Conditioned media from human umbilical cord blood-derived mesenchymal stem cells stimulate rejuvenation function in human skin

Developing treatments that inhibit skin aging is an important research project. Rejuvenation, which focuses on prevention of skin aging, is one of the major issues. Recent studies suggested that mesenchymal stem cells (MSCs) secrete many cytokines, which are important in wound healing. In this study, we investigated the effect of human umbilical cord blood-derived mesenchymal stem cells conditioned media (USC-CM) in cutaneous wound healing and collagen synthesis. We found that USC-CM has many useful growth factors associated with skin rejuvenation, such as Epithelial Growth Factor (EGF), basic Fibroblast Growth Factor (bFGF), Platelet Derived Growth Factor (PDGF), Hepatocyte Growth Factor (HGF), Collagen type 1, and especially, one of the rejuvenation factors, the growth differentiation factor-11 (GDF-11). Our in vitro results showed that USC-CM stimulate growth and extracellular matrix (ECM) production of Human Dermal Fibroblasts (HDFs) compared to those of other MSCs conditioned media (CM) from different origins. Moreover, we evaluated the roles of GDF-11. The results showed that GDF-11 accelerates growth, migration and ECM production of HDFs. Our In vivo results showed that topical treatment of USC-CM showed anti-wrinkle effect and significantly increased dermal density in women. In conclusion, USC-CM has various useful growth factors including GDF-11 that can stimulate skin rejuvenation by increasing growth and ECM production of HDFs.     

CD14 monocytes promote the immunosuppressive effect of human umbilical cord matrix stem cells

Here, the effect of CD14⁺ monocytes on human umbilical cord matrix stem cell (hUC-MSC)-mediated immunosuppression was studied in vitro. hUC-MSCs exerted a potent inhibitory effect on the proliferation and interferon-γ (IFN-γ) secretion capacities of CD4⁺ and CD8⁺ T cells in response to anti-CD3/CD28 stimulation. Transwell co-culture system revealed that the suppressive effect was primarily mediated by soluble factors. Addition of prostaglandin synthesis inhibitors (indomethacin or NS-398) almost completely abrogated the immunosuppression activity of hUC-MSCs, identifying prostaglandin E₂ (PGE₂) as an important soluble mediator. CD14⁺ monocytes were found to be able to enhance significantly the immunosuppressive effect of hUC-MSCs in a dose-dependent fashion. Moreover, the inflammatory cytokine IL-1β, either exogenously added or produced by CD14⁺ monocytes in culture, could trigger expression of high levels of PGE₂ by hUC-MSCs, whereas inclusion of the IL-1 receptor antagonist (IL-1RA) in the culture down-regulated not only PGE₂ expression, but also reversed the promotional effect of CD14⁺ monocytes and partially restored CD4⁺ and CD8⁺ T cell proliferation and IFN-γ secretion. Our data demonstrate an important role of monocytes in the hUC-MSC-induced immunomodulation, which may have important implications in future efforts to explore the clinical potentials of hUC-MSCs.     

In vitro differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocyte-like cells

In addition to long-term self-renewal capability, human mesenchymal stem cells (MSCs) possess versatile differentiation potential ranging from mesenchyme-related multipotency to neuroectodermal and endodermal competency. Of particular concern is hepatogenic potential that can be used for liver-directed stem cell therapy and transplantation. In this study, we have investigated whether human umbilical cord blood (UCB)-derived MSCs are also able to differentiate into hepatocyte-like cells. MSCs isolated from UCB were cultured under the pro-hepatogenic condition similar to that for bone marrow (BM)-derived MSCs. Expression of a variety of hepatic lineage markers was analyzed by flow cytometry, RT-PCR, Western blot, and immunofluorescence. The functionality of differentiated cells was assessed by their ability to incorporate DiI-acetylated low-density lipoprotein (DiI-Ac-LDL). As the cells were morphologically transformed into hepatocyte-like cells, they expressed Thy-1, c-Kit, and Flt-3 at the cell surface, as well as albumin, alpha-fetoprotein, and cytokeratin-18 and 19 in the interior. Moreover, about a half of the cells were found to acquire the capability to transport DiI-Ac-LDL. Based on these observations, and taking into account immense advantages of UCB over other stem cell sources, we conclude that UCB-derived MSCs retain hepatogenic potential suitable for cell therapy and transplantation against intractable liver diseases.     

Angiogenic CXC chemokine expression during differentiation of human mesenchymal stem cells towards the osteoblastic lineage

The potential role of ELR(+) CXC chemokines in early events in bone repair was studied using human mesenchymal stem cells (hMSCs). Inflammation, which occurs in the initial phase of tissue healing in general, is critical to bone repair. Release of cytokines from infiltrating immune cells and injured bone can lead to recruitment of MSCs to the region of repair. CXC chemokines bearing the Glu-Leu-Arg (ELR) motif are also released by inflammatory cells and serve as angiogenic factors stimulating chemotaxis and proliferation of endothelial cells. hMSCs, induced to differentiate with osteogenic medium (OGM) containing ascorbate, beta-glycerophosphate (beta-GP), and dexamethasone (DEX), showed an increase in mRNA and protein secretion of the ELR(+) CXC chemokines CXCL8 and CXCL1. CXCL8 mRNA half-life studies reveal an increase in mRNA stability upon OGM stimulation. Increased expression and secretion is a result of DEX in OGM and is dose-dependent. Inhibition of the glucocorticoid receptor with mifepristone only partially inhibits DEX-stimulated CXCL8 expression indicating both glucocorticoid receptor dependent and independent pathways. Treatment with signal transduction inhibitors demonstrate that this expression is due to activation of the ERK and p38 mitogen-activated protein kinase (MAPK) pathways and is mediated through the G(alphai)-coupled receptors. Angiogenesis assays demonstrate that OGM-stimulated conditioned media containing secreted CXCL8 and CXCL1 can induce angiogenesis of human microvascular endothelial cells in an in vitro Matrigel assay.     

Sphingosine-1-phosphate promotes the differentiation of human umbilical cord mesenchymal stem cells into cardiomyocytes under the designated culturing conditions

Background  

It is of growing interest to develop novel approaches to initiate differentiation of mesenchymal stem cells (MSCs) into cardiomyocytes. The purpose of this investigation was to determine if Sphingosine-1-phosphate (S1P), a native circulating bioactive lipid metabolite, plays a role in differentiation of human umbilical cord mesenchymal stem cells (HUMSCs) into cardiomyocytes. We also developed an engineered cell sheet from these HUMSCs derived cardiomyocytes by using a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) cell sheet technology.     

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。     

Direct intracardiac injection of umbilical cord-derived stromal cells and umbilical cord blood-derived endothelial cells for the treatment of ischemic cardiomyopathy

The development of new therapeutic strategies is necessary to reduce the worldwide social and economic impact of cardiovascular disease, which produces high rates of morbidity and mortality. A therapeutic option that has emerged in the last decade is cell therapy. The aim of this study was to compare the effect of transplanting human umbilical cord-derived stromal cells (UCSCs), human umbilical cord blood-derived endothelial cells (UCBECs) or a combination of these two cell types for the treatment of ischemic cardiomyopathy (IC) in a Wistar rat model. IC was induced by left coronary artery ligation, and baseline echocardiography was performed seven days later. Animals with a left ventricular ejection fraction (LVEF) of ≤40% were selected for the study. On the ninth day after IC was induced, the animals were randomized into the following experimental groups: UCSCs, UCBECs, UCSCs plus UCBECs, or vehicle (control). Thirty days after treatment, an echocardiographic analysis was performed, followed by euthanasia. The animals in all of the cell therapy groups, regardless of the cell type transplanted, had less collagen deposition in their heart tissue and demonstrated a significant improvement in myocardial function after IC. Furthermore, there was a trend of increasing numbers of blood vessels in the infarcted area. The median value of LVEF increased by 7.19% to 11.77%, whereas the control group decreased by 0.24%. These results suggest that UCSCs and UCBECs are promising cells for cellular cardiomyoplasty and can be an effective therapy for improving cardiac function following IC.     

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.     

In vitro labeling of human umbilical cord mesenchymal stem cells with superparamagnetic iron oxide nanoparticles

Human umbilical cord mesenchymal stem cells (hUC‐MSCs) transplantation has been shown to promote regeneration and neuroprotection in central nervous system (CNS) injuries and neurodegenerative diseases. To develop this approach into a clinical setting it is important to be able to follow the fates of transplanted cells by noninvasive imaging. Neural precursor cells and hematopoietic stem cells can be efficiently labeled by superparamagnetic iron oxide (SPIO) nanoparticle. The purpose of our study was to prospectively evaluate the influence of SPIO on hUC‐MSCs and the feasibility of tracking for hUC‐MSCs by noninvasive imaging. In vitro studies demonstrated that magnetic resonance imaging (MRI) can efficiently detect low numbers of SPIO‐labeled hUC‐MSCs and that the intensity of the signal was proportional to the number of labeled cells. After transplantation into focal areas in adult rat spinal cord transplanted SPIO‐labeled hUC‐MSCs produced a hypointense signal using T2‐weighted MRI in rats that persisted for up to 2 weeks. This study demonstrated the feasibility of noninvasive imaging of transplanted hUC‐MSCs. J. Cell. Biochem. 108: 529–535, 2009. © 2009 Wiley‐Liss, Inc.     

人脐带间充质干细胞对1型糖尿病鼠肝脏损伤的保护作用

目的探讨人脐带间充质干细胞（hUCMSCs）对初发1型非肥胖型糖尿病（NOD）小鼠肝脏损伤的保护作用。 方法雌性NOD小鼠共33只,饲养9周后,将成模的21只小鼠随机分为糖尿病组和干细胞组,每组10只,其中干细胞（MSCs）组发病后第3天尾静脉注射hUCMSCs 1?次;另取10只未发病小鼠为正常对照组。各组小鼠每周检测随机血糖（GLU）水平,8周后处死小鼠,取肝脏,HE染色后观察肝脏结构改变,ELISA法检测糖基化终末产物（AGEs）水平,Real-time PCR法检测糖基化终末产物受体（RAGE）、NF-κB P65、白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α） mRNA的表达水平。采用单因素方差分析和SNK-q检验进行统计学分析。 结果MSCs治疗8周后,MSCs组小鼠随机血糖（8.46±1.37）mmol/L比T1DM组（32.82±0.59）?mmol/L降低,差异具有统计学意义（P < 0.05）。同时T1DM组肝脏细胞形态异常,炎症细胞浸润,而MSCs组的较T1DM组明显改善。MSCs组小鼠肝脏组织的AGEs浓度（0.72±0.10）μg/ml低于T1DM组（1.35±0.22）μg/ml;同时MSCs组的NF-κB P65、IL-6、TNF-α、RAGE mRNA水平（分别为10.08±1.94、9.31±1.67、11.92±1.82、3.87±0.27）,均低于T1DM组（分别为15.46±3.09、18.04±1.69、22.12±3.23、5.12±0.26）,差异具有统计学意义（P < 0.05）。 结论hUCMSCs可以降低糖尿病小鼠血糖水平,改善肝脏微观病理状态,降低AGEs浓度及某些炎性因子的水平以减轻肝脏损伤。     

人脐带间充质干细胞移植治疗脊髓损伤的研究进展

脊髓损伤（SCI）由于复杂病理生理和神经修复再生困难,至今仍旧是难以攻克的医学难题,而干细胞因其神经再生和神经保护特性被认为是治疗SCI最有希望的方法。其中人脐带间充质干细胞（HUC-MSCs）近年培养分化方法不断改进、神经修复机制初步阐明,联合移植等综合治疗方案也不断实践,使HUC-MSCs移植治疗效果提高。另外关于HUC-MSCs治疗SCI的临床试验逐渐开展,术后患者神经功能恢复改善且无严重并发症出现,表明干细胞移植应用于人体是安全有效的。本文就HUC-MSCs治疗SCI的研究状况及进展进行综述。     

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.     

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.     

Me2SO- and serum-free cryopreservation of human umbilical cord mesenchymal stem cells using electroporation-assisted delivery of sugars

Cryopreservation is the universal technology used to enable long-term storage and continuous availability of cell stocks and tissues for regenerative medicine demands. The main components of standard freezing media are dimethyl sulfoxide (hereinafter Me₂SO) and fetal bovine serum (FBS). However, for manufacturing of cells and tissue-engineered products in accordance with the principles of Good Manufacturing Practice (GMP), current considerations in regenerative medicine suggest development of Me₂SO- and serum-free biopreservation strategies due to safety concerns over Me₂SO-induced side effects and immunogenicity of animal serum.In this work, the effect of electroporation-assisted pre-freeze delivery of sucrose, trehalose and raffinose into human umbilical cord mesenchymal stem cells (hUCMSCs) on their post-thaw survival was investigated. The optimal strength of electric field at 8 pulses with 100 μs duration and 1 Hz pulse repetition frequency was determined to be 1.5 kV/cm from permeabilization (propidium iodide uptake) vs. cell recovery data (resazurin reduction assay).Using sugars as sole cryoprotectants with electroporation, concentration-dependent increase in cell survival was observed. Irrespective of sugar type, the highest cell survival (up to 80%) was achieved at 400 mM extracellular concentration and electroporation. Cell freezing without electroporation yielded significantly lower survival rates. In the optimal scenario, cells were able to attach 24 h after thawing demonstrating characteristic shape and sugar-loaded vacuoles. Application of 10% Me₂SO/90% FBS as a positive control provided cell survival exceeding 90%. Next, high glass transition temperatures determined for optimal concentrations of sugars by differential scanning calorimetry (DSC) suggest the possibility to store samples at −80 °C. In summary, using electroporation to incorporate cryoprotective sugars into cells is an effective strategy towards Me₂SO- and serum-free cryopreservation and may pave the way for further progress in establishing clinically safe biopreservation strategies for efficient long-term biobanking of cells.     

Cardiac potential of stem cells from whole human umbilical cord tissue

We investigated the role of stem cells from human umbilical cord tissue in cardiomyocyte regeneration. The umbilical cord stem cells were initially characterized and differentiated in a myocardial differentiation medium containing 5‐azacytidine for 24 h. Differentiation into cardiomyocytes was determined by expression of cardiac specific markers, like cardiac α‐actin, connexin43, myosin, Troponin T, and ultrastructural analysis. In vivo, the transplanted umbilical cord stem cells were sprouting from local injection and differentiated into cardiomyocyte‐like cells in a rat myocardial infarction model. Echocardiography revealed increasing left ventricular function after umbilical cord stem cell transplantation. These results demonstrate that umbilical cord stem cells can differentiate into cardiomyocyte‐like cells both in vitro and in vivo. Therefore, human umbilical cord might represent a source of stem cells useful for cellular therapy and myocardial tissue engineering. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon. J. Cell. Biochem. 107: 926–932, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

一种大量快速分离脐带间充质干细胞的新方法

目的:探讨体外快速大量分离脐带间充质干细胞的新方法。方法:采用复合胶原NB4、dispaseII、透明质酸酶三种酶消化3h,加入PBSA溶液稀释,离心获得脐带间充质干细胞,培养;用流式细胞仪对P3代细胞进行表面标记的鉴定,用化学诱导的方法使第3代细胞向脂肪、骨、软骨细胞分化,2~4周后,分别行oilred、Safranin'O和茜素红染色,倒置显微镜下观察诱导结果。结果:经3种酶消化和PBSA稀释,短时间内从脐带中获得了大量间充质干细胞;伴随着细胞的传代,形态逐渐均一,传至第3代,细胞的形态已基本相似;流式细胞仪鉴定,细胞强表达间充质细胞的特异性标记CD90,CD73,CD105,而不表达造血系或内皮系细胞的标记CD45、CD14、CD11、CD34、CD19,也不表达主要组织相容性抗原HLA-DR;向脂肪细胞诱导后第4周,oilred染色见细胞内大量红染的脂滴;向软骨细胞诱导后第4周,Safranin'O染色见多数切片呈阳性,细胞团块中存在大量软骨特异性的陷窝样结构;向骨细胞诱导后第4周,茜素红染色发现肉眼可见的广泛散在的红色阳性钙结节。结论:本研究所采用的3种酶消化结合PBSA稀释的方法可以快速获得脐带间...