Embryonic stem cells: similarities and differences between human and murine embryonic stem cells

The derivation of murine embryonic stem (mES) cell lines was reported for the first time in 1981 (Nature, 1981; 292:154-156; Proc Natl Acad Sci U S A, 1981; 78:7634-7638), and they have since proved to be a very useful tool with which to study mammalian development, which is characterized by pluripotency and differentiation. About 20 years later, the successful generation of human embryonic stem (hES) cell lines was described (Science, 1998; 282:1145-1147). Although mES and hES are derived from mammals, they cannot be looked at as being one and the same. While basic information for hES can be derived from mES, such information does not correspond on a one-to-one basis. This review gives an overview of the characteristics of embryonic stem cells with the main focus on the similarities and differences between human and mES cells.     

Differentiation of human embryonic stem cells into bipotent mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors that can be found in many connective tissues, including fat, bone, cartilage, and muscle. We report here a method to reproducibly differentiate human embryonic stem cells (hESCs) into MSCs that does not require the use of any feeder layer. The cells obtained with this procedure are morphologically similar to bone marrow MSCs, are contact-inhibited, can be grown in culture for about 20 to 25 passages, have an immunophenotype similar to bone marrow MSCs (negative for CD34 and CD45 and positive for CD13, CD44, CD71, CD73, CD105, CD166, human leukocyte antigen [HLA]-ABC, and stage-specific embryonic antigen [SSEA]-4), can differentiate into osteocytes and adipocytes, and can be used as feeder cells to support the growth of undifferentiated hESCs. The ability to produce MSCs from hESCs should prove useful to produce large amounts of genetically identical and genetically modifiable MSCs that can be used to study the biology of MSCs and for therapeutic applications.     

The derivation of mesenchymal stem cells from human embryonic stem cells

Human embryonic stem cells (hESCs) hold promise for tissue regeneration therapies by providing a potentially unlimited source of cells capable of undergoing differentiation into specified cell types. Several preclinical studies and a few clinical studies use human bone marrow stromal cells (hBMSCs) to treat skeletal diseases and repair damaged tissue. However, hBMSCs have limited proliferation and differentiation capacity, suggesting that an alternate cell source is desirable, and hESCs may serve this purpose. Here we describe a protocol for the reproducible derivation of mesenchymal stem cells from hESCs (hES-MSCs). The hES-MSCs have a similar immunophenotype to hBMSCs, specifically they are CD73+, STRO-1+ and CD45-, and are karyotypically stable. The derived hES-MSCs are also capable of differentiating into osteoblasts and adipocytes. When the hES-MSCs were genetically modified with the lineage-specific Col2.3-GFP lentivirus and cultured in osteogenic medium, increased GFP expression was detected over time, indicating the hES-MSCs have the capacity to differentiate down the osteogenic lineage and had progressed toward a mature osteoblast phenotype.     

Characterization and in vivo testing of mesenchymal stem cells derived from human embryonic stem cells

Mesenchymal stem cells (MSCs) have been shown to contribute to the recovery of tissues through homing to injured areas, especially to hypoxic, apoptotic, or inflamed areas and releasing factors that hasten endogenous repair. In some cases genetic engineering of the MSC is desired, since they are excellent delivery vehicles. We have derived MSCs from the human embryonic stem cell (hESC) line H9 (H9-MSCs). They expressed CD105, CD90, CD73, and CD146, and lacked expression of CD45, CD34, CD14, CD31, and HLA-DR, the hESC pluripotency markers SSEA-4 and Tra-1-81, and the hESC early differentiation marker SSEA-1. Marrow-derived MSCs showed a similar phenotype. H9-MSCs did not form teratoma in our initial studies, whereas the parent H9 line did so robustly. H9-MSCs differentiated into bone, cartilage, and adipocytes in vitro, and displayed increased migration under hypoxic conditions. Finally, using a hindlimb ischemia model, H9-MSCs were shown to home to the hypoxic muscle, but not the contralateral limb, by 48?h after IV injection. In summary, we have defined methods for differentiation of hESCs into MSCs and have defined their characteristics and in vivo migratory properties.     

Regeneration of peripheral nerves by transplanted sphere of human mesenchymal stem cells derived from embryonic stem cells

In cell therapy, the most important factor for therapeutic efficacy is the stable supply of cells with best engraftment efficiency. To meet this requirement, we have developed a culture strategy such as three-dimensional sphere of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) in serum-free medium. To investigate the in?vivo therapeutic efficacy of hESC-MSC spheres in nerve injury model, we transected the sciatic nerve in athymic nude mice and created a 2-mm gap. Transplantation of hESC-MSC as sphere repaired the injured nerve significantly better than transplantation of hESC-MSC as suspended single cells in regard to 1) nerve conduction (sphere; 28.81?±?3.55 vs. single cells; 18.04?±?2.10, p?相似文献   

Functional markers and the 'homogeneity' of human mesenchymal stem cells

Stem cells are characterized by the abilities to self-renew, generate large numbers of progeny and differentiate into at least one mature cell type. Bone marrow serves as a reservoir for several classes of adult stem cells. In addition to haematopoietic stem cells (HSCs), which can reconstitute the haematopoietic system of a myeloablated host, bone marrow contains a diverse population of marrow stromal cells ( Herzog et al. 2003 ). Included among these are mesenchymal stem cells (MSCs), which ex vivo can be isolated as a relatively homogeneous and undifferentiated cell population that produces multiple mature cell types including fat, bone and cartilage ( Pittenger et al. 1999 ). Under appropriate, but ill-defined conditions, human MSCs (hMSCs) can also differentiate into other cell types, including excitable cells with neuronal-, myogenic and cardiomyogenic-like phenotypes. Because MSCs are multipotent and readily expandable in vitro , these cells have already been employed in early clinical studies, including the treatment of human myocardial infarction. Transplantation of autologous or allogeneic MSCs therefore represents a novel form of cellular therapy, which shows substantial promise for the treatment of a number of human diseases.     

人脐带间充质干细胞表达胚胎干细胞相关转录因子

背景：Nanog、Oct4和Sox2通过调节胚胎干细胞的基因转录,对其多潜能性和自我更新的能力具有关键性的调控作用,脐带间充质干细胞中这些胚胎干细胞相关转录因子的表达情况如何还不太清楚。 目的：研究脐带间充质干细胞中Nanog、Oct4和Sox2等这些胚胎干细胞相关转录因子的表达情况。 方法：胶原酶和胰酶消化法培养脐带间充质干细胞;mTeSRTM1体系进行无滋养层培养人胚胎干细胞,定量PCR比较上述两种细胞中Nanog、Oct4和Sox2 mRNA表达量的差异;免疫荧光检测上述两种细胞中Nanog、Oct4和Sox2的表达情况。 结果与结论：间充质干细胞表达胚胎干细胞标记Nanog、Oct4和Sox2,但Oct4主要表达在胞浆,且以Oct4B为主。脐带间充质干细胞Nanog、Oct4A和Sox2的表达量明显低于胚胎干细胞,其mRNA表达量分别为胚胎干细胞的20%,0.3%,10%左右。通过了解两种细胞Nanog、Oct4和Sox2的表达差异,可为优化脐带间充质干细胞重编程提供依据,也为进一步研究胚胎干细胞相关转录因子在成体干细胞表达起何种作用提供参考。     

Maintenance of human embryonic stem cells in mesenchymal stem cell-conditioned media augments hematopoietic specification

The realization of human embryonic stem cells (hESC) as a model for human developmental hematopoiesis and in potential cell replacement strategies relies on an improved understanding of the extrinsic and intrinsic factors regulating hematopoietic-specific hESC differentiation. Human mesenchymal stem cells (hMSCs) are multipotent cells of mesodermal origin that form a part of hematopoietic stem cell niches and have an important role in the regulation of hematopoiesis through production of secreted factors and/or cell-to-cell interactions. We have previously shown that hESCs may be successfully maintained feeder free using hMSC-conditioned media (MSC-CM). Here, we hypothesized that hESCs maintained in MSC-CM may be more prone to differentiation toward hematopoietic lineage than hESCs grown in standard human foreskin fibroblast-conditioned media. We report that specification into hemogenic progenitors and subsequent hematopoietic differentiation and clonogenic progenitor capacity is robustly enhanced in hESC lines maintained in MSC-CM. Interestingly, co-culture of hESCs on hMSCs fully abrogates hematopoietic specification of hESCs, thus suggesting that the improved hematopoietic differentiation is mediated by MSC-secreted factors rather than by MSC-hESC physical interactions. To investigate the molecular mechanism involved in this process, we analyzed global (LINE-1) methylation and genome-wide promoter DNA methylation. hESCs grown in MSC-CM showed a decrease of 17% in global DNA methylation and a promoter DNA methylation signature consisting of 45 genes commonly hypomethylated and 102 genes frequently hypermethylated. Our data indicate that maintenance of hESCs in MSC-CM robustly augments hematopoietic specification and that the process seems mediated by MSC-secreted factors conferring a DNA methylation signature to undifferentiated hESCs which may influence further predisposition toward hematopoietic specification.     

Hematopoietic engraftment of human embryonic stem cell-derived cells is regulated by recipient innate immunity

Human embryonic stem cells (hESCs) provide an important means to characterize early stages of hematopoietic development. However, the in vivo potential of hESC-derived hematopoietic cells has not been well defined. We demonstrate that hESC-derived cells are capable of long-term hematopoietic engraftment when transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Human CD45(+) and CD34(+) cells are identified in the mouse bone marrow (BM) more than 3 months after injection of hESCs that were allowed to differentiate on S17 stromal cells for 7-24 days. Secondary engraftment studies further confirm long-term repopulating cells derived from hESCs. We also evaluated two mechanisms that may inhibit engraftment: host immunity and requirement for homing to BM. Treatment with anti-ASGM1 antiserum that primarily acts by depletion of natural killer cells in transplanted mice leads to improved engraftment, likely due to low levels of HLA class I expressed on hESCs and CD34(+) cells derived from hESCs. Intra-BM injection also provided stable engraftment, with hematopoietic cells identified in both the injected and contra-lateral femur. Importantly, no teratomas are evident in animals injected with differentiated hESCs. These results demonstrate that SCID-repopulating cells, a close surrogate for hematopoietic stem cells, can be derived from hESCs. Moreover, both adaptive and innate immune effector cells may be barriers to engraftment of these cells.     

Microvascular mural cell functionality of human embryonic stem cell-derived mesenchymal cells

Microvascular mural or perivascular cells are required for the stabilization and maturation of the remodeling vasculature. However, much less is known about their biology and function compared to large vessel smooth muscle cells. We have developed lines of multipotent mesenchymal cells from human embryonic stem cells (hES-MC); we hypothesize that these can function as perivascular mural cells. Here we show that the derived cells do not form teratomas in SCID mice and independently derived lines show similar patterns of gene expression by microarray analysis. When exposed to platelet-derived growth factor-BB, the platelet-derived growth factor receptor β is activated and hES-MC migrate in response to a gradient. We also show that in a serum-free medium, transforming growth factor β1 (TGFβ1) induces robust expression of multiple contractile proteins (α smooth muscle actin, smooth muscle myosin heavy chain, smooth muscle 22α, and calponin). TGFβ1 signaling is mediated through the TGFβR1/Alk5 pathway as demonstrated by inhibition of α smooth muscle actin expression by treatment of the Alk5-specific inhibitor SB525334 and stable retroviral expression of the Alk5 dominant negative (K232R). Coculture of human umbilical vein endothelial cell (HUVEC) with hES-MC maintains network integrity compared to HUVEC alone in three-dimensional collagen I-fibronectin by paracrine signaling. Using high-resolution laser confocal microscopy, we show that hES-MC also make direct contact with HUVEC. This demonstrates that hESC-derived mesenchymal cells possess the molecular machinery expected in a perivascular progenitor cells and can play a functional role in stabilizing EC networks in in vitro three-dimensional culture.     

Induction of cardiomyogenesis in human embryonic stem cells by human embryonic stem cell-derived definitive endoderm

We previously reported that chick anterolateral endoderm (AL endoderm) induces cardiomyogenesis in mouse embryoid bodies. However, the requirement to micro-dissect AL endoderm from gastrulation-stage embryos precludes its use to identify novel cardiomyogenic factors, or to scale up cardiomyocyte numbers for therapeutic experiments. To circumvent this problem we have addressed whether human definitive endoderm (hDE) cells, which can be efficiently generated in large numbers from human embryonic stem cells (hESCs), can mimic the ability of AL endoderm to induce cardiac myogenesis. Results demonstrate that both hDE cells and medium conditioned by them induce cardiac myogenesis in pluripotent hESCs, as indicated by rhythmic beating and immunohistochemical/quantitative polymerase chain reaction monitoring of marker gene expression. The cardiomyogenic effect of hDE is enhanced when pluripotent hESCs are preinduced to the mes-endoderm state. Because this approach is tractable and scalable, it may facilitate identification of novel hDE-secreted factors for inclusion in defined cardiomyogenic cocktails.     

Functional human artificial chromosomes are generated and stably maintained in human embryonic stem cells

We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC), which behave as autonomous endogenous host chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore, and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines, but never in stem cells, thus limiting their potential therapeutic application. In this work, we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency, which were stably maintained without selection for 3 months. Importantly, no integration of the HAC DNA was observed in the hESc lines, compared with the fibrosarcoma-derived control cells, where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency, differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc, and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications.     

The enhancement of chondrogenic differentiation of human mesenchymal stem cells by enzymatically regulated RGD functionalities

A thiol-acrylate photopolymerization was used to incorporate enzymatically cleavable peptide sequences into PEG hydrogels to induce chondrogenic differentiation of encapsulated human mesenchymal stem cells (hMSCs). An adhesive sequence, RGD, was designed with an MMP-13 specific cleavable linker. RGD promotes survival of hMSCs encapsulated in PEG gels and has shown to induce early stages of chondrogenesis, while its persistence can limit complete differentiation. Therefore, an MMP-13 cleavage site was incorporated into the peptide sequence to release RGD mimicking the native differentiation timeline. Active MMP-13 production of encapsulated hMSCs was seen to increase from day 9 to 14 and only in chondrogenic differentiating cultures. Seeded hMSCs attached to the material prior to enzymatic cleavage, but a significant population of the cells detach after cleavage and release of RGD. Finally, hMSCs encapsulated in RGD-releasing gels produce 10 times as much glycosaminoglycan as cells with uncleavable RGD functionalities, by day 21 of culture. Furthermore, 75% of the cells stain positive for collagen type II deposition where RGD is cleavable, as compared to 19% for cultures where RGD persists. Collectively, these data provide evidence that temporal regulation of integrin-binding peptides is important in the design of niches in differentiating hMSCs to chondrocytes.     

胚胎干细胞和间充质干细胞分化为内皮细胞的研究现状

内皮细胞对于维持血管正常功能具有重要作用,然而其来源有限.因此,寻找内皮细胞的其他丰富来源一直为研究者们所关注.干细胞在体外培养具有能大量增殖、并在合适的条件下定向分化的特性.近年来,各种类型的干细胞诱导分化为内皮细胞的研究不断深入并各有优缺点.对胚胎干细胞和各种来源的间充质干细胞诱导分化为内皮细胞的研究现状作一综述.     

Enrichment of neural-related genes in human mesenchymal stem cells from neuroblastoma patients

Neuroblastoma (NB) is one of the most common pediatric solid tumors and, like most human cancers, is characterized by a broad variety of genomic alterations. Although mesenchymal stem cells (MSCs) are known to interact with cancer cells, the relationship between MSCs and metastatic NB cancer cells in bone marrow (BM) is unknown. To obtain genetic evidence about this interaction, we isolated ΒΜ-derived MSCs from children with NB and compared their global expression patterns with MSCs obtained from normal pediatric donors, using the Agilent 44K microarrays. Significance analysis of microarray results with a false discovery rate (FDR)?<5% identified 496 differentially expressed genes showing either a 2-fold upregulation or downregulation between both groups of samples. Comparison of gene ontology categories of differentially expressed genes revealed the upregulation of genes categorized as 'neurological system process', 'cell adhesion', 'apoptosis', 'cell surface receptor linked signal transduction', 'intrinsic to membrane' and 'extracellular region'. Among the downregulated genes, several immunology-related terms were the most abundant. These findings provide preliminary genetic evidence of the interaction between MSCs and NB cancer cells in ΒΜ as well as identify relevant biological processes potentially altered in MSCs in response to NB.     

High-potential human mesenchymal stem cells

Bone marrow-derived stromal mesenchymal stem cells (MSCs) have been characterized in vitro by their growth characteristics, the expression of a panel of surface antigens, and their potential to differentiate into mesenchymal lineages. They can be separated by physical methods as well as by immunological or chemical separation or cultivation. Different protocols are used in different laboratories, making the comparison of various reported MSC populations difficult. Here we describe a population of bone marrow-derived adult stem cells that has been separated on a Percoll gradient with low density. It is characterized by an extraordinary high proliferative potential and a conserved phenotype characteristic of MSCs that retain their plutipotentiality in culture, as evidenced by their ability to differentiate into osteo-, chondro-, and adipogenic lineages. Separation of these cells provide an effective and convenient method for rapid expansion of pluripotential human MSCs for clinical use where large amounts of stem cells are needed.