Differentiation and isolation of hepatic-like cells from human embryonic stem cells

Human embryonic stem cells are pluripotent cells that can serve as a cell source for transplantation medicine, and as a tool to study human embryogenesis. We investigate here the potential of human embryonic stem cells to differentiate into hepatic cells. We have characterized the expression level of liver-enriched genes in undifferentiated and differentiated human embryonic stem cells by DNA microarrays. Our analysis revealed a subset of fetal hepatic enriched genes that are expressed in human embryonic stem cells upon differentiation into embryoid bodies. In order to isolate the hepatic-like cells, we introduced a reporter gene regulated by a hepatocyte-specific promoter into human embryonic stem cells. We isolated clones of human embryonic stem cells that express enhanced green fluorescent protein upon in vitro differentiation. Through immunostaining, we showed that most of these cells express albumin, while some cells still express the earlier expressed protein alpha-fetoprotein. Using fluorescence activated cell sorter, we were able to sort out the fluorescent differentiated cells and expand them for a few more weeks. This is the first report to demonstrate the possibility of purifying differentiated derivatives of human embryonic stem cells and culturing them further. Through confocal microscopy, we detected clusters of hepatic-like cells in 20-day-old embryoid bodies and in teratomas. As observed during embryonic development, we showed that in teratomas, the hepatic-like endodermal cells develop next to cardiac mesodermal cells. In order to examine the secreted factors involved in the induction of hepatic differentiation, human embryonic stem cells were grown in the presence of various growth factors, demonstrating the potential involvement of acidic fibroblast growth factor in the differentiation. In conclusion, given certain growth conditions and genetic manipulation, we can now differentiate and isolate hepatic-like cells from human embryonic stem cells.     

M-CSF诱导人骨髓间充质干细胞分化为肝样细胞的分子机制

本研究主要目的是明确M-CSF诱导骨髓间充质干细胞分化为肝样细胞的分子机制,为临床中的肝移植和治疗肝病提供新思路。对取自于本院骨科治疗的患者的股骨骨髓间充质干细胞进行提取、分离、传代培养及鉴定。流式细胞仪检测BMSCs的表面表型。为了诱导BMSCs的肝分化,本研究将BMSCs加入到培养基中。骨髓间充质干细胞诱导21 d后,BMSCs表达了肝细胞特异性标志物a-蛋白(AFP)和细胞角蛋白18(CK18),通过免疫荧光染色证实了分化与为分化的BMSCs表达的差异性。分化的BMSCs还显示了肝细胞的体外功能特征,包括白蛋白产生、尿素分泌和糖原储存。本研究结果表明,BMSCs在M-CSF诱导下可分化为功能性肝细胞样细胞,可作为肝病治疗的细胞来源。     

Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo

Embryonic stem cells (ES cells), bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, and hepatic stem cells in liver have been known as a useful source that can induce to differentiate into hepatocytes. In this study, we examined whether human adipose tissue-derived stromal cells (hADSC) can differentiate into hepatic lineage in vitro. hADSC, that were induced to differentiate into hepatocyte-like cells by the treatment of HGF and OSM, had morphology similar to hepatocytes. Addition of DMSO enhanced differentiation into hepatocytes. RT-PCR and immunocytochemical analysis showed that hADSC express albumin and alpha-fetoprotein during differentiation. Differentiated hADSC showed LDL uptake and production of urea. Additionally, transplanted hADSC to CCl4-injured SCID mouse model were able to be differentiated into hepatocytes and they expressed albumin in vivo. Mesenchymal stem cells isolated from human adipose tissue are immunocompatible and are easily isolated. Therefore, hADSC may become an alternative source to hepatocyte regeneration or liver cell transplantation.     

人源诱导多能干细胞体外定向分化为功能性肝细胞的方法研究

摘要 目的：研究开发一种简易、快速在体外使多能诱导干细胞（induced pluripotent stem cells,iPSCs）定向分化为功能性肝样细胞的培养方法。方法：根据正常肝细胞在体内的发育规律,设计简化诱导方法使iPS细胞定向分化为内胚层细胞,应用qPCR和流式细胞术鉴定其纯度后进一步诱导分化为肝样细胞,并通过qPCR、ELISA、免疫荧光等技术鉴定肝细胞的性状和功能。结果：iPS细胞诱导7天后, OCT4和NANOG的表达水平显著下降,内胚层细胞相关基因CXCR4、FOXA2和HNF4A表达水平明显升高。内胚层细胞继续诱导培养15天后,肝细胞特异性标志基因ALB、TDO2、RBP4、G6PC和肝药酶基因CYPs等显著上调,同时产生高水平的白蛋白和尿素;PAS糖原染色为阳性,能主动摄取和释放吲哚菁绿,证实诱导成的肝样细胞具备正常肝细胞的部分功能。结论：该诱导方案能够在体外使iPS细胞遵循正常肝脏发育通路简易、高效地分化为功能性肝细胞。本研究为大量获得iPS来源的肝细胞及其在细胞疗法和药筛模型中的运用提供了可能性。     

Embryonic stem cell-derived neurogenesis

Embryonic stem (ES) cells are able to differentiate in vitro into endodermal, mesodermal, and ectodermal cell types. However. the spontaneous development of neuronal cells from ES cells is rather limited. Therefore, specific protocols to increase the differentiation of neuronal cells have been established, such as retinoic acid (RA) induction and lineage selection of neuronal cells. High concentrations of RA resulted in efficient neuronal differentiation paralleled by the expression of tissue-specific genes, proteins, ion channels, and receptors in a developmentally controlled manner. Because the developmental pattern and survival capacity of RA-induced neuronal cells were limited, specific differentiation protocols by lineage selection of neuronal cells have been established using growth and extracellular matrix factors. After formation of cells of the three primary germ layers, mesodermal differentiation was inhibited by serum depletion, and neural precursor cells were generated by addition of basic fibroblast growth factor, followed by differentiation induction by neuronal differentiation factors. Further application of survival-promoting factors such as neurotrophic factors and cytokines at terminal stages resulted in a significant increase, survival, and maintenance of dopaminergic neurons. In the future, these cellular systems will be applicable: (1) for studying commitment and neuronal specification in vitro, (2) as pharmacological assays for drug screening, and (3) for the selective isolation of differentiated neuronal cells which may be used as a source for cell and tissue grafts.     

Human amniotic mesenchymal stem cells promote endometrium regeneration in a rat model of intrauterine adhesion

Human amniotic transplantation has been proposed to improve the therapeutic efficacy of intrauterine adhesions (IUAs). Human amniotic mesenchymal stem stromal cells (hAMSCs) can differentiate into multiple tissue types. This study aimed to investigate the mechanism by which hAMSCs transplantation promotes endometrial regeneration. The rat models with IUA were established through mechanical and infective methods, and PKH26-labeled hAMSCs were transplanted through the tail vein (combined with/without estrogen). Under three different conditions, hAMSCs differentiated into endometrium-like cells. HE and Mason staining assays, and immunohistochemistry were used to compare the changes in rat models treated with hAMSCs and/or estrogen transplantation. To define the induction of hAMSCs to endometrium-like cells in vitro, an induction medium (cytokines, estrogen) was used to investigate the differentiation of hAMSCs into endometrium-like cells. qRT-polymerase chain reaction (PCR) and western blotting were performed to detect the differentiation of hAMSCs into endometrium-like cells. A greater number of glands, fewer endometrial fibrotic areas, and stronger expression of vascular endothelial growth factor and cytokeratin in the combined group (hAMSCs transplantation combined with estrogen) than in the other treatment groups were observed. hAMSCs could be induced into endometrium-like cells by cytokine treatment (TGF-β1, EGF, and PDGF-BB). Transplantation of hAMSCs is an effective alternative for endometrial regeneration after injury in rats. The differentiation protocol for hAMSCs will be useful for further studies on human endometrial regeneration.     

Differentiation of putative hepatic stem cells derived from adult rats into mature hepatocytes in the presence of epidermal growth factor and hepatocyte growth factor

Oval cells, putative hepatic stem cells, can differentiate into a wide range of cell types including hepatocytes, bile epithelial cells, pancreatic cells and intestinal epithelial cells. In this study, we used different growth factor combinations to induce oval cells to differentiate into mature hepatocytes. We isolated and purified oval cells utilizing selective enzymatic digestion and density gradient centrifugation. Oval cells were identified by their morphological characteristics and the strong expressions of OV-6, albumin, cytokeratin (CK)-19 and CK-7. Using a 2-step induction protocol, we demonstrated that oval cells first changed into small hepatocytes, then differentiated into mature hepatocytes. Small hepatocytes were distinguished from oval cells by their morphological features (e.g. round shape and nuclei) and the lack of CK-19 mRNA expression. Mature hepatocytes were identified by their ultrastructural traits and their expressions of albumin, CK-18, tyrosine aminotransferase (TAT), and alpha-1-antitrypsin (alpha-1-AT). Differentiated cells acquired the functional attributes of hepatocytes in that they secreted albumin and synthesized urea at a high level throughout differentiation. Oval cells can thus differentiate into cells with the morphological, phenotypic and functional characteristics of hepatocytes. This 2-step induction procedure could provide an abundant source of hepatocytes for cell transplantation and tissue engineering.     

Neural differentiation of adipose-derived stem cells isolated from GFP transgenic mice

Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have recently reported that adipose-derived stromal cells (ASCs) could differentiate into mesenchymal lineages in vitro. In this study, we performed neural induction using ASCs from GFP transgenic mice and were able to induce these ASCs into neuronal and glial cell lineages. Most of the neurally induced cells showed bipolar or multipolar appearance morphologically and expressed neuronal markers. Electron microscopy revealed their neuronal morphology. Some cells also showed glial phenotypes, as shown immunocytochemically. The present study clearly shows that ASCs derived from GFP transgenic mice differentiate into neural lineages in vitro, suggesting that these cells might provide an ideal source for further neural stem cell research with possible therapeutic application for neurological disorders.     

Regulation of FAS ligand expression by chemokine ligand 2 in human endometrial cells

Human endometrium is a dynamic tissue under the influence of numerous hormones, growth factors, and cytokines interacting to maintain a balance of cellular growth, differentiation, and apoptosis. We have previously demonstrated that several factors including interleukin-8, extracellular matrix, and steroid hormones modulate FASLG, one of the apoptotic molecules, in human endometrium. Chemokine ligand 2 (CCL2), a monocyte chemoattractant and activating factor, is a cytokine involved in endometrial function. CCL2 is elevated in the peritoneal fluid of women with endometriosis. We hypothesize that increased levels of CCL2 in the endometriotic environment may upregulate FASLG expression in human endometrial stromal cells and induce a local immunotolerance in endometriosis. To test our hypothesis, we studied the in vitro regulation of FASLG expression and apoptosis by CCL2 in endometrial stromal cells. Western blot analysis revealed that CCL2 upregulated FASLG protein expression in cultured endometrial stromal cells. Based on semiquantitative RT-PCR analysis, CCL2 did not alter either FAS or FASLG mRNA expression in endometrial stromal cells. Immunocytochemistry results from the same cells treated with CCL2 demonstrated upregulation of FASLG protein expression. CCL2 did not change rate of apoptosis in endometrial stromal cells as evaluated by TUNEL assay. However, an increased apoptotic rate was detected in Jurkat (T lymphocytes) cells cocultured with endometrial stromal cells previously treated with CCL2. We speculate that increased FASLG expression by CCL2 may induce apoptosis of T lymphocytes and thus produce an immunotolerant environment for the development of ectopic implants.     

Expression of hepatocyte-like phenotypes in bone marrow stromal cells after HGF induction

Bone marrow comprises heterogeneous cell populations, of which certain progenitors have demonstrated the ability to differentiate into multiple mesenchymal cell lineages. This study demonstrates the bone marrow stromal cells (BMSCs) with intrinsic plasticity to differentiate into hepatocyte-like phenotypes under in vitro induction of hepatocyte growth factor (HGF). BMSCs isolated from rat femurs and tibias were cultured and passaged 3-4 times in the presence of HGF. Cells were harvested on days 0, 10, and 20 and subjected to examination of any hepatocyte characteristics by flow cytometry, RT-PCR, Western blot, and immunocytochemistry. Expression of albumin and alpha-fetoprotein at both mRNA and protein levels was detectable on day 10. By contrast, c-Met mRNA was significantly decreased in BMSC in the course of HGF induction. Here BMSC was shown to differentiate into hepatocyte-like phenotypes given the HGF induction, as an alternative source for adult stem cell transplantation in liver repair.     

Bone marrow-derived cells from male donors do not contribute to the endometrial side population of the recipient

Accumulated evidence demonstrates the existence of bone marrow-derived cells origin in the endometria of women undergoing bone marrow transplantation (BMT). In these reports, cells of a bone marrow (BM) origin are able to differentiate into endometrial cells, although their contribution to endometrial regeneration is not yet clear. We have previously demonstrated the functional relevance of side population (SP) cells as the endogenous source of somatic stem cells (SSC) in the human endometrium. The present work aims to understand the presence and contribution of bone marrow-derived cells to the endometrium and the endometrial SP population of women who received BMT from male donors. Five female recipients with spontaneous or induced menstruations were selected and their endometrium was examined for the contribution of XY donor-derived cells using fluorescent in situ hybridization (FISH), telomapping and SP method investigation. We confirm the presence of XY donor-derived cells in the recipient endometrium ranging from 1.7% to 2.62%. We also identify 0.45-0.85% of the donor-derived cells in the epithelial compartment displaying CD9 marker, and 1.0-1.83% of the Vimentin-positive XY donor-derived cells in the stromal compartment. Although the percentage of endometrial SP cells decreased, possibly being due to chemotherapy applied to these patients, they were not formed by XY donor-derived cells, donor BM cells were not associated with the stem cell (SC) niches assessed by telomapping technique, and engraftment percentages were very low with no correlation between time from transplant and engraftment efficiency, suggesting random terminal differentiation. In conclusion, XY donor-derived cells of a BM origin may be considered a limited exogenous source of transdifferentiated endometrial cells rather than a cyclic source of BM donor-derived stem cells.     

FGF-2 suppresses cellular senescence of human mesenchymal stem cells by down-regulation of TGF-beta2

Human mesenchymal stem cells (hMSCs) are able to both self-replicate and differentiate into a variety of cell types. Fibroblast growth factor-2 (FGF-2) stimulates the growth of hMSCs in vitro, but its mechanisms have not been clarified yet. In this study, we investigated whether cellular senescence was involved in the stimulation of hMSCs growth by FGF-2 and the expression levels of transforming growth factor-beta1 and -beta2 (TGF-betas). Because hMSCs were induced cellular senescence due to long-term culture, FGF-2 decreased the percentage of senescent cells and suppressed G1 cell growth arrest through the suppression of p21(Cip1), p53, and p16(INK4a) mRNA expression levels. Furthermore, the levels of TGF-betas mRNA expression in hMSCs were increased by long-term culture, but FGF-2 suppressed the increase of TGF-beta2 mRNA expression due to long-term culture. These results suggest that FGF-2 suppresses the hMSCs cellular senescence dependent on the length of culture through down-regulation of TGF-beta2 expression.     

Neural Differentiation of Rat Adipose-Derived Stem Cells in Vitro

It is reported that adipose-derived stem cells (ADSCs) had multilineage differentiation potential, and could differentiate into neuron-like cells induced by special induction media, which may provide a new idea for restoration of erectile dysfunction (ED) after cavernous nerve injury. The aim of this research was to explore the neuronal differentiation potential of ADSCs in vitro. ADSCs isolated from inguinal adipose tissue of rat were characterized by flow cytometry, and results showed that ADSCs were positive for mesenchymal stem cell markers CD90 and CD44, but negative for hematopoietic stem cell markers. ADSCs maintained self-renewing capacity and could differentiate into adipocytes and neurocytes under special culture condition. In this research, two methods were used to induce ADSCs. In method 1, ADSCs were treated with the preinduction medium including epithelium growth factor, basic fibroblast growth factor, and brain derived neurotrophic factor (BDNF) for 3?days, then with the neurogenic induction medium containing isobutylmethylxanthine, indomethacin, and insulin. While in method 2, BDNF was not used to treat ADSCs. After induction, neuronal differentiation of ADSCs was evaluated. Neuronal markers, glial fibrillary acidic protein (GFAP), and ??-tubulin III (Tuj-1) were detected by immunofluorescence and Western Blot analyses. The expressions of GFAP and Tuj-1 in method 1 were obviously higher then those in method 2. In addition, the positive rate of the neuron-like cells was higher in method 1. It suggested that ADSCs are able to differentiate into neural-like cells in vitro, and the administration of BDNF in the preinduction medium may provide a new way to modify the culture method for getting more neuron-like cells in vitro.     

Niche-independent symmetrical self-renewal of a mammalian tissue stem cell

Pluripotent mouse embryonic stem (ES) cells multiply in simple monoculture by symmetrical divisions. In vivo, however, stem cells are generally thought to depend on specialised cellular microenvironments and to undergo predominantly asymmetric divisions. Ex vivo expansion of pure populations of tissue stem cells has proven elusive. Neural progenitor cells are propagated in combination with differentiating progeny in floating clusters called neurospheres. The proportion of stem cells in neurospheres is low, however, and they cannot be directly observed or interrogated. Here we demonstrate that the complex neurosphere environment is dispensable for stem cell maintenance, and that the combination of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) is sufficient for derivation and continuous expansion by symmetrical division of pure cultures of neural stem (NS) cells. NS cells were derived first from mouse ES cells. Neural lineage induction was followed by growth factor addition in basal culture media. In the presence of only EGF and FGF-2, resulting NS cells proliferate continuously, are diploid, and clonogenic. After prolonged expansion, they remain able to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. Colonies generated from single NS cells all produce neurons upon growth factor withdrawal. NS cells uniformly express morphological, cell biological, and molecular features of radial glia, developmental precursors of neurons and glia. Consistent with this profile, adherent NS cell lines can readily be established from foetal mouse brain. Similar NS cells can be generated from human ES cells and human foetal brain. The extrinsic factors EGF plus FGF-2 are sufficient to sustain pure symmetrical self-renewing divisions of NS cells. The resultant cultures constitute the first known example of tissue-specific stem cells that can be propagated without accompanying differentiation. These homogenous cultures will enable delineation of molecular mechanisms that define a tissue-specific stem cell and allow direct comparison with pluripotent ES cells.     

Bovine endometrial stromal cells display osteogenic properties

The endometrium is central to mammalian fertility. The endometrial stromal cells are very dynamic, growing and differentiating throughout the estrous cycle and pregnancy. In humans, stromal cells appear to have progenitor or stem cell capabilities and the cells can even differentiate into bone. It is not clear whether bovine endometrial stromal cells exhibit a similar phenotypic plasticity. So, the present study tested the hypothesis that bovine endometrial stromal cells could be differentiated along an osteogenic lineage. Pure populations of bovine stromal cells were isolated from the endometrium. The endometrial stromal cell phenotype was confirmed by morphology, prostaglandin secretion, and susceptibility to viral infection. However, cultivation of the cells in standard endometrial cell culture medium lead to a mesenchymal phenotype similar to that of bovine bone marrow cells. Furthermore, the endometrial stromal cells developed signs of osteogenesis, such as alizarin positive nodules. When the stromal cells were cultured in a specific osteogenic medium the cells rapidly developed the characteristics of mineralized bone. In conclusion, the present study has identified that stromal cells from the bovine endometrium show a capability for phenotype plasticity similar to mesenchymal progenitor cells. These observations pave the way for further investigation of the mechanisms of stroma cell differentiation in the bovine reproductive tract.