Embryoid Bodies Research Articles

Generation and characterization of induced pluripotent stem cell lines from two patients with recessive dystrophic epidermolysis Bullosa

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare and severe genetic disease responsible for blistering of the skin and mucosa caused by a wide variety of mutations in COL7A1 encoding type VII collagen. We have generated Induced Pluripotent Stem Cells (iPSCs) from two RDEB patients’ fibroblasts harboring homozygous recurrent mutations in COL7A1. Their pluripotent state was confirmed by gene and protein expression of stem cell markers OCT4, SOX2, TRA1/60 and SSEA4. Embryoid body formation followed by immunostaining and TaqMan scorecard analysis confirmed the capacity of RDEB iPSCs to differentiate into cell types from the three germ layers in vitro.

Bioinformatic analysis predicts that ethanol exposure during early development causes alternative splicing alterations of genes involved in RNA post-transcriptional regulation.

Prenatal ethanol exposure is associated with neurodevelopmental defects and long-lasting cognitive deficits, which are grouped as fetal alcohol spectrum disorders (FASD). The molecular mechanisms underlying FASD are incompletely characterized. Alternative splicing, including the insertion of microexons (exons of less than 30 nucleotides in length), is highly prevalent in the nervous system. However, whether ethanol exposure can have acute or chronic deleterious effects in this process is poorly understood. In this work, we used the bioinformatic tools VAST-TOOLS, rMATS, MAJIQ, and MicroExonator to predict alternative splicing events affected by ethanol from available RNA sequencing data. Experimental protocols of ethanol exposure included human cortical tissue development, human embryoid body differentiation, and mouse development. We found common genes with predicted differential alternative splicing using distinct bioinformatic tools in different experimental designs. Notably, Gene Ontology and KEGG analysis revealed that the alternative splicing of genes related to RNA processing and protein synthesis was commonly affected in the different ethanol exposure schemes. In addition, the inclusion of microexons was also affected by ethanol. This bioinformatic analysis provides a reliable list of candidate genes whose splicing is affected by ethanol during nervous system development. Furthermore, our results suggest that ethanol particularly modifies the alternative splicing of genes related to post-transcriptional regulation, which probably affects neuronal proteome complexity and brain function.

Establishment of iPSC lines and zebrafish with loss-of-function AHDC1 variants: Models for Xia-Gibbs syndrome

Xia-Gibbs syndrome (XGS) is a syndromic form of intellectual disability caused by heterozygous AHDC1 variants, but the pathophysiological mechanisms underlying this syndrome are still unclear. In this manuscript, we describe the development of two different functional models: three induced pluripotent stem cell (iPSC) lines with different loss-of-function (LoF) AHDC1 variants, derived by reprogramming peripheral blood mononuclear cells from XGS patients, and a zebrafish strain with a LoF variant in the ortholog gene (ahdc1) obtained through CRISPR/Cas9-mediated editing. The three iPSC lines showed expression of pluripotency factors (SOX2, SSEA-4, OCT3/4, and NANOG). To verify the capacity of iPSC to differentiate into the three germ layers, we obtained embryoid bodies (EBs), induced their differentiation, and confirmed the mRNA expression of ectodermal, mesodermal, and endodermal markers using the TaqMan hPSC Scorecard. The iPSC lines were also approved for the following quality tests: chromosomal microarray analysis (CMA), mycoplasma testing, and short tandem repeat (STR) DNA profiling. The zebrafish model has an insertion of four base pairs in the ahdc1 gene, is fertile, and breeding between heterozygous and wild-type (WT) animals generated offspring in a genotypic proportion in agreement with Mendelian law. The established iPSC and zebrafish lines were deposited on the hpscreg.eu and zfin.org platforms, respectively. These biological models are the first for XGS and will be used in future studies that investigate the pathophysiology of this syndrome, unraveling its underlying molecular mechanisms.

FOXK1 regulates Wnt signalling to promote cardiogenesis.

Congenital heart disease (CHD) is the most common genetic birth defect, which has considerable morbidity and mortality. We focused on deciphering key regulators that govern cardiac progenitors and cardiogenesis. FOXK1 is a forkhead/winged helix transcription factor known to regulate cell cycle kinetics and is restricted to mesodermal progenitors, somites, and heart. In the present study, we define an essential role for FOXK1 during cardiovascular development. We used the mouse embryoid body system to differentiate control and Foxk1 KO embryonic stem cells into mesodermal, cardiac progenitor cells and mature cardiac cells. Using flow cytometry, immunohistochemistry, cardiac beating, transcriptional and chromatin immunoprecipitation quantitative polymerase chain reaction assays, bulk RNA sequencing (RNAseq) and assay for transposase-accessible chromatin using sequencing (ATACseq) analyses, FOXK1 was observed to be an important regulator of cardiogenesis. Flow cytometry analyses revealed perturbed cardiogenesis in Foxk1 KO embryoid bodies (EBs). Bulk RNAseq analysis at two developmental stages showed a significant reduction of the cardiac molecular program in Foxk1 KO EBs compared to the control EBs. ATACseq analysis during EB differentiation demonstrated that the chromatin landscape nearby known important regulators of cardiogenesis was significantly relaxed in control EBs compared to Foxk1 KO EBs. Furthermore, we demonstrated that in the absence of FOXK1, cardiac differentiation was markedly impaired by assaying for cardiac Troponin T expression and cardiac contractility. We demonstrate that FOXK1 is an important regulator of cardiogenesis by repressing the Wnt/β-catenin signalling pathway and thereby promoting differentiation. These results identify FOXK1 as an essential transcriptional and epigenetic regulator of cardiovascular development. Mechanistically, FOXK1 represses Wnt signalling to promote the development of cardiac progenitor cells.

Visualization of basement membranes by a nidogen-based fluorescent reporter in mice

Basement membranes (BMs) are thin, sheet-like extracellular structures that cover the basal side of epithelial and endothelial tissues and provide structural and functional support to adjacent cell layers. The molecular structure of BMs is a fine meshwork that incorporates specialized extracellular matrix proteins. Recently, live visualization of BMs in invertebrates demonstrated that their structure is flexible and dynamically rearranged during cell differentiation and organogenesis. However, the BM dynamics in mammalian tissues remain to be elucidated. We developed a mammalian BM imaging probe based on nidogen-1, a major BM-specific protein. Recombinant human nidogen-1 fused with an enhanced green fluorescent protein (Nid1-EGFP) retains its ability to bind to other BM proteins, such as laminin, type IV collagen, and perlecan, in a solid-phase binding assay. When added to the culture medium of embryoid bodies derived from mouse ES cells, recombinant Nid1-EGFP accumulated in the BM zone of embryoid bodies, and BMs were visualized in vitro. For in vivo BM imaging, a knock-in reporter mouse line expressing human nidogen-1 fused to the red fluorescent protein mCherry (R26-CAG-Nid1-mCherry) was generated. R26-CAG-Nid1-mCherry showed fluorescently labeled BMs in early embryos and adult tissues, such as the epidermis, intestine, and skeletal muscles, whereas BM fluorescence was unclear in several other tissues, such as the lung and heart. In the retina, Nid1-mCherry fluorescence visualized the BMs of vascular endothelium and pericytes. In the developing retina, Nid1-mCherry fluorescence labeled the BM of the major central vessels; however, the BM fluorescence were hardly observed in the peripheral growing tips of the vascular network, despite the presence of endothelial BM. Time-lapse observation of the retinal vascular BM after photobleaching revealed gradual recovery of Nid1-mCherry fluorescence, suggesting the turnover of BM components in developing retinal blood vessels. To the best of our knowledge, this is the first demonstration of in vivo BM imaging using a genetically engineered mammalian model. Although R26-CAG-Nid1-mCherry has some limitations as an in vivo BM imaging model, it has potential applications in the study of BM dynamics during mammalian embryogenesis, tissue regeneration, and pathogenesis.

HIGH ANTIOXIDATION-CAPACITY STEM CELLS ENHANCE CARTILAGE REGENERATION

Stem cells are known to have low levels of intracellular reactive oxygen species (ROS) and high levels of glutathione. ROS are thought to interact with several pathways that affect the transcription machinery required for stem cell differentiation, and are critical for maintaining stem cell function. In this study, we are developing a new fluorescent probe that rapidly and reversibly reacts with glutathione (GSH), the most abundant non-protein thiol in living cells that acts as an antioxidant and redox regulator.Multipotent perivascular progenitor cells derived from human ESCs (hESC-PVPCs): Differentiated ESCs as embryoid bodies in the presence of BMP4 to induce mesoderm differentiation followed by a simple cell selection strategy using attachment of single cells onto collagen-coated dishes. Differential gene expression profiling was performed among H9 hESCs, EBs induced by BMP4 and naturally selected CD140B+CD44+ population at Day 7 (PVPCs). Colony-forming assay: GSHhigh and GSHlow PVPCs were plated on 10-cm tissue culture-treated polystyrene dishes in triplicate in growth medium and cultured for 14 days. Transwell migration assay: GSHhigh and GSHlow PVPCs at passage 4 were resuspended at 1 × 106/mL in the migration medium and seeded in the upper chamber. The following human recombinant SDF-1 and PDGF-AA proteins were used as chemoattractants in the lower compartment.Probe-GSH conjugate shows shifts in fluorescence excitation and emission spectra that enables ratiometric measurement of GSH levels. Using these properties, stem cells can be purified by FACS-based technology according to intracellular GSH level. We are developing a protocol both for comparing GSH level in stem cell from different culture conditions and for preparing stem cells with high-GSH level . Our results reveal that GSHhigh PVPC purified by FACS show increased colony forming ability compared with that GSHlow PVPC, indicating that intracellular GSH contributes to the maintenance of stemness. Moreover, transplantation of GSHlow PVPC is more effective than that of GSHlow PVPC for cartilage regeneration in osteochondral defect.This technique enable FACS-based sorting of stem cells according to intracellular GSH levels and thus investigation of functional role of GSH (high antioxidant capacity) in the stem cell maintenance and chondrogenic differentiation.

Abstract 2919: A novel method for clinical scale production of natural killer cells from clonal master induced pluripotent stem cells with CISH knockout for next generation, off-the-shelf cancer immunotherapy

Abstract Natural killer (NK) cells are innate immune cells that play a key role in in tumor immune surveillance. NK cells are self-tolerant to healthy cells and can kill tumor cells in both hematological and solid malignancies. Recent advances allow for the derivation of immune cells such as NK cells, from human induced pluripotent stem cells, that can be utilized for cancer immunotherapy. Recent clinical trials suggest that high doses of NK cells (approx. 0.5-5 x 109Jaya cells per dose) and multiple doses are both safe and likely necessary for clinical efficacy. Manufacturing large number of NK cells from a clonal master iPSC line provides a promising strategy to enable next generation, off-the-shelf, affordable, cancer immunotherapies. We have developed a novel method to produce high purity, clinical scale iNK cells and improved the process for the expansion of NK cells efficiently and consistently. This method does not require cell sorting step and the usage of murine derived stromal cells. Briefly, hematopoietic progenitor cells were induced using an improved spin embryoid body (EB) method and the hematopoietic progenitor cells were subsequently differentiated into mature iNK cells in the absence of cell sorting and independent of stroma cells support. Using this novel method, differentiated NK cells could be expanded more than 8,000-fold to enable us to potentially produce 1 x 1012 iNK cells starting from 1 x 106 undifferentiated iPSC. This cell production scale can supply hundreds of doses in clinic from one cGMP manufacturing campaign. iNK cells produced using this method display the typical phenotype of NK cell activating receptor including NKG2D, NKp44, NKp46, DNAM-1 etc. Importantly, these iNK cells demonstrate better anti-tumor activities and higher levels of IFNγ and TNFα, compared to primary peripheral blood-derived NK cells isolated from healthy donors. Moreover, this protocol has been adapted and optimized for clinical scale manufacturing of iNK cells from genetically engineered, clonal master iPSC cell line with knock out of CISH (CISH KO iNK), a key intracellular checkpoint of NK cell anti-tumor immunity. CISH KO iNK cells produced using this optimized method show significantly better anti-tumor activities against multiple liquid and solid tumor cell lines including K562, Raji, Daudi, SUP-B15, MOLT-4, SKOV-3, OVCAR-4, HCC-827, ZR-75, and BT-474 etc. in vitro and in vivo in an AML xenograft mouse model, compared with unmodified iNK cells. Overall, this novel cell production strategy paves the way for clinical trials using higher doses of iPSC-derived NK cells with increased potency, creating next-generation NK cell-based immunotherapies. Citation Format: Davide Bernareggi, Caryn Gonsalves, Max Schabla, Alejandra Gárate-Carrillo, Duygu Ozmadenci, Jaya Thangaraj, Qin Li, Leah Mitchell, Dan S. Kaufman, Robert Hollingsworth, Huang Zhu. A novel method for clinical scale production of natural killer cells from clonal master induced pluripotent stem cells with CISH knockout for next generation, off-the-shelf cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2919.

Stiffness-Tunable Hydrogel-Sandwich Culture Modulates the YAP-Mediated Mechanoresponse in Induced-Pluripotent Stem Cell Embryoid Bodies and Augments Cardiomyocyte Differentiation.

Microenvironmental factors, including substrate stiffness, regulate stem cell behavior and differentiation. However, the effects of substrate stiffness on the behavior of induced pluripotent stem cell- (iPSC) derived embryoid bodies (EB) remain unclear. To investigate the effects of mechanical cues on iPSC-EB differentiation, we developed a three-dimensional hydrogel-sandwich culture (HGSC) system that controls the microenvironment surrounding iPSC-EBs using a stiffness-tunable polyacrylamide hydrogel assembly. Mouse iPSC-EBs were seeded between upper and lower polyacrylamide hydrogels of differing stiffness (Young's modulus [E'] = 54.3 ± 7.1kPa [hard], 28.1 ± 2.3kPa [moderate], and 5.1 ± 0.1kPa [soft]) and cultured for 2 days. HGSC induced stiffness-dependent activation of the yes-associated protein (YAP) mechanotransducer and actin cytoskeleton rearrangement in the iPSC-EBs. Moreover, moderate-stiffness HGSC specifically upregulated the mRNA and protein expression of ectoderm and mesoderm lineage differentiation markers in iPSC-EBs via YAP-mediated mechanotransduction. Pretreatment of mouse iPSC-EBs with moderate-stiffness HGSC promoted cardiomyocyte differentiation and structural maturation of myofibrils. Our proposed HGSC system provides a viable platform for investigating the role of mechanical cues on the pluripotency and differentiation of iPSCs that could be beneficial for research into tissue regeneration and engineering. This article is protected by copyright. All rights reserved.

Case report of schizophrenia in a patient with de novo mutation in the SLC6A1 gene

IntroductionSchizophrenia is a severe mental disorder mainly caused by genetic risk factors. Many studies have demonstrated that both multiple genetic variants and rare mutations are associated with schizophrenia risk. The next step is to study the causal effect of the gene on the phenotype. Recently, a large family-based study identified de novo mutations, which may increase liability to schizophrenia (Rees et al 2020). In particular, a mutation in the GABA transporter (SLC6A1) gene (rs756927822 C/T) was identified in one patient from our subsample.ObjectivesHere, we present a case report of this patient and describe the procedure of derivation of induced pluripotent stem cells (iPSCs) from fibroblast cultures.MethodsClinical, psychometric and neuropsychological methods were used. iPSCs were derived from patients’ and both unaffected parents’ fibroblasts. Human fibroblasts, cultured in fibroblast medium, are infected with lentivirus vectors expressing the transcription factors Oct4, Sox2, c-Myc, and KLF4. All iPSCs were immunocytochemical stained for intracellular (Oct4, Sox2) and extracellular (SSEA4, Tra-1-81, Tra-1-60) pluripotency markers. An qPCR analysis for pluripotency markers (TDGF1, Sox2, Oct4, REX1, LIN28, NANOG, KLF4, GDF3, DPPA4, DNMT3) was performed. All four iPSC lines formed embryoid bodies before the differentiating into three germ layers. Differentiation was confirmed by immunostaining for mesoderm (aSMA), ectoderm (Nestin, Desmin) andendoderm (FoxA2, Pax6) markers.ResultsA 47-year-old male patient was presented to psychiatry at the age of 16. There was no personal or family history of psychiatric disorder, the premorbid functioning was normal, the patient had no somatic diseases, showed high performance in sport (mountain skiing). On his first admission, he was diagnosed with schizoaffective psychosis. The patient showed signs of mania and catatonia. Neuropsychological testing revealed a decrease of cognitive functioning (short-term and associative memory). The patient was followed up for more than 20 years. The diagnosis was changed for schizophrenia at the age of 43 years. There was a deterioration in cognitive function (the apparent decrease in performance on neurocognitive tests (attention, memory, executive functions) from the first examination (1997) till last one (2019). The patient refused or was not able to perform most of the tasks. During follow-up, the patient shows good adherence to treatment.ConclusionsFor this patient, obtained lines might be valuable for investigating the disease mechanisms and screening candidate drugs.Disclosure of InterestNone Declared

ETV2 and VEZF1 interaction and regulation of the hematoendothelial lineage during embryogenesis.

Ets variant 2 (Etv2), a member of the Ets factor family, has an essential role in the formation of endothelial and hematopoietic cell lineages during embryonic development. The functional role of ETS transcription factors is, in part, dependent on the interacting proteins. There are relatively few studies exploring the coordinated interplay between ETV2 and its interacting proteins that regulate mesodermal lineage determination. In order to identify novel ETV2 interacting partners, a yeast two-hybrid analysis was performed and the C2H2 zinc finger transcription factor VEZF1 (vascular endothelial zinc finger 1) was identified as a binding factor, which was specifically expressed within the endothelium during vascular development. To confirm this interaction, co-immunoprecipitation and GST pull down assays demonstrated the direct interaction between ETV2 and VEZF1. During embryoid body differentiation, Etv2 achieved its peak expression at day 3.0 followed by rapid downregulation, on the other hand Vezf1 expression increased through day 6 of EB differentiation. We have previously shown that ETV2 potently activated Flt1 gene transcription. Using a Flt1 promoter-luciferase reporter assay, we demonstrated that VEZF1 co-activated the Flt1 promoter. Electrophoretic mobility shift assay and Chromatin immunoprecipitation established VEZF1 binding to the Flt1 promoter. Vezf1 knockout embryonic stem cells had downregulation of hematoendothelial marker genes when undergoing embryoid body mediated mesodermal differentiation whereas overexpression of VEZF1 induced the expression of hematoendothelial genes during differentiation. These current studies provide insight into the co-regulation of the hemato-endothelial lineage development via a co-operative interaction between ETV2 and VEZF1.

Exosomes induce neurogenesis of pluripotent P19 cells

Exosomes play a role in tissue/organ development and differentiation. Retinoic acid induces differentiation of P19 cells (UD-P19) to P19 neurons (P19N) that behave like cortical neurons and express characteristic neuronal genes such as NMDA receptor subunits. Here we report P19N exosome-mediated differentiation of UD-P19 to P19N. Both UD-P19 and P19N released exosomes with characteristic exosome morphology, size, and common protein markers. P19N internalized significantly higher number of Dil-P19N exosomes as compared to UD-P19 with accumulation in the perinuclear region. Continuous exposure of UD-P19 to P19N exosomes for six days induced formation of small-sized embryoid bodies that differentiated into MAP2-/GluN2B-positive neurons recapitulating RA-induction of neurogenesis. Incubation with UD-P19 exosomes for six days did not affect UD-P19. Small RNA-seq identified enrichment of P19N exosomes with pro-neurogenic non-coding RNAs (ncRNAs) such as miR-9, let-7, MALAT1 and depleted with ncRNAs involved in maintenance of stem cell characteristics. UD-P19 exosomes were rich with ncRNAs required for maintenance of stemness. P19N exosomes provide an alternative method to genetic modifications for cellular differentiation of neurons. Our novel findings on exosomes-mediated differentiation of UD-P19 to P19 neurons provide tools to study pathways directing neuron development/differentiation and develop novel therapeutic strategies in neuroscience.Graphical

The differentiation of human induced pluripotent stem cells into hematopoietic stem cells on 3D bone scaffold in a dynamic culture system

Hematopoietic stem cell transplantation is used for cell-based therapy for many hematological disorders. However, difficulty in finding proper donors has limited this source of stem cells. For clinical application, the generation of these cells from induced pluripotent stem cells (iPSs) is a fascinating and endless source. One of the experimental methods to generate HSCs from iPSs is the mimicking of the hematopoietic niche. In the current study, as the first phase of differentiation, embryoid bodies were formed from iPSs. They were then cultured in different dynamic conditions in order to determine the appropriate settings for their differentiation into HSCs. The dynamic culture was composed of DBM Scaffold with or without growth factor. After ten days, the specific HSC markers (CD34, CD133, CD31 and CD45) were assessed using flow-cytometry. Our findings demonstrated that the dynamic conditions were significantly suitable than static ones. In addition, in 3D scaffold and dynamic system the expression of CXCR4, as a homing marker, was increased. These results suggest that the 3D culture bioreactor with DBM scaffold could provide a new approach for differentiation of iPSs into HSCs. Moreover, this system could provide maximum mimicry of bone marrow niche.

Embryoid Body Cells from Human Embryonic Stem Cells Overexpressing Dopaminergic Transcription Factors Survive and Initiate Neurogenesis via Neural Rosettes in the Substantia Nigra

Transplantation of immature dopaminergic neurons or neural precursors derived from embryonic stem cells (ESCs) into the substantia nigra pars compacta (SNpc) is a potential therapeutic approach for functional restitution of the nigrostriatal pathway in Parkinson's disease (PD). However, further studies are needed to understand the effects of the local microenvironment on the transplanted cells to improve survival and specific differentiation in situ. We have previously reported that the adult SNpc sustains a neurogenic microenvironment. Non-neuralized embryoid body cells (EBCs) from mouse ESCs (mESCs) overexpressing the dopaminergic transcription factor Lmx1a gave rise to many tyrosine hydroxylase (Th+) cells in the intact and damaged adult SNpc, although only for a short-term period. Here, we extended our study by transplanting EBCs from genetically engineered naive human ESC (hESC), overexpressing the dopaminergic transcription factors LMX1A, FOXA2, and OTX2 (hESC-LFO), in the SNpc. Unexpectedly, no graft survival was observed in wild-type hESC EBCs transplants, whereas hESC-LFO EBCs showed viability in the SNpc. Interestingly, neural rosettes, a developmental hallmark of neuroepithelial tissue, emerged at 7- and 15-days post-transplantation (dpt) from the hESC-LFO EBCs. Neural rosettes expressed specification dopaminergic markers (Lmx1a, Otx2), which gave rise to several Th+ cells at 30 dpt. Our results suggest that the SNpc enables the robust initiation of neural differentiation of transplanted human EBCs prompted to differentiate toward the midbrain dopaminergic phenotype.

Microfluidic device for differentiation induction of iPS cells derived embryoid bodies with local chemical stimulation

AbstractThis paper reports a microfluidic device that allows us to measure the effect of local differentiation stimulation on embryoid bodies for the purpose of elucidating the role of the cell‐cell interaction of iPS cells in differentiation induction. We developed the local stimulation device having two chambers separated by thin polydimethyl siloxane (PDMS) membrane with through‐holes of the diameter smaller than single cell. Embryoid bodies of iPS cells are formed on the membrane and seal the holes, separating the aqueous environments of two chambers without leakage. This configuration brings localized exposure to chemicals for differentiation induction at the area defined by the holes. In this paper, we demonstrated the function of local stimulation using fluorescent solution.

YAP1 is essential for self-organized differentiation of pluripotent stem cells

Induced pluripotent stem cells (iPSCs) form aggregates that recapitulate aspects of the self-organization in early embryogenesis. Within few days, cells undergo a transition from epithelial-like structures to organized three-dimensional embryoid bodies (EBs) with upregulation of germ layer-specific genes. However, it is largely unclear, which signaling cascades regulate self-organized differentiation. The Yes-associated protein 1 (YAP1) is a downstream effector of the Hippo pathway and essential mechanotransducer. YAP1 has been suggested to play a crucial role for early embryo development, but the relevance for early germ layer commitment of human iPSCs remains to be elucidated. To gain insights into the function of YAP1 in early cell-fate decisions, we generated YAP1 knockout (YAP−/−) iPSC lines with CRISPR/Cas9 technology and analyzed transcriptomic and epigenetic modifications. YAP−/− iPSCs showed increased expression of several YAP1 targets and of NODAL, an important regulator of cell differentiation. Furthermore, YAP1 deficiency evoked global DNA methylation changes. Directed differentiation of adherent iPSC colonies towards endoderm, mesoderm, and ectoderm could be induced, albeit endodermal and ectodermal differentiation showed transcriptomic and epigenetic changes in YAP−/− lines. Notably, in undirected self-organized YAP−/− EBs germ layer specification was clearly impaired. This phenotype was rescued via lentiviral overexpression of YAP1 and also by NODAL inhibitors. Our results demonstrate that YAP1 plays an important role during early germ layer specification of iPSCs, particularly for the undirected self-organization of EBs, and this is at least partly attributed to activation of the NODAL signaling.

Genes Involved in DNA Repair and Mitophagy Protect Embryoid Bodies from the Toxic Effect of Methylmercury Chloride under Physioxia Conditions.

The formation of embryoid bodies (EBs) from human pluripotent stem cells resembles the early stages of human embryo development, mimicking the organization of three germ layers. In our study, EBs were tested for their vulnerability to chronic exposure to low doses of MeHgCl (1 nM) under atmospheric (21%O2) and physioxia (5%O2) conditions. Significant differences were observed in the relative expression of genes associated with DNA repair and mitophagy between the tested oxygen conditions in nontreated EBs. When compared to physioxia conditions, the significant differences recorded in EBs cultured at 21% O2 included: (1) lower expression of genes associated with DNA repair (ATM, OGG1, PARP1, POLG1) and mitophagy (PARK2); (2) higher level of mtDNA copy number; and (3) higher expression of the neuroectodermal gene (NES). Chronic exposure to a low dose of MeHgCl (1 nM) disrupted the development of EBs under both oxygen conditions. However, only EBs exposed to MeHgCl at 21% O2 revealed downregulation of mtDNA copy number, increased oxidative DNA damage and DNA fragmentation, as well as disturbances in SOX17 (endoderm) and TBXT (mesoderm) genes expression. Our data revealed that physioxia conditions protected EBs genome integrity and their further differentiation.

The Effect of Oxygen Tension on the Differentiation of Outgrowth Cells from Embryoid Bodies Produced by Mouse Induced Pluripotent Stem Cells

The purpose of this study was to investigate the effects of oxygen tension on mouse induced pluripotent stem (iPS) cells by assessing differentiation in terms of embryoid body (EB) size and mRNA and protein expression levels of collagen type 1 and Runx2. EBs and outgrowth cells (OGCs) were cultured in conditions of hypoxia (2%), normoxia (21%) and hyperoxia (35%). Significantly larger EBs were observed in the hyperoxia and normoxia groups compared to the hypoxia group on Days 8 and 10. The hyperoxia group had significantly higher mRNA and protein expression levels of collagen type 1 and Runx2 compared to the hypoxia and normoxia groups on Days 10 and 26, and these expression levels in the hypoxia group were significantly lower than the normoxia group on Days 10 and 26. These results suggest that hyperoxia accelerates the enlargement in EBs and the differentiation of iPS cells.

The Mechanism of Houttuynia cordata Embryotoxicity Was Explored in Combination with an Experimental Model and Network Pharmacology.

Houttuynia cordata (H. cordata) is the most common herb as a food and traditional Chinese medicine. Currently, studies on its toxicity have mainly focused on hepatotoxicity. However, its potential embryotoxicity by long-term exposure is often overlooked. Objective: To investigate the effects of H. cordata on embryonic development and its toxicity mechanism by combining network pharmacology, molecular docking, and in vitro experimental methods. Methods: The effects of H. cordata on embryos were evaluated. Zebrafish embryos and embryoid bodies were administered to observe the effects of H. cordata on embryonic development. Based on network pharmacological analysis, it was found that the main active agents producing toxicity in H. cordata were oleanolic acid, lignan, and aristolactam AII. H. cordata can affect PI3K-Akt, MAPK, and Ras signaling pathways by regulating targets, such as AKT1, EGFR, CASP3, and IGF-1. RT-PCR and immunohistochemistry results showed that the expression of AKT1 and PI3K in the embryoid body was significantly reduced after drug administration (p < 0.05). Conclusions: The results of network pharmacology and in vitro experiments suggest that H. cordata may affect embryonic development by influencing the PI3K-Akt signaling pathway.

Reflow-molded deep concave microwell arrays for robust and large-scale production of embryoid bodies.

Embryonic stem cell (ESC)-derived aggregates, called embryoid bodies (EBs), are powerful in vitro models used to study human development and disease. However, the cost-effective and large-scale production of homogeneous EBs still remains a challenge. Here, we report a rapid, straightforward method for fabricating closely arrayed deep concave microwells, enabling the mass production of uniform EBs from single cell suspensions. By simply combining micromilling, caramel replica molding, and thermal reflow, we generate convex micromolds with high aspect ratios and excellent surface smoothness. Benefitting from the nature of reflow, this method can produce rounded bottom polydimethylsiloxane (PDMS) microwells, which are not easily achieved with standard soft lithography techniques but critical to producing spherical EBs. To achieve optimal concave microwells, we investigated the effect of thermal reflow temperature and time on the surface smoothness and roundness of the finished microwells. In addition, to further improve the utility of this method, we also investigated the effect of microwell aspect ratio (AR) on the loss of EBs during medium manipulation. The capability of this deep concave microwell system was validated by rapidly generating a large number of human embryonic stem cell (hESC)-derived EBs and then efficiently differentiating them into a cardiac lineage. The proposed fabrication method and deep concave microwell platform are highly practical, and thus will benefit the mass production of EBs for potential tissue regeneration and cell therapy applications.

Suppression of P-cadherin expression as a key regulatory element for embryonic stem cell stemness.

In embryonic stem (ES) cell colonies, a small subpopulation that changes cell shape and loses pluripotency often appears in two-dimensional (2D) cultures, even in the presence of a stemness factor. We have previously shown that membrane translocation of the syntaxin4, t-SNARE protein contributes to this phenomenon. Here, we show that ES cells in three-dimensional (3D) aggregates do not succumb to extruded syntaxin4 owing to suppressed expression of P-cadherin protein. While extracellular expression of syntaxin4 led to the striking upregulation of P-cadherin mRNA in both 2D and 3D-ES cells, morphological changes and appreciable expression of P-cadherin protein were detected only in 2D-ES cells. Importantly, the introduction of an expression cassette for P-cadherin practically reproduced the effects induced by extracellular syntaxin4, where the transgene product was clearly detected in 2D-, but not 3D-ES cells. An expression construct for P-cadherin-Venus harboring an in-frame insertion of the P2A sequence at the joint region gave fluorescent signals only in the cytoplasm of 2D-ES cells, demonstrating translational regulation of P-cadherin. These results provide the mechanistic insight into the uncontrollable differentiation in 2D-ES cells and shed light on the validity of the "embryoid body protocol commonly used for ES cell handling" for directional differentiation.Key words: differentiation, embryoid body, ES cells, P-cadherin, syntaxin4.