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.     

Introducing pulsed low-intensity ultrasound to culturing human umbilical cord-derived mesenchymal stem cells

The human umbilical cord (hUC) is a source of adult tissue-derived mesenchymal stem cells (MSCs). A pulsed low-intensity ultrasound (PLIUS) method is described for increasing the yield of MSCs from whole hUC without enzymatic digestion or growth factor supplementation. Analysis of the immunophenotype of cells and a differentiation study were performed to show the compatibility of MSCs. The mean number of cells recovered from primocultures of hUC was 6 × 10⁵ cells/cm. PLIUS resulted in a 3.3-fold increase in MSC yield at passage 0. PLIUS exposure increases the yield of hUC-MSCs by promoting release and enhancing proliferation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Intrastriatal grafts of mesenchymal stem cells in adult intact rats can elevate tyrosine hydroxylase expression and dopamine levels

MSCs (mesenchymal stem cells) derived from the bone marrow have shown to be a promising source of stem cells in a therapeutic strategy of neurodegenerative disorder. Also, MSCs can enhance the TH (tyrosine hydroxylase) expression and DA (dopamine) content in catecholaminergic cells by in vitro co‐culture system. In the present study, we investigated the effect of intrastriatal grafts of MSCs on TH protein and gene levels and DA content in adult intact rats. When MSCs were transplanted into the striatum of normal rats, the grafted striatum not only had significantly higher TH protein and mRNA levels, but also significantly higher DA content than the untransplanted striatum. Meanwhile, the grafted MSCs differentiated into neurons, astrocytes and oligodendrocytes; however, TH‐positive cells could not be detected in our study. These experimental results offer further evidence that MSCs are a promising candidate for treating neurodegenerative diseases such as Parkinson's disease.     

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.     

Effects of hepatocyte growth factor overexpressed bone marrow‐derived mesenchymal stem cells on prevention from left ventricular remodelling and functional improvement in infarcted rat hearts

Bone marrow‐derived mesenchymal stem cells (BM‐MSCs ) transplantation has been reported to be a promising therapy for myocardial infarction (MI). However, low survival rate of BM‐MSCs in infarcted heart is one of the major limitations for the perspective clinical application. In this study, we aimed to investigate the effect of hepatocyte growth factor (HGF) on left ventricular function improvement of HGF gene‐modified BM‐MSCs (HGF‐MSCs) after its delivery into the infarcted rat hearts. BM‐MSCs were isolated with fibroblast‐like morphology and expressed CD44+CD29+CD90+/CD34‐CD45‐CD31‐CD11a. After 5‐azacytidine induction in vitro, 20%–30% of the cells were positively stained for desmin, cardiac‐specific cardiac troponin I and connexin‐43. Histological staining revealed that 2 weeks after MI is an optimal time point with decreased neutrophil infiltration and increased vascular number. Minimal infarct size and best haemodynamic analysis were also observed after cell injection at 2 weeks compared with that of 1 h, 1 week or 4 weeks. Echocardiogram confirmed that transplantation with HGF‐MSCs significantly improved left ventricular function compared with other groups in rat MI models. MSCs and HGF‐MSCslabelled with DAPI were detected 4 weeks after MI in the infarcted area. Decreased infarcted scar area and increased angiogenesis formation could be found in HGF‐MSCs group than in other groups as demonstrated by hematoxylin and eosin (H&E) staining and factor VIII staining. These results indicate that HGF‐MSCs transplantation could enhance the contractile function and attenuate left ventricular remodelling efficiently in rats with MI. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterization of mesenchymal stem cells isolated from the human umbilical cord

Numerous papers have reported that mesenchymal stem cells (MSCs) can be isolated from various sources such as bone marrow, adipose tissue and others. Nonetheless it is an open question whether MSCs isolated from different sources represent a single cell lineage or if cells residing in different organs are separate members of a family of MSCs. Subendothelial tissue of the umbilical cord vein has been shown to be a promising source of MSCs. The aim of this study was to isolate and characterize cells derived from the subendothelial layer of umbilical cord veins as regards their clonogenicity and differentiation potential. The results from these experiments show that cells isolated from the umbilical cord vein displayed fibroblast-like morphology and grew into colonies. Immunophenotyping by flow cytometry revealed that the isolated cells were negative for the hematopoietic line markers HLA-DR and CD34 but were positive for CD29, CD90 and CD73. The isolated cells were also positive for survivin, Bcl-2, vimentin and endoglin, as confirmed by RT-PCR and immunofluorescence. These cells can be induced to differentiate into osteogenic and adipogenic cells, but a new finding is that these cells can be induced to differentiate into endothelial cells expressing CD31, vWF and KDR-2, and also form vessel-like structures in Matrigel. The differentiated cells stopped expressing survivin, thus showing a diminished proliferative potential. It can be assumed that the subendothelial layer of the umbilical cord vein contains a population of cells with the overall characteristics of MSCs, with the additional capability to transform into endothelial cells.     

The similar effect of transplantation of marrow-derived mesenchymal stem cells with or without prior differentiation induction in experimental myocardial infarction

Marrow-derived mesenchymal stem cells (MSCs) have been heralded as a source of great promise for the regeneration of the infarcted heart. There is no clear data indicating whether or not in vitro differentiation of MSCs into major myocardial cells can increase the beneficial effects of MSCs. The aim of this study is to address this issue. To induce MSCs to transdifferentiate into cardiomyocyte-like and endothelial-like cells, 5-azacytidine and vascular endothelial growth factor (VEGF) were used, respectively. Myocardial infarction in rabbits was generated by ligating the left anterior descending coronary artery. Animals were divided into three experimental groups: I, control group; II, undifferentiated mesenchymal stem cell transplantation group; III, differentiated mesenchymal stem cell transplantation group; which respectively received peri-infarct injections of culture media, autologous undifferentiated MSCs and autologous differentiated MSCs. General pathology, immunohistochemistry, electron microscopy and echocardiography were performed in order to search for myocardial regeneration and improvement of cardiac function. In Groups II and III, implanted cells transdifferentiate into myocardial cells within 28 days post injection in a similar manner, and well-developed ultra structures formed within transplanted cells. Improvements in left ventricular function and reductions in infarcted area were observed in both cell-transplanted groups to the same degree. Vascular density was similar in Groups II and III and significantly higher in these groups compared with the control group. There is no need for prior differentiation induction of marrow-derived MSCs before transplantation and peri-infarct implantation of MSCs can efficiently regenerate the infarcted myocardium and improve cardiac function.     

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...     

Transplantation of mesenchymal stem cells overexpressing interleukin‐10 induces autophagy response and promotes neuroprotection in a rat model of TBI

Autophagy, including mitophagy, is critical for neuroprotection in traumatic brain injury (TBI). Transplantation of mesenchymal stem cells (MSCs) provides neuroprotection and induces autophagy by increasing anti‐inflammatory cytokines, such as interleukin‐10 (IL‐10). To evaluate these effects of IL10 that are released by MSCs, we genetically engineered MSCs to overexpress IL10 and compared their effects to unaltered MSCs following transplantation near the site of induced TBIs in rats. Adult, male Sprague‐Dawley rats were divided into four groups: Sham + vehicle, TBI + vehicle, TBI + MSCs‐IL‐10 and TBI + MSCs‐GFP. Thirty‐six hours post‐TBI, the first two groups received vehicle (Hanks balance salt solution), whereas last two groups were transplanted with MSCs‐IL‐10 or MSCs‐GFP. Three weeks after transplantation, biomarkers for neurodegenerative changes, autophagy, mitophagy, cell death and survival markers were measured. We observed a significant increase in the number of dead cells in the cortex and hippocampus in TBI rats, whereas transplantation of MSCs‐IL‐10 significantly reduced their numbers in comparison to MSCs alone. MSCs‐IL‐10 rats had increased autophagy, mitophagy and cell survival markers, along with decreased markers for cell death and neuroinflammation. These results suggest that transplantation of MSCs‐IL‐10 may be an effective strategy to protect against TBI‐induced neuronal damage.     

移植骨髓间充质干细胞治疗大鼠糖尿病的研究

目的 通过移植骨髓间充质干细胞（mesenchymal stem cell,MSC）的方法试治疗大鼠糖尿病。方法 贴壁生长的MSC与大鼠胰腺的细胞共培养以检测其向胰岛细胞分化的潜能。并将体外培养扩增的MSC移植入糖尿病大鼠体内,观测其能否改善糖尿病病情及其在大鼠体内微环境中的分化情况。结果 共培养法可使MSC分化为胰岛样细胞。对大鼠的MSC移植能明显缓解糖尿病病情。结论 MSC移植的方法对大鼠糖尿病有一定的治疗作用。     

In vitro culture of Keratinocytes from human umbilical cord blood mesenchymal stem cells: the Saigonese culture

There have been many attempts to acquire and culture human keratinocytes for clinical purposes including from keratotome slices in media with fetal calf serum (FCS) or pituitary extract (PE), from skin specimens in media with feeder layers, from suction blister epidermal roofs’ in serum-free culture and from human umbilical cord blood (hUCB) mesenchymal stem cells (MSCs) in media with skin feeder layers. Conversely this study was designed to investigate whether keratinocytes could be obtained directly from hUCB MSCs in vitro. It is widely established that mesenchymal stem cells from human umbilical cord blood have multipotent capacity and the ability to differentiate into disparate cell lineages hUCB MSCs were directly induced to differentiate into keratinocytes by using a specific medium composed of primary culture medium (PCM) and serum free medium (SFM) in a ratio 1:9 for a period of 7 days and tested by immunostain p63 and K1-K10. Cells thus cultured were positive in both tests, confirming the possibility to directly obtain keratinocytes from MSCs hUCB in vitro.     

Endometrial stem cell transplantation restores dopamine production in a Parkinson’s disease model

Parkinson’s disease (PD) is a neurodegenerative disorder caused by the loss of dopaminergic neurons. Adult human endometrial derived stem cells (HEDSC), a readily obtainable type of mesenchymal stem‐like cell, were used to generate dopaminergic cells and for transplantation. Cells expressing CD90, platelet derived growth factor (PDGF)‐Rβ and CD146 but not CD45 or CD31 were differentiated in vitro into dopaminergic neurons that exhibited axon projections, pyramidal cell bodies and dendritic projections that recapitulate synapse formation; these cells also expressed the neural marker nestin and tyrosine hydroxylase, the rate‐limiting enzyme in dopamine synthesis. Whole cell patch clamp recording identified G‐protein coupled inwardly rectifying potassium current 2 channels characteristic of central neurons. A 1‐methyl 4‐phenyl 1,2,3,6‐tetrahydro pyridine induced animal model of PD was used to demonstrate the ability of labelled HEDSC to engraft, migrate to the site of lesion, differentiate in vivo and significantly increase striatal dopamine and dopamine metabolite concentrations. HEDSC are a highly inducible source of allogenic stem cells that rescue dopamine concentrations in an immunocompetent PD mouse model.     

Bone marrow and adipose mesenchymal stem cells attenuate cardiac fibrosis induced by methotrexate in rats

Mesenchymal stem cells (MSCs) are an ideal adult stem cell with capacity for self‐renewal and differentiation with an extensive tissue distribution. The present study evaluates the therapeutic effects of bone marrow mesenchymal stem cells (BM‐MSCs) or adipose‐derived mesenchymal stem cells (AD‐MSCs) against the development of methotrexate (MTX)‐induced cardiac fibrosis versus dexamethasone (DEX). Rats were allocated into five groups; group 1, received normal saline orally; group 2, received MTX (14 mg/kg/week for 2 weeks); groups 3 and 4, treated once with 2 × 10 ⁶ cells of MTX + BM‐MSCs and MTX + AD‐MSCs, respectively; and group 5, MTX + DEX (0.5 mg/kg, for 7 days, P.O.). MTX induced cardiac fibrosis as marked changes in oxidative biomarkers and elevation of triglyceride, cholesterol, aspartate aminotransferase, gamma‐glutamyl transferase, creatine kinase, and caspase‐3, as well as deposited collagen. These injurious effects were antagonized after treatment with MSCs. So, MSCs possessed antioxidant, antiapoptotic, as well antifibrotic effects, which will perhaps initiate them as notable prospective for the treatment of cardiac fibrosis.     

Improving the efficacy of type 1 diabetes therapy by transplantation of immunoisolated insulin-producing cells

Type 1 diabetes occurs when pancreatic islet β-cells are damaged and are thus unable to secrete insulin. Pancreas- or islet-grafting therapy offers highly efficient treatment but is limited by inadequate donor islets or pancreases for transplantation. Stem-cell therapy holds tremendous potential and promises to enhance treatment efficiency by overcoming the limitations of traditional therapies. In this study, we evaluated the efficiency of preclinical diabetic treatment. Diabetes was induced in mice by injections of streptozotocin. Mesenchymal stem cells (MSCs) were derived from mouse bone marrow or human umbilical cord blood and subsequently differentiated into insulin-producing cells. These insulin-producing cells were encapsulated in an alginate membrane to form capsules. Finally, these capsules were grafted into diabetic mice by intraperitoneal injection. Treatment efficiency was evaluated by monitoring body weight and blood glucose levels. Immune reactions after transplantation were monitored by counting total white blood cells. Allografting or xenografting of encapsulated insulin-producing cells (IPCs) reduced blood glucose levels and increased body weight following transplantation. Encapsulation with alginate conferred immune isolation and prevented graft rejection. These results provide further evidence supporting the use of allogeneic or xenogeneic MSCs obtained from bone marrow or umbilical cord blood for treating type 1 diabetes.     

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     

Mesenchymal stromal cells from human perinatal tissues: From biology to cell therapy

Cell-based regenerative medicine is of growing interest in biomedical research. The role of stem cells in this context is under intense scrutiny and may help to define principles of organ regeneration and develop innovative therapeutics for organ failure. Utilizing stem and progenitor cells for organ replacement has been conducted for many years when performing hematopoietic stem cell transplantation. Since the first successful transplantation of umbilical cord blood to treat hematological malignancies, non-hematopoietic stem and progenitor cell populations have recently been identified within umbilical cord blood and other perinatal and fetal tissues. A cell population entitled mesenchymal stromal cells (MSCs) emerged as one of the most intensely studied as it subsumes a variety of capacities: MSCs can differentiate into various subtypes of the mesodermal lineage, they secrete a large array of trophic factors suitable of recruiting endogenous repair processes and they are immunomodulatory.Focusing on perinatal tissues to isolate MSCs, we will discuss some of the challenges associated with these cell types concentrating on concepts of isolation and expansion, the comparison with cells derived from other tissue sources, regarding phenotype and differentiation capacity and finally their therapeutic potential.     

骨髓间充质干细胞源神经细胞移植治疗帕金森病大鼠模型

目的探讨骨髓间充质干细胞（mesenchymal stemcells,MSCs）源神经细胞脑内移植对帕金森病（Parkinson s disease,PD）大鼠的治疗作用。方法贴壁培养法分离、培养大鼠骨髓MSCs,脑匀浆上清诱导第3代MSCs向神经细胞分化,采用免疫细胞化学法鉴定诱导分化后细胞的性质,激光共聚焦显微镜检测诱导前后细胞Ca2＋浓度变化,6只PD大鼠行纹状体内MSCs源神经细胞移植作为细胞移植组,6只PD大鼠作为对照组。细胞移植术后4周检测PD大鼠的行为变化,观察移植细胞在脑内的分布情况。结果倒置显微镜下可见MSCs呈纺锤形和多角形,有1～2个核仁,MSCs经脑匀浆上清诱导后其胞体折光性增强,发出数个细长突起,互相交织成网,有的似轴突。诱导后细胞表达神经元特异性标志物神经元特异性烯醇化酶（NSE）和神经丝蛋白（NF）,胞质Ca2＋荧光强度显著增强,可推测诱导后的细胞为MSCs源神经细胞,将BrdU标记的MSCs源神经细胞移植到PD大鼠纹状体治疗4周后,可见细胞散在分布于注射侧脑组织,有少量细胞可迁移到对侧脑组织,PD大鼠的旋转行为得到显著改善。结论MSCs源神经细胞移植治疗帕金森病大鼠可使其旋转行为得到改善。