Enhanced chondrogenic differentiation of embryonic stem cells by coculture with hepatic cells

Enhancing the specific differentiation of pluripotent embryonic stem (ES) cells has been a challenge in the field of tissue engineering. Previously, hepatic cells have been shown to secrete various soluble morphogenic factors to direct mesodermal differentiation of ES cells. In this study, we hypothesized that factors secreted by hepatic cells possess chondrogenic-differentiating effects, and, therefore, the co-culture of hepatic cells would enhance chondrogenesis of ES cells. ES-derived cells(ESDCs) were co-cultured with hepatic cells (HEPA-1C1c7) in three-dimensional bilayered hydrogels. After 3 weeks culture, the histological and biochemical analysis of the HEPA-co-cultured ESDCs revealed a four-fold increase in glycosaminoglycan (GAG) compared to ESDCs cultured alone. This result was supported by real-time PCR analysis, which demonstrated an 80-fold increase in aggrecan expression in co-cultured ESDCs. Additionally, type IIB collagen expression was observed only with co-cultured ESDCs, and immunohistochemical analysis resulted in significantly more positive type II collagen staining with co-cultured ESDCs. Moreover, at day 21, gene expression of other lineages in HEPA-co-cultured ESDCs was either comparable to or lower than those of ESDCs cultured alone. These results indicated that co-culture of ESDCs with hepatic cells significantly enhanced specific chondrogenic differentiation of ESDCs.     

Generation of tyrosine hydroxylase positive neurons from human embryonic stem cells after coculture with cellular substrates and exposure to GDNF

Tyrosine hydroxylase (TH)-positive neurons were generated from human embryonic stem (hES) cells by coculturing on astrocytes or PA6 stromal cells. After 3 to 4 weeks in culture, TH-positive cells with neuronal morphology developed. Coculture with astrocytes from the embryonic striatum produced a larger number of TH-positive cells than did coculture with astrocytes from embryonic mesencephalon (329 +/- 149 versus 33 +/- 16 TH-positive cells per well, p < .05). In other experiments using PA6 cells as a substrate, glial-derived neurotrophic factor (GDNF) was added to the media of differentiating hES cells, and this led to a doubling of the number of TH-positive cells (PA6: 443 +/- 105 TH-positive cells per well versus PA6 + GDNF: 934 +/- 136, p < .05). We conclude that substrates of striatal astrocytes and PA6 cells can promote differentiation of human embryonic stem cells to a TH-positive phenotype and that GDNF can increase the number of cells expressing that phenotype.     

Generation of dopaminergic neurons in vitro from human embryonic stem cells treated with neurotrophic factors

The aim of this study was to produce dopaminergic neurons in vitro from human embryonic stem (hES) cells following treatment of various neurotrophic factors. MB03 hES cells were induced by retinoic acid (RA) or basic fibroblast growth factor (bFGF), which were further treated with brain derived neurotrophic factor (BDNF) or transforming growth factor (TGF)-alpha in each induction method during neuron differentiation days. At the final differentiation stage (21 days), all treatment groups revealed very similar levels (bFGF, 76-78%; RA, 70-74%) of mature neurons (anti-NF-200) in two induction methods irrespective of the addition of BDNF or TGF-alpha. In addition, immunostaining and HPLC analyses revealed higher levels of tyrosine hydroxylase (20+/-2.3%) and dopamine (265.5+/-62.8 pg/ml) in the bFGF- and TGF-alpha-treated hES cells than in RA- or BDNF-treated hES cells. These data are one of the first reports on the generation of dopaminergic neurons of hES cells in vitro. Also, our results indicate that TGF-alpha may be successfully used in the bFGF induction protocol and yield higher numbers of dopaminergic neurons from hES cells.     

维甲酸诱导小鼠iPS细胞向神经细胞分化

目的:研究小鼠诱导性多能干细胞(induced pluripotent stem cells,iP S细胞)在体外维甲酸(retinoic acid,RA)的诱导下能否分化成功能性的神经细胞。方法:首先将iPS细胞悬浮培养形成拟胚体,利用RA将其诱导分化成神经前体细胞,然后利用免疫荧光染色技术观察小鼠iPS细胞在体外分化成神经细胞以及突触发生的形态特征。结果:小鼠iPS细胞在RA的诱导下不但能够分化为成熟神经元与胶质细胞还可以观察到树突棘及突触连接的形成。结论:RA作为一种作用很强的分化诱导剂,可以有效的诱导小鼠iPS细胞分化为功能性的神经元和神经胶质细胞,这对其进行后续的神经发育的机制、药物筛选以及自体iPS细胞移植的研究具有重要意义。     

Directed differentiation of ventral spinal progenitors and motor neurons from human embryonic stem cells by small molecules

Specification of distinct cell types from human embryonic stem cells (hESCs) is key to the potential application of these na?ve pluripotent cells in regenerative medicine. Determination of the nontarget differentiated populations, which is lacking in the field, is also crucial. Here, we show an efficient differentiation of motor neurons ( approximately 50%) by a simple sequential application of retinoid acid and sonic hedgehog (SHH) in a chemically defined suspension culture. We also discovered that purmorphamine, a small molecule that activates the SHH pathway, could replace SHH for the generation of motor neurons. Immunocytochemical characterization indicated that cells differentiated from hESCs were nearly completely restricted to the ventral spinal progenitor fate (NKX2.2+, Irx3+, and Pax7-), with the exception of motor neurons (HB9+) and their progenitors (Olig2+). Thus, the directed neural differentiation system with small molecules, even without further purification, will facilitate basic and translational studies using human motoneurons at a minimal cost.     

Generation of peripheral sensory and sympathetic neurons and neural crest cells from human embryonic stem cells

Human embryonic stem cells (hESCs) have been directed to differentiate into neuronal cells using many cell-culture techniques. Central nervous system cells with clinical importance have been produced from hESCs. To date, however, there have been no definitive reports of generation of peripheral neurons from hESCs. We used a modification of the method of Sasai and colleagues for mouse and primate embryonic stem cells to elicit neuronal differentiation from hESCs. When hESCs are cocultured with the mouse stromal line PA6 for 3 weeks, neurons are induced that coexpress (a) peripherin and Brn3a, and (b) peripherin and tyrosine hydroxylase, combinations characteristic of peripheral sensory and sympathetic neurons, respectively. In vivo, peripheral sensory and sympathetic neurons develop from the neural crest (NC). Analysis of expression of mRNAs identified in other species as NC markers reveals that the PA6 cells induce NC-like cells before neuronal differentiation takes place. Several NC markers, including SNAIL, dHAND, and Sox9, are increased at 1 week of coculture relative to naive cells. Furthermore, the expression of several NC marker genes known to be downregulated upon in vivo differentiation of NC derivatives, was observed to be present at lower levels at 3 weeks of PA6-hESC coculture than at 1 week. Our report is the first on the expression of molecular markers of NC-like cells in primates, in general, and in humans, specifically. Our results suggest that this system can be used for studying molecular and cellular events in the almost inaccessible human NC, as well as for producing normal human peripheral neurons for developing therapies for diseases such as familial dysautonomia.     

Stage-dependent Olig2 expression in motor neurons and oligodendrocytes differentiated from embryonic stem cells

Although embryonic stem (ES) cells are capable of forming any cell type in the body, the mechanisms that control cell type-specific differentiation are largely unknown. In the present study, we examined the process of differentiation to motor neurons and oligodendrocytes from mouse (Olig2GFP) ES cells. Mouse ES cells undergo a sequential process of differentiation over a 3-week period to generate motor neurons and oligodendrocytes. At day 7 of differentiation, Olig2-expressing cells are biased to a neuronal lineage. However, further differentiation (day 32) resulted in the majority of Olig2-expressing cells exhibiting an oligodendrocyte phenotype as well as a reduced ability to make motor neurons. Exposure of human ES cells to Sonic hedgehog (Shh) likewise resulted in enhanced motor neuron differentiation. Our results establish the requirements for directing ES cells to become motor neurons and oligodendrocytes and show that ES cell-derived Olig2 + cells can give rise to both motor neurons and oligodendrocytes, depending on the time at which differentiation is initiated.     

Ultrastructure of human embryonic stem cells and spontaneous and retinoic acid-induced differentiating cells

Ultrastructural and immunohistochemical studies of 4 groups of cells-(human embryonic stem cells (hES), embryoid bodies (EB), and spontaneously and retinoic acid (RA)-induced differentiating cells)-were carried out to investigate their detailed phenotype. Immunohistochemically, the EB cells showed strong immunoreactivity for CD34, CD117, and nestin. Differentiating cells expressed pancytokertin, vimentin, CD31, CD56, GFAP, nestin, and NeuN as well as CD34, and c-Kit. However, synaptophysin and neurofilaments were not present in these same differentiating cells. Transmission electron microscopy showed that hES and EB cells were very similar to germ cells or cells of the inner cell mass. Spontaneously and RA-induced differentiating cells exhibited epithelial, mesenchymal, endodermal, and neuronal phenotypes. The perikarya of the neuronal cells had rich RERs (Nissl substance) and long cytoplasmic processes filled with numerous neural tubules. However, neither synaptic junctions nor synaptic vesicles were developed. In our study, RA treatment with brain-derived growth factor and TGFalpha in neuron differentiation medium induced not only neuronal differentiation but also pluripotential differentiation. Full neuronal differentiation did not occur after 2 weeks in culture, as no synaptic junctions and synaptic vesicles developed.     

Uni-directional differentiation of mouse embryonic stem cells into neurons by the neural stem sphere method

We previously showed that our neural stem sphere (NSS) method promotes the neuronal differentiation of mouse, monkey and human embryonic stem (ES) cells. Here we analyzed changes in expression of marker genes and proteins during neuronal differentiation. When cultured in astrocyte-conditioned medium (ACM) under free-floating conditions, colonies of ES cells formed floating cell spheres, which, within 4 days, gave rise to NSSs. In the spheres, the expression of ES cell marker genes was consistently down-regulated, while expression of an epiblast marker was transiently up-regulated, beginning on day 2, and the expression of neuroectoderm, neural stem cell and neuron markers was up-regulated, beginning on days 3, 4 and 6, respectively. The expression of the marker genes was consistent with that of marker proteins. The time course of expression of these markers in the spheres resembled that of neuronal differentiation from the inner cell mass (ICM) cells of blastula. In contrast, the expression of endoderm, mesoderm, epidermis, astrocyte and oligodendrocyte markers was low and not up-regulated during differentiation. Only a small number of apoptotic cells were present in the spheres. These results suggest that mouse ES cells uni-directionally differentiate into neurons via epiblast cells, neuroectodermal cells and neural stem cells.     

Human motor neuron differentiation from human embryonic stem cells

The therapeutic potential of embryonic stem (ES) cells is promising, but in many cases limited by our inability to promote their differentiation to specific cell types, such as motor neurons. Here we provide the first report of the successful differentiation of human ES cells to cells of a motor neuron phenotype. A renewable source of neuroepithelial cells was generated from human ES cells. Extracellular signals were then employed to induce motor neuron differentiation and related gene expression by these cells. OLIG2 and HLXB9 gene expression increased upon the addition of basic fibroblast growth factor, retinoic acid, and sonic hedgehog, as a motor neuron phenotype expressing Islet1 and choline acetyltransferase (ChAT) developed. This study demonstrates that neuroepithelial cells derived from human ES cells are renewable progenitors capable of generating motor neurons at levels that may be therapeutically useful. Sonic hedgehog, basic fibroblast growth factor, and retinoic acid differentially influence human motor neuron differentiation by mechanisms that remain to be defined.     

大鼠脊髓源神经干细胞与运动神经元的差异蛋白质组学

目的 探讨大鼠脊髓源神经干细胞与运动神经元的差异蛋白质组学,寻找出重要的差异蛋白质.方法 采用双向电泳分离两种细胞的蛋白质,用DeCyder软件分析蛋白表达差异,并采用质谱(HPLC-ESI-MS/MS)进行鉴定.结果 脊髓源神经干细胞与运动神经元蛋白质凝胶分析获得1 300余个清晰的蛋白质斑点,在比国产分析的87个差异点中,初步鉴定出44个差异表达蛋白,其中24个在神经干细胞高表达,20个在运动神经元高表达.结论 脊髓源神经干细胞与运动神经元有各自特定的蛋白质表达,某些差异蛋白可能在神经干细胞向运动神经元分化中发挥关键作用.     

Generation of insulin-producing islet-like clusters from human embryonic stem cells

Recent success in pancreatic islet transplantation has energized the field to discover an alternative source of stem cells with differentiation potential to beta cells. Generation of glucose-responsive, insulin-producing beta cells from self-renewing, pluripotent human ESCs (hESCs) has immense potential for diabetes treatment. We report here the development of a novel serum-free protocol to generate insulin-producing islet-like clusters (ILCs) from hESCs grown under feeder-free conditions. In this 36-day protocol, hESCs were treated with sodium butyrate and activin A to generate definitive endoderm coexpressing CXCR4 and Sox17, and CXCR4 and Foxa2. The endoderm population was then converted into cellular aggregates and further differentiated to Pdx1-expressing pancreatic endoderm in the presence of epidermal growth factor, basic fibroblast growth factor, and noggin. Soon thereafter, expression of Ptf1a and Ngn3 was detected, indicative of further pancreatic differentiation. The aggregates were finally matured in the presence of insulin-like growth factor II and nicotinamide. The temporal pattern of pancreas-specific gene expression in the hESC-derived ILCs showed considerable similarity to in vivo pancreas development, and the final population contained representatives of the ductal, exocrine, and endocrine pancreas. The hESC-derived ILCs contained 2%-8% human C-peptide-positive cells, as well as glucagon- and somatostatin-positive cells. Insulin content as high as 70 ng of insulin/mug of DNA was measured in the ILCs, representing levels higher than that of human fetal islets. In addition, the hESC-derived ILCs contained numerous secretory granules, as determined by electron microscopy, and secreted human C-peptide in a glucose-dependent manner. Disclosure of potential conflicts of interest is found at the end of this article.     

Immunosuppression by embryonic stem cells

Embryonic stem cells or their progeny inevitably differ genetically from those who might receive the cells as transplants. We tested the barriers to engraftment of embryonic stem cells and the mechanisms that determine those barriers. Using formation of teratomas as a measure of engraftment, we found that semiallogeneic and fully allogeneic embryonic stem cells engraft successfully in mice, provided a sufficient number of cells are delivered. Successfully engrafted cells did not generate immunological memory; unsuccessfully engrafted cells did. Embryonic stem cells reversibly, and in a dose-dependent manner, inhibited T-cell proliferation to various stimuli and the maturation of antigen-presenting cells induced by lipopolysaccharide. Inhibition of both was owed at least in part to production of transforming growth factor-beta by the embryonic stem cells. Thus, murine embryonic stem cells exert "immunosuppression" locally, enabling engraftment across allogeneic barriers.     

Generation of novel adipocyte monolayer cultures from embryonic stem cells

Here we show a simplified and improved method to produce large quantities of evenly distributed monolayer cultures that display major characteristics of adipocytes. These cultures are applicable for quantitative analysis for biochemical and molecular events in adipogenesis during development and may provide a useful system for high-throughput drug screening assays of antiobesity drugs. In our method, we treated embryoid bodies (EBs) with all-trans retinoic acid (ATRA) for 3 days, 1 day after they attached to the gelatin-coated culture plates without further transfer. The cells were maintained in insulin and trioiodothyronine (T(3))-containing medium until day 12, when they were dispersed by enzymatic digestion and replated onto multiple culture plates. Two days later, adipocyte induction factors were added for 6 days and examined 6 days later. The amount of lipid droplet-laden adipocytes in the culture reached approximately 80%, with a nearly five-fold increase in GPDH activity. The cells expressed high levels of adipose-specific proteins (adipocyte markers), including PPARgamma2, ALBP, LPL, HSL, perilipin, and DGAT1. The adipocytes are functionally active, as evidenced by their response to lipolytic agents, such as forskolin, Bt2-cAMP, and isoproterenol, with more than 20-fold increases in glycerol release.     

Astrocyte-derived factors instruct differentiation of embryonic stem cells into neurons

Pluripotent embryonic stem (ES) cells may differentiate into neurons in vitro. This is valuable in the study of neurogenesis and in the generation of donor cells for neuronal transplantation. Here we show that astrocyte-derived factors instruct mouse and primate ES cells to differentiate into neurons. Cultured in astrocyte-conditioned medium (ACM) under free-floating conditions, within 4 days, colonies of undifferentiated mouse ES cells give rise to floating spheres of concentric stratiform structure with a periphery of neural stem cells, which are termed Neural Stem Spheres. Culturing the spheres on an adhesive substrate in ACM promotes neurogenesis, and cells in the spheres differentiate into neurons within 5 days, including dopaminergic neurons. In contrast, neither astrocytes nor oligodendrocytes are formed. The procedure developed for mouse ES cells can be applied to monkey ES cells. This neurogenesis pathway provides a new insight into mechanisms of specification of cell fates in early development and also provides a simple procedure for fast and efficient generation of a vast number of neural stem cells and neurons.