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Dax1 ( Nr0b1 ; Dosage-sensitive sex reversal-adrenal hypoplasia congenital on the X-chromosome gene-1) is an important component of the transcription factor network that governs pluripotency in mouse embryonic stem cells (ESCs). Functional evaluation of alternative splice variants of pluripotent transcription factors has shed additional insight on the maintenance of ESC pluripotency and self-renewal. Dax1 splice variants have not been identified and characterized in mouse ESCs. We identified 18 new transcripts of Dax1 with putative protein-coding properties and compared their protein structures with known Dax1 protein (Dax1-472). The expression pattern analysis showed that the novel isoforms were cotranscribed with Dax1-472 in mouse ESCs, but they had transcriptional heterogeneity among single cells and the subcellular localization of the encoded proteins differed. Cell function experiments indicated that Dax1-404 repressed Gata6 transcription and functionally replaced Dax1-472, while Dax1-38 and Dax1-225 partially antagonized Dax1-472 transcriptional repression. This study provided a comprehensive characterization of the Dax1 splice variants in mouse ESCs and suggested complex effects of Dax1 variants in a self-renewal regulatory network.

Haploinsufficiency of genes that participate in telomere elongation and maintenance processes, such as telomerase RNA component ( Terc ) and telomere reverse transcriptase ( Tert ), often leads to premature aging-related diseases such as dyskeratosis congenita and aplastic anemia. Previously, we reported that when mouse Terc +/- tail tip fibroblasts (TTFs) were used as donor cells for somatic cell nuclear transfer (SCNT, also known as cloning), the derivative embryonic stem cells (ntESCs) had elongated telomeres. In the present work, we are interested to know if an additional round of SCNT, or recloning, could lead to further elongation of telomeres. Terc +/- TTFs were used to derive the first-generation (G1) ntESCs, followed by a second round of SCNT using G1- Terc +/- ntESCs as donor cells to derive G2- Terc +/- ntESCs. Multiple lines of G1- and G2- Terc +/- ntESCs were efficiently established, and all expressed major pluripotent markers and supported efficient chondrocyte differentiation in vitro. Compared with donor TTFs, telomere lengths of G1 ntESCs were elongated to the level comparable with that in wild-type ntESCs. Interestingly, recloning did not further elongate the telomere lengths of Terc +/- ntESCs. Together, our work demonstrates that while a single round of SCNT is a viable means to reprogram Terc haploinsufficient cells to the ESC state, and to elongate these cells' telomere lengths, a second round of SCNT does not necessarily further elongate the telomeres.

Patient-specific stem cells derived from somatic cell nuclear transfer (SCNT) embryos or from induced pluripotent stem cells (iPSCs) could be used to treat various diseases with minimal immune rejection. Many studies using these cells have been conducted in rats and mice; however, there exist numerous dissimilarities between the rodents and humans limiting the clinical predictive power and experimental utility of rodent experiments alone. Nonhuman primates (NHPs) share greater homology to human than rodents in all respects, including genomics, physiology, biochemistry, and the immune system. Thus, experimental data obtained from monkey studies would be more predictive for designing an effective cell replacement therapy in humans. Unfortunately, there are few iPSC lines and even fewer SCNT lines that have been derived in NHPs, hampering broader studies in regenerative medicine. One promising potential therapy would be the replacement of dopamine neurons that are lost in Parkinson's disease. After dopamine depletion by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the African green monkey (Chlorocebus sabaeus) shows the most complete model of Parkinsonism compared with other species and brain pathology and behavioral changes are almost identical to those in humans after accidental exposure to MPTP. Therefore, we have developed a SCNT procedure to generate multiple pluripotent stem cell lines in this species for studies of possible treatment of Parkinsonism and for comparing with cells derived from iPSCs. Using 24 female monkeys as egg donors and 7 somatic cell donor monkeys, we have derived 11 SCNT embryonic stem cell lines that expressed typical stemness genes and formed all three germ layer derivatives. We also derived two iPSC lines using an episome-mediated reprogramming factor delivery system. This report describes the process for deriving these cell lines and proving their pluripotency for differentiation into various potentially therapeutic cells.

Pluripotent stem cells are promising candidates for cell-based regenerative therapies. To avoid rejection of transplanted cells, several approaches are being pursued to reduce immunogenicity of the cells or modulate the recipient's immune response. These include gene editing to reduce the antigenicity of cell products, immunosuppression of the host, or using major histocompatibility complex-matched cells from cell banks. In this context, we have investigated the antigenicity of H-Y antigens, a class of minor histocompatibility antigens encoded by the Y chromosome, to assess whether the gender of the donor affects the cell's antigenicity. In a murine transplant model, we show that the H-Y antigen in undifferentiated embryonic stem cells (ESCs), as well as ESC-derived endothelial cells, provokes T- and B cell responses in female recipients.

Mammalian pluripotent stem cells are thought to exist in two states: naive and primed. Generally, unlike those in rodents, pluripotent stem cells in primates, including humans, are regarded as being in the primed pluripotent state. Recently, several groups reported the existence of naive pluripotent stem cells in humans. In this study, we report the conversion of primed state embryonic stem cells from common marmoset, a New World monkey, to the naive state using transgenes. The cells showed typical naive state features, including dome-like colony morphology, growth factor requirement, gene expression profile, X chromosome activation state, and energy metabolic status. Moreover, interspecies chimeric embryo formation ability with mouse embryos was increased in the naive state. This technique can be applied in basic medical research using nonhuman primates, such as preclinical use of naive pluripotent stem cells and generating genetically modified primates.

Glaucoma is characterized by retinal ganglion cell (RGC) degeneration and is the second leading cause of blindness worldwide. However, current treatments such as eye drop or surgery have limitations and do not target the loss of RGC. Regenerative therapy using embryonic stem cells (ESCs) holds a promising option, but ethical concern hinders clinical applications on human subjects. In this study, we employed spermatogonial stem cells (SSCs) as an alternative source of ESCs for cell-based regenerative therapy in mouse glaucoma model. We generated functional RGCs from SSCs with a two-step protocol without applying viral transfection or chemical induction. SSCs were first dedifferentiated to embryonic stem-like cells (SSC-ESCs) that resemble ESCs in morphology, gene expression signatures, and stem cell properties. The SSC-ESCs then differentiated toward retinal lineages. We showed SSC-ESC-derived retinal cells expressed RGC-specific marker Brn3b and functioned as bona fide RGCs. To allow in vivo RGC tracing, Brn3b-EGFP reporter SSC-ESCs were generated and the derived RGCs were subsequently transplanted into the retina of glaucoma mouse models by intravitreal injection. We demonstrated that the transplanted RGCs could survive in host retina for at least 10 days after transplantation. SSC-ESC-derived RGCs can thus potentially be a novel alternative to replace the damaged RGCs in glaucomatous retina.

Transplantation of dopaminergic (DA) cells into the striatum can rescue from dopamine deficiency in a Parkinson's disease condition, but this is not a suitable procedure for regaining the full control of motor activity. The minimal condition toward recovering the nigrostriatal pathway is the proper innervation of transplanted DA neurons or their precursors from the substancia nigra pars compacta (SNpc) to their target areas. However, functional integration of transplanted cells would require first that the host SNpc is suitable for their survival and/or differentiation. We recently reported that the intact adult SNpc holds a strong neurogenic environment, but primed embryonic stem cells (ie, embryoid body cells, EBCs) could not derive into DA neurons. In this study, we transplanted into the intact or lesioned SNpc, EBCs derived from embryonic stem cells that were prompt to differentiate into DA neurons by the forced expression of Lmx1a in neural precursor cells (R1B5/NesE-Lmx1a). We observed that, 6 days posttransplantation (dpt), R1B5 or R1B5/NesE-Lmx1a EBCs gave rise to Nes + and Dcx + cells within the host SNpc, but a large number of Th + cells derived only from EBCs exogenously expressing Lmx1a . In contrast, when transplantation was carried out into the 6-hydroxidopamine-lesioned SNpc, the emergence of Th + cells from EBCs was independent of exogenous Lmx1a expression, although these cells were not found by 15 dpt. These results suggest that the adult SNpc is not only a permissive niche for initiation of DA differentiation of non-neuralized cells but also releases factors upon damage that promote the acquisition of DA characteristics by transplanted EBCs.

Propagation of pluripotent cells from early stage embryos in mouse and human highly depend on leukemia inhibitory factor (LIF)/signal transducer and activator of transcription 3 (STAT3) and FGF2/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways. However, mechanisms for maintaining pluripotency in embryonic stem cells using various combinations of growth factors (targeting LIF or FGF2 pathways) and inhibitors (targeting WNT/GSK3 or FGF2 pathways) still have to be deciphered in other models, including the domestic cat. Our objective was to understand how cytokines influence pluripotency in the cat inner cell mass (ICM) outgrowths. Cat ICM was isolated from in vitro-produced embryos and outgrowths were cultured for up to 6 days with single or combined cytokines. Cell proliferation was enhanced with almost all single growth factors and cytokine combinations. Based on gene expression and presence of NANOG, POU5F1, and Sex-determining region Y box 2 (SOX2) as cell state markers, single growth factors could not maintain similar levels in outgrowths as in the original ICMs, which is different from the response in mouse and human. In our conditions, cytokine combinations involving LIF, GSK3 inhibitor, and MEK inhibitor resulted in the most robust expression levels and allowed single-cell dissociation and propagation. However, further characterization of embryonic cells derived from ICM indicated that the pluripotent state was not fully preserved. The absence of detectable transcripts for BMP2-receptor and SMAD4, and very low levels of LIF-receptor and STAT3 in the cat ICM indicated that pluripotency regulatory machinery appear to be different in the cat from the predominant mouse and human models.

Primordial germ cells (PGCs), the precursors of gametes, have regulatory mechanisms involving transcription factors and epigenetic modifications. The transcription factor NANOG is a key regulator of germ cell and embryonic stem cell characteristics. However, the epigenetic regulation of NANOG with a histone deacetylase (HDAC) complex in PGCs has not been studied. In this study, we investigated the epigenetic regulation, and in particular the histone acetylation, of NANOG in chicken PGCs. Intriguingly, although NANOG was highly activated in chicken PGCs, the upstream region of its promoter was moderately suppressed by histone deacetylation. HDAC inhibition induced histone H3 lysine 9 acetylation (H3K9ac) and derepressed NANOG expression. Furthermore, knockdown studies revealed that HDAC complex members, such as RE1-silencing transcription factor (REST) and REST corepressor 3 (RCOR3), are important epigenetic modulators of NANOG expression in chicken PGCs. We demonstrate that moderate regulation of NANOG by the REST/CoREST/HDAC complex might be crucial for maintaining the integrity of PGCs.

Pluripotent stem cells may serve as an alternative source of beta-like cells for replacement therapy of type 1 diabetes; however, the beta-like cells generated in many differentiation protocols are immature. The maturation of endogenous beta cells involves an increase in insulin expression starting in late gestation and a gradual acquisition of the abilities to sense glucose and secrete insulin by week 2 after birth in mice; however, what molecules regulate these maturation processes are incompletely known. In this study, we aim to identify small molecules that affect immature beta cells. A cell-based assay, using pancreatic beta-like cells derived from murine embryonic stem (ES) cells harboring a transgene containing an insulin 1-promoter driven enhanced green fluorescent protein reporter, was used to screen a compound library (NIH Clinical Collection-003). Cortisone, a glucocorticoid, was among five positive hit compounds. Quantitative reverse transcription-polymerase chain reaction analysis revealed that glucocorticoids enhance the gene expression of not only insulin 1 but also glucose transporter-2 (Glut2; Slc2a2) and glucokinase (Gck), two molecules important for glucose sensing. Mifepristone, a pharmacological inhibitor of glucocorticoid receptor (GR) signaling, reduced the effects of glucocorticoids on Glut2 and Gck expression. The effects of glucocorticoids on ES-derived cells were further validated in immature primary islets. Isolated islets from 1-week-old mice had an increased Glut2 and Gck expression in response to a 4-day treatment of exogenous hydrocortisone in vitro. Gene deletion of GR in beta cells using rat insulin 2 promoter-driven Cre crossed with GR flox/flox mice resulted in a reduced gene expression of Glut2, but not Gck, and an abrogation of insulin secretion when islets were incubated in 0.5 mM d-glucose and stimulated by 17 mM d-glucose in vitro. These results demonstrate that glucocorticoids positively regulate glucose sensors in immature murine beta-like cells.

Cultured mouse spermatogonial stem cells (SSCs), also known as germline stem cells (GSCs), revert back to pluripotent state either spontaneously or upon being modified genetically. However, the reprogramming efficiencies are low, and the underlying mechanism remains poorly understood. In the present study, we conducted transcriptomic analysis and found that many transcription factors and epigenetic modifiers were differentially expressed between GSCs and embryonic stem cells. We failed in reprogramming GSCs to pluripotent state using the Yamanaka 4 Factors, but succeeded when Nanog and Tet1 were included. More importantly, reprogramming was also achieved with Nanog alone in a p53-deficient GSC line with an efficiency of 0.02‰. These GSC-derived-induced pluripotent stem cells possessed in vitro and in vivo differentiation abilities despite the low rate of chimera formation, which might be caused by abnormal methylation in certain paternally imprinted genes. Together, these results show that GSCs can be reprogrammed to pluripotent state via multiple avenues and contribute to our understanding of the mechanisms of GSC reprogramming.

A well-formed and robust vasculature is critical to the health of most organ systems in the body. However, the endothelial cells (ECs) forming the vasculature can exhibit a number of distinct functional subphenotypes like arterial or venous ECs, as well as angiogenic tip and stalk ECs. In this study, we investigate the in vitro differentiation of EC subphenotypes from embryonic stem cells (ESCs). Using our staged induction methods and chemically defined mediums, highly angiogenic EC subpopulations, as well as less proliferative and less migratory EC subpopulations, are derived. Furthermore, the EC subphenotypes exhibit distinct surface markers, gene expression profiles, and positional affinities during sprouting. While both subpopulations contained greater than 80% VE-cad + /CD31 + cells, the tip/stalk-like EC contained predominantly Flt4 + /Dll4 + /CXCR4 + /Flt-1 - cells, while the phalanx-like EC was composed of higher numbers of Flt-1 + cells. These studies suggest that the tip-specific EC can be derived in vitro from stem cells as a distinct and relatively stable EC subphenotype without the benefit of its morphological positioning in the sprouting vessel.

Pluripotent stem cells have demonstrated the potential to generate large numbers of functional cardiomyocytes (CMs) from different cell sources. Besides Wnt signaling, additional pathways are involved in early cardiac development and function. To date however, no study exists showing the effects of perturbing the canonical Wnt pathway using nonhuman primate embryonic stem (ES) cells. In this study, we investigated the effect of canonical Wnt inhibition during differentiation of nonhuman primate ES cell-derived CMs under defined, growth factor conditions. Rhesus monkey ES (rES) cells were differentiated into spontaneously beating CMs in the absence (control) or presence (treated) of Wnt inhibitor Dickkopf1 (DKK1), vascular endothelial growth factor, and basic fibroblast growth factor combined or added in a sequential manner during differentiation. Quantification and functional characterization of CMs were assessed by molecular and electrophysiological techniques. Analysis revealed no difference in average ratio of spontaneously beating clusters in both control and treated groups. However, the percentage of CMs was significantly reduced and the expressions of specific cardiac markers tested were also decreased in the treated group. Interestingly, we found that in CMs obtained from treated group, β-adrenergic receptors (β-ARs) were less expressed, their function was altered and electrophysiological studies revealed differences in action potential responsiveness to β-AR stimulation. We demonstrated that the Wnt/β-catenin pathway inhibitor, DKK1 associated with other growth factors repressed functional expression of β-ARs in rES cell-derived CMs. Thus, control of this pathway in each cell line and source is important for proper basic research and further cell therapy applications.

To date, little is known about the mechanisms underlying the self-renewal of embryonic stem (ES) cells from fish species. In this study, we report that the leukemia inhibitory factor (LIF; named as OnLif) from a teleost fish, Nile tilapia (Oreochromis niloticus), is essential for the proliferation, survival, and pluripotency maintenance of Nile tilapia ES cells (TES1) by activating the signal transducer and activator of transcription 3 (Stat3). This protein has 221 amino acid residues with similar sequence features to mammalian LIF. By fusing to a small ubiquitin-related modifier and inducing expression at 16°C, the soluble tag-free protein had been successfully obtained. Further investigation indicates that OnLif could significantly enhance the proliferation and survival of TES1. Moreover, it contributed to the pluripotency maintenance of TES1 characteristic of high expression of pluripotency genes, no or low expression of differentiation genes, and strong alkaline phosphatase activity. Notably, it mediated Stat3 phosphorylation, whose inhibitor treatment could lead to apoptosis. In addition, OnLif significantly enhanced the proliferation of ES cells from medaka (Oryzias latipes), suggesting its potential role in other fish ES cells. These data first suggest that Lif/Stat3 signaling has an essential role in the self-renewal of ES cells from fish, just like that in the ground state pluripotency maintenance of mouse and human ES cells. Our study would not only be helpful for the understanding of molecular mechanisms underlying self-renewal of ES cells from the perspective of evolution but also facilitate ES-based biotechnology application in fishery.

Neural crest (NC) cells are a migratory, multipotent population giving rise to numerous lineages in the embryo. Their plasticity renders attractive their use in tissue engineering-based therapies, but further knowledge on their in vivo behavior is required before clinical transfer may be envisioned. We here describe the isolation and characterization of a new mouse embryonic stem (ES) line derived from Wnt1-CRE-R26 Rosa TomatoTdv blastocyst and show that it displays the characteristics of typical ES cells. Further, these cells can be efficiently directed toward an NC stem cell-like phenotype as attested by concomitant expression of NC marker genes and Tomato fluorescence. As native NC progenitors, they are capable of differentiating toward typical derivative phenotypes and interacting with embryonic tissues to participate in the formation of neo-structures. Their specific fluorescence allows purification and tracking in vivo. This cellular tool should facilitate a better understanding of the mechanisms driving NC fate specification and help identify the key interactions developed within a tissue after in vivo implantation. Altogether, this novel model may provide important knowledge to optimize NC stem cell graft conditions, which are required for efficient tissue repair.

Human embryonic stem cells (hESCs) represent a promising tool to study functions of genes during development, to model diseases, and to even develop therapies when combined with gene editing techniques such as CRISPR/CRISPR-associated protein-9 nuclease (Cas9) system. However, the process of disruption of gene expression by generation of null alleles is often inefficient and tedious. To circumvent these limitations, we developed a simple and efficient protocol to permanently downregulate expression of a gene of interest in hESCs using CRISPR/Cas9. We selected p53 for our proof of concept experiments. The methodology is based on series of hESC transfection, which leads to efficient downregulation of p53 expression even in polyclonal population (p53 Low cells), here proven by a loss of regulation of the expression of p53 target gene, microRNA miR-34a. We demonstrate that our approach achieves over 80% efficiency in generating hESC clonal sublines that do not express p53 protein. Importantly, we document by a set of functional experiments that such genetically modified hESCs do retain typical stem cells characteristics. In summary, we provide a simple and robust protocol to efficiently target expression of gene of interest in hESCs that can be useful for laboratories aiming to employ gene editing in their hESC applications/protocols.

The derivation of functional cardiomyocytes (CMs) from human embryonic stem cells (hESCs) represents a unique way of studying human cardiogenesis, including the development of CM subtypes. In this study, we investigated the development and organization of hESC-derived cardiomyocytes (hESC-CMs) and examined how the expression levels of CM subtypes correspond to human in vivo cardiogenesis. Beating clusters were used to determine cardiac differentiation, which was evaluated by the expression of cardiac genes GATA4 and TNNT2 and subcellular localization of GATA4 and NKX2.5. Sharp electrode recordings to determine action potentials (APs) further revealed spatial organization of intracluster CM subtypes (ie, complex clusters). Nodal-, atrial-, and ventricular-like AP morphologies were detected within distinct regions of complex clusters. The ability of different CM subtypes to self-organize was documented by immunohistochemical analyses and a differential spatial expression of β-III tubulin, myosin light chain 2v (MLC-2V), and α-smooth muscle actin (α-SMA). Furthermore, all hESC-CM subtypes formed expressed primary cilia, which are known to coordinate cellular signaling pathways during cardiomyogenesis and heart development. This study expands the foundation for studying regulatory pathways for spatial and temporal CM differentiation during human cardiogenesis.

Amniotic fluid represents an abundant source of multipotent stem cells, referred as broadly multipotent given their differentiation potential and expression of pluripotency-related genes. However, the origin of this broadly multipotent cellular fraction is not fully understood. Several sources have been proposed so far, including embryonic and extraembryonic tissues. In this regard, the ovine developmental model uniquely allows for direct comparison of fetal fluid-derived cells from two separate fetal fluid cavities, the allantois and the amnion, over the entire duration of gestation. As allantoic fluid mainly collects fetal urine, cells originating from the efferent urinary tract can directly be compared with cells deriving from the extraembryonic amniotic tissues and the fetus. This study shows isolation of cells from the amniotic [ovine amniotic fluid cells (oAFCs)] and allantoic fluid [ovine allantoic fluid cells (oALCs)] in a strictly paired fashion with oAFCs and oALCs derived from the same fetus. Both cell types showed cellular phenotypes comparable to standard mesenchymal stem cells (MSCs), with trilineage differentiation potential, and expression of common ovine MSC markers. However, the expression of MSC markers per single cell was higher in oAFCs as measured by flow cytometry. oAFCs exhibited higher proliferative capacities and showed significantly higher expression of pluripotency-related genes OCT4, STAT3, NANOG, and REX1 by quantitative real-time polymerase chain reaction compared with paired oALCs. No significant decrease of pluripotency-related gene expression was noted over gestation, implying that cells with high differentiation potential may be isolated at the end of pregnancy. In conclusion, this study suggests that cells with highest stem cell characteristics may originate from the fetus itself or the amniotic fetal adnexa rather than from the efferent urinary tract or the allantoic fetal adnexa.

It was previously reported that WD repeat domain 5 (Wdr5), a core member of the mammalian Trithorax complex, is a key regulator for the maintenance of embryonic stem (ES) cell pluripotency and somatic cell reprogramming. However, it remains unclear whether other factors are also involved in this process. Here, we show that CTCF is an upstream regulator of Wdr5: It physically associates with Wdr5 and further transcriptionally controls its expression by directly targeting the Wdr5 gene promoter. As a downstream effector, overexpression of Wdr5 can rescue ES cells from growth defects and decreased formation of induced pluripotent stem cells caused by CTCF knockdown. Our results reveal the functional relevance of CTCF and Wdr5, and they connect them to the re-establishment of pluripotency.

Acquisition of proper metabolomic fate is required to convert somatic cells toward fully reprogrammed pluripotent stem cells. The majority of induced pluripotent stem cells (iPSCs) are partially reprogrammed and have a transcriptome different from that of the pluripotent stem cells. The metabolomic profile and mitochondrial metabolic functions required to achieve full reprogramming of somatic cells to iPSC status have not yet been elucidated. Clarification of the metabolites underlying reprogramming mechanisms should enable further optimization to enhance the efficiency of obtaining fully reprogrammed iPSCs. In this study, we characterized the metabolites of human fully reprogrammed iPSCs, partially reprogrammed iPSCs, and embryonic stem cells (ESCs). Using capillary electrophoresis time-of-flight mass spectrometry-based metabolomics, we found that 89% of analyzed metabolites were similarly expressed in fully reprogrammed iPSCs and human ESCs (hESCs), whereas partially reprogrammed iPSCs shared only 74% similarly expressed metabolites with hESCs. Metabolomic profiling analysis suggested that converting mitochondrial respiration to glycolytic flux is critical for reprogramming of somatic cells into fully reprogrammed iPSCs. This characterization of metabolic reprogramming in iPSCs may enable the development of new reprogramming parameters for enhancing the generation of fully reprogrammed human iPSCs.

During ontogeny, fetal liver (FL) acts as a major site for hematopoietic stem cell (HSC) maturation and expansion, whereas HSCs in the adult bone marrow (ABM) are largely quiescent. HSCs in the FL possess faster repopulation capacity as compared with ABM HSCs. However, the molecular mechanism regulating the greater self-renewal potential of FL HSCs has not yet extensively been assessed. Recently, we published RNA sequencing-based gene expression analysis on FL HSCs from 14.5-day mouse embryo (E14.5) in comparison to the ABM HSCs. We reanalyzed these data to identify key transcriptional regulators that play important roles in the expansion of HSCs during development. The comparison of FL E14.5 with ABM HSCs identified more than 1,400 differentially expressed genes. More than 200 genes were shortlisted based on the gene ontology (GO) annotation term "transcription." By morpholino-based knockdown studies in zebrafish, we assessed the function of 18 of these regulators, previously not associated with HSC proliferation. Our studies identified a previously unknown role for tdg, uhrf1, uchl5, and ncoa1 in the emergence of definitive hematopoiesis in zebrafish. In conclusion, we demonstrate that identification of genes involved in transcriptional regulation differentially expressed between expanding FL HSCs and quiescent ABM HSCs, uncovers novel regulators of HSC function.

The transcription factor scleraxis is required for tendon development and is upregulated during embryonic stem cell (ESC) differentiation into tenocytes. However, its role beyond early embryonic development is not defined. We utilized a short hairpin RNA to knock down scleraxis expression in ESCs and adult and fetal tenocytes. No effect on growth or morphology was observed in two-dimensional cultures. However, scleraxis knockdown in fetal tenocytes significantly reduced COL1A1, COMP, and SOX9 gene expression. Scleraxis knockdown in adult tenocytes had no effect on the expression of these genes. Strikingly, differentiating ESCs and fetal tenocytes without scleraxis failed to reorganize a three-dimensional (3D) matrix and generate artificial tendons. This was associated with a significantly reduced survival. In contrast, there was no effect on the survival and remodeling capacity of adult tenocytes following scleraxis knockdown. Overexpression of scleraxis in fetal tenocytes rescued gene expression, cell survival in 3D, and subsequent matrix contraction. Together, these results demonstrate that scleraxis is not only essential for ESC differentiation into tenocytes but that it also has an active role in maintaining fetal tenocytes, which is then redundant in adult tenocytes.

Naïve and primed pluripotent stem cells (PSCs) reflect discrete pluripotent states that approximate the inner cell mass or the progressively lineage-restricted perigastrulation epiblast, respectively. Cells that occupy primed pluripotency have distinct epigenetic landscapes, transcriptional circuitry, and trophic requirements compared with their naïve counterparts. The existence of multiple pluripotent states has not been explored in dogs, which show promise as outbred biomedical models with more than 300 inherited diseases that also afflict humans. However, our understanding of canine embryogenesis and embryo-derived stem cells is limited. Herein, we converted leukemia inhibitory factor (LIF)-dependent and fibroblast growth factor 2 (FGF2)-dependent canine embryonic stem cells (cESCs) resembling primed PSCs toward a naïve pluripotent state using LIF and inhibitors of glycogen synthase kinase 3β and mitogen-activated protein kinase kinase 1/2 [called 2i and LIF (2iL)]. cESCs propagated in 2iL exhibited significant induction of genes associated with the naïve pluripotent state (eg, REX1, TBX3) and downregulation of primed pluripotency markers (eg, OTX2, FGF5) (P < 0.05). Differential phosphorylation of signal transducer and activator of transcription 3 (STAT3) and cell fate decisions on exposure to bone morphogenetic protein 4 (BMP4) suggested that a novel pluripotent identity has been established with 2iL. Accordingly, cESCs cultured with 2iL formed colonies at a greater efficiency than LIF-FGF2 cESCs following single-cell dissociation. Total genomic DNA methylation and histone H3 lysine 27 trimethylation signals were reduced in 2iL-treated cESCs. Our data suggest that 2iL culture conditions promote the conversion of cESCs toward an epigenetically distinct pluripotent state resembling naïve PSCs.

Nowadays, the low efficiency of small interfering RNA (siRNA) or plasmid DNA (pDNA) transfection is a critical issue in genetic manipulation of human embryonic stem (hES) cells. Development of an efficient transfection method for delivery of siRNAs and plasmids into hES cells becomes more and more imperative. In this study, we tried to modify the traditional transfection protocol by introducing two crucial processes, single-cell plating and starvation, to increase the transfection efficiency in hES cells. Furthermore, we comparatively examined the transfection efficiency of some commercially available siRNA or pDNA transfection reagents in hES cells. Our results showed that the new developed method markedly enhanced the transfection efficiency without influencing the proliferation and pluripotency of hES cells. Lipofectamine RNAiMAX exhibited much higher siRNA transfection efficiency than the other reagents, and FuGENE HD was identified as the best suitable reagent for efficient pDNA transfection of hES cells among the tested reagents.

Chicken pluripotent stem cells (PSCs), such as embryonic stem cells and blastoderm cells, have been used to study development and differentiation in chicken. However, chicken PSCs are not widely used because they are hard to maintain in long-term culture. Recent reports suggest that chicken somatic cells can be reprogrammed to pluripotent state by defined factors to form induced pluripotent stem cells (iPSCs). These chicken iPSCs showed pluripotent differentiation potential and could be maintained in long-term culture. However, intracytoplasmic remodeling during reprogramming of chicken cells remains largely unknown. In this study, we generated chicken iPS-like cells (ciPSLCs) from chicken embryonic fibroblasts using a retroviral expression system encoding human reprogramming factors. These ciPSLCs could be maintained for more than 10 passages and expressed the endogenous chicken pluripotency markers, cNonog and cSox2. Moreover, the ciPSLCs showed higher nucleus to cytoplasm ratio and contained globular mitochondria with immature cristae. This morphology was similar to that of mammalian PSCs, but different from that of avian somatic cells, which showed lower nucleus to cytoplasm ratio and mature mitochondria. These results suggest that intracytoplasmic organelles in differentiated somatic cells could be successfully remodeled into the pluripotent state during reprogramming in chicken.

The vast majority of cellular ATP is produced by the oxidative phosphorylation (OXPHOS) system, which comprises the four complexes of the electron transfer chain plus the ATP synthase. Complex I is the largest of the OXPHOS complexes, and mutation of the genes encoding either the subunits or assembly factors of Complex I can result in Complex I deficiency, which is the most common OXPHOS disorder. Mutations in the Complex I gene NDUFS4 lead to Leigh syndrome, which is the most frequent presentation of Complex I deficiency in children presenting with progressive encephalopathy shortly after birth. Symptoms include motor and intellectual retardation, often accompanied by dystonia, ataxia, and growth retardation, and most patients die by 3 years of age. To understand the origins of this disease, we have generated a series of mouse embryonic stem cell lines from blastocysts that were wild type, heterozygous, and homozygous for the deletion of the Ndufs4 gene. We have demonstrated their pluripotency and potential to differentiate into all cell types of the body. Although the loss of Ndufs4 did not affect the stability of the mitochondrial and nuclear genomes, there were significant differences in patterns of chromosomal gene expression following both spontaneous differentiation and directed neural differentiation into astrocytes. The defect also affected the potential of the cells to generate beating embryoid bodies. These outcomes demonstrate that defects associated with Complex I deficiency affect early gene expression patterns, which escalate during early and later stages of differentiation and are mediated by the defect and not other chromosomal or mitochondrial DNA defects.

Murine bone marrow (BM)-derived very small embryonic-like stem cells (BM VSELs), defined by a lineage-negative (Lin(-)), CD45-negative (CD45(-)), Sca-1-positive (Sca-1(+)) immunophenotype, were previously reported as postnatal pluripotent stem cells (SCs). We developed a highly efficient method for isolating Lin(-)CD45(-)Sca-1(+) small cells using enzymatic treatment of murine bone. We designated these cells as bone-derived VSELs (BD VSELs). The incidences of BM VSELs in the BM-derived nucleated cells and that of BD VSELs in bone-derived nucleated cells were 0.002% and 0.15%, respectively. These BD VSELs expressed a variety of hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and endothelial cell markers. The gene expression profile of the BD VSELs was clearly distinct from those of HSCs, MSCs, and ES cells. In the steady state, the BD VSELs proliferated slowly, however, the number of BD VSELs significantly increased in the bone after acute liver injury. Moreover, green fluorescent protein-mouse derived BD VSELs transplanted via tail vein injection after acute liver injury were detected in the liver parenchyma of recipient mice. Immunohistological analyses suggested that these BD VSELs might transdifferentiate into hepatocytes. This study demonstrated that the majority of the Lin(-)CD45(-)Sca-1(+) VSEL phenotypic cells reside in the bone rather than the BM. However, the immunophenotype and the gene expression profile of BD VSELs were clearly different from those of other types of SCs, including BM VSELs, MSCs, HSCs, and ES cells. Further studies will therefore be required to elucidate their cellular and/or SC characteristics and the potential relationship between BD VSELs and BM VSELs.

Rat pluripotent stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) as mouse and human ones have a great potential for studying mammalian early development, disease modeling, and evaluation of regenerative medicine approaches. However, data on pluripotency realization and self-renewal maintenance in rat cells are still very limited, and differentiation protocols of rat ESCs (rESCs) and iPSCs to study development and obtain specific cell types for biomedical applications are poorly developed. In this study, the RNA-Seq technique was first used for detailed transcriptome characterization in rat pluripotent cells. The rESC and iPSC transcriptomes demonstrated a high similarity and were significantly different from those in differentiated cells. Additionally, we have shown that reprogramming of rat somatic cells to a pluripotent state was accompanied by X-chromosome reactivation. There were two active X chromosomes in XX rESCs and iPSCs, which is one of the key attributes of the pluripotent state. Differentiation of both rESCs and iPSCs led to X-chromosome inactivation (XCI). The dynamics of XCI in differentiating rat cells was very similar to that in mice. Two types of facultative heterochromatin described in various mammalian species were revealed on the rat inactive X chromosome. To explore XCI dynamics, we established a new monolayer differentiation protocol for rESCs and iPSCs that may be applied to study different biological processes and optimized for directed derivation of specific cell types.

Heart disease is the leading cause of human death in the 21st century. Heart transplantation is a promising way to treat this. Because donor resources are limited, cell-based therapy has been developed as an alternative. Therefore, genes that trigger cardiogenesis could have potential in the treatment of heart disease. Fibroblast growth factor 1 (FGF1) is reported to stimulate cardiomyocyte proliferation under conditions of myocardial infarction, but little is known about its function during cardiac differentiation. In this study, we established an in vitro cardiogenesis model through a reliable chemical induction protocol to determine whether FGF1 and its gene expression are involved in cardiogenesis. Oxytocin, not only a well-known hormone but also a cardiac differentiation inducer, was used in a mouse embryonic stem cell line, E14Tg2a, to achieve cardiac differentiation. After differentiation, beating cell clusters appeared and the expression of FGF1B mRNA was upregulated in the late differentiation stage (differentiation days 8-14). Interestingly, FGF1B expression patterns during cardiac differentiation were similar to those of a mature cardiomyocyte marker, troponin T2, cardiac. The blockage of FGF1-FGF receptor (FGFR) signaling reduced not only the appearance of beating cluster formation but also the expression levels of cardiomyocyte-associated genes. Moreover, by investigating FGF1 downstream signaling cascades, we observed that the efficiency of beating cluster formation was mainly regulated through the FGF1-FGFR-PKC signaling axis. Taken together, we provide evidence to support that FGF1 could regulate cardiogenesis primarily through the protein kinase C signaling, but not through the mitogen-activated protein kinase signaling, pathway.

Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na(+) and K(+) currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for retinal replacement therapies.

Promoting both endogenous and exogenous neural stem cells' (NSCs) survival in the hostile host environments is essential to cell replacement therapy for central nervous system (CNS) disorders. Type 4 metabotropic glutamate receptor (mGluR4), one of the members of mGluRs, has been shown to protect neurons from acute and chronic excitotoxic insults in various brain damages. The present study investigated the preventive effects of mGluR4 on NSC injury induced by oxidative stress. Under challenge with H2O2, loss of cell viability was observed in cultured rat NSCs, and treatment with selective mGluR4 agonist VU0155041 conferred protective effects against the loss of cellular viability in a concentration-dependent manner, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Pretreatment of VU0155041 (30 μM) also inhibited the excessive NSC death induced by H2O2, and group III mGluRs antagonist (RS)-a-methylserine-O-phosphate (MSOP) or gene-targeted knockdown abolished the protective action of mGluR4, indicated by propidium iodide-Hoechst and terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) staining. Western blot assay demonstrated that mGluR4 activation reversed the decreased procaspase-8/9/3and the destructed Bcl-2/Bax expressing balance, and likewise, MSOP and mGluR4 knockdown abrogated the action of mGluR4 activity. Furthermore, inhibition of JNK and p38 mitogen-activated protein kinases (MAPKs) were observed after mGluR4 activation, and as paralleling control, JNK-specific inhibitor SP600125 and p38-specific inhibitor SB203580 significantly rescued the H2O2-mediated NSC apoptosis and cleavage of procaspase-3. We suggest that activation of mGluR4 prevents oxidative stress-induced NSC death and apoptotic-associated protein activities with involvement of inhibiting the JNK and p38 pathways in cell culture. Our findings may help to develop strategies for enhancing the resided and transplanted NSC survival after oxidative stress insult of CNS.

Excitatory commissural V3 interneurons (INs) of the ventral spinal cord have been shown to balance locomotor rhythm regularity and robustness in vivo. Unfortunately, due to the scarcity of these cells in the spinal cord, in vitro studies of dissociated V3 INs have yet to be reported. In this study, we developed an induction protocol for V3 INs from mouse embryonic stem cells. The effect of the concentration of a strong sonic hedgehog (Shh) agonist (smoothened agonist [SAG]) and retinoic acid (RA) on expression of progenitor p3 and postmitotic V3 IN transcription factor markers (ie, Nkx2.2 and Sim1) was examined. Cells were differentiated toward a more ventral fate by increasing the duration of SAG exposure from 4 days in a previously established motoneuron induction protocol to 6 days. At the end of the induction period, transcription factor expression was assessed using quantitative real-time polymerase chain reaction, immunocytochemistry, in situ hybridization, and flow cytometry. Lower concentrations of RA and a longer duration of SAG exposure led to increased levels of p3 and V3 marker expression. This novel induction protocol reveals the importance of Shh signaling duration in the dorsal-ventral patterning of the neural tube, and it provides a method to obtain V3 INs for future studies to allow better understanding their role in rewiring and regeneration after spinal cord injury.

Primordial germ cells (PGCs) share many properties with embryonic stem cells (ESCs) and innately express several key pluripotency-controlling factors, including OCT4, NANOG, and LIN28. Therefore, PGCs may provide a simple and efficient model for studying somatic cell reprogramming to induced pluripotent stem cells (iPSCs), especially in determining the regulatory mechanisms that fundamentally define pluripotency. Here, we report a novel model of PGC reprogramming to generate iPSCs via transfection with SOX2 and OCT4 using integrative lentiviral. We also show the feasibility of using nonintegrative approaches for generating iPSC from PGCs using only these two factors. We show that human PGCs express endogenous levels of KLF4 and C-MYC protein at levels similar to embryonic germ cells (EGCs) but lower levels of SOX2 and OCT4. Transfection with both SOX2 and OCT4 together was required to induce PGCs to a pluripotent state at an efficiency of 1.71%, and the further addition of C-MYC increased the efficiency to 2.33%. Immunohistochemical analyses of the SO-derived PGC-iPSCs revealed that these cells were more similar to ESCs than EGCs regarding both colony morphology and molecular characterization. Although leukemia inhibitory factor (LIF) was not required for the generation of PGC-iPSCs like EGCs, the presence of LIF combined with ectopic exposure to C-MYC yielded higher efficiencies. Additionally, the SO-derived PGC-iPSCs exhibited differentiation into representative cell types from all three germ layers in vitro and successfully formed teratomas in vivo. Several lines were generated that were karyotypically stable for up to 24 subcultures. Their derivation efficiency and survival in culture significantly supersedes that of EGCs, demonstrating their utility as a powerful model for studying factors regulating pluripotency in future studies.

Human embryonic stem cells (hESCs) have long been considered as a promising source for cell replacement therapy. However, one major obstacle for the use of these cells is immune compatibility. Histocompatible human parthenogenetic ESCs have been reported as a new method for generating human leukocyte antigen (HLA)-matched hESCs. To further investigate the possibility of obtaining histocompatible stem cells from uniparental embryos, we tried to produce androgenetic haploid human embryos by injecting a single spermatozoon into enucleated human oocyte, and establish human androgenetic embryonic stem (hAGES) cell lines from androgenetic embryos. In the present study, a diploid hAGES cell line has been established, which exhibits typical features of human ESCs, including the expression of pluripotency markers, having differentiation potential in vitro and in vivo, and stable propagation in an undifferentiated state (>P40). Bisulfite sequencing of the H19, Snrpn, Meg3, and Kv imprinting control regions suggested that hAGES cells maintained to a certain extent a sperm methylation pattern. Genome-wide single nucleotide polymorphism, short tandem repeat, and HLA analyses revealed that the hAGES cell genome was highly homozygous. These results suggest that hAGES cells from spermatozoon could serve as a useful tool for studying the mechanisms underlying genomic imprinting in humans. It might also be used as a potential resource for cell replacement therapy as parthenogenetic stem cells.

The commonly used inhalation anesthetic, isoflurane, can permeate rapidly through the placental barrier and thus cause toxicity to the central nervous system of the developing fetus. In this study, we treated pregnant mice with clinically relevant concentration of isoflurane early on in development (days 3.5-6.5), and then found that the fetus growth was inhibited by isoflurane. We further used the mouse embryonic stem cell (mES cell) to be the early development model to investigate the mechanism of the embryotoxicity of isoflurane and found that isoflurane inhibited self-renewal of mES cells. In addition, neuronal differentiation from the mES cells treated with isoflurane was also inhibited. Overexpression of E-cadherin attenuated the effects of isoflurane on self-renewal and the subsequent neuronal differentiation. We also found that miR-9 can be upregulated by isoflurane. Overexpression of miR-9 inhibited the self-renewal and subsequent neuronal differentiation. E-cadherin was directly targeted by miR-9. Overexpression of E-cadherin can abolish the function of miR-9 or isoflurane on self-renewal and subsequent neuronal differentiation. These data suggested that isoflurane inhibits self-renewal and neuronal differentiation of mES cells, possibly by regulating the miR-9-E-cadherin signaling. The result of the current study may provide a novel idea for preventing the toxicity of inhalation anesthetics in the developing fetal brain in clinical practice when pregnant women accept nonobstetric surgery under inhalation general anesthesia.

Human embryonic stem cells (hESCs) exhibiting skewed X chromosome inactivation (XCI) have been reported. The copy number variations (CNVs), loss of heterozygosity (LOH), or single-nucleotide variant (SNV) events in those epigenetically distinct cells remain unknown, and whether such genetic abnormalities will influence the XCI status of hESCs is unclear. In this study, three hESCs with skewed XCI, three with random XCI, and two male hESC lines at different passages were analyzed for CNVs and LOH levels using a high-resolution genotyping microarray. Whole-exome sequencing was used to investigate the potentially damaging SNVs. On average, 17.6 CNVs and 5.3 cases of LOH were identified in the skewed hESCs, which were similar to the rates observed in random hESCs. Five recurrent CNV regions were uniquely identified in the skewed hESCs, but all of them were considered polymorphisms. With the exception of a nongenic CNV, no additional CNVs were detected on the X chromosome in the skewed hESCs. Although the XCI status in two hESC lines was observed to be changed from random to skewed, no significant CNV difference was identified before and after the XCI change. SNV analysis indicated that normal alleles are maintained for most genes within copy-neutral LOH regions. Three types of expression patterns were observed in heterozygous alleles, and the damaging SNVs in skewed hESCs favored the expression of the wild-type alleles. In conclusion, in the present study, we did not find genetic differences in the CNV and LOH levels between hESCs with and without skewed XCI. Wild-type allele expression in the presence of damaging SNVs on the X chromosome in skewed hESCs might alleviate adverse effects in those hESCs.

A comparative analysis of mouse and human pancreatic development may reveal common mechanisms that control key steps as organ morphogenesis and cell proliferation and differentiation. More specifically, understanding beta cell development remains an issue, despite recent progress related to their generation from human embryonic and induced pluripotent stem cells. In this study, we use an integrated approach, including prospective isolation, organ culture, and characterization of intermediate stages, and report that cells from human and mouse fetal pancreas can be expanded in the long term and give rise to hollow duct-like structures in 3D cultures. The expanded cells express a combination of markers (E-cadherin, PDX1, NKX6-1, SOX9, and HNF1β) that reveals pancreatic progenitor identity. Proliferation of embryonic progenitors was stimulated by the Wnt agonist R-spondin1 (RSPO1), FGF10, and EGF. This combination of growth factors allowed maintaining human fetal pancreatic progenitors in culture for many passages, a finding not reported previously. Importantly, in the absence of EGF, proliferation was reduced, while endocrine differentiation was significantly enhanced. We conclude that modulation of EGF signaling affects in vitro expansion and differentiation of progenitors from embryonic pancreas of both mice and man.

Oct4 pseudogenes and isoforms seriously confuse the detection of the pluripotency-associated Oct4A expression in somatic cells, which in many cases was not accurately determined. This confusion has recently been discussed, but the wrong conclusions have continuously been made. Most studies failed to detect the expression of Oct4 pseudogenes and isoforms in somatic cells but detected only Oct4A, for which the detection signals incorrectly came from its pseudogenes and isoforms. Some studies detected the expression of only Oct4 pseudogenes in somatic cells but failed to detect Oct4A. The other studies failed to detect the expression of any Oct4 genes. Oct4A is more homologous to its pseudogenes than its isoforms, and it is much more difficult to distinguish Oct4A from its pseudogenes, so this study focused on them. In this study, the strict experimental procedures were followed. Three pairs of Oct4A-specific polymerase chain reaction (PCR) primers were carefully designed and tested by sequencing reverse transcription-polymerase chain reaction (RT-PCR) clones, which showed that only one of them was truly specific to Oct4A. RT-PCR was also performed with the primers amplifying both Oct4A and its pseudogenes, and several hundreds of PCR clones from each cell type were sequenced to reliably distinguish the low-abundant Oct4A from its high-abundant pseudogenes. Western blot, immunocytochemistry, and flow cytometric analyses were performed with three Oct4 antibodies to confirm the results of Oct4 mRNA expression. This study undoubtedly made the correct conclusions about Oct4 expression in human somatic cells and showed that all the tested human somatic cells expressed both Oct4A and its pseudogenes but expressed Oct4A at much lower levels compared with its pseudogenes.

Reprogramming is one of the most essential areas of research in stem cell biology. Despite this importance, the mechanism and correlates of reprogramming remain largely unknown. In this study, we investigated the cytoplasmic remodeling and changes in metabolism that occur during reprogramming and differentiation of pluripotent stem cells. Specifically, we examined the cellular organelles of three pluripotent stem cells, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and epiblast stem cells (EpiSCs), by electron microscopy. We found that the cellular organelles of primed pluripotent EpiSCs were more similar to those of naive pluripotent ESCs and iPSCs than somatic cells. EpiSCs, as well as ESCs and iPSCs, contain large nuclei, poorly developed endoplasmic reticula, and underdeveloped cristae; however, their mitochondria were still mature relative to the mitochondria of ESCs and iPSCs. Next, we differentiated these pluripotent stem cells into neural stem cells (NSCs) in vitro and compared the morphology of organelles. We found that the morphology of organelles of NSCs differentiated from ESCs, iPSCs, and EpiSCs was indistinguishable from brain-derived NSCs. Finally, we examined the changes in energy metabolism that accompanied mitochondrial remodeling during reprogramming and differentiation. We found that the glycolytic activity of ESCs and iPSCs was greater compared with EpiSCs, and that the glycolytic activity of EpiSCs was greater compared with NSCs differentiated from ESCs, iPSCs, and EpiSCs. These results suggest that a change in the cellular state is accompanied by dynamic changes in the morphology of cytoplasmic organelles and corresponding changes in energy metabolism.

The 3' untranslated region (UTR)-associated RNAs (uaRNAs) have important roles in various biological processes, especially in development. However, since they overlap with protein-coding mRNAs, uaRNAs are difficult to study by RNA interference techniques. We recently identified a chemical molecule, 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO), that could efficiently induce human embryonic stem cells (hESCs) differentiation, and meanwhile selectively and efficiently downregulate the uaRNA FLJ11812. By acting as a competing endogenous RNA, downregulated FLJ11812 by 3BDO further increased miR-4459 level in hESCs. miR-4459 could decrease the expression of its targets, CDC20B and ATG13, and thus altered stemness via cell cycle and autophagy. Our results revealed that FLJ11812 played a key role in maintenance of stemness of hESCs for the first time. The findings provide new clues and a powerful tool for investigating the action mechanism of FLJ11812 in early development.

The generation of beta-thalassemia (β-Thal) patient-specific induced pluripotent stem cells (iPSCs), subsequent homologous recombination-based gene correction of disease-causing mutations/deletions in the β-globin gene (HBB), and their derived hematopoietic stem cell (HSC) transplantation offers an ideal therapeutic solution for treating this disease. However, the hematopoietic differentiation efficiency of gene-corrected β-Thal iPSCs has not been well evaluated in the previous studies. In this study, we used the latest gene-editing tool, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), to correct β-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and full pluripotency as human embryonic stem cells (hESCs) showed no off-targeting effects. Then, we evaluated the differentiation efficiency of the gene-corrected β-Thal iPSCs. We found that during hematopoietic differentiation, gene-corrected β-Thal iPSCs showed an increased embryoid body ratio and various hematopoietic progenitor cell percentages. More importantly, the gene-corrected β-Thal iPSC lines restored HBB expression and reduced reactive oxygen species production compared with the uncorrected group. Our study suggested that hematopoietic differentiation efficiency of β-Thal iPSCs was greatly improved once corrected by the CRISPR/Cas9 system, and the information gained from our study would greatly promote the clinical application of β-Thal iPSC-derived HSCs in transplantation.

Embryonic stem cells (ESCs) undergoing neural differentiation form radial arrays of neural stem cells, termed neural rosettes. These structures manifest many of the properties associated with embryonic and adult neurogenesis, including cell polarization, interkinetic nuclear migration (INM), and a gradient of neuronal differentiation. We now identify novel rosette structural features that serve to localize key regulators of neurogenesis. Cells within neural rosettes have specialized basal as well as apical surfaces, based on localization of the extracellular matrix receptor β1 integrin. Apical processes of cells in mature rosettes terminate at the lumen, where adherens junctions are apparent. Primary cilia are randomly distributed in immature rosettes and tightly associated with the neural stem cell's apical domain as rosettes mature. Components of two signaling pathways known to regulate neurogenesis in vivo and in rosettes, Hedgehog and Notch, are apically localized, with the Hedgehog effector Smoothened (Smo) associated with primary cilia and the Notch pathway γ-secretase subunit Presenilin 2 associated with the adherens junction. Increased neuron production upon treatment with the Notch inhibitor DAPT suggests a major role for Notch signaling in maintaining the neural stem cell state, as previously described. A less robust outcome was observed with manipulation of Hedgehog levels, though consistent with a role in neural stem cell survival or proliferation. Inhibition of both pathways resulted in an additive effect. These data support a model by which cells extending a process to the rosette lumen maintain neural stem cell identity whereas release from this association, either through asymmetric cell division or apical abscission, promotes neuronal differentiation.

Embryonic stem cells (ESCs) are maintained in an undifferentiated state through expression of the core transcriptional factors Nanog, Oct4, and Sox2. However, the epigenetic regulation of pluripotency is poorly understood. Differentiation of ESCs is accompanied by a global reduction of panacetylation of histones H3 and H4 suggesting that histone acetylation plays an important role in maintenance of ESC pluripotency. Acetylated lysine residues on histones are read by members of the bromodomain family that includes BET (bromodomain and extraterminal domain) proteins for which highly potent and selective inhibitors have been developed. In this study we demonstrate that the pan-BET bromodomain inhibitor JQ1 induces rapid spontaneous differentiation of murine ESCs by inducing marked transcriptional downregulation of Nanog as well as the stemness markers Lefty1 and Lefty2, but not Myc, often used as a marker of BET inhibitor activity in cancer. We show that the effects of JQ1 are recapitulated by knockdown of the BET family member BRD4 implicating this protein in Nanog regulation. These data are also supported by chromatin immunoprecipitation experiments which confirm BRD4 binding at the Nanog promoter that is known to require acetylation by the histone acetyltransferase MOF for transcriptional activity. In further support of our findings, we show that JQ1 antagonizes the stem cell-promoting effects of the histone deacetylase inhibitors sodium butyrate and valproic acid. Our data suggest that BRD4 is critical for the maintenance of ESC pluripotency and that this occurs primarily through the maintenance of Nanog expression.

Vascular smooth muscle cells (SMCs), which arise from multiple embryonic progenitors, have unique lineage-specific properties and this diversity may contribute to spatial patterns of vascular diseases. We developed in vitro methods to generate distinct vascular SMC subtypes from human pluripotent stem cells, allowing us to explore their intrinsic differences and the mechanisms involved in SMC development. Since Notch signaling is thought to be one of the several key regulators of SMC differentiation and function, we profiled the expression of Notch receptors, ligands, and downstream elements during the development of origin-specific SMC subtypes. NOTCH3 expression in our in vitro model varied in a lineage- and developmental stage-specific manner so that the highest expression in mature SMCs was in those derived from paraxial mesoderm (PM). This pattern was consistent with the high expression level of NOTCH3 observed in the 8-9 week human fetal descending aorta, which is populated by SMCs of PM origin. Silencing NOTCH3 in mature SMCs in vitro reduced SMC markers in cells of PM origin preferentially. Conversely, during early development, NOTCH3 was highly expressed in vitro in SMCs of neuroectoderm (NE) origin. Inhibition of NOTCH3 in early development resulted in a significant downregulation of specific SMC markers exclusively in the NE lineage. Corresponding to this prediction, the Notch3-null mouse showed reduced expression of Acta2 in the neural crest-derived SMCs of the aortic arch. Thus, Notch3 signaling emerges as one of the key regulators of vascular SMC differentiation and maturation in vitro and in vivo in a lineage- and temporal-dependent manner.

Vascular derivatives of human embryonic stem cells (hESC) are being developed as sources of tissue-specific cells for organ regeneration. However, identity of developmental pathways that modulate the specification of endothelial cells is not known yet. We studied phosphatidylinositol 3-kinase (PI3K)-Forkhead box O transcription factor 1A (FOXO1A) pathways during differentiation of hESC toward endothelial lineage and on proliferation, maturation, and cell death of hESC-derived endothelial cells (hESC-EC). During differentiation of hESC, expression of FOXO1A transcription factor was linked to the expression of a cluster of angiogenesis- and vascular remodeling-related genes. PI3K inhibitor LY294002 activated FOXO1A and induced formation of CD31(+) hESC-EC. In contrast, differentiating hESC with silenced FOXO1A by small interfering RNA (siRNA) showed lower mRNA levels of CD31 and angiopoietin2. LY294002 decreased proliferative activity of purified hESC-EC, while FOXO1A siRNA increased their proliferation. LY294002 inhibits migration and tube formation of hESC-EC; in contrast, FOXO1A siRNA increased in vitro tube formation activity of hESC-EC. After in vivo conditioning of cells in athymic nude rats, cells retain their low FOXO1A expression levels. PI3K/FOXO1A pathway is important for function and survival of hESC-EC and in the regulation of endothelial cell fate. Understanding these properties of hESC-EC may help in future applications for treatment of injured organs.

Blastocyst formation represents the first lineage specification by segregation of the trophectoderm from the inner cell mass in early embryonic development. Transcriptional regulation of Cdx2, which is selectively expressed in and essential for the specification of trophectoderm, has been extensively studied. However, post-transcriptional regulation of Cdx2 remains largely unknown. In this study, we report that RNA-binding protein motif 46 (Rbm46), an RNA-binding motif protein with unknown function, directly binds to and stabilizes Cdx2 mRNA in early mouse embryos. In addition, knockdown of Rbm46 using RNA interference downregulated the majority of trophectoderm markers in mouse embryonic stem cells and blocked the allocation of blastomere cells to the trophectoderm in mouse embryos. Our study revealed a novel mechanism by which Rbm46 regulates trophectoderm specification through stabilizing Cdx2 mRNA in early mouse embryos.

Protein tyrosine phosphatases have been recognized as critical components of multiple signaling regulators of fundamental cellular processes, including differentiation, cell death, and migration. In this study, we show that dual specificity phosphatase 4 (DUSP4) is crucial for neuronal differentiation and functions in the neurogenesis of embryonic stem cells (ESCs). The endogenous mRNA and protein expression levels of DUSP4 gradually increased during mouse development from ESCs to postnatal stages. Neurite outgrowth and the expression of neuron-specific markers were markedly reduced by genetic ablation of DUSP4 in differentiated neurons, and these effects were rescued by the reintroduction of DUSP4. In addition, DUSP4 knockdown dramatically enhanced extracellular signal-regulated kinase (ERK) activation during neuronal differentiation. Furthermore, the DUSP4-ERK pathway functioned to balance calcium signaling, not only by regulating Ca(2+)/calmodulin-dependent kinase I phosphorylation, but also by facilitating Cav1.2 expression and plasma membrane localization. These data are the first to suggest a molecular link between the MAPK-ERK cascade and calcium signaling, which provides insight into the mechanism by which DUSP4 modulates neuronal differentiation.

Dysregulation of forkhead box protein A2 (Foxa2) expression in fetal ventral mesencephalon (VM)-derived neural precursor cells (NPCs) appears to be associated with the loss of their potential to differentiate into dopaminergic (DA) neurons after mitogenic expansion in vitro, hindering their efficient use as a transplantable cell source. Here, we report that epigenetic activation of Foxa2 in VM-derived NPCs by inducing histone hyperacetylation rescues the mitogenic-expansion-dependent decrease of differentiation potential to DA neurons. The silencing of Foxa2 gene expression after expansion is accompanied by repressive histone modifications, including hypoacetylation of histone H3 and H4 and trimethylation of H3K27 on the Foxa2 promoter, as well as on the global level. In addition, histone deacetylase 7 (HDAC7) is highly expressed during differentiation and recruited to the Foxa2 promoter. Induction of histone acetylation in VM-derived NPCs by either knockdown of HDAC7 or treatment with the HDAC inhibitor apicidin upregulates Foxa2 expression via hyperacetylation of H3 and a decrease in H3K27 trimethylation on the promoter regions, leading to the expression of DA neuron developmental genes and enhanced differentiation of DA neurons. These effects are antagonized by the expression of shRNAs specific for Foxa2 but enhanced by shRNA for HDAC7. Collectively, these findings indicate that loss of differentiation potential of expanded VM-derived NPCs is attributed to a decrease in Foxa2 expression and suggest that activation of the endogenous Foxa2 gene by epigenetic regulation might be an approach to enhance the generation of DA neurons.

The primary function of Leydig cells is to secrete testosterone, which is critical in the regulation of male reproduction and development. Low levels of testosterone will lead to male hypogonadism. Stem cell-derived Leydig cell transplantation may be a promising alternative therapy for male hypogonadism. Thus far, others have reported that Leydig-like cells can be derived from mesenchymal stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells. However, the efficiency of the differentiating Leydig cells remains low, and progress toward generating functional adult Leydig cells (ALCs) is limited. Herein, we describe a robust method of directing differentiation of mouse embryonic stem cells (mESCs) into Leydig-like cells in vitro by overexpression of the transcription factor steroidogenic factor-1 (SF-1) and treatment with a combination of 8-Bromoadenosine-3',5'-cyclic monophosphate and forskolin. These differentiated cells express mRNA encoding the steroidogenic enzymes and produce progesterone and testosterone. Importantly, when transplanted into male rats that had their original Leydig cells selectively eliminated by ethylene dimethanesulfonate, these in vitro-derived Leydig-like cells further developed into functional ALCs that rescued serum testosterone levels. These data provide evidence that mESCs can be induced to differentiate into Leydig-like cells in vitro, which can develop in the in vivo microenvironment.

Retinoic acid (RA) signaling is important to normal development. However, the function of the different RA receptors (RARs)--RARα, RARβ, and RARγ--is as yet unclear. We have used wild-type and transgenic zebrafish to examine the role of RARγ. Treatment of zebrafish embryos with an RARγ-specific agonist reduced somite formation and axial length, which was associated with a loss of hoxb13a expression and less-clear alterations in hoxc11a or myoD expression. Treatment with the RARγ agonist also disrupted formation of tissues arising from cranial neural crest, including cranial bones and anterior neural ganglia. There was a loss of Sox 9-immunopositive neural crest stem/progenitor cells in the same anterior regions. Pectoral fin outgrowth was blocked by RARγ agonist treatment. However, there was no loss of Tbx-5-immunopositive lateral plate mesodermal stem/progenitor cells and the block was reversed by agonist washout or by cotreatment with an RARγ antagonist. Regeneration of the caudal fin was also blocked by RARγ agonist treatment, which was associated with a loss of canonical Wnt signaling. This regenerative response was restored by agonist washout or cotreatment with the RARγ antagonist. These findings suggest that RARγ plays an essential role in maintaining stem/progenitor cells during embryonic development and tissue regeneration when the receptor is in its nonligated state.