Human bone marrow-derived mesenchymal stem cells differentiate into epidermal-like cells in vitro

Human bone marrow-derived mesenchymal stem cells (hMSCs) are a population of pluripotent cells. They can differentiate into different embryonic layer cells as osteoblasts, adipocytes, chondrocytes, myoblasts, neurocytes, etc. However, there are only few reports with regard to differentiate hMSCs into epidermal cells in vitro. In this study, we want to investigate the feasibility of inducing hMSCs into epidermal-like cells under specific medium in vitro. hMSCs in specific inducing medium expressed the early markers of epidermal cell lineage, P63, cytokeratin19 (CK19), the late differentiated marker, the pan-cytokeratin, and another early marker, the beta1-integrin, which up-regulated remarkably in inducing medium. Their morphologies were changed from spindle-like fibroblastic appearances to oblate or irregular shapes under phase contrast microscopy. The hemidesmosome structure was found using the transmission electron microscope. All these data suggested that, under certain conditions, hMSCs have the potential to differentiate into epidermal-like cells. It will be of great accordance in the study of the multipotential property of hMSCs.     

Ear mesenchymal stem cells (EMSC) can differentiate into spontaneously contracting muscle cells

We have previously shown that cells isolated from the outer ears of adult mice are a source of mesenchymal stem cells that can be induced to differentiate into adipo-, osteo-, and chondrocytes. In this study, we demonstrate that ear mesenchymal stem cells (EMSC) express stromal cell-associated markers (CD44, CD73) and stem cell marker Sca-1 and can be differentiated into spontaneously contracting muscle cells. Treatment of cells with epidermal growth factor (EGF) change their morphology from fibroblast shapes into stick-like structures that show repeated spontaneous contractions. Under conditions that promote myogenic differentiation, EMSC expressed mRNA for myoD and ventricular specific myosin light chain (MLC-2v) and protein for connexin 43, sarcomeric alpha-actinin, myocyte enhancer factor 2c (MEF2c), myosin heavy chain (MyHC), myogenin, and sarco-endoplasmic reticulum Ca(2+)ATPase (SERCA) 1. However, the cells were negative for Nkx2.5, GATA4, and ANP. Intracellular Ca(2+) transients in spontaneously beating EMSC, visualized by Fluo-3AM, showed a frequency of Ca(2+) oscillations ranging over 28-59/min (mean 41.17 +/- SEM 1.54). We also demonstrated that small pieces of ear tissues (ear punches) collected from live mice provide sufficient numbers of EMSC to isolate, culture and differentiate them into myocytes. Due to the ease of acquiring an expanding repertoire of differentiated EMSC cell types by a noninvasive surgical procedure, we conclude that the ear may prove to be a potential source of autologous cells for regenerative medicine, as supported by the fact that ears are one of the best sources of cells for somatic cell nuclear transfer (SCNT).     

Bone marrow-derived mesenchymal stem cells differentiation into tubular epithelial-like cells in vitro

The possibility of differentiating bone marrow‐derived mesenchymal stem cells (BMSCs) into tubular epithelial‐like cells is explored in vitro. Purified BMSCs from Sprague–Dawley rats were obtained by density gradient centrifugation. Third generation BMSCs were divided into six groups and were cultured under different conditions. The expression of alkaline phosphatase and cytokeratin (CK)‐18 protein was detected through staining and immunocytochemistry, respectively, and the expression of E‐cadherin proteins was recorded through immunofluorescence. Some cells in ischemia/reperfusion (I/R), all‐trans retinoic acid (ATRA), epidermal growth factor (EGF) and bone morphogenetic protein‐7 (BMP‐7) groups turned positive, whereas the positive cells in the combined group significantly increased compared with the other groups. Compared with the control group, the positive expression rates of CK‐18 in the I/R, ATRA, EGF, BMP‐7 and the combined group were 11·50% ± 3·84%, 27·40% ± 2·70%, 29·60% ± 4·51%, 26·80% ± 5·00% and 44·00% ± 3·16%, respectively, and CK‐18 mRNA expression in the combined group was obviously higher than that in the other groups (P < 0·01). Immunofluorescence detection showed that E‐cadherin expression was not detectable in the control group, whereas the positive expression rates of E‐cadherin in the I/R, ATRA, EGF, BMP‐7 and the combined group were 6·75% ± 2·13%, 16·40% ± 2·69%, 18·25% ± 3·50%, 16·06% ± 2·00% and 30·26% ± 5·16%, respectively. The addition of ATRA, EGF and BMP‐7 induces BMSCs differentiation into tubular epithelial‐like cells in stimulated acute renal failure microenvironment in vitro. Copyright © 2011 John Wiley & Sons, Ltd.     

Clonal mesenchymal stem cells derived from human bone marrow can differentiate into hepatocyte‐like cells in injured livers of SCID mice

There is increasing evidence that human mesenchymal stem cells (hMSCs) can be a valuable, transplantable source of hepatocytes. Most of the hMSCs preparations used in these studies were likely heterogeneous cell populations, isolated by adherence to plastic surfaces or by density gradient centrifugation. Therefore, the participation of other unknown trace cell populations cannot be rigorously discounted. Here we report the isolation and establishment of a cloned human MSC line (chMSC) from human bone marrow primary culture, through which we confirmed the hepatic differentiation capability of authentic hMSCs. chMSCs expressed markers of mesenchymal cells, but not markers of hematopoietic stem cells. In vitro, chMSCs can differentiate into either mesenchymal cells or cells exhibiting hepatocyte‐like phenotypes. When transplanted intrasplentically into carbon tetrachloride‐injured livers of SCID mice, EGFP‐tagged chMSCs engrafted into the host liver parenchyma, exhibited typical hepatocyte morphology, form a three‐dimensional architecture, and differentiate into hepatocyte‐like cells expressing human albumin and α‐1‐anti‐trypsin. By confocal microscopy, ultrafine intercellular nanotubular structures were visible between adjacent transplanted and host hepatocytes. We postulate that these structures may assist in the phenotype conversion of chMSCs, possibly by exchange of cytoplasmic components between native hepatocytes and transplanted cells. Thus, a clonal pure population of hMSCs, which can be expanded in culture, may have potential as a cellular source for substitution damaged cells in hepatic injury. J. Cell. Biochem. 108: 693–704, 2009. © 2009 Wiley‐Liss, Inc.     

In vitro and in vivo differentiation of human umbilical cord derived stem cells into endothelial cells

The successful use of tissue-engineered transplants is hampered by the need for vascularization. Recent advances have made possible the using of stem cells as cell sources for therapeutic angiogenesis, including the vascularization of engineered tissue grafts. The goal of this study was to examine the endothelial potential of human umbilical cord-derived stem (UCDS) cells. UCDS cells were initially characterized and differentiated in an endothelial differentiation medium containing VEGF and bFGF. Differentiation into endothelial cells was determined by acetylated low-density lipoprotein incorporation and expression of endothelial-specific proteins, such as PECAM and CD34. In vivo, the transplanted UCDS cells were sprouting from local injection and differentiated into endothelial cells in a hindlimb ischemia mouse model. These findings indicate the presence of a cell population within the human umbilical cord that exhibits characteristics of endothelial progenitor cells. Therefore, human umbilical cord might represent a source of stem cells useful for therapeutic angiogenesis and re-endothelialization of engineered tissue grafts.     

Mesenchymal stem cells: a double-edged sword in regulating immune responses

Mesenchymal stem cells (MSCs) have been employed successfully to treat various immune disorders in animal models and clinical settings. Our previous studies have shown that MSCs can become highly immunosuppressive upon stimulation by inflammatory cytokines, an effect exerted through the concerted action of chemokines and nitric oxide (NO). Here, we show that MSCs can also enhance immune responses. This immune-promoting effect occurred when proinflammatory cytokines were inadequate to elicit sufficient NO production. When inducible nitric oxide synthase (iNOS) production was inhibited or genetically ablated, MSCs strongly enhance T-cell proliferation in vitro and the delayed-type hypersensitivity response in vivo. Furthermore, iNOS(-/-) MSCs significantly inhibited melanoma growth. It is likely that in the absence of NO, chemokines act to promote immune responses. Indeed, in CCR5(-/-)CXCR3(-/-) mice, the immune-promoting effect of iNOS(-/-) MSCs is greatly diminished. Thus, NO acts as a switch in MSC-mediated immunomodulation. More importantly, the dual effect on immune reactions was also observed in human MSCs, in which indoleamine 2,3-dioxygenase (IDO) acts as a switch. This study provides novel information about the pathophysiological roles of MSCs.     

An improved protocol that induces human embryonic stem cells to differentiate into neural cells in vitro

Human embryonic stem (ES) cells have the capacity for self-renewal and are able to differentiate into any cell type. However, obtaining high-efficient neural differentiation from human ES cells remains a challenge. This study describes an improved 4-stage protocol to induce a human ES cell line derived from a Chinese population to differentiate into neural cells. At the first stage, embryonic bodies (EBs) were formed in a chemically-defined neural inducing medium rather than in traditional serum or serum-replacement medium. At the second stage, rosette-like structures were formed. At the third stage, the rosette-like structures were manually selected rather than enzymatically digested to form floating neurospheres. At the fourth stage, the neurospheres were further differentiated into neurons. The results show that, at the second stage, the rate of the formation of rosette-like structures from EBs induced by noggin was 88+/-6.32%, higher than that of retinoic acid 55+/-5.27%. Immunocytochemistry staining was used to confirm the neural identity of the cells. These results show a major improvement in obtaining efficient neural differentiation of human ES cells.     

Retention of differentiated characteristics in human fetal keratinocytes in vitro

Summary Many of the morphologic and biochemical changes that occur during human fetal skin development have been described, yet there has been little experimental analysis of the processes that regulate the development of human fetal skin. This is due in part to difficulties in culturing human fetal epidermal keratinocytes. We have successfully cultured fetal keratinocytes in two different in vitro systems; in a serum-free keratinocyte growth medium (KGM) on tissue culture plastic and cocultured with dermal fibroblasts as spheroidal aggregates. To characterize these fetal keratinocytes in vitro we have assessed their ability to express several markers of epidermal differentiation. Human fetal keratinocytes grown on plastic in KGM stratify and express some of the components of the differentiated epidermis, such as involucrin and the high molecular weight keratins. However, these keratinocytes co-express keratins and vimentin and do not form a structured basement membrane. More characteristics of fetal skin are preserved in mixed aggregates of epidermal keratinocytes and dermal fibroblasts including epidermal stratification, synthesis of basement membrane components, tissue-specific expression of intermediate filaments, involucrin, and expression of high molecular weight keratins. The maintenance of human fetal epidermal keratinocytes in these two in vitro systems and their ability to express many differentiated characteristics suggests that these cultures will be valuable for studies of the molecular mechanisms that regulate the regionally specific differentiation of the human fetal epidermis. This work was supported by the Dermatology Foundation Fellowships funded by Herbert Laboratories and The Upjohn Company and awarded to A. R. H., NIH Training Program in Dermatological Research #5T32AR07472, and NIH grant #5R01HD20996 to A. T. L. Publication no. 74 of the Dermatology Department, University of Rochester, Rochester, NY.     

Fibromodulin gene is expressed in human epidermal keratinocytes in culture and in human epidermis in vivo

Fibromodulin is a small leucine-rich proteoglycan that has a central role in the maintenance of collagen fibrils structure, and in regulation of TGF-β biological activity. Although, it is mainly found in cartilage and tendon, little is known regarding the expression of the fibromodulin gene in other cell types. By RT-PCR, real time PCR and immunohistochemistry, we describe the expression of the fibromodulin gene and the presence of the protein in human epidermal keratinocytes (HEK), both in culture and in normal human epidermis. Our results show, for the first time, that fibromodulin gene is constantly expressed in HEK during culture time. Immunostaining showed that fibromodulin is located intracytoplasmically in basal and stratified keratinocytes of the growing colonies, confluent cultures, and epidermis in vivo. The expression and intracellular localization of fibromodulin in HEK is a new finding and opens new possible biological roles for the SLRP family.     

WJSC 6th Anniversary Special Issues（2）:Mesenchymal stem cells Mesenchymal stem cells in the treatment of spinal cord injuries:A review

With technological advances in basic research,the intricate mechanism of secondary delayed spinal cord injury(SCI)continues to unravel at a rapid pace.However,despite our deeper understanding of the molecular changes occurring after initial insult to the spinal cord,the cure for paralysis remains elusive.Current treatment of SCI is limited to early administration of high dose steroids to mitigate the harmful effect of cord edema that occurs after SCI and to reduce the cascade of secondary delayed SCI.R ecent evident-based clinical studies have cast doubt on the clinical benefit of steroids in SCI and intense focus on stem cell-based therapy has yielded some encouraging results.An array of mesenchymal stem cells(MSCs)from various sources with novel and promising strategies are being developed to improve function after SCI.In this review,we briefly discuss the pathophysiology of spinal cord injuries and characteristics and the potential sources of MSCs that can be used in the treatment of SCI.We will discuss the progress of MSCs application in research,focusing on the neuroprotective properties of MSCs.Finally,we will discuss the results from preclinical and clinical trials involving stem cell-based therapy in SCI.     

Salidroside induces rat mesenchymal stem cells to differentiate into dopaminergic neurons

Parkinson's disease (PD) is a neurodegenerative disorder characterised by the loss of substantia nigra dopaminergic neurons that leads to a reduction in striatal dopamine (DA) levels. Replacing lost cells by transplanting dopaminergic neurons has potential value to repair the damaged brain. Salidroside (SD), a phenylpropanoid glycoside isolated from plant Rhodiola rosea, is neuroprotective. We examined whether salidroside can induce mesenchymal stem cells (MSCs) to differentiate into neuron‐like cells, and convert MSCs into dopamine neurons that can be applied in clinical use. Salidroside induced rMSCs to adopt a neuronal morphology, upregulated the expression of neuronal marker molecules, such as gamma neuronal enolase 2 (Eno2/NSE), microtubule‐associated protein 2 (Map2), and beta 3 class III tubulin (Tubb3/β‐tubulin III). It also increased expression of brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3) and nerve growth factor (NGF) mRNAs, and promoted the secretion of these growth factors. The expression of dopamine neurons markers, such as dopamine‐beta‐hydroxy (DBH), dopa decarboxylase (DDC) and tyrosine hydroxylase (TH), was significantly upregulated after treatment with salidroside for 1–12 days. DA steadily increased after treatment with salidroside for 1–6 days. Thus salidroside can induce rMSCs to differentiate into dopaminergic neurons.     

Mesenchymal stem cells isolated from human uterine cervix cancer tissues

In the present study, MSCs (mesenchymal stem cells) were successfully isolated and identified from hUCC (human uterine cervix cancer) tissues. The morphological appearance, immunophenotype, growth curve, cell cycle, cytogenetic features and differentiation potential of these cells were investigated. Results showed that cells isolated from the uterine cervix cancer tissues displayed fibroblast‐like morphology and grew into colonies. Immunophenotyping by flow cytometry revealed that the isolated cells were positive for CD13, CD29, CD44, CD105 and HLA‐I, while negative for CD10, CD14, CD31, CD34, CD38 and HLA‐DR. The cells kept a normal karyotype by chromosome analysis. At the third passage, the percentages of cells in G₀‐/G₁‐, 2‐/M‐ and S‐phase were 84.94, 8.36 and 6.71%, respectively. Under appropriate induction conditions, these cells can differentiate into osteogenic, adipogenic cells and hepatocytes. Taken together, MSCs were confirmed to exist in hUCC tissues, which may provide a new target for clinical cancer therapy.     

Putative epidermal stem cell convert into corneal epithelium-like cell under corneal tissue in vitro

Rhesus putative epidermal stem cells are being investigated for their potential use in regenerative corneal epithelium-like cells, which may provide a practical source of autologous seed cells for the construction of bioengineered corneas. The goal of this study was to investigate the potential of epi-dermal stem cells for trans-differentiation into corneal epithelium-like cells. Rhesus putative epidermal stem cells were isolated by type IV collagen attachment method. Flow cytometry analysis, immuno-histology and RT-PCR were conducted to identify the expression of specific markers (β1, α6 integrin, K15, K1/K10, K3/K12 and CD71) on the isolated rapid attaching cells. The isolated cells were cocultured with human corneal limbal stroma and corneal epithelial cells. After coculture, the expression of the same specific markers was evaluated in order to identify expression difference caused by the coculture conditions. K3/K12 expression was analyzed in coculture cells on day 2, 4, 6, 8 and 10. Putative epi-dermal stem cells in conditioned culture media were used as control. Putative epidermal stem cells were predominant in rapid attaching cells by type IV collagen attachment isolation. Before being co-cultured, the rhesus putative epidermal stem cells expressed K15, α6 and β1 integrin, but no CD71, K1/K10 and K3/K12. After coculture, these cells expressed K3/K12 (a marker of corneal epithelial cells), K15 and β 1 integrin, but no K1/K10. Cells being not coculture converted into terminally differentiated cells expressing K1/K10. These results indicate that rhesus putative epidermal stem cells can trans-differentiate into corneal epithelium-like cells and, therefore, may have potential therapeutic application as autologous seed cells for the construction of bioengineered corneas.     

人多能干细胞向红细胞的诱导分化

目前,体外生成人红细胞的实验技术较为复杂,为优化诱导步骤,采用两步法将人多能干细胞诱导分化为红细胞。首先,利用人多能干细胞(包括Rh阴性A型的脐带间充质干细胞(hUCMSC~(Rh-A))和人iPS(hiPS)细胞)在BVF培养液中进行诱导分化得到CD31~~+和CD34~+的阳性细胞群。经PCR和流式细胞仪检测CD31和CD34的表达发现,hUCMSC~(Rh-A)细胞诱导得到的CD31和CD34阳性细胞率分别是5.3%和22.7%;hiPS细胞诱导得到的CD31和CD34阳性细胞率分别是31.2%和8.2%。第二步,将获得的CD31~+和CD34~+的阳性细胞群在多种生长因子的作用下经过36 d诱导,分化为成熟红细胞。经吉姆萨染色检测得到的红细胞在形态和大小上与正常人红细胞相近,且存在血细胞去核的现象。荧光定量RT-PCR检测到了globin的表达,其中β-globin的表达量占20%以上。将得到的红细胞收集到离心管中,自然沉降后可见红色的红细胞沉淀。上述研究为大量制备人红细胞提供了新的有效的技术方法。