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The peri-implantation window of mammalian development is the crucial window for primordial germ cell (PGC) specification. Whereas pre-implantation dynamics are relatively conserved between species, the implantation window marks a stage of developmental divergence between key model organisms, and thus potential variance in the cell and molecular mechanisms for PGC specification. In humans, PGC specification is very difficult to study in vivo To address this, the combined use of human and nonhuman primate embryos, and stem cell-based embryo models are essential for determining the origin of PGCs, as are comparative analyses to the equivalent stages of mouse development. Understanding the origin of PGCs in the peri-implantation embryo is crucial not only for accurate modeling of this essential process using stem cells, but also in determining the role of global epigenetic reprogramming upon which sex-specific differentiation into gametes relies., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Single-cell RNA sequencing (scRNA-seq) has broad applications across biomedical research. One of the key challenges is to ensure that only single, live cells are included in downstream analysis, as the inclusion of compromised cells inevitably affects data interpretation. Here, we present a generic approach for processing scRNA-seq data and detecting low quality cells, using a curated set of over 20 biological and technical features. Our approach improves classification accuracy by over 30 % compared to traditional methods when tested on over 5,000 cells, including CD4+ T cells, bone marrow dendritic cells, and mouse embryonic stem cells.

Chromatin profiling provides a versatile means to investigate functional genomic elements and their regulation. However, current methods yield ensemble profiles that are insensitive to cell-to-cell variation. Here we combine microfluidics, DNA barcoding and sequencing to collect chromatin data at single-cell resolution. We demonstrate the utility of the technology by assaying thousands of individual cells and using the data to deconvolute a mixture of ES cells, fibroblasts and hematopoietic progenitors into high-quality chromatin state maps for each cell type. The data from each single cell are sparse, comprising on the order of 1,000 unique reads. However, by assaying thousands of ES cells, we identify a spectrum of subpopulations defined by differences in chromatin signatures of pluripotency and differentiation priming. We corroborate these findings by comparison to orthogonal single-cell gene expression data. Our method for single-cell analysis reveals aspects of epigenetic heterogeneity not captured by transcriptional analysis alone.

The availability of and access to quality genetically defined, health-status known mouse resources is critical for biomedical research. By ensuring that mice used in research experiments are biologically, genetically, and health-status equivalent, we enable knowledge transfer, hypothesis building based on multiple data streams, and experimental reproducibility based on common mouse resources (reagents). Major repositories for mouse resources have developed over time and each has significant unique resources to offer. Here we (a) describe The International Mouse Strain Resource that offers users a combined catalog of worldwide mouse resources (live, cryopreserved, embryonic stem cells), with direct access to repository sites holding resources of interest and (b) discuss the commitment to nomenclature standards among resources that remain a challenge in unifying mouse resource catalogs.

The International Knockout Mouse Consortium (IKMC; http://www.mousephenotype.org ) has generated mutations in almost every protein-coding mouse gene and is completing the companion Cre driver resource to expand tissue-specific conditional mutagenesis. Accordingly, the IKMC has carried out high-throughput gene trapping and targeting producing conditional mutations in murine embryonic stem cells in more than 18,500 genes, from which at least 4900 mutant mouse lines have been established to date. This resource is currently being upgraded with more powerful tools, such as visualization and manipulation cassettes that can be easily introduced into IKMC alleles for multifaceted functional studies. In addition, we discuss how existing IKMC products can be used in combination with CRISPR technology to accelerate genome engineering projects. All information and materials from this extraordinary biological resource together with coordinated phenotyping efforts can be retrieved at www.mousephenotype.org . The comprehensive IKMC knockout resource in combination with an extensive set of modular gene cassettes will continue to enhance functional gene annotation in the future and solidify its impact on biomedical research.

The apical domain of embryonic (radial glia) and adult (B1 cells) neural stem cells (NSCs) contains a primary cilium. This organelle has been suggested to function as an antenna for the detection of morphogens or growth factors. In particular, primary cilia are essential for Hedgehog (Hh) signaling, which plays key roles in brain development. Their unique location facing the ventricular lumen suggests that primary cilia in NSCs could play an important role in reception of signals within the cerebrospinal fluid. Surprisingly, ablation of primary cilia using conditional alleles for genes essential for intraflagellar transport [kinesin family member 3A (Kif3a) and intraflagellar transport 88 (Ift88)] and Cre drivers that are activated at early [Nestin; embryonic day 10.5 (E10.5)] and late [human glial fibrillary acidic protein (hGFAP); E13.5] stages of mouse neural development resulted in no apparent developmental defects. Neurogenesis in the ventricular-subventricular zone (V-SVZ) shortly after birth was also largely unaffected, except for a restricted ventral domain previously known to be regulated by Hh signaling. However, Kif3a and Ift88 genetic ablation also disrupts ependymal cilia, resulting in hydrocephalus by postnatal day 4. To directly study the role of B1 cells' primary cilia without the confounding effects of hydrocephalus, we stereotaxically targeted elimination of Kif3a from a subpopulation of radial glia, which resulted in ablation of primary cilia in a subset of B1 cells. Again, this experiment resulted in decreased neurogenesis only in the ventral V-SVZ. Primary cilia ablation led to disruption of Hh signaling in this subdomain. We conclude that primary cilia are required in a specific Hh-regulated subregion of the postnatal V-SVZ.

Human embryonic stem cells (hESCs) are generally induced to differentiate by forming spherical structures termed embryoid bodies (EBs) in the presence of soluble growth factors. hEBs are generated by suspending small clumps of hESC colonies; however, the resulting hEBs are heterogeneous because this method lacks the ability to control the number of cells in individual EBs. This heterogeneity affects factors that influence differentiation such as cell-cell contact and the diffusion of soluble factors, and consequently, the differentiation capacity of each EB varies. Here, we fabricated size-tunable concave microwells to control the physical environment, thereby regulating the size of EBs formed from single hESCs. Defined numbers of single hESCs were forced to aggregate and generate uniformly sized EBs with high fidelity, and the size of the EBs was controlled using concave microwells of different diameters. Differentiation patterns in H9- and CHA15-hESCs were affected by EB size in both the absence and presence of growth factors. By screening EB size in the presence of various BMP4 concentrations, a two-fold increase in endothelial cell differentiation was achieved. Because each hESC line has unique characteristics, the findings of this study demonstrate that concave microwells could be used to screen different EB sizes and growth factor concentrations to optimize differentiation for each hESC line., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Mouse embryonic stem (ES) cells are prototypical stem cells that remain undifferentiated in culture for long periods, yet maintain the ability to differentiate into essentially all cell types. Previously, we have reported that ES cells oscillate between two distinct states, which can be distinguished by the transient expression of Zscan4 genes originally identified for its specific expression in mouse two-cell stage embryos. Here, we report that the nascent protein synthesis is globally repressed in the Zscan4-positive state of ES cells, which is mediated by the transient expression of newly identified eukaryotic translation initiation factor 1A (Eif1a)-like genes. Eif1a-like genes, clustered on Chromosome 12, show the high sequence similarity to the Eifa1 and consist of 10 genes (Eif1al1-Eif1al10) and 9 pseudogenes (Eif1al-ps1-Eif1al-ps9). The analysis of the expressed sequence tag database showed that Eif1a-like genes are expressed mostly in the two-cell stage mouse embryos. Microarray analyses and quantitative real-time polymerase chain reaction analyses show that Eif1a-like genes are expressed specifically in the Zscan4-positive state of ES cells. These results indicate a novel mechanism to repress protein synthesis by Eif1a-like genes and a unique mode of protein synthesis regulation in ES cells, which undergo a transient and reversible repression of global protein synthesis in the Zscan4-positive state.

Embryonic (ES) and epiblast (EpiSC) stem cells are pluripotent but committed to an embryonic lineage fate. Conversely, trophoblast (TS) and extraembryonic endoderm (XEN) stem cells contribute predominantly to tissues of the placenta and yolk sac, respectively. Here we show that each of these four stem cell types is defined by a unique DNA methylation profile. Despite their distinct developmental origin, TS and XEN cells share key epigenomic hallmarks, chiefly characterized by robust DNA methylation of embryo-specific developmental regulators, as well as a subordinate role of 5-hydroxymethylation. We also observe a substantial methylation reinforcement of pre-existing epigenetic repressive marks that specifically occurs in extraembryonic stem cells compared to in vivo tissue, presumably due to continued high Dnmt3b expression levels. These differences establish a major epigenetic barrier between the embryonic and extraembryonic stem cell types. In addition, epigenetic lineage boundaries also separate the two extraembryonic stem cell types by mutual repression of key lineage-specific transcription factors. Thus, global DNA methylation patterns are a defining feature of each stem cell type that underpin lineage commitment and differentiative potency of early embryo-derived stem cells. Our detailed methylation profiles identify a cohort of developmentally regulated sequence elements, such as orphan CpG islands, that will be most valuable to uncover novel transcriptional regulators and pivotal "gatekeeper" genes in pluripotency and lineage differentiation., (Copyright © 2012 AlphaMed Press.)

As stem cells are capable of self-renewal and can generate differentiated progenies for organ development, they are considered as potential source for regenerative medicine and tissue replacement after injury or disease. Along with this capacity, stem cells have the therapeutic potential for treating human diseases including cancers. According to the origins, stem cells are broadly classified into two types: embryonic stem cells (ESCs) and adult stem cells. In terms of differentiation potential, ESCs are pluripotent and adult stem cells are multipotent. Amnion, which is a membranous sac that contains the fetus and amniotic fluid and functions in protecting the developing embryo during gestation, is another stem cell source. Amnion-derived stem cells are classified as human amniotic membrane-derived epithelial stem cells, human amniotic membrane-derived mesenchymal stem cells and human amniotic fluid-derived stem cells. They are in an intermediate stage between pluripotent ESCs and lineage-restricted adult stem cells, non-tumorigenic, and contribute to low immunogenicity and anti-inflammation. Furthermore, they are easily available and do not cause any controversial issues in their recovery and applications. Not only are amnion-derived stem cells applicable in regenerative medicine, they have anticancer capacity. In non-engineered stem cells transplantation strategies, amnion-derived stem cells effectively target the tumor and suppressed the tumor growth by expressing cytotoxic cytokines. Additionally, they also have a potential as novel delivery vehicles transferring therapeutic genes to the cancer formation sites in gene-directed enzyme/prodrug combination therapy. Owing to their own advantageous properties, amnion-derived stem cells are emerging as a new candidate in anticancer therapy.

'In this report, we use single cell gene expression to identify transcriptional patterns emerging during the differentiation of human embryonic stem cells (hESCs) into the endodermal lineage. Endoderm-specific transcripts are highly variable between individual CXCR4(+) endodermal cells, suggesting that either the cells generated from in vitro differentiation are distinct or that these embryonic cells tolerate a high degree of transcript variability. Housekeeping transcripts, on the other hand, are far more consistently expressed within the same cellular population. However, when we compare the levels of housekeeping transcripts between hESCs and derived endoderm, patterns emerge that can be used to clearly separate the two embryonic cell types. We further compared four additional human cell types, including 293T, induced pluripotent stem cell (iPSC), HepG2, and endoderm-derived iPSC. In each case, the relative levels of housekeeping transcripts defined a particular cell fate. Interestingly, we find that three transcripts, LDHA, NONO, and ACTB, contribute the most to this diversity and together serve to segregate all six cell types. Overall, this suggests that levels of housekeeping transcripts, which are expressed within all cells, can be leveraged to distinguish between human cell types and thus may serve as important biomarkers for stem cell biology and other disciplines., (Copyright © 2011 AlphaMed Press.)

Many types of epigenetic profiling have been used to classify stem cells, stages of cellular differentiation, and cancer subtypes. Existing methods focus on local chromatin features such as DNA methylation and histone modifications that require extensive analysis for genome-wide coverage. Replication timing has emerged as a highly stable cell type-specific epigenetic feature that is regulated at the megabase-level and is easily and comprehensively analyzed genome-wide. Here, we describe a cell classification method using 67 individual replication profiles from 34 mouse and human cell lines and stem cell-derived tissues, including new data for mesendoderm, definitive endoderm, mesoderm and smooth muscle. Using a Monte-Carlo approach for selecting features of replication profiles conserved in each cell type, we identify "replication timing fingerprints" unique to each cell type and apply a k nearest neighbor approach to predict known and unknown cell types. Our method correctly classifies 67/67 independent replication-timing profiles, including those derived from closely related intermediate stages. We also apply this method to derive fingerprints for pluripotency in human and mouse cells. Interestingly, the mouse pluripotency fingerprint overlaps almost completely with previously identified genomic segments that switch from early to late replication as pluripotency is lost. Thereafter, replication timing and transcription within these regions become difficult to reprogram back to pluripotency, suggesting these regions highlight an epigenetic barrier to reprogramming. In addition, the major histone cluster Hist1 consistently becomes later replicating in committed cell types, and several histone H1 genes in this cluster are downregulated during differentiation, suggesting a possible instrument for the chromatin compaction observed during differentiation. Finally, we demonstrate that unknown samples can be classified independently using site-specific PCR against fingerprint regions. In sum, replication fingerprints provide a comprehensive means for cell characterization and are a promising tool for identifying regions with cell type-specific organization.

Prolonged in vitro culture of human embryonic stem (hES) cells can result in chromosomal abnormalities believed to confer a selective advantage. This potential occurrence has crucial implications for the appropriate use of hES cells for research and therapeutic purposes. In view of this, time-point karyotypic evaluation to assess genetic stability is recommended as a necessary control test to be carried out during extensive 'passaging'. Standard techniques currently used for the cytogenetic assessment of ES cells include G-banding and/or Fluorescence in situ Hybridization (FISH)-based protocols for karyotype analysis, including M-FISH and SKY. Critical for both banding and FISH techniques are the number and quality of metaphase spreads available for analysis at the microscope. Protocols for chromosome preparation from hES and human induced pluripotent stem (hiPS) cells published so far appear to differ considerably from one laboratory to another. Here we present an optimized technique, in which both the number and the quality of chromosome metaphase spreads were substantially improved when compared to current standard techniques for chromosome preparations. We believe our protocol represents a significant advancement in this line of work, and has the required attributes of simplicity and consistency to be widely accepted as a reference method for high quality, fast chromosomal analysis of human ES and iPS cells.

Background Aims: Fetal membrane from human placenta tissue has been described as a potential source of stem cells. Despite abundant literature on amnion stem cells, there are limited studies on the stem cell properties of chorion-derived stem cells., Methods: The main aim was to determine the stemness properties of serial-passaged human chorion-derived stem cells (hCDSC). Quantitative polymerase chain reaction (PCR) was performed to reveal the following stemness gene expression in serial-passaged hCDSC: Oct-4, Sox-2, FGF-4, Rex-1, TERT, Nanog (3), Nestin, FZD-9, ABCG-2 and BST-1. Cell growth rate was evaluated from passage (P) 1 until P5. The colony-forming unit-fibroblast (CFU-F) frequency of P3 and P5 cells and multilineage differentiation potential of P5 cells were determined. The immunophenotype of hCDSC was compared using the surface markers CD9, CD31, CD34, CD44, CD45, CD73, CD90, CD117, HLA-ABC and HLA-DR, -DP and -DQ. Immunostaining for trophoblast markers was done on P0, P1, P3 and P5 cells to detect the contamination of trophoblasts in culture, while chromosomal abnormality was screened by cytogenetic analysis of P5 cells., Results: The surface markers for mesenchymal lineage in hCDSC were more highly expressed at P5 compared with P3 and P0, indicating the increased purity of these stem cells after serial passage. Indeed, all the stemness genes except TERT were expressed at P1, P3 and P5 hCDSC. Furthermore, human chorion contained high clonogenic precursors with a 1:30 CFU-F frequency. Successful adipogenic, chondrogenic and osteogenic differentiation demonstrated the multilineage potential of hCDSC. The karyotyping analysis showed hCDSC maintained chromosomal stability after serial passage., Conclusions: hCDSC retain multipotent potential even at later passages, hence are a promising source for cell therapy in the future.

Human embryonic stem cells are elusive, recalcitrant entities that resist characterization and standardization. Without agreements about what the cells are and how best to systematize cell culture and testing, data cannot be extracted meaningfully, the nascent field will be slow to stabilize, and significantly, there may be safety risks for patients. I discuss efforts to characterize cells definitively and standardize practices across uniquely derived lines, labs, and researchers. I argue that such efforts are made more complicated by layered identities imposed on them by classification conventions, interactions with researchers and laboratory environments, and inheritances from genetic ancestry. The need to understand and possibly capitalize on such distinct, cumulative identities is in tension with the desire to stabilize the field under conditions of political and scientific uncertainty. The article links STS work on standardization with anthropological perspectives on identity and material culture in science.

Sox2 is expressed by neural stem and progenitor cells, and a sox2 enhancer identifies these cells in the forebrains of both fetal and adult transgenic mouse reporters. We found that an adenovirus encoding EGFP placed under the regulatory control of a 0.4 kb sox2 core enhancer selectively identified multipotential and self-renewing neural progenitor cells in dissociates of human fetal forebrain. Upon EGFP-based fluorescence-activated cell sorting (FACS), the E/sox2:EGFP(+) isolates were propagable for up to 1 year in vitro, and remained multilineage competent throughout. E/sox2:EGFP(+) cells expressed more telomerase enzymatic activity than matched E/sox2:EGFP-depleted populations, and maintained their telomeric lengths with successive passage. Gene expression analysis of E/sox2:EGFP-sorted neural progenitor cells, normalized to the unsorted forebrain dissociates from which they derived, revealed marked overexpression of genes within the notch and wnt pathways, and identified multiple elements of each pathway that appear selective to human neural progenitors. Sox2 enhancer-based FACS thus permits the prospective identification and direct isolation of a telomerase-active population of neural stem cells from the human fetal forebrain, and the elucidation of both the transcriptome and dominant signaling pathways of these critically important cells.

Alphab-crystallin (CRYAB) is a small heat shock protein with a chaperoning activity that is present in the postnatal healthy human brain in oligodendrocytes and in a few astrocytes. The involvement of CRYAB in cell differentiation, proliferation, signaling, cytoskeletal assembly, and apoptosis in various model systems has suggested that it might also play a role in the developing human brain. We analyzed the distribution and the levels of this molecular chaperone in healthy and polygenetically compromised (Down syndrome [DS]) human telencephalon at midgestation. We demonstrate that CRYAB is expressed in a temporospatial pattern by numerous radial glial cells and some early oligodendrocyte progenitors, including dividing cells, as well as a few astroglial cells in both healthy and DS fetal brains. We also found abundant phosphorylation of CRYAB at Ser-59, which mediates its antiapoptotic and cytoskeletal functions. There was only marginal phosphorylation at Ser-45.In contrast to our earlier study in young DS subjects, upregulation of phosphorylated CRYAB occurred rarely in DS fetuses. The distribution, the timing of appearance, and the results of colocalization studies suggest that CRYAB assists in the biological processes associated with developmental remodeling/differentiation and proliferation of select subpopulations of progenitor cells in human fetal brain at midgestation.

We have developed a combined micro-channel and micro-well system for easy cell loading, culture and post-culture operation on a chip. To demonstrate the reliability of the system, on chip cell culture and differentiation were performed with different types of substrates made of culture dish, glass cover slide and polydimethylsiloaxe (PDMS). As expected, mouse embryo fibroblasts (MEF) showed different adhesion and growth rate on different substrates. When embryonic stem (ES) cells were co-cultured with MEFs, the formation of ES colonies is efficient on both glass and Petri dish, although PDMS could also be used. Finally, ES cell differentiation with neuron growth factors was performed on different substrates, showing clear advantages of using culture Petri dish over both glass and PDMS.

Prolonged culture of human embryonic stem cells (hESCs) can lead to adaptation and the acquisition of chromosomal abnormalities, underscoring the need for rigorous genetic analysis of these cells. Here we report the highest-resolution study of hESCs to date using an Affymetrix SNP 6.0 array containing 906,600 probes for single nucleotide polymorphisms (SNPs) and 946,000 probes for copy number variations (CNVs). Analysis of 17 different hESC lines maintained in different laboratories identified 843 CNVs of 50 kb-3 Mb in size. We identified, on average, 24% of the loss of heterozygosity (LOH) sites and 66% of the CNVs changed in culture between early and late passages of the same lines. Thirty percent of the genes detected within CNV sites had altered expression compared to samples with normal copy number states, of which >44% were functionally linked to cancer. Furthermore, LOH of the q arm of chromosome 16, which has not been observed previously in hESCs, was detected.

Embryonic stem cells (ESCs) are potentially powerful tools for regenerative medicine and establishment of disease models. The recent progress in ESC technologies is noteworthy, but ESC differentiation into renal lineages is relatively less established. The present study aims to differentiate mouse ESCs (mESCs) into a renal progenitor pool, the intermediate mesoderm (IM), without addition of exogenous cytokines and embryoid formation. First, we treated mESCs with a combination of small molecules (Janus-associated tyrosine kinase inhibitor 1, LY294002, and CCG1423) and differentiated them into BMP7-positive cells, BMP7 being the presumed inducing factor for IM. When these cells were cultured with adding retinoic acid, expression of odd-skipped related 1 (Osr1), which is essential to IM differentiation, was enhanced. To simplify the differentiation protocol, the abovementioned four small molecules (including retinoic acid) were combined and added to the culture. Under this condition, more than one-half of the cells were positive for Osr1, and at the same time, Pax2 (another IM marker) was detected by real-time PCR. Expressions of ectodermal marker and endodermal marker were not enhanced, while mesodermal marker changed. Moreover, expression of genes indispensable to kidney development, i.e., Lim1 and WT1, was detected by RT-PCR. These results indicate the establishment of a specific, effective method for differentiation of the ESC monolayer into IM using a combination of small molecules, resulting in an attractive cell source that could be experimentally differentiated to understand nephrogenic mechanisms and cell-to-cell interactions in embryogenesis., (Copyright 2010 Elsevier Inc. All rights reserved.)

For human embryonic stem cells (hESCs) to be used clinically, it is imperative that immune responses evoked by hESCs and their derivates after transplantation should be prevented. Human leukocyte antigens (HLA) and ABO blood group antigens are important histocompatibility factors in graft rejection. HLA matching between recipient and unrelated donors, in particular, is important in improving outcomes in hematopoietic cell transplantation (HCT). We have established and successfully maintained 29 hESC lines and analyzed the HLA and ABO genotypes of these lines. HLA-A, -B, -C and -DR (DRB1) genotyping was performed by polymerase chain reaction (PCR) sequence-based typing and ABO genotyping was carried out by PCR restriction fragment length polymorphism methods. To determine what proportion of the Korean population would be covered by these cell lines in organ transplantation, 27 cell lines with HLA-A, -B, and -DR data were evaluated for HCT (cord blood) donors and 28 cell lines with HLA-DR and ABO data were evaluated for solid organ (kidney) transplantation donors, and then compared the data with those from 6,740 donated cord bloods. When 2 HLA mismatches are allowed for HCT, as currently accepted for cord blood transplantation, it was estimated that about 16% and 25% of the possible recipients can find one or more donor cell lines with ≤2 mismatches at A, B, DRB1 allele level and at A, B antigen/DRB1 allele level, respectively. When HLA-DR antigen level matching and ABO compatibility was considered for solid organ (kidney) transplantation, it was estimated that about 29% and 96% of the possible recipients can find one or more ABO-compatible donor cell lines with 0 and 1 DR mismatches, respectively. We provided the first report on the HLA and ABO genotypes of hESC lines, and estimated the degree of HLA and ABO matching in organ transplantation for the Korean population.

Throughout the developing central nervous system, pre-patterning of the ventricular zone into discrete neural progenitor domains is one of the predominant strategies used to produce neuronal diversity in a spatially coordinated manner. In the retina, neurogenesis proceeds in an intricate chronological and spatial sequence, yet it remains unclear whether retinal progenitor cells (RPCs) display intrinsic heterogeneity at any given time point. Here, we performed a detailed study of RPC fate upon temporally and spatially confined inactivation of Pax6. Timed genetic removal of Pax6 appeared to unmask a cryptic divergence of RPCs into qualitatively divergent progenitor pools. In the more peripheral RPCs under normal circumstances, Pax6 seemed to prevent premature activation of a photoreceptor-differentiation pathway by suppressing expression of the transcription factor Crx. More centrally, Pax6 contributed to the execution of the comprehensive potential of RPCs: Pax6 ablation resulted in the exclusive generation of amacrine interneurons. Together, these data suggest an intricate dual role for Pax6 in retinal neurogenesis, while pointing to the cryptic divergence of RPCs into distinct progenitor pools.

Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal and adult sources have been called stem cells, even though they range from pluripotent cells-typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation-to adult stem cell lines, which can generate a far more limited repertoire of differentiated cell types. The rapid increase in reports of new sources of stem cells and their anticipated value to regenerative medicine has highlighted the need for a general, reproducible method for classification of these cells. We report here the creation and analysis of a database of global gene expression profiles (which we call the 'stem cell matrix') that enables the classification of cultured human stem cells in the context of a wide variety of pluripotent, multipotent and differentiated cell types. Using an unsupervised clustering method to categorize a collection of approximately 150 cell samples, we discovered that pluripotent stem cell lines group together, whereas other cell types, including brain-derived neural stem cell lines, are very diverse. Using further bioinformatic analysis we uncovered a protein-protein network (PluriNet) that is shared by the pluripotent cells (embryonic stem cells, embryonal carcinomas and induced pluripotent cells). Analysis of published data showed that the PluriNet seems to be a common characteristic of pluripotent cells, including mouse embryonic stem and induced pluripotent cells and human oocytes. Our results offer a new strategy for classifying stem cells and support the idea that pluripotency and self-renewal are under tight control by specific molecular networks.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM) and the epiblast, and have been suggested to be a homogeneous population with characteristics intermediate between them. These cells express Oct3/4 and Rex1 genes, which have been used as markers to indicate the undifferentiated state of ES cells. Whereas Oct3/4 is expressed in totipotent and pluripotent cells in the mouse life cycle, Rex1 expression is restricted to the ICM, and is downregulated in pluripotent cell populations in the later stages, i.e. the epiblast and primitive ectoderm (PrE). To address whether ES cells comprise a homogeneous population equivalent to a certain developmental stage of pluripotent cells or a heterogeneous population composed of cells corresponding to various stages of differentiation, we established knock-in ES cell lines in which genes for fluorescent proteins were inserted into the Rex1 and Oct3/4 gene loci to visualize the expression of these genes. We found that undifferentiated ES cells included at least two different populations, Rex1(+)/Oct3/4(+) cells and Rex1(-)/Oct3/4(+) cells. The Rex1(-)/Oct3/4(+) and Rex1(+)/Oct3/4(+) populations could convert into each other in the presence of LIF. In accordance with our assumption that Rex1(+)/Oct3/4(+) cells and Rex1(-)/Oct3/4(+) cells have characteristics similar to those of ICM and early-PrE cells, Rex1(+)/Oct3/4(+) cells predominantly differentiated into primitive ectoderm and contributed to chimera formation, whereas Rex1(-)/Oct3/4(+) cells differentiated into cells of the somatic lineage more efficiently than non-fractionated ES cells in vitro and showed poor ability to contribute to chimera formation. These results confirmed that undifferentiated ES cell culture contains subpopulations corresponding to ICM, epiblast and PrE.

Embryonic stem (ES) cells represent a unique cellular model to recapitulate in vitro early steps of embryonic development and an unlimited cellular source in therapy for many diseases, as well as targets for drug discovery and toxicology screens. Although previous studies have reported epidermal differentiation of mouse and human embryonic stem (huES) cells, the heterogeneity of the resulting cell culture impairs the evaluation of differentiated cells for cell therapy. We report here the reproducible isolation of a homogenous ectodermal cell population, IT1, from human ES cells. Like primary cells, IT1 cells remain homogenous over 15 passages, expand up to 60 population doublings, and then die through senescence. Accordingly, IT1 cells display a normal karyotype and a somatic cell cycle kinetics and do not produce teratoma in nude mice. The production of K14-expressing epithelial cells driven by p63 expression strengthens the ectodermal nature of IT1 cells. Since IT1 can be isolated from different huES cell lines, it may provide a ready source of ectodermal progenitors for the development of a toxicology cell model, new-drug-screening strategies, and cell therapy transplantation.

In the developing mouse, vascular endothelial cell (EC) and hematopoietic cell (HPC) lineages are two initial cell lineages that diverge from mesodermal cells, which have been roughly subdivided into three subtypes according to their geographical location: the organizer, embryonic mesoderm in the primitive streak, and extraembryonic mesoderm during gastrulation. Although the initial progenitors that become the two lineages appear in both vascular endothelial growth factor receptor 2(+) (VEGFR2(+)) lateral and extraembryonic mesoderm, little is known about the underlying molecular events that regulate the derivation of ECs and HPCs. Here, we describe an experimental system consisting of two types of embryonic stem cell lines capable of distinguishing between organizer and the middle section of the primitive streak region. Using this system, we were able to establish a defined culture condition that can separately induce distinct types of mesoderm. Although we were able to differentiate ECs from all mesoderm subsets, however, the potential of HPCs was restricted to the VEGFR2(+) cells derived from primitive streak-type mesodermal cells. We also show that the culture condition for the progenitors of primitive erythrocytes is separated from that for the progenitors of definitive erythrocytes. These results suggest the dominant role of extrinsic regulation during diversification of mesoderm.

Cell therapy has been perceived as the main or ultimate goal of human embryonic stem (ES) cell research. Where are we now and how are we going to get there? There has been rapid success in devising in vitro protocols for differentiating human ES cells to neuroepithelial cells. Progress has also been made to guide these neural precursors further to more specialized neural cells such as spinal motor neurons and dopamine-producing neurons. However, some of the in vitro produced neuronal types such as dopamine neurons do not possess all the phenotypes of their in vivo counterparts, which may contribute to the limited success of these cells in repairing injured or diseased brain and spinal cord in animal models. Hence, efficient generation of neural subtypes with correct phenotypes remains a challenge, although major hurdles still lie ahead in applying the human ES cell-derived neural cells clinically. We propose that careful studies on neural differentiation from human ES cells may provide more immediate answers to clinically relevant problems, such as drug discovery, mechanisms of disease and stimulation of endogenous stem cells.

Nanog is a critical homeodomain factor responsible for maintaining embryonic stem (ES) cell self-renewal and pluripotency. Of interest, Nanog expression is not homogeneous in the conventional culture of murine ES cells. A Nanog-high population expresses markers for pluripotent ES cells, whereas a Nanog-low population expresses markers for primitive endoderm, such as Gata6. Since the inner cell mass of early blastocysts has recently been reported to be heterogeneous in terms of Nanog and Gata6 expression, ES cells appear to closely resemble the developing stage from which they originate. We further demonstrate that Nanog can directly repress Gata6 expression through its binding to the proximal promoter region of the Gata6 gene and that overexpression of Nanog reduces heterogeneity during ES cell maintenance. Interestingly, Nanog heterogeneity does not correlate with the heterogeneous expression of stage-specific embryonic antigen-1, suggesting that multiple but overlapping levels of heterogeneity may exist in ES cells. These findings provide insight into the factors that control ES cell self-renewal and the earliest lineage commitment to primitive endoderm while also suggesting methods to promote homogeneity during ES cell maintenance. Disclosure of potential conflicts of interest is found at the end of this article.

Human embryonic stem cells offer a renewable source of a wide range of cell types for use in research and cell-based therapies. Characterizing these cells provides important information about their current state and affords relevant details for subsequent downstream manipulation. Prior to considering therapeutic applications, it is crucial that the cells are surveyed at a genetic and proteomic level during the extensive propagation, expansion and differentiation. Hence, a set of characterization tests to measure stem cell stability and identity--genomic, epigenomic and mitochondrial markers, as well as functional measures of utility, need to be developed. Thus, we outline a plan of standard assays that can be afforded by multiple laboratories to unambiguously test the quality of human embryonic stem cells. In this manuscript, we describe a comprehensive characterization of ReliCell hES1, the only human embryonic stem cell line reported from the Indian subcontinent. Our study employs gene expression analysis using quantitative reverse transcription-polymerase chain reaction and microarray, mitochondrial DNA sequencing, microRNA analysis, immunophenotyping and teratoma formation, in addition to demonstrating its capacity to propagate under feeder-free conditions.

The possibility of using stem cells for tissue engineering has greatly encouraged scientists to design new platforms in the field of regenerative and reconstructive medicine. Stem cells have the ability to rejuvenate and repair damaged tissues and can be derived from both embryonic and adult sources. Among cell types suggested as a cell source for tissue engineering (TE), human embryonic stem cells (hESCs) are one of the most promising candidates. Isolated from the inner cell mass of preimplantation stage blastocysts, they possess the ability to differentiate into practically all adult cell types. In addition, their unlimited self-renewal capacity enables the generation of sufficient amount of cells for cell-based TE applications. Yet, several important challenges are to be addressed, such as the isolation of the desired cell type and gaining control over its differentiation and proliferation. Ultimately, combing scaffolding and bioactive stimuli, newly designed bioengineered constructs, could be assembled and applied to various clinical applications. Here we define the culture conditions for the derivation of connective tissue lineage progenitors, design strategies, and highlight the special considerations when using hESCs for TE applications.

The generation of hepatocytes from embryonic stem cells (ESCs) holds considerable promise for basic and applied research. However, the unequivocal identification of hepatocytes in ESC differentiation strategies has been hampered by a lack of hepatocyte-specific markers. Recent studies are beginning to address this issue with the identification of hepatocyte-specific genes and the production of hepatocytes from intermediate cell types like definitive endoderm. Assuming the successful identification of ESC-derived hepatocytes, the next challenge will be in balancing the proliferation and differentiation of these cells in order to generate usable numbers of functional hepatocytes in vitro.

Human embryonic stem cells (hESCs) provide great opportunities for regenerative medicine, pharmacological and toxicological investigation, and the study of human embryonic development. These applications require proper derivation, maintenance, and extensive characterization of undifferentiated cells before being used for differentiation into cells of interest. Undifferentiated hESCs possess several unique features, including their extensive proliferation capacity in the undifferentiated state, ability to maintain a normal karyotype after long-term culture, expression of markers characteristic of stem cells, high constitutive telomerase activity, and capacity to differentiate into essentially all somatic cell types. This chapter will summarize the current development in culture conditions and provide technical details for the evaluation and characterization of hESCs.

Restoration of cardiac function by replacement of diseased myocardium with functional cardiac myocytes may offer a potential cure for cardiac disease and will likely revolutionize treatment methods. During the past 20 years, we have seen the development of tissue engineering; among these types of tissue engineering is cardiac tissue engineering. This type of cardiac tissue engineering includes growing neonatal cardiomyocytes on preformed polymers, liquid collagen, and temperature-responsive surfaces. It also includes the application of neonatal rat or chick cardiomyocytes to skeletal myoblasts, mesenchymal stem cells and embryonic stem cells, static culture, and bioreactor and stretching cultivation. Progress has come step-by-step, but, in recent years, with great technological advances, the progress has been accelerating, moving this area of research from dream to reality. The engineered cardiac tissue not only reproduces in vitro, but it can also be shaped so that it will, at some time, be able to form valves or endothelial lining. This chapter describes the currently used protocols for cardiac tissue engineering: liquid collagen-based cardiac tissue engineering and cell sheet-based cardiac tissue engineering, especially cardiac tissue engineering using cardiomyocytes derived from embryonic stem cells.

The worldwide status of rapidly increasing demand for organ and tissue transplantation has promoted tissue engineering as a promising alternative, in particular the use of stem cells. Human embryonic stem cells (hESCs) have the advantage of differentiating to all cell types in the body and high proliferation capabilities. Considering their ability to organize into complex multi-cell-type structures during embryonic-like differentiation, hESCs can potentially provide a source of cells for tissue engineering applications and meet the growing demand for viable human tissue structures in therapeutic clinical application. This chapter describes first steps toward realizing this goal gathered from our experience in growing and differentiating hESCs in three dimensions.

Gene trapping in embryonic stem cells (ESCs) generates random, sequence-tagged insertional mutations, which can often report the gene expression pattern of the mutated gene. This mutagenesis strategy has often been coupled to expression or function-based assays in gene discovery screens. The availability of the mouse genome sequence has shifted gene trapping from a gene discovery platform to a high-throughput mutagenesis platform. At present, a concerted worldwide effort is underway to develop a library of loss-of-function mutations in all mouse genes. The International Gene Trap Consortium (IGTC) is leading the way by making a first pass of the genome by random mutagenesis before a high-throughput gene targeting program takes over. In this chapter, we provide a methods guidebook to exploring and using the IGTC resource, explain the different kinds of vectors and insertions that reside in the different libraries, and provide advice and methods for investigators to design novel expression-based "cottage industry" screens.

Our project was to determine whether embryonic stem (ES) cells could be induced to differentiate into corneal epithelia by superficial corneoscleral limbal stroma. To achieve this goal, ES-GFP cell line D3 was pre-induced by retinoic acid (RA). The pre-induced cells were seeded on deepithelialized superficial corneoscleral slices (SCSS) to form a monolayer, and divided into three groups. Group 1 was cultured and passaged in vitro for direct detection. Group 2 was exposed to air-liquid interfaces for 10 days and implanted into the subcutaneous layer of nude mice for 2 weeks for further induction in vivo. Group 3 was cultured in vitro without any inducing factors for control. There were no teratomas found in nude mice which were implanted with differentiated ES cells after two weeks. The differentiated cells showed an appearance of epithelia both in vitro and in vivo. Expression of CK3, P63 and PCNA was detected by immunohistochemical staining in the differentiated cells in group 1 and 2. Microvillis and zonula occludens were observed on the surface of the differentiated cells under an electron microscope. In the control group, ES cells differentiated freely without any inducing factors. Most cells were shed and formed a neuronal dendrite-like structure, and a minority of cells appeared polymorphic. These results demonstrate that ES cells can differentiate into corneal epithelia on the surface of SCSS under the controlled condition. Differentiated ES cells could be used as epithelial seeding cells for the reconstruction of ocular surface and corneal tissue engineering in the future.