Enrichment of corneal epithelial stem/progenitor cells using cell surface markers, integrin alpha6 and CD71

Corneal epithelial stem cells are believed to reside in the basal layer of the limbal epithelium, but no definitive cell surface markers have been identified. For keratinocytes, stem/progenitor cells are known to be enriched by cell surface markers, integrin α₆ and CD71, as a minor subpopulation which shows high integrin α₆ and low CD71 expressions (α₆^bri/CD71^dim). In the present study, we investigated the possibility that corneal epithelial stem cells can be enriched by integrin α₆ and CD71. The α₆^bri/CD71^dim cells were separated by fluorescence-activated cell sorting, as a minor subpopulation of the limbal epithelial cells. They were enriched for relatively small cells, showing a higher clonogenic capacity and expression of stem cell markers, but a lower expression of differentiation markers, compared to other cell populations. The cells were localized immunohistochemically in the basal region of the limbal epithelium. These results indicate that the α₆^bri/CD71^dim subpopulation enriched corneal epithelial stem cells.     

Side populations from cervical-cancer-derived cell lines have stem-cell-like properties

The target cells for the transforming mutations caused by high-risk human papillomavirus (HPV) infection could be the stem cells of the uterine cervical epithelium, generating particular cancer stem cells (CSCs). The aim of this study was to identify and characterize the CSCs from cervical-cancer-derived cell lines. The ability of SiHa, CaLo, and C-33A cell lines to efflux Hoechst 33342 was evaluated by flow cytometry and cells from the corresponding side populations (SPs) and nonside populations (NSPs) were analyzed for their cell-cycle status (pyronin Y) and their mRNA levels of ABC transporter family members (with qPCR). Specific markers (α₆-integrin^bri/CD71^dim, CK17) of normal epithelial stem cells were evaluated by flow cytometry. The biological properties of these cells were analyzed, including their colony heterogeneity, repopulation, and anchorage-independent colony formation. We identified SPs (around 3 %) in the SiHa and CaLo cell lines, more than 70 % of which were in G₀ phase and strongly expressed ABC transporters (predominantly ABCG2 and ABCB1). The SP from CaLo cells showed an α₆-integrin^bri/CD^dim pattern, whereas the SP from the SiHa cells showed an α₆-integrin^?/CD^dim pattern. Recultured cells from the SPs of both cell lines generated both SPs and NSPs, and had higher clonogenic potential to form mainly holoclones and greater colony-forming efficiency under anchorage-independent growth conditions than the cells from the NSPs or total cell populations. Interestingly, we identified no SP in the HPV-uninfected C-33A cell line, and it did not express ABCG2 or other members of the ABC transporters (ABCB1, ABCC1, or ABCA3).     

Effect of the in vivo application of granulocyte colony‐stimulating factor on NK cells in bone marrow and peripheral blood

Granulocyte colony‐stimulating factor (G‐CSF) has been widely used in the field of allogeneic haematopoietic stem cell transplantation (allo‐HSCT) for priming donor stem cells from the bone marrow (BM) to peripheral blood (PB) to collect stem cells more conveniently. Donor‐derived natural killer (NK) cells have important antitumour functions and immune regulatory roles post‐allo‐HSCT. The aim of this study was to evaluate the effect of G‐CSF on donors' NK cells in BM and PB. The percentage of NK cells among nuclear cells and lymphocyte was significantly decreased and led to increased ratio of T and NK cells in BM and PB post‐G‐CSF in vivo application. Relative expansion of CD56^bri NK cells led to a decreased ratio of CD56^dim and CD56^bri NK subsets in BM and PB post‐G‐CSF in vivo application. The expression of CD62L, CD54, CD94, NKP30 and CXCR4 on NK cells was significantly increased in PB after G‐CSF treatment. G‐CSF treatment decreased the IFN‐γ‐secreting NK population (NK1) dramatically in BM and PB, but increased the IL‐13‐secreting NK (NK2), TGF‐β‐secreting NK (NK3) and IL‐10‐secreting NK (NKr) populations significantly in BM. Clinical data demonstrated that higher doses of NK1 infused into the allograft correlated with an increased incidence of chronic graft‐vs‐host disease post‐transplantation. Taken together, our results show that the in vivo application of G‐CSF can modulate NK subpopulations, leading to an increased ratio of T and NK cells and decreased ratio of CD56^dim and CD56^bri NK cells as well as decreased NK1 populations in both PB and BM.     

The effects of adenoviral transfection of the keratinocyte growth factor gene on epidermal stem cells: An in vitro study

Epidermal stem cells (ESCs) are characterized as slow-cycling, multi-potent, and self-renewing cells that not only maintain somatic homeostasis but also participate in tissue regeneration and repair. To examine the feasibility of adenoviral vector-mediated keratinocyte growth factor (KGF) gene transfer into in vitro-expanded ESCs, ESCs were isolated from samples of human skin, cultured in vitro, and then transfected with recombinant adenovirus (Ad) carrying the human KGF gene (AdKGF) or green fluorescent protein gene (AdGFP). The effects of KGF gene transfer on cell proliferation, cell cycle arrest, cell surface antigen phenotype, and β-catenin expression were investigated. Compared to ESCs transfected with AdGFP, AdKGF-transfected ESCs grew well, maintained a high proliferative capacity in keratinocyte serum-free medium, and expressed high levels of β-catenin. AdKGF infection increased the number of ESCs in the G0/G1 phase and promoted ESCs entry into the G2/M phase, but had no effect on cell surface antigen phenotype (CD49f⁺/CD71⁻). The results suggest that KGF gene transfer can stimulate ESCs to grow and undergo cell division, which can be applied to enhance cutaneous wound healing.     

CD45 Isoform Profile Identifies Natural Killer (NK) Subsets with Differential Activity

The leucocyte-specific phosphatase CD45 is present in two main isoforms: the large CD45RA and the short CD45RO. We have recently shown that distinctive expression of these isoforms distinguishes natural killer (NK) populations. For example, co-expression of both isoforms identifies in vivo the anti tumor NK cells in hematological cancer patients. Here we show that low CD45 expression associates with less mature, CD56^bright, NK cells. Most NK cells in healthy human donors are CD45RA⁺CD45RO^-. The CD45RA^-RO⁺ phenotype, CD45RO cells, is extremely uncommon in B or NK cells, in contrast to T cells. However, healthy donors possess CD45RA^dimRO^- (CD45RA^dim cells), which show immature markers and are largely expanded in hematopoietic stem cell transplant patients. Blood borne cancer patients also have more CD45RA^dim cells that carry several features of immature NK cells. However, and in opposition to their association to NK cell progenitors, they do not proliferate and show low expression of the transferrin receptor protein 1/CD71, suggesting low metabolic activity. Moreover, CD45RA^dim cells properly respond to in vitro encounter with target cells by degranulating or gaining CD69 expression. In summary, they are quiescent NK cells, with low metabolic status that can, however, respond after encounter with target cells.     

Ferulic acid maintains the self-renewal capacity of embryo stem cells and adipose-derived mesenchymal stem cells in high fat diet-induced obese mice

Self-renewal is required for embryo stem cells (ESCs) and adipose-derived mesenchymal stem cells (ADMSCs). This study examined the ability of ferulic acid in mouse ESCs and ADMSCs, in a high fat diet-induced mouse model. Initially, five natural compounds of ferulic acid, xanthohumol, curcumin, ascorbic acid, and quercetin were screened in ESCs using an alkaline phosphate ⁺(AP⁺) assay, as a self-renewal biomarker. A ferulic acid treatment was the highest AP⁺ staining in hop-hit screening compounds. Also a ferulic acid increased Nanog mRNA levels in ESCs. The in vivo effects of ferulic acid were next examined in an obese mouse model. C57BL/6 J male mice were fed either a high fat diet (HFD) or control diet with ferulic acid (5 g/kg diet) for 8 weeks. Ferulic acid exhibited weight loss and improved glucose homeostasis, lipid profiling, and hepatic steatosis in a HFD-induced mouse model. Next, ADMSCs (Sca-1⁺CD45⁻), a hallmark of fat stem cells, were then isolated and quantified from mouse abdominal adipose tissue. A HFD decreased the Sca-1⁺CD45⁻ cell population of ADMSCs, but HFD-induced obese mice given ferulic acid showed an increased the Sca-1⁺CD45⁻ cell population of ADMSCs. Moreover, ferulic acid enhanced NANOG mRNA levels in human ADMSCs and its related gene mRNA expression. Overall, this study suggests that ferulic acid preserves self-renewal in ESCs, and contributes to ADMSCs self-renewal and effective weight control in obesity.     

人树突状细胞的大量培养及鉴定

摘要 目的：探讨人树突状细胞体外大量培养及鉴定方法。方法：采用免疫磁珠法分离纯化CD34⁺干细胞;采用含有TPO、SCF、Flt3L和IL-3的扩增培养基培养1周,以及含有SCF、Flt3L、GM-CSF和IL-4的分化培养基培养2-3周,获得CD34⁺细胞来源树突状细胞。采用普通光学显微镜观察细胞形态,牛鲍氏血细胞计数板进行细胞计数,荧光抗体标记、流式细胞仪检测细胞纯度和细胞表面共刺激分子的表达情况。结果：以含有TPO、SCF、Flt3L和IL-3的培养基扩展培养一周,及含有SCF、Flt3L、GM-CSF和IL-4的培养基诱导分化3周,可获得大量悬浮细胞;细胞数目扩增倍数约达50倍;普通光学显微镜下可见悬浮细胞有明显的树突状凸起;流式细胞术检测结果显示悬浮细胞中CD141和CD11c双阳性细胞（等同于单核细胞来源树突状细胞）比例达30%,此群细胞高表达HLA-DR和CD209,低表达共刺激分子CD80和CD86;细胞寿命较短,40天时培养体系中悬浮细胞和CD34⁺细胞来源树突状细胞数目急剧减少。结论：采用多细胞因子联合刺激可获得大量的树突状细胞,为树突状细胞的特性及功能学研究奠定了基础。     

Hematopoietic stem cell and mesenchymal stem cell population size in bone marrow samples depends on patient’s age and harvesting technique

Mesenchymal stem cells (MSCs) are heterogeneous population of cells with great potential for regenerative medicine. MSCs are relatively easy to expand in a cell culture, however determination of their concentration in harvested tissue is more complex and is not implemented as routine procedure. To identify MSCs collected from bone marrow we have used two combinations of cell markers (CD45^?/CD73⁺/CD90⁺/CD105⁺ and CD45^?/CD271⁺) and fibroblast colony-forming unit (CFU-F) assay. Further, in donors of various ages, mesenchymal stem cell concentration was compared with the result of CFU-F assay and with hematopoietic stem cell concentration, determined by a standardized flow cytometric assay. A positive correlation of MSC populations to the CFU-F numbers is observed, the population of the CD45^?/CD271⁺ cells correlates better with CFU-F numbers than the population of the CD45^?/CD73⁺/CD90⁺/CD105⁺ cells. The relationship between the hematopoietic CD45^dim/CD34⁺ cell concentration and mesenchymal CFU-Fs or CD45^?/CD271⁺ cells shows a positive linear regression. An age-related quantitative reduction of hematopoietic CD45^dim/CD34⁺, mesenchymal CD45^?/CD73⁺/CD90⁺/CD105⁺ and CD45^?/CD271⁺ stem cells, and CFU-F numbers were noted. Additionally, statistically significant higher CFU-F numbers were observed when bone marrow samples were harvested from three different sites from the anterior iliac crest instead of harvesting the same sample amount only from one site.     

Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells

Background

Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).

Methodology/Principal Findings

We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184⁺/CD271⁻/CD44⁻/CD24⁺ from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184⁻/CD44⁻/CD15^LOW/CD24⁺ and a population of glia that was CD184⁺/CD44⁺ were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo.

Conclusions/Significance

These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations.     

Characterization of Liver Monocytic Myeloid-Derived Suppressor Cells and Their Role in a Murine Model of Non-Alcoholic Fatty Liver Disease

Myeloid-derived suppressor cells (MDSCs) are potent suppressors of T cell immunity in tumors and inflammatory diseases. They are identified by surface expression of CD11b⁺Gr1⁺ in mice, and CD11b⁺Gr1⁺ cells accumulate in the livers of obese mice. However, many myeloid cells share these CD11b⁺Gr1⁺ markers. Accordingly, the aim of this study was to identify the authentic phenotype of MDSCs and investigate their functions in non-alcoholic fatty liver disease (NAFLD). C57BL/6J mice were divided into 2 diet groups: a normal control group and high-fat group to induce NAFLD. We demonstrated that monocytic CD11b⁺Gr1^dim cells could be further divided into 2 populations based on side scatter (SSC) during flow cytometry. We found that SSC^lowCD11b⁺Gr1^dim cells accumulated in the livers of NAFLD mice over time, and that these cells were recruited by the chemokine CCL2 and its receptor CCR2 and might expand in the liver via macrophage colony-stimulating factor stimulation. Furthermore, SSC^lowCD11b⁺Gr1^dim cells had a strong suppressive ability on T cells; this effect was not observed for SSC^highCD11b⁺Gr1^dim cells, and was dependent on nitric oxide production by inducible nitric oxide synthase. Our findings demonstrate that SSC^lowCD11b⁺Gr1^dim cells represent authentic MDSCs in NAFLD livers, and might serve an important negative feedback function in liver inflammation.     

Human gingival fibroblasts: Isolation,characterization, and evaluation of CD146 expression

Gingival fibroblasts (GFs) that exhibit adult stem cell-like characteristics are known as gingival mesenchymal stem cells (GMSCs). Specific mesenchymal stem cell (MSC) markers have not been identified to distinguish GMSCs from GFs. Recently, the cell surface molecule known as cluster of differentiation (CD) 146 has been identified as a potential MSC surface marker. In the present study, we investigated the differentiation potential of GMSCs based on CD146 expression.GFs were isolated by two techniques: tissue explants or enzymatic digestion. GFs were cultured and expanded then magnetically sorted according to CD146 expression. CD146^low and CD146^high cells were collected, expanded, and then tested for stem cell markers by flow cytometry as well as osteogenic and chondrogenic differentiation potential. The differentiation of these cells was analyzed after 21 days using histology, immunofluorescence, real-time quantitative PCR (qPCR), and glycosaminoglycan (GAG) to DNA ratio (GAG/DNA) assays. Positive histological staining indicated osteogenic differentiation of all groups regardless of the isolation techniques utilized. However, none of the groups demonstrated chondrogenic differentiation, confirmed by the lack of collagen type II in the extracellular matrix (ECM) of GF aggregates. Our data suggest that identification of gingival stem cells based solely on CD146 is not sufficient to properly carry out translational research using gingival fibroblasts for novel therapeutic methods of treating oral disease.     

Evaluation of mobilized peripheral stem cells according to CD34 and aldehyde dehydrogenase expression and effect of SSClo ALDHbr cells on hematopoietic recovery

Background aimsWe evaluated hematopoietic stem cells according to CD34 expression and aldehyde dehydrogenase (ALDH) activity in peripheral blood and apheresis product samples from patients after mobilization with granulocyte–colony-stimulating factor (G-CSF) alone or G-CSF after high-dose cyclophosphamide (4 g/m² once daily, intravenously on day 1). We also investigated the relationship between the number of SSC^lo CD45^dim CD34^hi cells, SSC^lo ALDH^br cells and engraftment.MethodsThirty patients (20 males and 10 females), who were candidates for autologous peripheral blood stem cell transplantation, were included in the study. Cyclophosphamide + G-CSF was used for 17 and G-CSF alone for 24 mobilizations. Primary diagnoses were multiple myeloma (n% = 14), Hodgkin's lymphoma (n% = 7), non-Hodgkin's lymphoma (n% = 2), acute myloid leukemia (n% = 2), chronic lymphocytic leukemia (n% = 1) and germ cell testis tumor (n% = 1).ResultsNumbers of SSC^lo CD45^dim CD34^hi cells and SSC^lo ALDH^br cells were highly correlated in both peripheral blood and apheresis products (P < 0.001). We could not find a relationship between the transplanted SSC^lo CD45^dim CD34^hi cell dose or SSC^lo ALDH^br cell dose and platelet or neutrophil recovery. The optimal thresholds for SSC^lo CD45^dim CD34^hi cells were 5.40 × 10⁶/kg for neutrophil recovery and 7.22 × 10⁶/kg for platelet recovery. The optimal thresholds for SSC^lo ALDH^br cells were 6.53 × 10⁶/kg for neutrophil recovery and 8.72 × 10⁶/kg platelet recovery.ConclusionsAccording to our data, numbers of SSC^lo ALDH^br cells are in very good agreement with numbers of SSC^lo CD45^dim CD34^hi cells and can be a predictor of stem cell mobilization.     

Challenges in αCD38-chimeric antigen receptor (CAR)-expressing natural killer (NK) cell-based immunotherapy in multiple myeloma: Harnessing the CD38dim phenotype of cytokine-stimulated NK cells as a strategy to prevent fratricide

Background aimsAdoptive cell therapy with chimeric antigen receptor (CAR)-expressing natural killer (NK) cells is an emerging approach that holds promise in multiple myeloma (MM). However, the generation of CAR-NK cells targeting CD38 is met with obstacles due to the expression of CD38 on NK cells. Knock-out of CD38 is currently explored as a strategy, although the consequences of the lack of CD38 expression with regards to engraftment and activity in the bone marrow microenvironment are not fully elucidated. Here, we present an alternative approach by harnessing the CD38^dim phenotype occurring during long-term cytokine stimulation of primary NK cells.MethodsPrimary NK cells were expanded from peripheral blood mononuclear cells by long-term IL-2 stimulation. During expansion, the CD38 expression was monitored in order to identify a time point when introduction of a novel affinity-optimized αCD38-CAR confered optimal viability, i.e. prevented fratricide. CD38^dim NK cells were trasduced with retroviral vectors encoding for the CAR trasngene and their functionality was assessed in in vitro activation and cytotoxicity assays.ResultsWe verified the functionality of the αCD38-CAR-NK cells against CD38⁺ cell lines and primary MM cells. Importantly, we demonstrated that αCD38-CAR-NK cells derived from patients with MM have increased activity against autologous MM samples ex vivo.ConclusionsOverall, our results highlight that incorporation of a functional αCD38-CAR construct into a suitable NK-cell expansion and activation protocol results in a potent and feasible immunotherapeutic strategy for the treatment of patients with MM.     

Human Natural Killer Cell Maturation Defect Supports In Vivo CD56bright to CD56dim Lineage Development

Two populations of human natural killer (NK) cells can be identified in peripheral blood. The majority are CD3⁻CD56^dim cells while the minority exhibits a CD3⁻CD56^bright phenotype. In vitro evidence indicates that CD56^bright cells are precursors of CD56^dim cells, but in vivo evidence is lacking. Here, we studied NK cells from a patient that suffered from a melanoma and opportunistic fungal infection during childhood. The patient exhibited a stable phenotype characterized by a reduction in the frequency of peripheral blood CD3⁻CD56^dim NK cells, accompanied by an overt increase in the frequency and absolute number of CD3⁻CD56^bright cells. These NK cells exhibited similar expression of perforin, CD57 and CD158, the major activating receptors CD16, NKp46, NKG2D, DNAM-1, and 2B4, as well as the inhibitory receptor CD94/NKG2A, on both CD56^bright and CD56^dim NK cells as healthy controls. Also, both NK cell subpopulations produced IFN-γ upon stimulation with cytokines, and CD3⁻CD56^dim NK cells degranulated in response to cytokines or K562 cells. However, upon stimulation with cytokines, a substantial fraction of CD56^dim cells failed to up-regulate CD57 and CD158, showed a reduction in the percentage of CD16⁺ cells, and CD56^bright cells did not down-regulate CD62L, suggesting that CD56^dim cells could not acquire a terminally differentiated phenotype and that CD56^bright cells exhibit a maturation defect that might result in a potential altered migration pattern. These observations, support the notion that NK cells of this patient display a maturation/activation defect that precludes the generation of mature NK cells at a normal rate accompanied by CD56^dim NK cells that cannot completely acquire a terminally differentiated phenotype. Thus, our results provide evidence that support the concept that in vivo CD56^bright NK cells differentiate into CD56^dim NK cells, and contribute to further understand human NK cell ontogeny.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript> production within tumor microenvironment inversely correlated with infiltration of CD56<Superscript>dim</Superscript> NK cells in gastric and esophageal cancer: possible mechanisms of NK cell dysfunction

Human NK cells can be divided into two subsets, CD56^dimCD16(+)NK and CD56^brightCD16(−)NK cells, based on their expression of CD56 and CD16. In the present study, we analyzed the relationship between CD56^dim/CD56^bright NK cells and H₂O₂ in tumor-infiltrating NK cells in patients with gastric (n = 50) and esophageal (n = 35) cancer. The ratio of CD56^dim NK cells infiltrating tumors gradually decreased according to disease progression. H₂O₂ was abundantly produced within tumor microenvironments, and there was an inverse correlation between CD56^dim NK cell infiltration and H₂O₂ production. CD56^dim NK cells are more sensitive to apoptosis induced by physiological levels of H₂O₂ than CD56^bright NK cells. Furthermore, the exposure of NK cells to H₂O₂ resulted in the impairment of ADCC activity. In conclusion, H₂O₂ produced within tumor microenvironments inversely correlated with the infiltration of CD56^dim NK cells, possibly due to their preferentially induced cell death. These observations may explain one of the mechanisms behind NK cell dysfunction frequently observed in tumor microenvironments.     

Towards expansion of human hair follicle stem cells in vitro

Objectives: Multipotential human hair follicle stem cells can differentiate into various cell lineages and thus are investigated here as potential autologous sources for regenerative medicine. Towards this end, we have attempted to expand these cells, directly isolated from minimal amounts of hair follicle explants, to numbers more suitable for stem‐cell therapy. Materials and methods: Two types of human follicle stem cells, commercially available and directly isolated, were cultured using an in‐house developed medium. The latter was obtained from bulge areas of hair follicles by mechanical and enzymatic dissociation, and was magnetically enriched for its CD200⁺ fraction. Isolated cells were cultured for up to 4 weeks, on different supports: blank polystyrene, laminin‐ and Matrigel^TM‐coated surfaces. Results: Two‐fold expansion was found, highlighting the slow‐cycling nature of these cells. Flow cytometry characterization revealed: magnetic enrichment increased the proportion of CD200⁺ cells from initially 43.3% (CD200+, CD34: 25.8%; CD200+, CD34+: 17.5%) to 78.2% (CD200+, CD34: 41.5%; CD200+, CD34+: 36.7%). Enriched cells seemed to have retained and passed on their morphological and molecular phenotypes to their progeny, as isolated CD200⁺ presenting cells expanded in our medium to a population with 80% of cells being CD200⁺: 51.5% (CD200⁺, CD34^?) and 29.6% (CD200⁺, CD34⁺). Conclusions: This study demonstrates the possibility of culturing human hair follicle stem cells without causing any significant changes to phenotypes of the cells.     

山羊胚胎干细胞的分离与培养

对关中奶山羊配种后6～7天的桑椹胚和囊胚,分别采用全胚培养法、酶消化法和免疫外科法进行处理.将处理后的胚胎培养于小鼠胎儿成纤维细胞(MEF)饲养层上,分离培养山羊胚胎干细胞(Embryonic stem cell,ESC).对分离传代的山羊ESCs分别进行免疫组化染色,RT-PCR检测和体外诱导分化试验.结果表明.全胚培养法易于胚胎贴壁形成原代集落,采用全胚培养法获得的ESCs有一株目前已传至18代.山羊ESCs Nanong、Oct4、SSEA-3免疫组化染色呈阳性,SSEA-1免疫组化染色呈弱阳性,SSEA-4免疫组化染色呈阴性,RT-PCR检测显示其表达Nanog、Oct4、端粒酶、CD117.山羊ESCs经DMSO体外诱导可以向心肌细胞分化.这些试验均表明该细胞具有ESCs的生物学特性.     

Robust and Highly-Efficient Differentiation of Functional Monocytic Cells from Human Pluripotent Stem Cells under Serum- and Feeder Cell-Free Conditions

Monocytic lineage cells (monocytes, macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3×10⁶±0.3×10⁶ floating monocytes from approximately 30 clusters of ESCs/iPSCs 5–6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.