Embryonic stem cells can form germ cells in vitro

Knock-in embryonic stem (ES) cells, in which GFP or lacZ was expressed from the endogenous mouse vasa homolog (Mvh), which is specifically expressed in differentiating germ cells, were used to visualize germ cell production during in vitro differentiation. The appearance of MVH-positive germ cells depended on embryoid body formation and was greatly enhanced by the inductive effects of bone morphogenic protein 4-producing cells. The ES-derived MVH-positive cells could participate in spermatogenesis when transplanted into reconstituted testicular tubules, demonstrating that ES cells can produce functional germ cells in vitro. In vitro germ cell differentiation provides a paradigm for studying the molecular basis of germ line establishment, as well as for developing new approaches to reproductive engineering.     

Embryonic stem cells as a model for cardiogenesis

Embryonic stem (ES) cells are derived from the inner cell mass of mouse blastocysts. These cells, when placed upon a suitable fibroblast feeder layer, continue to proliferate without overt differentiation and remain totipotent. Cells in this state are competent for gene targeting via homologous recombination. Hence, they hold the possibility of developing defined animal models of human cardiovascular disease. When removed from the feeder layer, ES cells undergo differentiation and development into large, multicellular structures, termed embryoid bodies (EBs). Morphologic, biochemical, and molecular genetic analyses indicate that during EB development some early aspects of cardiogenesis are recapitulated. Thus, EB development in culture is useful for studying certain early cardiogenic events.     

Bone-marrow stem cells as a source for cell therapy

Bone marrow stroma contains a subgroup of cells which can be guided in vitro to differentiate, and express cardiomyocyte phenotype. In vivo, these cells can become cardiomyocytes when implanted into the myocardium, in response to signals from the microenvironment. They appear to participate in the physiologic healing process of tissue injury, such as myocardial infarction, by being recruited from the bone marrow, traffic via the circulation and home in to the injured site. Thus such cells may be employed to therapeutically augment the myocardial repair for patients who suffer cardiac damages, which may lead to heart failure. Optimization of the cell implant strategy, and further exploration of the preliminary findings that such adult stem cells may be uniquely immuno-tolerant and thus may be used as universal donors, will further enhance the clinical significance of adult stem cell-based regenerative therapy for heart failure.     

Embryonic stem cell-derived hematopoietic stem cells

Despite two decades of studies documenting the in vitro blood-forming potential of murine embryonic stem cells (ESCs), achieving stable long-term blood engraftment of ESC-derived hematopoietic stem cells in irradiated mice has proven difficult. We have exploited the Cdx-Hox pathway, a genetic program important for blood development, to enhance the differentiation of ESCs along the hematopoietic lineage. Using an embryonic stem cell line engineered with tetracycline-inducible Cdx4, we demonstrate that ectopic Cdx4 expression promotes hematopoietic mesoderm specification, increases hematopoietic progenitor formation, and, together with HoxB4, enhances multilineage hematopoietic engraftment of lethally irradiated adult mice. Clonal analysis of retroviral integration sites confirms a common stem cell origin of lymphoid and myeloid populations in engrafted primary and secondary mice. These data document the cardinal stem cell features of self-renewal and multilineage differentiation of ESC-derived hematopoietic stem cells.     

Embryonic stem cell-derived endothelial cells may lack complete functional maturation in vitro

Stem cell therapies will only become clinically relevant if the stem cells differentiated in vitro function as their in vivo counterparts. Here, we employed our previously developed techniques for deriving endothelial cells (>96% purity) from mouse embryonic stem cells (ESC) and compared these with mouse aortic endothelial cells (MAEC) obtained from thoracic aortas. Immunocytochemical analysis of ESC-derived endothelial cells (EC) demonstrates that both cell types are positive for the EC markers endothelial nitric oxide synthase (eNOS), Flk-1, Flt-1, vascular endothelial cadherin (VEcad), platelet-endothelial cell adhesion molecule-1 (PECAM-1), and CD34. However, ESC-derived EC express slightly lower levels of PECAM-1 and VE-cadherin, and significantly lower levels of acetylated low-density lipoprotein (LDL) uptake and von Willebrand factor. Although ESC-derived EC do express VE-cadherin, the VE-cadherin in the ESC-derived EC did not localize as well at the cell-cell junctions as in the MAEC. Interestingly, ESC-derived EC express much greater levels of the endothelial and hematopoietic stem cell marker CD34 and vasculogenic and angiogenic sprouting than MAEC. These results indicate that ESC-derived EC share some key characteristics of 'mature' EC, while retaining markers of alternate phenotypes including immature endothelium.     

Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes

Recent observations indicate that several stem cells can differentiate into hepatocytes; thus, cell-based therapy is a potential alternative to liver transplantation. The goal of the present study was to examine the in vitro hepatic differentiation potential of adipose tissue-derived mesenchymal stem cells (AT-MSCs). We used AT-MSCs from different age patients and found that, after incubation with specific growth factors (hepatocyte growth factor [HGF], fibroblast growth factor [FGF1], FGF4) the CD105(+) fraction of AT-MSCs exhibited high hepatic differentiation ability in an adherent monoculture condition. CD105(+) AT-MSC-derived hepatocyte-like cells revealed several liver-specific markers and functions, such as albumin production, low-density lipoprotein uptake, and ammonia detoxification. More importantly, CD105(+) AT-MSC-derived hepatocyte-like cells, after transplantation into mice incorporated into the parenchyma of the liver. CONCLUSION: Adipose tissue is a source of multipotent stem cells that can be easily isolated, selected, and induced into mature, transplantable hepatocytes. The fact that they are easy to procure ex vivo in large numbers makes them an attractive tool for clinical studies in the context of establishing an alternative therapy for liver dysfunction.     

Inhibitory effects of a synthetic pentapeptide on hemopoietic stem cells in vitro and in vivo

A synthetic analogue of a pentapeptide associated with mature granulocytes has been investigated for biological effects on stem cell activity in vitro and in vivo. When tested on bone marrow cells from female C3H mice, a short incubation in vitro in doses from 10(-9) to 10(-5) M inhibited myelopoietic colony formation (CFU-C). A maximum of 80% reduction of colony yield was found at 10(-7) M. An oxidized form of the molecule had a stimulatory effect on colony formation, but the inhibitory effect was restored by treatment with a reducing substance (mercaptoethanol). The peptide was nontoxic during continuous exposure of liquid cultures of bone marrow cells for up to 24 h at a dose range of 10(-11) to 10(-4) M. When injected into mice, a dose-dependent inhibitory effect on CFU-C was seen. Maximal effect was obtained by continuous infusion of 1.4 micrograms/h for six days, where only one-fifth of the normal CFU-C number per femur could be retrieved. Prolonged exposure to the same dose level resulted in less reduction. A significant, but less pronounced reduction of spleen colony formation (CFU-S) per femur was seen in the same dose range. Inhibition of both CFU-S and CFU-C was in all cases reversible and mostly accompanied by an overshoot of up to 75% above normal level. In addition, primarily noninhibitory doses led to a secondary increase in the numbers of CFU-S. The total cell number per femur was moderately but significantly reduced, and a prolonged reduction of granulocyte numbers in peripheral blood resulted. No direct toxic effects were seen in vivo on 271 mice given up to 9 mg of the peptide. The results indicate that the peptide may have a regulatory function for stem cell activity in vivo.     

Embryonic stem cells. Future perspectives

Embryonic stem cells (ES cells) are able to differentiate into any cell type, and therefore represent an excellent source for cellular replacement therapies in the case of widespread diseases, for example heart failure, diabetes, Parkinson's disease and spinal cord injury. A major prerequisite for their efficient and safe clinical application is the availability of pure populations for direct cell transplantation or tissue engineering as well as the immunological compatibility of the transplanted cells. The expression of human surface markers under the control of cell type specific promoters represents a promising approach for the selection of cardiomyocytes and other cell types for therapeutic applications. The first human clinical trial using ES cells will start in the United States this year.     

Pluripotent stem cells as source of dendritic cells for immune therapy

Dendritic cells (DC) are the most potent antigen-presenting cells. In vivo transfer of antigen-bearing DC has proven efficient in priming T cell responses specific to the antigen. DC-based cellular vaccination is now regarded as a powerful means for immunotherapy, especially for anti-cancer immunotherapy. Clinical trials of therapy with DC pulsed with peptide antigens or genetically modified to present antigens are currently carried out in many institutions. In addition, antigen-specific negative regulation of immune response by DC is considered to be a promising approach for treatments of autoimmune diseases and also for regulation of allo-reactive immune response causing graft rejection and GVHD in transplantation medicine. DC for transfer therapy are now generated by in vitro differentiation of peripheral blood monocytes of the patients. However, there is a limitation in the number of available monocytes, and the DC-differentiation potential of monocytes varies depending on the blood donor. Embryonic stem (ES) cells possess both pluripotency and infinite propagation capacity. We consider ES cells to be an ideal source for DC to be used in immunotherapy. Several groups, including us, have developed methods to generate DC from ES cells. This review introduces the studies on generation, characterization, and genetic modification of DC derived from ES cells or induced pluripotent stem (iPS) cells. The issues to be resolved before clinical application of pluripotent stem cell-derived DC will also be discussed.     

Embryonic and embryonic-like stem cells in heart muscle engineering

Cardiac muscle engineering is evolving rapidly and may ultimately be exploited to (1) model cardiac development, physiology, and pathology; (2) identify and validate drug targets; (3) assess drug safety and efficacy; and (4) provide therapeutic substitute myocardium. The ultimate success in any of these envisioned applications depends on the utility of human cells and their assembly into myocardial equivalents with structural and functional properties of mature heart muscle. Embryonic stem cells appear as a promising cell source in this respect, because they can be cultured reliably and differentiated robustly into cardiomyocytes. Despite their unambiguous cardiogenicity, data on advanced maturation and seamless myocardial integration of embryonic stem cell-derived cardiomyocytes in vivo are sparse. Additional concerns relate to the limited control over cardiomyogenic specification and cardiomyocyte maturation in vitro as well as the risk of teratocarcinoma formation and immune rejection of stem cell implants in vivo. Through the invent of embryonic-like stem cells - such as parthenogenetic stem cells, male germline stem cells, and induced pluripotent stem cells - some but certainly not all of these issues may be addressed, albeit at the expense of additional concerns. This review will discuss the applicability of embryonic and embryonic-like stem cells in myocardial tissue engineering and address issues that require particular attention before the potential of stem cell-based heart muscle engineering may be fully exploited. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".     

Differentiation of mouse embryonic stem cells into hepatocytes in vitro and in vivo

OBJECTIVE: Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages. Cell‐to‐cell contact is important for cell differentiation. Mouse ES cells were cocultured with mouse fetal liver cells and the green fluorescent protein (GFP) positive ES cells were transplanted into rats liver through the portal vein in order to investigate their potential to differentiate into hepatocytes. METHODS: Mouse ES cells were cocultured with the mouse fetal liver cell line, BNL.CL₂. They did not make direct contact; instead the culture media was exchanged freely. After coculture for 48 h, albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4 and SEK1 mRNA were assayed by RT‐PCR, and alpha‐fetoprotein by immunohistochemistry. The morphology was investigated by microscopy. After transplantion of the GFP‐positive ES cells, the whole liver was removed from a rat every four days. The liver slices were examined under a fluorescent microscope to detect the GFP‐positive cells. Albumin was detected on the same slices by immunohistochemistry. RESULTS: After coculture with BNL.CL₂ cells, the differentiated ES cells had the same morphology as the BNL.CL₂ cells, and albumin, transthyretin, glucose 6 phosphates and SEK‐1 mRNA were found by RT‐PCR, and alpha‐fetoprotein was detected immuno­histochemically. The transplanted GFP‐positive ES cells were found in the rats’ liver slices by GFP fluorescence, and development of teratomas was not observed. The immunohistochemistry results indicated that the transplanted GFP‐positive ES cells retained an albumin‐producing ability. CONCLUSIONS: Cell‐to‐cell contact is important for the differentiation of ES cells. Mouse embryonic stem cells can differentiate into hepatocytes directly either in vitro or in vivo.     

Embryonic beginnings of adult hematopoietic stem cells

Hematopoietic stem cells (HSC) are at the foundation of the adult hematopoietic system. HSC give rise to all blood cells through a complex series of proliferation and differentiation events that occur throughout the lifespan of the individual. Because of their clinical importance in transplantation protocols, recent research has focused on the developmental origins and potential of embryonic HSC. In both mammalian and non-mammalian vertebrate embryos, two independent anatomical sites have been found to generate hematopoietic cells. The yolk sac (or its equivalent in amphibians, the ventral blood islands) participates in a first transient wave of hematopoiesis by producing primitive erythrocytes. Importantly, adult-type HSCs emerge autonomously in a second wave of hematopoietic generation in an intraembryonic region surrounding the dorsal aorta, the aorta-gonads-mesonephros (AGM) region. In this review, we will discuss research advances in the field of developmental hematopoiesis, with a particular emphasis on the cellular origins of AGM HSC and their regulation by the embryonic hematopoietic microenvironment.     

乳腺成体干细胞体内及体外特性的实验研究

目的观察来源于乳腺癌患者的乳腺成体干细胞的体外生长特性。并将其移植到裸鼠体内,研究其体内的发展及变化趋势。方法以免疫磁珠分离乳腺成体干细胞进行体外培养。30只裸鼠,随机分为2组,以胸壁皮下细胞注射法移植入裸鼠体内,并以移植入MCF-7乳腺癌细胞的裸鼠作对照。于术后行大体观察,术后6w行组织学检查。结果乳腺成体干细胞在体外培养可以分化成导管上皮细胞及肌上皮细胞,将其移植裸鼠体内后形成的组织与肿瘤组织有明显差别。在组织切片中可见形成类似乳腺腺管样结构,未形成癌巢组织,并由结缔组织分隔成若干小叶,可见到大量的脂肪组织。结论经免疫磁珠分离的乳腺成体干细胞具有继续增殖及分化的特性,可以诱导裸鼠体内形成类似乳腺样组织,为乳腺形成及发育的研究提供一种新的方法。     

Embryonic stem cells and somatic cells differ in mutation frequency and type

Pluripotent embryonic stem (ES) cells have been used to produce genetically modified mice as experimental models of human genetic diseases. Increasingly, human ES cells are being considered for their potential in the treatment of injury and disease. Here we have shown that mutation in murine ES cells, heterozygous at the selectable Aprt locus, differs from that in embryonic somatic cells. The mutation frequency in ES cells is significantly lower than that in mouse embryonic fibroblasts, which is similar to that in adult cells in vivo. The distribution of spontaneous mutagenic events is remarkably different between the two cell types. Although loss of the functional allele is the predominant mutation type in both cases, representing about 80% of all events, mitotic recombination accounted for all loss of heterozygosity events detected in somatic cells. In contrast, mitotic recombination in ES cells appeared to be suppressed and chromosome loss/reduplication, leading to uniparental disomy (UPD), represented more than half of the loss of heterozygosity events. Extended culture of ES cells led to accumulation of cells with adenine phosphoribosyltransferase deficiency and UPD. Because UPD leads to reduction to homozygosity at multiple recessive disease loci, including tumor suppressor loci, in the affected chromosome, the increased risk of tumor formation after stem cell therapy should be viewed with concern.     

Effects of sargentgloryvine stem extracts on HepG-2 cells in vitro and in vivo

AIM:To observe the effects of sargentgloryvine stem extracts (SSE) on the hepatoma cell line HepG-2 in vitro andin vivo and determine its mechanisms of action.METHODS:Cultured HepG-2 cells treated with SSE were analysed by 3-(4,5-Dimethyl-thiazol-2-yl)-2,5Diphenyltetrazolium bromide and clone formation assay.The cell cycle and apoptosis analysis were conducted by flow cytometric,TdT-Mediated dUTP Nick End Labeling and acridine orange/ethidium bromide staining methods,and protein expression was examined by b...