of clonal propagation and germline transmission, making them a crucial research tool in organogenesis and disease modeling. Inhibition of PKC (PKCi) signaling was found to maintain an undifferentiated stage of pluripotency in both mouse and rat. Interestingly, when used along with a cocktail of inhibitors, the same inhibitor facilitated the derivation of naïve state pluripotency in human ESC. These findings lead us to a hypothesis that PKC signaling inhibition alone has a role in maintaining the naïve state of pluripotency. Hence, it has intrigued us to explore the complex network of mechanisms exerted by PKC signaling in dictating the transition of naïve to primed state of pluripotency.Methodology‐In that pursuit, we first observed the morphology of the mouse ESCs (mESCs) by phase‐contrast microscopy across passages and performed MTT assay to optimize the concentration used for further assay. Quantitative Real‐time PCR and western blotting were performed to determine the expression of transcription factors associated with naïve and primed states of pluripotency. Other techniques, including FACS, TiO2 phospho‐enrichment followed by Mass‐spectrometric (MS) analysis, Immunofluorescence assay, Immunoprecipitation assay, Nuclear‐cytoplasmic protein extraction and others were exploited to study the mechanism.Results‐We observed that PKCi could maintain naïve state of pluripotency in mESC even after prolonged culturing by reducing the expression of a critical primed transcription factor. The inhibition of phosphorylation of the PKCζ isoform is responsible for naïve state maintenance. Cell cycle analysis showed that more cells are in the G1 phase in the PKCi maintained condition than in the cells maintained in the presence of Leukemia Inhibitory Factor (LIF). Also, cell proliferation marker CDK1 expression was less in PKCi maintained cells, which probably is negatively regulated by the transcription factor KLF4. Increased nuclear expression of KLF4 was observed in PKCi maintained ESCs than that of ESCs cultured with LIF. Beside these transcription factor‐mediated regulations, PKCi exerts an epigenetic control over the ESCs absent in LIF maintained ESCs. PKCi mediated loss in phosphorylation of the epigenetic factor contributes to naïve state maintenance. Further, our data indicates towards PKCζ mediated NF‐κβ pathway activation, thereby dictating the transition of naïve to primed state of pluripotency.Conclusions‐PKCi maintained mESCs could maintain the naïve state of pluripotency. We have unraveled a multifaceted functionality of PKC signaling in the transition of naïve to primed state of pluripotency, which extends to both transcriptional and epigenetic regulation.

Stem cell-derived retinal pigment epithelium transplantation in age-related macular degeneration: recent advances and challenges.

Age-related macular degeneration (AMD) is one of the leading causes of irreversible vision loss in the world with more than 80% of the prevalence accounted for by the nonneovascular (NNAMD) or 'dry' form of the disease. NNAMD does not have any definitive treatment once vision loss has ensued and presents a major unmet medical need. This review will highlight stem cell-based therapies that are a promising form of treatment for advanced NNAMD. In the past decade, clinical trials utilizing both induced pluripotent stem cell-derived RPE and human embryonic stem cell-derived RPE have been aggressively pursued as potential treatments of RPE loss and prevention of overlying neurosensory atrophy. While promising preliminary results demonstrating safety and potential efficacy have been published, new challenges have also been identified. These include selecting the most appropriate cell-based therapy, identifying and managing potential immune response as well as characterizing anatomic and functional efficacy. In this review, we will discuss some of these challenges in light of the available data from several early phase clinical trials and discuss the strategies that are being considered to further advance the field. Cell-based therapies demonstrate promising potential to treat advanced stages of NNAMD. Several early phase clinical trials using both induced pluripotent stem cells (iPSC) and human embryonic stem cell derived (hESC) have demonstrated safety and preliminary signs of efficacy and highlighted remaining challenges which appear surmountable. These challenges include development of selection criteria for use of cell suspensions versus RPE sheets, especially in light of immunological properties of RPE that are intrinsic to the status of RPE differentiation in each of these cell formulations.

Effect of extracellular matrix proteins on the differentiation of human pluripotent stem cells into mesenchymal stem cells.

The transplantation of human mesenchymal stem cells (hMSCs), such as bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs), has shown beneficial effects in protecting transplanted tissues and cells against graft-versus-host disease (GVHD). Human pluripotent stem cell (hPSC)-derived mesenchymal stem cells (MSCs) can also be used to generate hMSCs with stable characteristics without limitations. Therefore, we differentiated human induced pluripotent stem cells (hiPSCs, H-M5) and human embryonic stem cells (hESCs, H9) into hMSCs on dishes coated with different extracellular matrix (ECM) proteins to study the effect of cell culture biomaterials on hPSC differentiation into hMSCs. hPSC-derived MSCs cultured on Matrigel (MAT)-coated, collagen (COL)-coated and laminin-521 (LN-521)-coated tissue culture polystyrene (TCP) dishes showed excellent proliferation speed and reduced aging over 10 passages. High MSC surface marker (CD44, CD73, CD90 and CD105) expression was also observed on hPSC-derived MSCs cultured on MAT-coated, COL-coated and LN-521-coated TCP dishes as well as uncoated TCP dishes. Analysis of late osteogenic differentiation by evaluation of mineral deposition revealed that hPSC-derived MSCs cultured on fibronectin (FN)-coated and LN-521-coated TCP dishes showed high osteogenic differentiation. ECM proteins are effective as coating materials on cell culture biomaterials to regulate the proliferation and differentiation fate of hPSC-derived MSCs.

Bidirectional cell-matrix interaction dictates neuronal network formation in a brain-mimetic 3D scaffold

Human pluripotent stem cells (hPSC) derived neurons are emerging as a powerful tool for studying neurobiology, disease pathology, and modeling. Due to the lack of platforms available for housing and growing hPSC-derived neurons, a pressing need exists to tailor a brain-mimetic 3D scaffold that recapitulates tissue composition and favourably regulates neuronal network formation. Despite the progress in engineering biomimetic scaffolds, an ideal brain-mimetic scaffold is still elusive. We bioengineered a physiologically relevant 3D scaffold by integrating brain-like extracellular matrix (ECM) components and chemical cues. Culturing hPSCs-neurons in hyaluronic acid (HA) gels and HA-chondroitin sulfate (HA-CS) composite gels showed that the CS component prevails as the predominant factor for the growth of neuronal cells, albeit to modest efficacy. Covalent grafting of dopamine (DA) moieties to the HA-CS gel (HADA-CS) enhanced the scaffold stability and stimulated the gel's remodeling properties by entrapping cell-secreted laminin, and binding brain-derived neurotrophic factor (BDNF). Neurons cultured in the scaffold expressed Col1, Col11, and ITGB4; important for cell adhesion and cell-ECM signaling. Thus, the HA-CS scaffold with integrated chemical cues (DA) supported neuronal growth and network formation. This scaffold offers a valuable tool for tissue engineering and disease modeling and helps in bridging the gap between animal models and human diseases by providing biomimetic neurophysiology. Statement of significanceDeveloping a brain mimetic 3D scaffold that supports neuronal growth could potentially be useful to study neurobiology, disease pathology, and disease modeling. However, culturing human induced pluripotent stem cells (hiPSC) and human embryonic stem cells (ESCs) derived neurons in a 3D matrix is extremely challenging as neurons are very sensitive cells and require tailored composition, viscoelasticity, and chemical cues. This article identified the key chemical cues necessary for designing neuronal matrix that trap the cell-produced ECM and neurotrophic factors and remodel the matrix and supports neurite outgrowth. The tailored injectable scaffold possesses self-healing/shear-thinning property which is useful to design injectable gels for regenerative medicine and disease modeling that provides biomimetic neurophysiology.

Modulating mesendoderm competence during human germ layer differentiation.

SUMMARYAs pluripotent human embryonic stem cells progress toward one germ layer fate, they lose the ability to adopt alternative fates. Using a low-dimensional reaction coordinate to monitor progression toward ectoderm, we show that a differentiating stem cell’s probability of adopting a mesendodermal fate given appropriate signals falls sharply at a point along the ectoderm trajectory. We use this reaction coordinate to prospectively isolate and profile differentiating cells based on their mesendoderm competence and analyze their RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) profiles to identify transcription factors that control the cell’s mesendoderm competence. By modulating these key transcription factors, we can expand or contract the window of competence to adopt the mesendodermal fate along the ectodermal differentiation trajectory. The ability of the underlying gene regulatory network to modulate competence is essential for understanding human development and controlling the fate choices of stem cells in vitro.

Development of a fully human assay combining NGN2-inducible neurons co-cultured with iPSC-derived astrocytes amenable for electrophysiological studies

Neurogenin 2 encodes a neural-specific transcription factor (NGN2) able to drive neuronal fate on somatic and stem cells. NGN2 is expressed in neural progenitors within the developing central and peripheral nervous systems. Overexpression of NGN2 in human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells has been shown to efficiently trigger conversion to neurons. Here we describe two gene-edited hiPSC lines harbouring a doxycycline (DOX)-inducible cassette in the AAVS1 locus driving expression of NGN2 (BIONi010-C-13) or NGN2-T2A-GFP (BIONi010-C-15). By introducing NGN2-expressing cassette, we reduce variability associated with conventional over-expression methods such as viral transduction, making these lines amenable for scale-up production and screening processes. DOX-treated hiPSCs convert to neural phenotype within one week and display the expression of structural neuronal markers such as Beta-III tubulin and tau. We performed functional characterization of NGN2-neurons co-cultured with hiPSC-derived astrocytes in a “fully-humanized” set up. Passive properties of NGN2-neurons were indistinguishable from mouse primary cells while displaying variable activity in extracellular recordings performed in multi-electrode arrays (MEAs). We demonstrate that hiPSC-derived astrocytes and neurons can be co-cultured and display functional properties comparable to the gold standard used in electrophysiology. Both lines are globally available via EBiSC repository at https://cells.ebisc.org/.

Specific epigenetic age acceleration patterns among four molecular subtypes of gastric cancer and their prognostic value.

Aims: To determine the association of the methylation age (Horvath epigenetic clock) of gastric cancer (GC) tissues with molecular subtypes and patient survival. Materials & methods: Multivariate regression models were used to determine the association of methylation age acceleration (AA) with the clinical and molecular characteristics of 333 GC patients. Results: Relative to the chromosomal instability subtype, the epigenetic AA was 49.8 (95% CI: 42.7-56.9) years for Epstein-Barr virus, 16.1 (10.6-21.6) years for microsatellite instability, and 6.05 (0.1-11.1) years for genomicstability subtype. GC patients with accelerated aging of tumor tissues had better outcomes (adjusted hazard ratio: 3.13; p =0.03). Differentially methylated probes in patients with accelerated and decelerated methylation aging enriched in pathways including BMP signaling, HMGB1 signaling, STAT3 signaling and human embryonic stem cell pluripotency. Conclusions: Our results highlight the prognostic value of epigenetic AA in GC and suggest that epigenetic AA is also an indicator of molecular subtype in GC.

Modulating Mesendoderm Competence During Human Germ Layer Differentiation

As pluripotent human embryonic stem cells progress towards one germ layer fate, they lose the ability to adopt alternative fates. It is unknown how the cells’ competence for these alternative fates changes along their developmental trajectory or if this competence can be modulated. Here, we show that a differentiating stem cell’s probability of adopting a mesendodermal fate given appropriate signals falls sharply at a point along the ectodermal differentiation trajectory, and demonstrate that this point can be moved using genetic perturbations. Using a low-dimensional reaction coordinate to monitor progression towards ectoderm, we can determine the probability that individual cells at different points along this path are competent to adopt mesendodermal fate upon BMP4 and Activin A signal exposure. Knowing this probability allows us to prospectively isolate and profile differentiating cells based on their mesendoderm competence. We then analyze RNA-seq and ATAC-seq profiles of these different populations to identify transcription factors that control the cell’s mesendoderm competence. By modulating these key transcription factors, we can expand the window of competence to adopt the mesendodermal fate along the ectodermal differentiation trajectory. The ability of the underlying gene regulatory network to modulate competence is essential for understanding human development and controlling the fate choices of stem cells in vitro.

OCT4 expression in human embryonic stem cells: spatio-temporal dynamics and fate transitions

The improved in vitro regulation of human embryonic stem cell (hESC) pluripotency and differentiation trajectories is required for their promising clinical applications. The temporal and spatial quantification of the molecular interactions controlling pluripotency is also necessary for the development of successful mathematical and computational models. Here we use time-lapse experimental data of OCT4-mCherry fluorescence intensity to quantify the temporal and spatial dynamics of the pluripotency transcription factor OCT4 in a growing hESC colony in the presence and absence of BMP4. We characterise the internal self-regulation of OCT4 using the Hurst exponent and autocorrelation analysis, quantify the intra-cellular fluctuations and consider the diffusive nature of OCT4 evolution for individual cells and pairs of their descendants. We find that OCT4 abundance in the daughter cells fluctuates sub-diffusively, showing anti-persistent self-regulation. We obtain the stationary probability distributions governing hESC transitions amongst the different cell states and establish the times at which pro-fate cells (which later give rise to pluripotent or differentiated cells) cluster in the colony. By quantifying the similarities between the OCT4 expression amongst neighbouring cells, we show that hESCs express similar OCT4 to cells within their local neighbourhood within the first two days of the experiment and before BMP4 treatment. Our framework allows us to quantify the relevant properties of proliferating hESC colonies and the procedure is widely applicable to other transcription factors and cell populations.