Characterizing endothelial cells derived from the murine embryonic stem cell line CCE

Embryonic stem cells (ESC) are defined by two main properties of self-renewal and their multipotency to differentiate into virtually all cell types of the body, including endothelial cells. ESCs have been widely regarded as an unlimited source of cells in regeneration medicine and also an ideal in vitro model to investigate complex developmental processes. Here, we report a simple and efficient in vitro model to derive a nearly pure population of endothelial cells from a murine ESC line. CCE ES cells are exposed to alpha-MEM medium containing 10% FBS for 4 days and then cultured in endothelial basal-2 medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), and 2% FBS for 42 days. The cells acquired a relatively uniform endothelial cell morphology and were able to propagate and expand in culture. When murine ES cell-derived endothelial cells (MESDECs) were cultured on Matrigel and incubated for 48 h, vessel-like tube structures consisting of CD31 (PECAM-1) or BS-1 immunoreactive cells were developed. Immunocytochemistry and RT-PCR analyses revealed that MESDECs express endothelial cell-specific marker proteins such as Flk-1, PECAM-1, Tie-1, and Tie-2, in which the expressions persist for long periods of time after differentiation. The cells were also capable of taking up acetylated low-density lipoprotein (LDL) in culture. Our data suggest that MESDECs could provide a suitable in vitro model to study molecular events involved in vascular development and open up a new therapeutic strategy in regeneration medicine of cardiovascular disorders.     

Endothelial cells derived from human embryonic stem cells

Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.     

Temporally restricted substrate interactions direct fate and specification of neural precursors derived from embryonic stem cells

It was, until now, not entirely clear how the nervous system attains its cellular phenotypic diversity and wired complexity during development. Here we describe how environmental interactions alone can modify the development of neurogenic precursor cells. Upon evaluating distinct growth-permissive substrates in an embryonic stem cell-neurogenesis assay, we found that laminin, fibronectin, and gelatin instruct neural fate and alter the functional specification of neurons when applied at distinct stages of development. Changes in phenotypic, electrophysiological, and molecular characteristics could resemble cellular events and interactions in the early embryonic brain and may explain why these extracellular matrix components transiently demarcate certain developing brain structures.     

Functional endothelial cells derived from rhesus monkey embryonic stem cells

We have used rhesus monkey embryonic stem (ES) cells to study endothelial cell development. Rhesus ES cells (R366.4 cell line) exposed to medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), and epidermal growth factor (EGF) assumed a relatively uniform endothelial cell morphology and could be propagated and expanded with a consistent phenotype and normal karyotype. When placed in Matrigel, these rhesus ES cell-derived endothelial cells (RESDECs) formed capillary-like structures characteristic of endothelial cells. Immunohistochemical and flow cytometric analysis of RESDECs showed that they take up acetylated low-density lipoprotein (LDL), express CD146, von Willebrand factor, and the integrin alpha v beta 3, and bind the lectin ulex europaeus agglutinin-1. These cells also express the VEGF receptor Flk-1 and secrete VEGF. When introduced in a Matrigel plug implanted subcutaneously in mice, RESDECs formed intact vessels and recruited new endothelial cell growth. In vivo function was demonstrated by coinjection of RESDECs with murine tumor cells subcutaneously into immunocompromised adult mice. RESDECs injected alone did not form measurable tumors. Tumor cells grew more rapidly and had increased vascularization when coinjected with the RESDECs. Immunohistochemical staining demonstrated that the RESDECs participated in forming the tumor neovasculature. RESDECs provide a novel means to examine the mechanisms of endothelial cell development, and may open up new therapeutic strategies.     

CD34+CD38- hematopoietic precursors derived from human embryonic stem cells exhibit an embryonic gene expression pattern

Gene expression patterns of CD34(+)CD38(-) cells derived from human embryonic stem cells (ESCs) were compared with those of cells isolated from adult human bone marrow (BM) using microarrays; 1692 and 1494 genes were expressed at levels at least 3-fold above background in cells from BM and ESCs, respectively. Of these, 494 showed similar levels of expression in cells from both sources, 791 genes were overexpressed in cells from BM (BM versus ESCs, at least 2-fold), and 803 genes were preferentially expressed in cells from ESCs (ESCs versus BM, at least 2-fold). The message of the flt-3 gene was markedly decreased in cells from ESCs, whereas there was substantial flt-3 expression in cells from BM. High levels of embryonic epsilon-globin expression were observed-but no adult beta-globin message-in CD34(+)CD38(-) cells from ESCs, whereas high levels of beta-globin expression-but no embryonic epsilon-globin message-could be detected in cells from BM. Furthermore, high levels of major histocompatibility complex (MHC) gene expression were demonstrated in cells from BM but very low levels of MHC message in corresponding cells from ESCs. These observations demonstrate that CD34(+)CD38(-) cells derived from ESCs correspond consistently to an early developmental stage at which the yolk sac and fetal liver are the primary sites of hematopoiesis.     

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.     

Human embryonic stem cells for myocardial regeneration

Terminally differentiated adult cardiomyocytes have limited regenerative capacity and therefore any significant cell loss may result in the development of progressive heart failure. Cell replacement therapy is a promising new approach for myocardial repair but has been limited by the paucity of cell sources for functional human cardiomyocytes. The recent establishment of the human pluripotent embryonic stem (ES) cell lines may present a novel solution for this cell-sourcing problem. The ES lines were derived from human blastocysts and were shown to be capable of continuous undifferentiated proliferation, in vitro, while retaining the capability to form derivatives of all three germ layers. More recently, a reproducible cardiomyocyte differentiation system was established using these unique cells. The current review describes the derivation and properties of human ES cells and the characteristics of the cardiomyocytes derived using this unique differentiating system. The possible applications in several research and clinical areas are discussed as well as the steps required to fully harness the potential of this new technology in the fields of myocardial cell replacement and tissue engineering.     

骨髓间充质干细胞在大鼠肝纤维化形成中的作用

研究显示,小鼠肝纤维化模型中给予一定数量的骨髓间充质干细胞(BMSC)能减轻肝纤维化程度,但也有研究持否定观点。本研究通过研究BMSC尾静脉移植后在纤维化肝脏的定位、分化来说明BMSC在肝纤维化大鼠的影响及其机制。     

Long-lasting in vitro hematopoiesis derived from primate embryonic stem cells

OBJECTIVE: Induction of hematopoietic cells from human embryonic stem (ES) cells has been reported recently. However, before cells derived from human ES cells can be used in the clinic, preclinical studies using these cells in experimental primates will be necessary. Therefore, we attempted to establish a method to induce hematopoietic cells robustly and abundantly from primate ES cells. METHODS: A primate ES cell line, CMK-6, derived from the cynomolgus monkey was used in this study. We adapted a method to induce hematopoiesis from CMK-6 cells on feeder cells, and tested the effectiveness of three kinds of feeder cell lines (OP9, C2C12, and C3H10T1/2). In addition, we tested the effect of vascular endothelial growth factor (VEGF) and insulin-like growth factor-II (IGF-II) on hematopoiesis induction from CMK-6 cells. RESULTS: VEGF and IGF-II showed an extremely strong synergistic effect to induce hematopoiesis from CMK-6 cells. C3H10T1/2 cells proved to be very useful for the induction of hematopoiesis from CMK-6 cells, and the production of blood cells on C3H10T1/2 cells has been maintained as long as 5 months. During this long period, ES cell derivatives continuously produced mature blood cells, including terminally differentiated cells. CONCLUSION: We have developed an original method to produce enriched blood cells abundantly from primate ES cells for an extremely long period. This method may represent a good in vitro model for studying primate hematopoiesis and related diseases. Furthermore, our method may be useful for preclinical studies of transfusion therapy using blood cells derived from ES cells in experimental primate systems.     

Adipose‐derived stem cells: A candidate for liver regeneration

The scarcity of donor livers and the impracticality of hepatocyte transplantation represent the biggest obstacles for the treatment of liver failure. Adipose‐derived stem cells, with their ability to differentiate into the hepatic lineage, provide a reliable alternative cell source with clear ethical and practical advantages. Moreover, adipose‐derived stem cells can effectively repair liver damage by the dominant indirect pattern and increase the number of hepatocytes by the secondary direct pattern. In recent years, the development of the indirect pattern, which mainly includes immunomodulatory and trophic effects, has become a hot topic in the field of cell engineering. Therefore, adipose‐derived stem cells are considered to be ideal therapeutic stem cells for human liver regeneration. In this article, we reviewed the advantages of adipose‐derived stem cells in liver regeneration, and explore their underlying mechanisms.     

Comparative gene expression in hematopoietic progenitor cells derived from embryonic stem cells

OBJECTIVE: The aim of this study was to characterize at the molecular level the hematopoietic progenitor cells derived from rhesus monkey embryonic stem (ES) cell differentiation. MATERIALS AND METHODS: We purified CD34(+) and CD34(+)CD38(-) cells from rhesus monkey ES cell cultures and examined the expression of a variety of genes associated with hematopoietic development, by semiquantitative polymerase chain reaction analysis. For comparison, we examined cell preparations from fresh or cultured rhesus monkey bone marrow (BM) and from mouse ES cells and BM. RESULTS: We observed a high degree of similarity in the expression patterns of these genes, with only a few exceptions. Most notably, the message of the flt3 gene was undetectable in rhesus monkey ES cell-derived CD34(+) and CD34(+)CD38(-) cells, whereas substantial flt3 expression was observed in the corresponding cells from fresh BM and in CD34(+) cells from cultured BM. The integrin alphaL and interleukin-6 (IL-6) receptor genes also were expressed in CD34(+)CD38(-) cells from BM, but there was little or no expression of these genes in CD34(+)CD38(-) cells derived from ES cells. Parallel analyses, using CD34(+)Lin(-) cells derived from murine ES cell cultures, showed no apparent expression of flt3, integrin alphaL, or IL-6 receptor, whereas corresponding cell preparations isolated from mouse BM expressed high levels of all of these genes. CONCLUSIONS: ES cell-derived hematopoietic progenitors, both from the rhesus monkey and from the mouse, exhibited the same alterations in gene expression compared with BM-derived cells from these animals. These observations could reflect the presence of different subpopulations in the cell fractions that were compared, or they may represent altered biologic properties of ES cell-derived hematopoietic stem cells.     

Monocytic cells derived from human embryonic stem cells and fetal liver share common differentiation pathways and homeostatic functions

The early emergence of macrophages and their large pattern of tissue distribution during development suggest that they may play a critical role in the initial steps of embryogenesis. In the present study, we show that monocytic cells derived from human embryonic stem cells (hESCs) and from fetal liver follow a differentiation pathway different to that of adult cells, leading to specific functions. Embryonic and fetal monocytic cells differentiated from a CD14(low)CD16(-) precursor to form CD14(high)CD16(+) cells without producing the CD14(high)CD16(-) cell population that predominates in adult peripheral blood. Both demonstrated an enhanced expression of genes encoding tissue-degrading enzymes, chemokines, and scavenger receptors, as was previously reported for M2 macrophages. Compared with adult blood monocytes, embryonic and fetal monocytic cells secreted high amounts of proteins acting on tissue remodeling and angiogenesis, and most of them expressed the Tie2 receptor. Furthermore, they promoted vascular remodeling in xenotransplanted human tumors. These findings suggest that the regulation of human fetal and embryonic monocytic cell differentiation leads to the generation of cells endowed mainly with anti-inflammatory and remodeling functions. Trophic and immunosuppressive functions of M2-polarized macrophages link fetus and tumor development, and hESCs offer a valuable experimental model for in vitro studies of mechanisms sustaining these processes.     

Development and functional analysis of eosinophils from murine embryonic stem cells

We have established a culture system for the development of eosinophils from murine embryonic stem (ES) cells. After transferring ES cells from embryonic fibroblast cells onto macrophage colony-stimulating factor-deficient stromal cells, OP9, ES cells were cultured in the presence of interleukin (IL)-5 with either IL-3 or granulocyte-macrophage colony stimulating factor (GM-CSF) for 20 d to obtain approximately 50% eosinophils. Electron microscopy confirmed the presence of crystallized major basic protein (MBP) in the granules of some of these cells. Neither IL-5, IL-3, GM-CSF nor eotaxin alone could induce eosinophils as efficiently as the conditions described above. Eotaxin induced eosinophil development in combination with either IL-3 or IL-5. Levels of GATA-1, Friend of GATA (FOG)-1, PU.1, CCAAT/enhancer binding protein (C/EBP)alpha, C/EBPbeta, IL-3 receptor alpha (IL-3Ralpha), GM-CSF receptor alpha (GM-CSFRalpha), and MBP mRNAs were increased in ES cells 10 d after transfer onto OP9 cells. In contrast, C/EBPepsilon, IL-5Ralpha, and eosinophil peroxidase mRNAs were induced in response to IL-3 and IL-5 after transfer onto OP9 cells. Eosinophils that developed in this system expressed Gr-1, F4/80, B220, CCR3, IL-3Ralpha, IL-5Ralpha, and DX5. Finally, eosinophils developed from ES cells produced reactive oxygen species in response to Leishmania as do peripheral blood eosinophils.