Conserved and divergent paths that regulate self-renewal in mouse and human embryonic stem cells

The past few years have seen remarkable progress in our understanding of embryonic stem cell (ES cell) biology. The necessity of examining human ES cells in culture, coupled with the wealth of genomic data and the multiplicity of cell lines available, has enabled researchers to identify critical conserved pathways regulating self-renewal and identify markers that tightly correlate with the ES cell state. Comparison across species has suggested additional pathways likely to be important in long-term self-renewal of ES cells including heterochronic genes, microRNAs, genes involved in telomeric regulation, and polycomb repressors. In this review, we have discussed information on molecules known to be important in ES cell self-renewal or blastocyst development and highlighted known differences between mouse and human ES cells. We suggest that several additional pathways required for self-renewal remain to be discovered and these likely include genes involved in antisense regulation, microRNAs, as well as additional global repressive pathways and novel genes. We suggest that cross species comparisons using large-scale genomic analysis tools are likely to reveal conserved and divergent paths required for ES cell self-renewal and will allow us to derive ES lines from species and strains where this has been difficult.     

Gene delivery methods and genome editing of human pluripotent stem cells

Induced pluripotent stem cells derived from normal somatic cells could be utilized to study tumorigenesis through overexpression of specific oncogenes, downregulation of tumor suppressors and dysregulation of other factors thought to promote tumorigenesis. Therefore, effective approaches that provide direct modifications of induced pluripotent stem cell genome are extremely needed. Emerging strategies are expected to provide the ability to more effectively introduce diverse genetic alterations, from as small as single-nucleotide modifications to whole gene amplification or deletion, all with a high degree of target specificity. To date, several techniques have been applied in stem cell studies to directly edit cell genome (ZFNs, TALENs or CRISPR/Cas9). In this review, we summarize specific gene delivery strategies that were applied to stem cell studies together with genome editing techniques, which enable a direct modification of endogenous DNA sequences in the context of cancer studies.     

Primary bone-derived cells induce osteogenic differentiation without exogenous factors in human embryonic stem cells

We developed a new and efficient method for osteoblastic differentiation of human embryonic stem cells (hESCs) using primary bone-derived cells (PBDs). Three days after embryoid body (hEB) formation, cells were allowed to adhere to culture surface where PBDs were pre-plated and mitomycin C-treated in DMEM/F12 medium supplemented with 5% knockout serum replacement. As early as 14 days, mineralization and formation of nodule-like structures in cocultured hEBs were prominent by von Kossa and Alizarin S staining, and expressions of osteoblast-specific markers including bone sialoprotein, alkaline phosphates, osteocalcin, collagen 1, and core binding factor alpha1 by RT-PCR. In addition, FACS analysis revealed that over 19% of the differentiated cells expressed osteocalcin. These results suggest that PBDs not only have osteogenic effects releasing osteogenic factors as bone morphogenic protein (BMP) 2 and BMP 4 but also have exerted other effects, whether chemical or physical, for the differentiation of hESCs.     

Differentiation of human embryonic stem cells into smooth muscle cells in adherent monolayer culture

Smooth muscle cell (SMC) plays critical roles in many human diseases, an in vitro system that recapitulates human SMC differentiation would be invaluable for exploring molecular mechanisms leading to the human diseases. We report a directed and highly efficient SMC differentiation system by treating the monolayer-cultivated human embryonic stem cells (hESCs) with all-trans retinoid acid (atRA). When the hESCs were cultivated in differentiation medium containing 10microM RA, more than 93% of the cells expressed SMC-marker genes along with the steadily accumulation of such SMC-specific proteins as SM alpha-actin and SM-MHC. The fully differentiated SMCs were stable in phenotype and capable of contraction. This inducible and highly efficient in vitro human SMC system could be an important resource to study the mechanisms of SMC phenotype determination in human.     

The role of FGF-signaling in early neural specification of human embryonic stem cells

The mechanisms that govern human neural specification are not completely characterized. Here we used human embryonic stem cells (hESCs) to study the role of fibroblast growth factor (FGF)-signaling in early human neural specification. Differentiation was obtained by culturing clusters of hESCs in chemically-defined medium. We show that FGF-signaling, which is endogenously active during early differentiation of hESCs, induces early neural specification, while its blockage inhibits neuralization. The early neuralization effect of FGF-signaling is not mediated by promoting the proliferation of existing neural precursors (NPs) or prevention of their apoptosis. The neural instructive effect of FGF-signaling occurs after an initial FGF-independent differentiation into primitive ectoderm-like fate. We further show that FGF-signaling can induce neuralization by a mechanism which is independent of modulating bone morphogenic protein (BMP)-signaling. Still, FGF-signaling is not essential for hESC neuralization which can occur in the absence of FGF and BMP-signaling. Collectively, our data suggest that human neural induction is instructed by FGF-signaling, though neuralization of hESCs can occur in its absence.     

Genetic recombination pathways and their application for genome modification of human embryonic stem cells

Human embryonic stem cells are pluripotent cells derived from early human embryo and retain a potential to differentiate into all adult cell types. They provide vast opportunities in cell replacement therapies and are expected to become significant tools in drug discovery as well as in the studies of cellular and developmental functions of human genes. The progress in applying different types of DNA recombination reactions for genome modification in a variety of eukaryotic cell types has provided means to utilize recombination-based strategies also in human embryonic stem cells. Homologous recombination-based methods, particularly those utilizing extended homologous regions and those employing zinc finger nucleases to boost genomic integration, have shown their usefulness in efficient genome modification. Site-specific recombination systems are potent genome modifiers, and they can be used to integrate DNA into loci that contain an appropriate recombination signal sequence, either naturally occurring or suitably pre-engineered. Non-homologous recombination can be used to generate random integrations in genomes relatively effortlessly, albeit with a moderate efficiency and precision. DNA transposition-based strategies offer substantially more efficient random strategies and provide means to generate single-copy insertions, thus potentiating the generation of genome-wide insertion libraries applicable in genetic screens.     

Isolation of multipotent stem cells in human periodontal ligament using stage-specific embryonic antigen-4

The periodontal ligament (PDL) comprises adult stem cells, which are responsible for periodontal tissue regeneration. In the present study, we investigated the specific profile of the stem cells in the human PDL. Microscopic analysis demonstrated that PDL cells showed a fibroblastic appearance, forming flat and loose aggregates. PDL cells expressed embryonic stem cell-associated antigens (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT4, NANOG, SOX2, and REX1, and alkaline phosphatase activity), as well as conventional mesenchymal stem cell markers. When PDL cells were cultured in the presence of all-trans-retinoic acid, the numbers of SSEA-3+ and SSEA-4+ PDL cells were significantly decreased, while that of SSEA-1+ was increased. SSEA-4+ PDL cells showed a greater telomere length and growth rate. SSEA-4+ PDL cells exhibited the potential to generate specialized cells derived from three embryonic germ layers: mesodermal (adipocytes, osteoblasts, and chondrocytes), ectodermal (neurons), and endodermal (hepatocytes) lineages. Our findings demonstrated that SSEA-4, a major antigen to distinguish human embryonic stem cells, could also be used to identify multipotent stem cells in the PDL. Hence, SSEA-4+ human PDL cells appear to be a promising source of stem cells for regenerative medicine.     

Early homing behavior of Stro-1 mesenchyme-like cells derived from human embryonic stem cells in an immunocompetent xenogeneic animal model

Mesenchymal stem cells (MSCs) have been induced to differentiate successfully from human embryonic stem cells (hES-MSCs), which could serve as an in vitro source of MSCs. However, the homing behaviors of such cells and their potential utility for liver regeneration in vivo have not been reported. We investigated factors that influenced early homing and the hepatic-directed differentiation potency of hES-MSCs in a mouse model of acute liver injury. The hES-MSCs could be detected 36 h after cell infusion and this was unaffected by the number of cell passages in culture. Pretreatment of hES-MSCs with TNF-α resulted in higher rates of homing of these cells to the injured liver. Interestingly most of the cells homing at an early stage expressed alpha-fetoprotein (AFP), indicating hepatic differentiation. Thus, hES-MSCs can home to the acutely injured liver at high efficiency and undergo hepatic differentiation, suggesting that these cells could be useful for treating acute human liver injury.     

A novel chemical-defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells

Traditionally, undifferentiated human embryonic stem cells (hESCs) are maintained on mouse embryonic fibroblast (MEF) cells or on matrigel with an MEF-conditioned medium (CM), which hampers the clinical applications of hESCs due to the contamination by animal pathogens. Here we report a novel chemical-defined medium using DMEM/F12 supplemented with N2, B27, and basic fibroblast growth factor (bFGF) [termed NBF]. This medium can support prolonged self-renewal of hESCs. hESCs cultured in NBF maintain an undifferentiated state and normal karyotype, are able to form embryoid bodies in vitro, and differentiate into three germ layers and extraembryonic cells. Furthermore, we find that hESCs cultured in NBF possess a low apoptosis rate and a high proliferation rate compared with those cultured in MEF-CM. Our findings provide a novel, simplified chemical-defined culture medium suitable for further therapeutic applications and developmental studies of hESCs.     

A unique epigenetic signature is associated with active DNA replication loci in human embryonic stem cells

The cellular epigenetic landscape changes as pluripotent stem cells differentiate to somatic cells or when differentiated cells transform to a cancerous state. These epigenetic changes are commonly correlated with differences in gene expression. Whether active DNA replication is also associated with distinct chromatin environments in these developmentally and phenotypically diverse cell types has not been known. Here, we used BrdU-seq to map active DNA replication loci in human embryonic stem cells (hESCs), normal primary fibroblasts and a cancer cell line, and correlated these maps to the epigenome. In all cell lines, the majority of BrdU peaks were enriched in euchromatin and at DNA repetitive elements, especially at microsatellite repeats, and coincided with previously determined replication origins. The most prominent BrdU peaks were shared between all cells but a sizable fraction of the peaks were specific to each cell type and associated with cell type-specific genes. Surprisingly, the BrdU peaks that were common to all cell lines were associated with H3K18ac, H3K56ac, and H4K20me1 histone marks only in hESCs but not in normal fibroblasts or cancer cells. Depletion of the histone acetyltransferases for H3K18 and H3K56 dramatically decreased the number and intensity of BrdU peaks in hESCs. Our data reveal a unique epigenetic signature that distinguishes active replication loci in hESCs from normal somatic or malignant cells.     

Renal ontogeny in the rhesus monkey (Macaca mulatta) and directed differentiation of human embryonic stem cells towards kidney precursors

The development of the metanephric kidney was studied immunohistochemically across gestation in monkeys to identify markers of cell specification, and to aid in developing experimental paradigms for renal precursor differentiation from human embryonic stem cells (hESC). PAX2, an important kidney developmental marker, was expressed at the tips of the ureteric bud, in the surrounding condensing mesenchyme, and in the renal vesicle. Vimentin, a mesenchymal and renal marker, was strongly expressed in the metanephric blastema then found to be limited to the glomerulus and interstitial cells of the medulla and cortex. A model of gene expression based on human and nonhuman primate renal ontogeny was developed and incorporated into studies of hESC differentiation. Spontaneous hESC differentiation revealed markers of metanephric mesenchyme (OSR1, PAX2, SIX2, WT1) that increased over time, followed by upregulation of kidney precursor markers (EYA1, LIM1, CD24). Directed hESC differentiation was also evaluated with the addition of retinoic acid, Activin-A, and BMP-4 or BMP-7, and using different culture substrate conditions. Of the culture substrates studied, gelatin most closely recapitulated the anticipated directed developmental pattern of renal gene expression. No differences were found when BMP-4 and BMP-7 were compared with baseline conditions. PAX2 and Vimentin immunoreactivity in differentiating hESC was also similar to the renal precursor patterns reported for human fetal kidneys and findings described in rhesus monkeys. The results of these studies are as follows: (1) provide additional data to support that rhesus monkey kidney development parallels that of humans, and (2) provide a useful model for hESC directed differentiation towards renal precursors.     

Competition between PRC2.1 and 2.2 subcomplexes regulates PRC2 chromatin occupancy in human stem cells

1. Download : Download high-res image (122KB)
2. Download : Download full-size image

     

Gap junctions modulate apoptosis and colony growth of human embryonic stem cells maintained in a serum-free system

We investigated the gap junctional properties of human embryonic stem cells (hESC) cultivated in a serum-free system using sphingosine-1-phosphate and platelet-derived growth factor (S1P/PDGF). We compared this condition to hESC grown on Matrigel in mouse embryonic fibroblast conditioned medium (MEF-CM) or unconditioned medium (UM). We show that in all culture systems, hESC express connexins 43 and 45. hESC maintained in S1P/PDGF conditions and hESC grown in presence of MEF-CM are coupled through gap junctions while hESC maintained on Matrigel in UM do not exhibit gap junctional intercellular communication. In this latter condition, coupling was retrieved by addition of noggin, suggesting that BMP-like activity in UM inhibits gap junctional communication. Last, our data indicate that the closure of gap junctions by the decoupling agent alpha-glycyrrhetinic acid increases cell apoptosis and inhibits hESC colony growth. Altogether, these results suggest that gap junctions play an important role in hESC maintenance.     

Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells

We tested a "standard" cryopreservation protocol (slow cooling with 10% DMSO) on the human embryonic stem cell (hESC) line H9 containing an Oct-4 (POU5F1) promoter-driven, enhanced green fluorescent protein (EGFP) reporter to monitor maintenance of pluripotency. Cells were cooled to -80 degrees C in cryovials and then transferred to a -80 degrees C freezer. Cells were held at -80 degrees C for 3 days ("short-term storage") or 3 months ("long-term storage"). Vials were thawed in a +36 degrees C water bath and cells were cultured for 3, 7, or 14 days. Propidium iodide (PI) was used to assess cell viability by flow cytometry. Control cells were passaged on the same day that the frozen cells were thawed. The majority of cells in control hESC cultures were Oct-4 positive and almost 99% of EGFP+ cells were alive as determined by exclusion of PI. In contrast, the frozen cells, even after 3 days of culture, contained only 50% live cells, and only 10% were EGFP-positive. After 7 days in culture, the proportion of dead cells decreased and there was an increase in the Oct-4-positive population but microscopic examination revealed large patches of EGFP-negative cells within clusters of colonies even after 14 days of culturing. After 3 months of storage at -80 degrees C the deleterious effect of freezing was even more pronounced: the samples regained a quantifiable number of EGFP-positive cells only after 7 days of culturing following thawing. It is concluded that new protocols and media are required for freezing hESC and safe storage at -80 degrees C as well as studies of the mechanisms of stress-related events associated with cell cryopreservation.