Activin a promotes hematopoietic fated mesoderm development through upregulation of brachyury in human embryonic stem cells

The development of the hematopoietic system involves multiple cellular steps beginning with the formation of the mesoderm from the primitive streak, followed by emergence of precursor populations that become committed to either the endothelial or hematopoietic lineages. A number of growth factors such as activins and fibroblast growth factors (FGFs) are known to regulate the early specification of hematopoietic fated mesoderm, notably in amphibians. However, the potential roles of these factors in the development of mesoderm and subsequent hematopoiesis in the human have yet to be delineated. Defining the cellular and molecular mechanisms by which combinations of mesoderm-inducing factors regulate this stepwise process in human cells in vitro is central to effectively directing human embryonic stem cell (hESC) hematopoietic differentiation. Herein, using hESC-derived embryoid bodies (EBs), we show that Activin A, but not basic FGF/FGF2 (bFGF), promotes hematopoietic fated mesodermal specification from pluripotent human cells. The effect of Activin A treatment relies on the presence of bone morphogenetic protein 4 (BMP4) and both of the hematopoietic cytokines stem cell factor and fms-like tyrosine kinase receptor-3 ligand, and is the consequence of 2 separate mechanisms occurring at 2 different stages of human EB development from mesoderm to blood. While Activin A promotes the induction of mesoderm, as indicated by the upregulation of Brachyury expression, which represents the mesodermal precursor required for hematopoietic development, it also contributes to the expansion of cells already committed to a hematopoietic fate. As hematopoietic development requires the transition through a Brachyury+ intermediate, we demonstrate that hematopoiesis in hESCs is impaired by the downregulation of Brachyury, but is unaffected by its overexpression. These results demonstrate, for the first time, the functional significance of Brachyury in the developmental program of hematopoietic differentiation from hESCs and provide an in-depth understanding of the molecular cues that orchestrate stepwise development of hematopoiesis in a human system.     

Hematopoiesis from human embryonic stem cells: overcoming the immune barrier in stem cell therapies

The multipotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source of stem cells for transplant therapies and of vital importance given the shortage in organ donation. Recent studies suggest some immune privilege associated with hESC-derived tissues. However, the adaptability of the immune system makes it unlikely that fully differentiated tissues will permanently evade immune rejection. One promising solution is to induce a state of immune tolerance to a hESC line using tolerogenic hematopoietic cells derived from it. This could provide acceptance of other differentiated tissues from the same line. However, this approach will require efficient multilineage hematopoiesis from hESCs. This review proposes that more efficient differentiation of hESCs to the tolerogenic cell types required is most likely to occur through applying knowledge gained of the ontogeny of complex regulatory signals used by the embryo for definitive hematopoietic development in vivo. Stepwise formation of mesoderm, induction of definitive hematopoietic stem cells, and the application of factors key to their self-renewal may improve in vitro production both quantitatively and qualitatively.     

Inability of human induced pluripotent stem cell-hematopoietic derivatives to downregulate microRNAs in vivo reveals a block in xenograft hematopoietic regeneration

Hematopoietic stem cells (HSCs) can regenerate the entire hematopoietic system in vivo, providing the most relevant criteria to measure candidate HSCs derived from human embryonic stem cell (hESC) or induced pluripotent stem cell (hiPSC) sources. Here we show that, unlike primitive hematopoietic cells derived from hESCs, phenotypically identical cells derived from hiPSC are more permissive to graft the bone marrow of xenotransplantation recipients. Despite establishment of bone marrow graft, hiPSC-derived cells fail to demonstrate hematopoietic differentiation in vivo. However, once removed from recipient bone marrow, hiPSC-derived grafts were capable of in vitro multilineage hematopoietic differentiation, indicating that xenograft imparts a restriction to in vivo hematopoietic progression. This failure to regenerate multilineage hematopoiesis in vivo was attributed to the inability to downregulate key microRNAs involved in hematopoiesis. Based on these analyses, our study indicates that hiPSCs provide a beneficial source of pluripotent stem cell-derived hematopoietic cells for transplantation compared with hESCs. Since use of the human-mouse xenograft models prevents detection of putative hiPSC-derived HSCs, we suggest that new preclinical models should be explored to fully evaluate cells generated from hiPSC sources.     

Abnormal CpG island methylation occurs during in vitro differentiation of human embryonic stem cells

Directed differentiation of human embryonic stem cells (hESCs) into specific somatic cells holds great promise for cell replacement therapies. However, it is unclear if in vitro hESC differentiation causes any epigenetic abnormality such as hypermethylation of CpG islands. Using a differential methylation hybridization method, we identified 65 CpG islands (out of 4608 CpG islands or 1.4%) that exhibited increased DNA methylation during the conversion of hESCs into neural progenitor/stem cells (NPCs). These methylated CpG islands belong to genes in cell metabolism, signal transduction and cell differentiation, which are distinctively different from oncogenic CpG island hypermethylation observed in cancer-related genes during tumorigenesis. We further determined that methylation in these CpG islands, which is probably triggered by de novo DNA methyltransferase Dnmt3a, is abnormally higher in hESC-NPCs than in primary NPCs and astrocytes. Correlating with hypermethylation in promoter CpG islands of metabolic enzyme gene CPT1A and axoneme apparatus gene SPAG6, levels of CPT1A and SPAG6 mRNAs are significantly reduced in hESC-NPCs when compared with hESCs or primary neural cells. Because CPT1A is involved in lipid metabolism and CPT1A deficiency in human is associated with the hypoketotic hypoglycemia disorder, the reduced CPT1A expression in hESC-NPCs raises a potential concern for the suitability of these cells in cell transplantation. Collectively, our data show that abnormal CpG island methylation takes place in a subset of genes during the differentiation/expansion of hESC derivatives under current culture conditions, which may need to be monitored and corrected in future cell transplantation studies.     

Chromatin insulator elements block transgene silencing in engineered human embryonic stem cell lines at a defined chromosome 13 locus

Lineage reporters of human embryonic stem cell (hESC) lines are useful for differentiation studies and drug screening. Previously, we created reporter lines driven by an elongation factor 1 alpha (EF1α) promoter at a chromosome 13q32.3 locus in the hESC line WA09 and an abnormal hESC line BG01V in a site-specific manner. Expression of reporters in these lines was maintained in long-term culture at undifferentiated state. However, when these cells were differentiated into specific lineages, reduction in reporter expression was observed, indicating transgene silencing. To develop an efficient and reliable genetic engineering strategy in hESCs, we used chromatin insulator elements to flank single-copy transgenes and integrated the combined expression constructs via PhiC31/R4 integrase-mediated recombination technology to the chromosome 13 locus precisely. Two copies of cHS4 double-insulator sequences were placed adjacent to both 5' and 3' of the promoter reporter constructs. The green fluorescent protein (GFP) gene was driven by EF1α or CMV early enhancer/chicken β actin (CAG) promoter. In the engineered hESC lines, for both insulated CAG-GFP and EF1α-GFP, constitutive expression at the chromosome 13 locus was maintained during prolonged culture and in directed differentiation assays toward diverse types of neurons, pancreatic endoderm, and mesodermal progeny. In particular, described here is the first normal hESC fluorescent reporter line that robustly expresses GFP in both the undifferentiated state and throughout dopaminergic lineage differentiation. The dual strategy of utilizing insulator sequences and integration at the constitutive chromosome 13 locus ensures appropriate transgene expression. This is a valuable tool for lineage development study, gain- and loss-of-function experiments, and human disease modeling using hESCs.     

Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy

We develop a label-free optical technique to image and discriminate undifferentiated human embryonic stem cells (hESCs) from their differentiating progenies in vitro. Using intrinsic cellular fluorophores, we perform fluorescence lifetime microscopy (FLIM) and phasor analysis to obtain hESC metabolic signatures. We identify two optical biomarkers to define the differentiation status of hESCs: Nicotinamide adenine dinucleotide (NADH) and lipid droplet-associated granules (LDAGs). These granules have a unique lifetime signature and could be formed by the interaction of reactive oxygen species and unsaturated metabolic precursor that are known to be abundant in hESC. Changes in the relative concentrations of these two intrinsic biomarkers allow for the discrimination of undifferentiated hESCs from differentiating hESCs. During early hESC differentiation we show that NADH concentrations increase, while the concentration of LDAGs decrease. These results are in agreement with a decrease in oxidative phosphorylation rate. Single-cell phasor FLIM signatures reveal an increased heterogeneity in the metabolic states of differentiating H9 and H1 hESC colonies. This technique is a promising noninvasive tool to monitor hESC metabolism during differentiation, which can have applications in high throughput analysis, drug screening, functional metabolomics and induced pluripotent stem cell generation.     

Differentiation of human embryonic stem cells into corneal epithelial-like cells by in vitro replication of the corneal epithelial stem cell niche

Human embryonic stem cells (hESCs) are pluripotent cells capable of differentiating into any cell type of the body. It has long been known that the adult stem cell niche is vital for the maintenance of adult stem cells. The cornea at the front of the eye is covered by a stratified epithelium that is renewed by stem cells located at its periphery in a region known as the limbus. These so-called limbal stem cells are maintained by factors within the limbal microenvironment, including collagen IV in basement membrane and limbal fibroblasts in the stroma. Because this niche is very specific to the stem cells (rather than to the more differentiated cells) of the corneal epithelium, it was hypothesized that replication of these factors in vitro would result in hESC differentiation into corneal epithelial-like cells. Indeed, here we show that culturing of hESC on collagen IV using medium conditioned by the limbal fibroblasts results in the loss of pluripotency and differentiation into epithelial-like cells. Further differentiation results in the formation of terminally differentiated epithelial-like cells not only of the cornea but also of skin. Scanning electron microscopy shows that some differences exist between hESC-derived and adult limbal epithelial-like cells, necessitating further investigation using in vivo animal models of limbal stem cell deficiency. Such a model of hESC differentiation is useful for understanding the early events of epithelial lineage specification and to the eventual potential application of epithelium differentiated from hESC for clinical conditions of epithelial stem cell loss. Disclosure of potential conflicts of interest is found at the end of this article.     

An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells

Human embryonic stem cells (hESCs) promise to revolutionize reparative medicine through their potential in developing cell replacement therapies for diseases like diabetes and parkinsonism. Most of the existing hESC lines available for research, including all National Institutes of Health-registered lines, have been derived and maintained on mouse embryonic fibroblast feeders in the presence of xenoproteins. For future clinical application, many more hESC lines derived and grown in current good manufacturing practice, good tissue culture practice, and xeno-free conditions need to be developed. Concurrently, effective cryopreservation methods that prevent or limit the accidental contact of hESCs with nonsterile liquid nitrogen during periods of long-term storage have to be formulated. We describe a safe, xeno-free cryopreservation protocol for hESCs involving vitrification in closed sealed straws using human serum albumin as opposed to fetal calf serum as the main protein source in the cryoprotectant and long-term storage in the vapor phase of liquid nitrogen. After thaw, hESCs exhibited high thaw-survival rates and low differentiation rates, remained pluripotent, and maintained normal diploid karyotypes throughout extended passage. The cryopreservation technique we describe here should complement xeno-free culture conditions for hESCs already in refinement and will prove very useful for the setting up of hESC banks throughout the world.     

Restriction landmark genome scanning identifies culture-induced DNA methylation instability in the human embryonic stem cell epigenome

Widespread provision of human embryonic stem cells (hESCs) for therapeutic use, drug screening and disease modelling will require cell lines sustainable over long periods in culture. Since the short-term, in vitro culture of mammalian embryos can result in DNA methylation changes, the epigenetic stability of hESCs warrants investigation. Existing hESC lines have been derived and cultured under diverse conditions, providing the potential for programming differential changes into the epigenome that may result in inter-line variability over and above that inherited from the embryo. By examining the DNA methylation profiles of > 2000 genomic loci by Restriction Landmark Genome Scanning, we identified substantial inter-line epigenetic distance between six independently derived hESC lines. Lines were found to inherit further epigenetic changes over time in culture, with most changes arising in the earliest stages post-derivation. The loci affected varied between lines. The majority of culture-induced changes (82.3-87.5%) were stably inherited both within the undifferentiated cells and post-differentiation. Adapting a line to a serum-free culture system resulted in additional epigenetic instability. Overall 80.5% of the unstable loci uncovered in hESCs have been associated previously with an adult tumour phenotype. Our study shows that current methods of hESC propagation can rapidly programme stable and unpredictable epigenetic changes in the stem cell genome. This highlights the need for (i) novel screening strategies to determine the experimental utility and biosafety of hESCs and (ii) optimization and standardization of procedures for the derivation and culture of hESC lines that minimize culture-induced instability.     

Human embryonic stem cells: lessons from stem cell niches in vivo

In vivo the stem cell niche is an essential component in controlling and maintaining the stem cells' ability to survive and respond to injury. Human embryonic stem cells (hESCs) appear to be an exception to this rule as they can be removed from their blastocytic microenvironment and maintained indefinitely in vitro. However, recent observations reveal the existence of an autonomously derived in vitro hESC niche. This provides a previously unappreciated mechanism to control hESC expansion and differentiation. Recognizing this, it may now be possible to take aspects of in vivo stem cell niches, namely extracellular matrices, paracrine signals and accessory cell types, and exploit them in order to gain fidelity in directed hESC differentiation. In doing so, routine customization of hESC lines and their application in regenerative therapies may be further enhanced using unique hESC niche-based approaches.