Wnt signaling promotes hematoendothelial cell development from human embryonic stem cells

Human embryonic stem cells (hESCs) provide an important means to effectively study soluble and cell-bound mediators that regulate development of early blood and endothelial cells in a human model system. Here, several complementary methods are used to demonstrate canonical Wnt signaling is important for development of hESC-derived cells with both hematopoietic and endothelial potential. Analyses using both standard flow cy-tometry, as well the more detailed high-throughput image scanning flow cytometry, characterizes sequential development of distinct early developing CD34(bright)CD31(+)Flk1(+) cells and a later population of CD34(dim)CD45(+) cells. While the CD34(bright)CD31(+)Flk1(+) have a more complex morphology and can develop into both endothelial cells and hematopoietic cells, the CD34(dim)CD45(+) cells have a simpler morphology and give rise to only hematopoietic cells. Treatment with dickkopf1 to inhibit Wnt signaling results in a dramatic decrease in development of cells with hematoendothelial potential. In addition, activation of the canonical Wnt signaling pathway in hESCs by coculture with stromal cells that express Wnt1, but not use of noncanonical Wnt5-expressing stromal cells, results in an accelerated differentiation and higher percentage of CD34(bright)CD31(+)Flk1(+) cells at earlier stages of differentiation. These studies effectively demonstrate the importance of canonical Wnt signaling to mediate development of early hematoendothelial progenitors during human development.     

Immortalized keratinocyte lines derived from human embryonic stem cells

Cells of the human embryonic stem (hES) cell line H9, when cultured in the form of embryoid bodies, give rise to cells with markers of the keratinocyte of stratified squamous epithelia. Keratinocytes also form in nodules produced in scid mice by injected H9 cells; the hES-derived keratinocytes could be recovered in culture, where their colonies underwent a peculiar form of fragmentation. Whether formed from embryoid bodies or in nodules, hES-derived keratinocytes differed from postnatal keratinocytes in their much lower proliferative potential in culture; isolated single keratinocytes could not be expanded into mass cultures. Although their growth was not improved by transduction with the hTERT gene, these keratinocytes were immortalized by transduction with the E6E7 genes of HPV16. Clonally derived lines isolated from E6E7-transduced keratinocytes continued to express markers of the keratinocyte lineage, but the frequency with which they terminally differentiated was reduced compared with keratinocytes cultured from postnatal human epidermis. If other hES-derived somatic cell types also prove to be restricted in growth potential, not identical to the corresponding postnatal cell types, and to require immortalization for clonal isolation and expansion, these properties will have to be considered in planning their therapeutic use.     

Derivation of germ-line-competent embryonic stem cell lines from preblastocyst mouse embryos

The first differentiation event of the mammalian embryo is thought to occur during blastulation and results in two populations of cells, the inner cell mass (ICM) and the trophectoderm. Most embryonic stem (ES) cell lines have been derived from the ICM or a further subset of ICM cells known as the epiblast. There appears to be a limited period of embryonic development during which pluripotent ES cells can be adapted from the cells of the blastocyst to culture. A method is presented here that allows ES cell lines to be isolated from preblastocyst mouse embryos. These lines were derived from 129S2/SvHsd mouse morulae and earlier cleavage stages with high efficiency. The lines expressed genes and antigens characteristic of pluripotent ES cells. XY cell lines remained karyotypically stable through extensive passaging and produced germ-line-competent chimeras upon blastocyst injection. These results suggest that true ES cells can be derived from embryos explanted at any stage of preimplantation development in the mouse. This finding raises the interesting question of whether ES cell lines derived from embryos at different stages of preimplantation development possess the same potential.     

Hematopoietic development of vav-/- mouse embryonic stem cells.

The vav protooncogene product is expressed nearly exclusively in hematopoietic lineages and contains several structural motifs (SH2/SH3 domains and a dbl-oncogene homology region) typical of proteins functioning in signaling pathways. To ascertain if vav expression is required for hematopoiesis we generated vav-negative mouse embryonic stem cells by gene targeting and examined the consequences of loss of vav function on erythroid and myeloid development in vitro and in vivo. In conflict with the conclusions drawn from expression of antisense vav RNA in embryonic stem cells [Wulf, G. M., Adra, C. N. & Lim, B. (1993) EMBO J. 12, 5065-5074], we observed erythroid and myeloid development in the absence of vav. These experiments demonstrate that vav expression is not absolutely required for hematopoietic development.     

Renin-angiotensin system and hemangioblast development from human embryonic stem cells

Human embryonic stem cells (hESCs) offer the opportunity to create a novel source of blood cells for transfusion, transplantation and cancer immunotherapy. Identification of sequential progenitors leading to blood development, as well as a detailed understanding of the molecular mechanisms of hematopoietic lineage specification and diversification from hESCs, will be critical to advance technologies for large-scale production of blood cells and in vitro generation of hematopoietic stem cells. Multiple lines of evidence suggest that hematopoiesis, both in vivo during embryogenesis and in vitro from hESCs, is initiated from hemangioblasts; cells with the potential to generate both hematopoietic and endothelial cells. However, the phenotypic and functional properties of hemangioblasts remain largely unknown. The paper from Zambidis et al. is the first demonstration that hemangioblasts generated from hESCs express angiotensin-converting enzyme (CD143). More importantly, the current study demonstrates that the renin-angiotensin system plays a critical role in the hemangioblast fate decision to produce either blood or endothelial cells. These findings could be exploited for developing novel cellular and drug therapies for hematological and vascular diseases.     

Hematopoietic stem cell development,aging and functional failure

Hematopoietic stem cells (HSCs) are found in yolk sac, fetal liver, umbilical cord blood, placenta, and amniotic fluid during mammalian embryonic development. In adults, HSCs reside in marrow cavity of long bones where they self-renew and differentiate to replenish short-lived mature blood cells. HSCs exist in very low frequencies within specific “niches” where they interact with the surrounding environment through molecular associations. Overall HSC function can last much longer than a normal lifetime, but HSCs do show functional senescence with characteristic features of decreased self-renewal, reduced clonal stability, reduced homing and engraftment, and biased lineage commitment. The progressive shortening of telomeres with increasing age, especially under conditions with specific mutations in the telomerase gene complex, could predispose patients to HSC dysfunction and bone marrow failure diseases. Continuous investigation into HSC biology should facilitate the utilization of HSCs as a therapeutic modality and helps to prevent HSC malfunction.     

Blood-forming endothelium in human ontogeny: lessons from in utero development and embryonic stem cell culture

During the early weeks of human gestation, hematopoietic cells first emerge within the extraembryonic yolk sac (primitive hematopoiesis) and secondarily within the truncal arteries of the embryo. This second wave includes the stem cells giving rise to adult-type lymphohematopoiesis. In both yolk sac blood islands and embryonic aorta, hematopoietic cells arise in the immediate vicinity of vascular endothelial cells. In vitro hematopoietic differentiation of endothelial cells stringently sorted from human embryonic and fetal blood-forming tissues has demonstrated that primitive endothelium lies at the origin of incipient hematopoiesis. These anatomically and temporally localized blood-forming endothelial cells are ultimately derived from a rare subset of mesodermal angio-hematopoietic stem cells, or hemangioblasts. The evidence for an early progenitor of blood-forming cells within the walls of human embryonic blood vessels concurs with parallel data obtained from lower vertebrate, avian, and murine models. Importantly, converging results have recently been obtained with in vitro differentiated human embryonic stem cells, in which we have modeled primitive and definitive hematopoiesis via an endothelium-like developmental intermediate.     

Transgenesis by means of blastocyst-derived embryonic stem cell lines.

This study demonstrates that blastocyst-derived embryonic stem cells (ES cells) can be used as a vehicle for transgenesis. The method is nearly as efficient as other methods, and the introduced neomycin phosphotransferase (neo) gene is stably transmitted through several generations with no apparent loss in G418 resistance. An important factor contributing to the efficiency of this process is the rigorous selection, before blastocyst injection, of genetically transformed cells for in vitro developmental pluripotency. One of the advantages of the ES cell route to transgenesis is that it provides investigators with the opportunity to screen for the desired genetic alterations before reintroducing the ES cells into the animal.     

PGD-derived human embryonic stem cell lines as a powerful tool for the study of human genetic disorders

Human embryonic stem (ES) cells are derived from the inner cell mass (ICM) of blastocyst embryos. They are established from spare embryos that have been obtained by in vitro fertilization (IVF) and donated for research purposes. The ICM-derived cell lines have two unique properties, they can be propagated indefinitely in culture and have the potential to develop into practically any cell type in vitro and in vivo. Human embryonic stem (hES) cells carrying specific mutations can be used as a valuable tool for studying genetic disorders in human. One favorable approach to obtain such mutant ES cell lines is their derivation from affected preimplantation genetic diagnosed (PGD) embryos. This review focuses on the importance of deriving human ES cell lines from genetically abnormal embryos, especially in cases where no good cellular and/or animal models exist.     

Hematopoietic differentiation of rhesus monkey embryonic stem cells

Several lines of embryonic stem cells (ESC) have been established from rhesus monkey blastocysts. We have examined two of these cell lines for their potential for generating hematopoietic progenitors in cell culture, and we identified culture conditions, including supplementation with bone morphogenetic proteins (BMP), that result in hematopoietic differentiation of rhesus ESC with high efficiency. We have also characterized the resulting hematopoietic progenitor cells for their patterns of gene expression, as compared to those of hematopoietic progenitor cells harvested from rhesus monkey bone marrow. Of more than 60 genes examined in this manner, CD34+/CD38- cells derived from embryonic stem cells and those obtained from bone marrow demonstrated very similar patterns of gene expression. However, with integrin alphaL, IL-6 receptor, and flt-3 gene expression was greatly diminished or absent in CD34+/CD38- cells derived from the ESC, whereas the bone marrow-derived progenitors showed substantial expression of all of these genes. When the same type of comparison was done with mouse (D3 and CCE) as well as human (H1) embryonic stem cells, in each case comparing ESC-derived hematopoietic progenitors with those harvested from bone marrow, the only consistent deficiency of gene expression was that of flt-3. In hematopoietic precursors derived from mouse ESC, globin-gene expression has previously been shown to be a useful index of the embryological maturity of the cells, and we also examined globin-gene expression in rhesus monkey ESC-derived hematopoietic precursor cells, using a semiquantitative technique. CD34+/CD38- cells demonstrated expression of the epsilon- and gamma-globin genes, but negligible levels of beta globin, suggesting that these cells were at the developmental stage in which the yolk sac and fetal liver are the primary sites of hematopoiesis.     

Integrative genomic and functional analyses reveal neuronal subtype differentiation bias in human embryonic stem cell lines

The self-renewal and differentiation potential of human embryonic stem cells (hESCs) suggests that hESCs could be used for regenerative medicine, especially for restoring neuronal functions in brain diseases. However, the functional properties of neurons derived from hESC are largely unknown. Moreover, because hESCs were derived under diverse conditions, the possibility arises that neurons derived from different hESC lines exhibit distinct properties, but this possibility remains unexplored. To address these issues, we developed a protocol that allows stepwise generation from hESCs of cultures composed of approximately 70-80% human neurons that exhibit spontaneous synaptic network activity. Comparison of neurons derived from the well characterized HSF1 and HSF6 hESC lines revealed that HSF1- but not HSF6-derived neurons exhibit forebrain properties. Accordingly, HSF1-derived neurons initially form primarily GABAergic synaptic networks, whereas HSF6-derived neurons initially form glutamatergic networks. microRNA profiling revealed significant expression differences between the two hESC lines, suggesting that microRNAs may influence their distinct differentiation properties. These observations indicate that although both HSF1 and HSF6 hESCs differentiate into functional neurons, the two hESC lines exhibit distinct differentiation potentials, suggesting that they are preprogrammed. Information on hESC line-specific differentiation biases is crucial for neural stem cell therapy and establishment of novel disease models using hESCs.     

Hematopoiesis from pluripotent stem cell lines

Embryonic stem cells (ESCs) can differentiate into various types of hematopoietic cells (HPCs) when placed in an appropriate environment. Various methods for the differentiation of ESCs into specific HPC lineages have been developed using mouse ESCs. These ESC-differentiation methods have been utilized also as an in vitro model to investigate hematopoiesis in embryos and they provided critical perceptions into it. These methods have been adapted for use with human ESCs, which have the possibility of being employed in regenerative medicine; further improvement of these methods may lead to the efficient production of HPCs for use in transfusions. The generation of transplantable hematopoietic stem cells is a medical goal that is still difficult to achieve. Recently, induced pluripotent stem (iPS) cells have been established from differentiated cells. Thereby, iPS cells have expanded further possibilities of the use of pluripotent stem cell lines in clinical application. Indeed, iPS cells have been established from cells with disease genes and those which have undergone reprogramming and targeting have generated phenotypically normal HPCs. Here, we mainly summarize the recent progress in research on hematopoiesis conducted with ESCs and iPS cells.