Platelet‐derived growth factor promotes the proliferation of human umbilical cord‐derived mesenchymal stem cells

This study was designed to investigate the effect of platelet‐derived growth factor (PDGF) on the proliferation of human umbilical cord mesenchymal stem cells (UC‐MSCs) and further explore the mechanism of PDGF in promoting the proliferation of UC‐MSCs. The human UC‐MSCs were treated with different concentrations of PDGF, and the effects were evaluated by counting the cell number, the cell viability, the expression of PDGF receptors analyzed by RT‐PCR, and the detection of the gene expression of cell proliferation, cell cycle and pluripotency, and Brdu assay by immunofluorescent staining and Quantitative real‐time (QRT‐PCR). The results showed that PDGF could promote the proliferation of UC‐MSCs in vitro in a dose‐dependent way, and 10 to 50 ng/ml PDGF had a significant proliferation effect on UC‐MSCs; the most obvious concentration was 50 ng/ml. Significant inhibition on the proliferation of UC‐MSCs was observed when the concentration of PDGF was higher than 100 ng/ml, and all cells died when the concentration reached 200 ng/ml PDGF. The PDGF‐treated cells had stronger proliferation and antiapoptotic capacity than the control group by Brdu staining. The expression of the proliferation‐related genes C‐MYC, PCNA and TERT and cell cycle–related genes cyclin A, cyclin 1 and CDK2 were up‐regulated in PDGF medium compared with control. However, pluripotent gene OCT4 was not significantly different between cells cultured in PDGF and cells analyzed by immunofluorescence and QRT‐PCR. The PDGF could promote the proliferation of human UC‐MSCs in vitro. Copyright © 2012 John Wiley & Sons, Ltd.     

骨髓间充质干细胞的免疫学研究进展

间充质干细胞（mesenchymal stem cells,MSCs）是骨髓中除造血干细胞以外的另一种成体干细胞,广泛分布于动物体内骨髓、肝脏、脂肪等多种组织中。MSCS具有强大的自我更新能力和多向分化潜能,是移植领域应用前景广阔的再生来源细胞;同时,MSCs是一种重要的免疫调节细胞,MSCs在炎症细胞因子刺激后对免疫系统表现出很强的抑制作用,所以MSCs有望应用于减少免疫排斥,延长移植物存活时间,治疗相关免疫失调症,如自身免疫疾病等方面。本文主要对间充质干细胞与免疫系统相互作用的研究做相关介绍。     

Induction of hepatocyte‐like cells from human umbilical cord‐derived mesenchymal stem cells by defined microRNAs

Generating functional hepatocyte‐like cells (HLCs) from mesenchymal stem cells (MSCs) is of great urgency for bio‐artificial liver support system (BALSS). Previously, we obtained HLCs from human umbilical cord‐derived MSCs by overexpressing seven microRNAs (HLC‐7) and characterized their liver functions in vitro and in vivo. Here, we aimed to screen out the optimal miRNA candidates for hepatic differentiation. We sequentially removed individual miRNAs from the pool and examined the effect of transfection with remainder using RT‐PCR, periodic acid—Schiff (PAS) staining and low‐density lipoprotein (LDL) uptake assays and by assessing their function in liver injury models. Surprisingly, miR‐30a and miR‐1290 were dispensable for hepatic differentiation. The remaining five miRNAs (miR‐122, miR‐148a, miR‐424, miR‐542‐5p and miR‐1246) are essential for this process, because omitting any one from the five‐miRNA combination prevented hepatic trans‐differentiation. We found that HLCs trans‐differentiated from five microRNAs (HLC‐5) expressed high level of hepatic markers and functioned similar to hepatocytes. Intravenous transplantation of HLC‐5 into nude mice with CCl₄‐induced fulminant liver failure and acute liver injury not only improved serum parameters and their liver histology, but also improved survival rate of mice in severe hepatic failure. These data indicated that HLC‐5 functioned similar to HLC‐7 in vitro and in vivo, which have been shown to resemble hepatocytes. Instead of using seven‐miRNA combination, a simplified five‐miRNA combination can be used to obtain functional HLCs in only 7 days. Our study demonstrated an optimized and efficient method for generating functional MSC‐derived HLCs that may serve as an attractive cell alternative for BALSS.     

The immunologic and hematopoietic profiles of mesenchymal stem cells derived from different sections of human umbilical cord

Mesenchymal stem cells （MSCs） have been widely used in allogeneic stem cell transplantation. We compared im- munologic and hematopoietic characteristics of MSCs derived from whole human umbilical cord （UC）, as well as from different sections of UCs, including the amniotic membrane （AM）, Wharton＇s jelly （WJ）, and umbilical vessel （UV）. Cell phenotypes were examined by flow cytometry. Lymphocyte transformation test and mixed lymphocyte reaction were performed to evaluate the immuno-modulatory activity of MSCs derived from UCs. The mRNA expression of cytokines was detected by real- time polymerase chain reaction. Hematopoietic function was studied by co-culturing MSCs with CD34＋ cells iso- lated from cord blood. Our results showed that MSCs separated from these four different sections including UC, W J, UV, and AM had similar biological characteristics. All of the MSCs had multi-lineage differentiation ability and were able to differentiate into osteoblasts, adipocytes, and chondrocytes. The MSCs also inhibited the proliferation of allogeneic T cells in a dose-dependent manner. The relative mRNA expression of cytokines was examined, and the results showed that UCMSCs had higher interleukin-6 （IL6）, ILll, stem cell factor, and FLT3 expression than MSCs derived from specific sections of UCs. CD34＋ cells had high propagation efficiencies when co-cultured with MSCs derived from different sections of UCs, among which UCMSCs are the most efficient feeding layer. Our study demonstrated that MSCs could be isolated from whole UC or specific sections of UC with similar immuno- modulation and hematopoiesis supporting characteristics.     

Optimization of primary culture condition for mesenchymal stem cells derived from umbilical cord blood with factorial design

Mesenchymal stem cells (MSCs) can not only support the expansion of hematopoietic stem cells in vitro, but also alleviate complications and accelerate recovery of hematopoiesis during hematopoietic stem cell transplantation. However, it proved challenging to culture MSCs from umbilical cord blood (UCB) with a success rate of 20–30%. Many cell culture parameters contribute to this outcome and hence optimization of culture conditions is critical to increase the probability of success. In this work, fractional factorial design was applied to study the effect of cell inoculated density, combination and dose of cytokines, and presence of serum and stromal cells. The cultured UCB‐MSC‐like cells were characterized by flow cytometry and their multilineage differentiation potentials were tested. The optimal protocol was identified achieving above 90% successful outcome: 2 × 10⁶ cells/mL mononuclear cells inoculated in Iscove's modified Dulbecco's medium supplied with 10% FBS, 15 ng/mL IL‐3, and 5 ng/mL Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). Moreover, the UCB‐MSC‐like cells expressed MSC surface markers of CD13, CD29, CD105, CD166, and CD44 positively, and CD34, CD45, and human leukocyte antigens‐DR (HLA‐DR) negatively. Meanwhile, these cells could differentiate into osteoblasts, chondrocytes, and adipocytes similarly to MSCs derived from bone marrow. In conclusion, we have developed an efficient protocol for the primary culture of UCB‐MSCs by adding suitable cytokines into the culture system. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Differentiation of human umbilical cord Wharton's jelly‐derived mesenchymal stem cells into germ‐like cells in vitro

Recent studies have demonstrated that mesenchymal stem cells could differentiate into germ cells under appropriate conditions. We sought to determine whether human umbilical cord Wharton's jelly‐derived mesenchymal stem cells (HUMSCs) could form germ cells in vitro. HUMSCs were induced to differentiate into germ cells in all‐trans retinoic acid, testosterone and testicular‐cell‐conditioned medium prepared from newborn male mouse testes. HUMSCs formed “tadpole‐like” cells after induction with different reagents and showed both mRNA and protein expression of germ‐cell‐specific markers Oct4 (POUF5), Ckit, CD49_f (α6), Stella (DDPA3), and Vasa (DDX4). Our results may provide a new route for reproductive therapy involving HUMSCs and a novel in vitro model to investigate the molecular mechanisms that regulate the development of the mammalian germ lineage. J. Cell. Biochem. 109: 747–754, 2010. © 2010 Wiley‐Liss, Inc.     

Human umbilical cord blood‐derived mesenchymal stem cells (hUCB‐MSC) inhibit the proliferation of K562 (human erythromyeloblastoid leukaemic cell line)

hUCB‐MSC (human umbilical cord blood‐derived mesenchymal stem cells) offer an attractive alternative to bone marrow‐derived MSC for cell‐based therapy by being less invasive a source of biological material. We have evaluated the effect of hUCB‐MSC on the proliferation of K562 (an erythromyeloblastoid cell line) and the cytokine secretion pattern of hUCB‐MSC. Co‐culturing of hUCB‐MSC and K562 resulted in inhibition of proliferation of K562 in a dose‐dependent manner. However, the anti‐proliferative effect was reduced in transwells, suggesting the importance of direct cell‐to‐cell contact. hUCB‐MSC inhibited proliferation of K562, arresting them in the G₀/G₁ phase. NO (nitric oxide) was not involved in the hUCB‐MSC‐mediated tumour suppression. The presence of IL‐6 (interleukin 6) and IL‐8 were obvious in the hUCB‐MSC conditioned media, but no significant increase was found in 29 other cytokines. Th1 cytokines, IFNα (interferon α), Th2 cytokine IL‐4 and Th17 cytokine, IL‐17 were not secreted by hUCB‐MSC. There was an increase in the number of hUCB‐MSC expressing the latent membrane‐bound form of TGFβ1 co‐cultured with K562. The anti‐proliferative effect of hUCB‐MSC was due to arrest of the growth of K562 in the G₀/G₁ phase. The mechanisms underlying increased IL‐6 and IL‐8 secretion and LAP (latency‐associated peptide; TGFβ1) by hUCB‐MSC remains unknown.     

Study of differentiated human umbilical cord–derived mesenchymal stem cells transplantation on rat model of advanced parkinsonism

The aim of this study was to explore the curative effect of differentiated human umbilical cord–derived mesenchymal stem cells (hUC‐MSCs) transplantation on rat of advanced Parkinson disease (PD) model. Human umbilical cord–derived mesenchymal stem cells were cultured and induced differentiation in vitro. The PD rats were established and allocated randomly into 2 groups: differentiated hUC‐MSCs groups and physiological saline groups (the control group). Rotation test and immunofluorescence double staining were done. The result showed that hUC‐MSCs could differentiate into mature dopamine neurons. Frequency of rotation was significantly less in differentiated hUC‐MSCs groups than in normal saline group. After we transplanted these cells into the unilateral lesioned substantia nigra induced by striatal injection of 6‐hydroxydopamine and performed in the medial forebrain bundle and ventral tegmental area, nigral tyrosine hydroxylase–positive cells were observed and survival of at least 2 months. In addition, transplantation of hUC‐MSCs could make an obviously therapeutic effect on PD rats.     

The umbilical cord matrix is a better source of mesenchymal stem cells (MSC) than the umbilical cord blood

Many studies have drawn attention to the emerging role of MSC (mesenchymal stem cells) as a promising population supporting new clinical concepts in cellular therapy. However, the sources from which these cells can be isolated are still under discussion. Whereas BM (bone marrow) is presented as the main source of MSC, despite the invasive procedure related to this source, the possibility of isolating sufficient numbers of these cells from UCB (umbilical cord blood) remains controversial. Here, we present the results of experiments aimed at isolating MSC from UCB, BM and UCM (umbilical cord matrix) using different methods of isolation and various culture media that summarize the main procedures and criteria reported in the literature. Whereas isolation of MSC were successful from BM (10:10) and (UCM) (8:8), only one cord blood sample (1:15) gave rise to MSC using various culture media [DMEM (Dulbecco's modified Eagle's medium) +5% platelet lysate, DMEM+10% FBS (fetal bovine serum), DMEM+10% human UCB serum, MSCGM®] and different isolation methods [plastic adherence of total MNC (mononuclear cells), CD3⁺/CD19⁺/CD14⁺/CD38⁺‐depleted MNC and CD133⁺‐ or LNGFR⁺‐enriched MNC]. MSC from UCM and BM were able to differentiate into adipocytes, osteocytes and hepatocytes. The expansion potential was highest for MSC from UCM. The two cell populations had CD90⁺/CD73⁺/CD105⁺ phenotype with the additional expression of SSEA4 and LNGFR for BM MSC. These results clearly exclude UCB from the list of MSC sources for clinical use and propose instead UCM as a rich, non‐invasive and abundant source of MSC.     

Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue

Human umbilical cord blood (CB) is a potential source for mesenchymal stem cells (MSC) capable of forming specific tissues, for example, bone, cartilage, or muscle. However, difficulty isolating MSC from CB (CB‐MSC) has impeded their clinical application. Using more than 450 CB units donated to two public CB banks, we found that successful cell recovery fits a hyper‐exponential function of time since birth with very high fidelity. Additionally, significant improvement in the isolation of CB‐MSC was achieved by selecting cord blood units having a volume ≥90 ml and time ≤2 h after donor's birth. This resulted in 90% success in isolation of CB‐MSC by density gradient purification and without a requirement for immunoaffinity methods as previously reported. Using MSC isolated from bone marrow (BM‐MSC) and adipose tissue (AT‐MSC) as reference controls, we observed that CB‐MSC exhibited a higher proliferation rate and expanded to the order of the 1 × 10⁹ cells required for cell therapies. CB‐MSC showed karyotype stability after prolonged expansion. Functionally, CB‐MSC could be more readily induced to differentiate into chondrocytes than could BM‐MSC and AT‐MSC. CB‐MSC showed immunosuppressive activity equal to that of BM‐MSC and AT‐MSC. Collectively, our data indicate that viable CB‐MSC could be obtained consistently and that CB should be reconsidered as a practical source of MSC for cell therapy and regenerative medicine using the well established CB banking system. J. Cell. Biochem. 112: 1206–1218, 2011. © 2011 Wiley‐Liss, Inc.     

Adipose‐derived mesenchymal stem cells and regenerative medicine

Adipose tissue‐derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β‐cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow‐derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs.     

Potential surrogate quantitative immunomodulatory potency assay for monitoring human umbilical cord-derived mesenchymal stem cells production

Mesenchymal stem cells (MSCs) play an important role as immune modulator through interaction with several immune cells, including macrophages. In this study, the immunomodulatory potency of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) was demonstrated in the in vivo middle cerebral artery occlusion (MCAo)-induced brain injury rat model and in vitro THP-1-derived macrophages model. At 24 h after induction of MCAo, hUC-MSCs was administered via tail vein as a single dose. Remarkably, hUC-MSCs could inhibit M1 polarization and promote M2 polarization of microglia in vivo after 14 days induction of MCAo. Compared with THP-1-derived macrophages which had been stimulated by lipopolysaccharide, the secretion of proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interferon-γ inducible protein (IP-10), were significantly reduced in the presence of hUC-MSCs. Moreover, the secretion of anti-inflammatory cytokine, interleukin-10 (IL-10), was significantly increased after cocultured with hUC-MSCs. Prostaglandins E2 (PGE2), secreted by hUC-MSCs, is one of the crucial immunomodulatory factors and could be inhibited in the presence of COX2 inhibitor, NS-398. PGE2 inhibition suppressed hUC-MSCs immunomodulatory capability, which was restored after addition of synthetic PGE2, establishing the minimum amount of PGE2 required for immunomodulation. In conclusion, our data suggested that PGE2 is a crucial potency marker involved in the therapeutic activity of hUC-MSCs through macrophages immune response modulation and cytokines regulation. This study provides the model for the development of a surrogate quantitative potency assay of immunomodulation in stem cells production.     

Mesenchymal stem cells derived from human placenta suppress allogeneic umbilical cord blood lymphocyte proliferation

Human placenta-derived mononuclear cells （MNC） were isolated by a Percoll density gradient and cultured in mesenchymal stem cell （MSC） maintenance medium. The homogenous layer of adherent cells exhibited a typical fibroblastlike morphology, a large expansive potential, and cell cycle characteristics including a subset of quiescent cells. In vitro differentiation assays showed the tripotential differentiation capacity of these cells toward adipogenic, osteogenic and chondrogenic lineages. Flow cytometry analyses and immunocytochemistry stain showed that placental MSC was a homogeneous cell population devoid of hematopoietic cells, which uniformly expressed CD29, CD44, CD73, CD105, CD166, laminin, fibronectin and vimentin while being negative for expression of CD31, CD34, CD45 and m-smooth muscle actin. Most importantly, immuno-phenotypic analyses demonstrated that these cells expressed class Ⅰ major histocompatibility complex （MHC-I）, but they did not express MHC-Ⅱ molecules. Additionally these cells could suppress umbilical cord blood （UCB） lymphocytes proliferation induced by cellular or nonspecific mitogenic stimuli. This strongly implies that they may have potential application in allograft transplantation. Since placenta and UCB are homogeneous, the MSC derived from human placenta can be transplanted combined with hematopoietic stem cells （HSC） from UCB to reduce the potential graft-versus-host disease （GVHD） in recipients.     

人脐带间充质干细胞作为维持人胚胎干细胞生长饲养层细胞的研究

目的寻找可以维持人胚胎干细胞未分化生长的人源性细胞作为饲养层细胞,从而解决使用鼠源性细胞作为饲养层带来的安全问题。方法尝试以人脐带间充质干细胞作为饲养层细胞来培养人胚胎干细胞,检验其是否可以维持人胚胎干细胞的未分化生长状态。用胶原酶消化法分离人脐带间充质干细胞,光镜下观察细胞形态;流式细胞仪检测其表面标志;诱导人脐带间充质干细胞向成骨细胞和脂肪细胞进行分化。将人胚胎干细胞系H1接种于丝裂霉素C灭活后的人脐带间充质干细胞上,每隔5d进行一次传代。培养20代后,对人胚胎干细胞特性进行相关检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达、分化能力。结果从人脐带中分离出的间充质干细胞为梭形,呈平行排列生长或漩涡状生长;细胞高表达CD44、CD29、CD73、CD105、CD90、CD86、CD147、CD117,不表达CD14、CD38、CD133、CD34、CD45、HLA-DR;具有分化成脂肪细胞和成骨细胞的潜能。人胚胎干细胞在人脐带间充质干细胞饲养层上培养20代后,继续保持人胚胎干细胞的典型形态,碱性磷酸酶染色为阳性,免疫荧光染色显示OCT4、Nanog、SSEA4、TRA-1-81、TRA-1-60的表达为阳性,SSEA1表达为阴性,体外悬浮培养可以形成拟胚体。结论人脐带间充质干细胞可以作为人胚胎干细胞的饲养层细胞,支持其生长,并维持其未分化生长状态。     

Immunomodulatory effects of mesenchymal stem cells for the treatment of cardiac allograft rejection

Heart transplantation continues to be the gold standard clinical intervention to treat patients with end-stage heart failure. However, there are major complications associated with this surgical procedure that reduce the survival prognosis of heart transplant patients, including allograft rejection, malignancies, infections, and other complications that arise from the use of broad-spectrum immunosuppression drugs. Recent studies have demonstrated the use of mesenchymal stem cells (MSCs) against allotransplantation rejection in both in vitro and in vivo settings due to their immunomodulatory properties. Therefore, utilization of MSCs provides new and exciting strategies to improve heart transplantation and potentially reduce the use of broad-spectrum immunosuppression drugs while alleviating allograft rejection. In this review, we will discuss the current research on the mechanisms of cardiac allograft rejection, the physiological and immunological characteristics of MSCs, the effects of MSCs on the immune system, and immunomodulation of heart transplantation by MSCs.     

Osteogenic differentiation of GFP-labeled human umbilical cord blood derived mesenchymal stem cells after cryopreservation

The osteogenic capacity of human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) has been demonstrated both in vitro and in vivo. Therefore, cell labeling and storage are becoming necessary for researching the potential therapeutic use of UCB-MSCs for bone tissue engineering. The aim of this study was to determine the effect of cryopreservation on the osteogenic differentiation of green fluorescent protein (GFP)-marked UCB-MSCs in vitro. MSCs were isolated from full-term human UCB, expanded, transfected with the GFP gene, and then cryopreserved in liquid nitrogen for 4 weeks. After thawing, cell surface antigen markers and osteogenic potential were analyzed, and the luminescence of these cells was observed by fluorescence microscopy. The results demonstrate that cryopreservation has no effect on the cell phenotype, GFP expression or osteogenic differentiation of UCB-MSCs, showing that cryopreserved GFP-labeled UCB-MSCs might be applied for bone tissue engineering.