Biologic properties of gadolinium diethylenetriaminepentaacetic acid-labeled and PKH26-labeled human umbilical cord mesenchymal stromal cells

Background aimsThis study was conducted to characterize gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA)-labeled and PKH26-labeled human umbilical cord mesenchymal stromal cells (HuMSCs) and to track them with magnetic resonance imaging (MRI) in vitro and in vivo.MethodsHuMSCs were isolated from umbilical cords and expanded in vitro. Cells were sequentially labeled with Gd-DTPA and PKH26. The labeling efficiency was determined by spectrophotometry measurements, and the longevity of Gd-DTPA maintenance was measured with MRI. The influence of double labeling on cellular biologic properties was assessed by cell proliferation, viability, differentiation, cycle and apoptosis. Transplantation of double-labeled HuMSCs or placebo was performed in 39 female Sprague-Dawley rats. Leak point pressure and maximal bladder capacity were measured in animals 6 weeks after injection.ResultsThe T1 values and signal intensity on T1-weighted imaging of labeled cells were significantly higher than the control group (P < 0.05). The signal intensity on T1-weighted imaging of labeled cells was retained >14 days in vitro and in vivo. There was no significant difference in the cell cycle, cell apoptosis, cell proliferation and cell viability between labeled and unlabeled HuMSCs (P > 0.05). After double labeling, HuMSCs were still capable of differentiating into osteoblasts and adipocytes. Periurethrally injected HuMSCs in the rats significantly improved leak point pressure and maximal bladder capacity.ConclusionsHuMSCs were successfully labeled with Gd-DTPA and PKH26. This labeling method is reliable and efficient and can be applied for tracking cells in vitro and in vivo without altering cellular biologic properties.     

PKH26标记的人羊膜间充质干细胞在宫腔粘连大鼠中的迁移示踪

为了观察PKH26标记的人羊膜间充质干细胞(hAMSCs)在宫腔粘连大鼠子宫内膜中的迁移情况,文中提取鉴定及PKH26标记hAMSCs,检测PKH26染色剂对hAMSCs生物学特性的影响;利用机械感染双重法建立大鼠宫腔粘连模型并经尾静脉移植PKH26标记的hAMSCs,荧光共聚焦显微镜下观察PKH26标记的hAMSCs移植后在大鼠子宫内膜中的分布情况。结果显示,PKH26染色剂对细胞的活性、周期、凋亡等无明显影响,PKH26标记的阳性细胞主要分布在大鼠受损的子宫内膜中。表明PKH26标记技术是一种安全有效的示踪方法,可用于hAMSCs移植在治疗宫腔粘连时的示踪研究。     

A protocol for umbilical cord tissue cryopreservation as a source of mesenchymal stem cells

Molecular Biology Reports - Mesenchymal stem cells (MSC) differentiate into different cell types and have immunomodulatory and paracrine effects. Cryopreservation of umbilical cord tissue as a...     

Phototoxicity of the fluorescent membrane dyes PKH2 and PKH26 on the human hematopoietic KG1a progenitor cell line.

BACKGROUND: The phototoxic effects of the well-known fluorescent membrane dyes PKH2 and PKH26 have been unknown, although their use in cell tracking experiments has increased dramatically. To eliminate the phototoxicity-induced alteration in cell function and morphology, it is essential to examine the suspicious phototoxicity of these dyes. METHODS: Chemical and phototoxic effects of PKH dyes on the human hematopoietic KG1a cell line were examined. To minimize phototoxicity in long-term cell tracking experiments lasting up to 18 h with a fluorescence microscope system, time-lapse monitoring with different time intervals and exposure times was introduced. RESULTS: There were no significant effects of the two PKH dyes on cell viability and growth when using dye concentrations up to 5 microM. However, when stained cells were exposed to excitation light, cell viability decreased dramatically, showing the phototoxicity of the PKH dyes. More than 60% of cells stained with 5 microM PKH26 died after 5 min of continuous light exposure. The phototoxic effect was more extensive in cells stained with higher concentrations of the dyes. CONCLUSIONS: We present guidelines for the optimal use of these dyes by using a defined hardware configuration.     

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。     

In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells

Cardiomyocyte loss in the ischemically injured human heart often leads to irreversible defects in cardiac function. Recently, cellular cardiomyoplasty with mesenchymal stem cells, which are multipotent cells with the ability to differentiate into specialized cells under appropriate stimuli, has emerged as a new approach for repairing damaged myocardium. In the present study, the potential of human umbilical cord-derived mesenchymal stem cells to differentiate into cells with characteristics of cardiomyocyte was investigated. Mesenchymal stem cells were isolated from endothelial/subendothelial layers of the human umbilical cords using a method similar to that of human umbilical vein endothelial cell isolation. Isolated cells were characterized by transdifferentiation ability to adipocytes and osteoblasts, and also with flow cytometry analysis. After treatment with 5-azacytidine, the human umbilical cord-derived mesenchymal stem cells were morphologically transformed into cardiomyocyte-like cells and expressed cardiac differentiation markers. During the differentiation, cells were monitored by a phase contrast microscope and their morphological changes were demonstrated. Immunostaining of the differentiated cells for sarcomeric myosin (MF20), desmin, cardiac troponin I, and sarcomeric alpha-actinin was positive. RT-PCR analysis showed that these differentiated cells express cardiac-specific genes. Transmission electron microscopy revealed a cardiomyocyte-like ultrastructure and typical sarcomers. These observations confirm that human umbilical cord-derived mesenchymal stem cells can be chemically transformed into cardiomyocytes and can be considered as a source of cells for cellular cardiomyoplasty.     

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.     

Differentiating characterization of human umbilical cord blood-derived mesenchymal stem cells in vitro

It has been demonstrated that the number and differentiating potential of bone marrow mesenchymal stem cells (MSCs) decrease with age. Therefore, the search for alternative sources of MSCs is of significant value. In the present study, MSCs were isolated from umbilical cord blood (UCB) by combining gradient density centrifugation with plastic adherence. Cultured cells were treated with ascorbate acid-2-phosphate, dexamethasone, beta-glycerophosphate dexamethasone, insulin, 1-methyl-3-isobutylxamthine, indomethacin, beta-mercaptoethanol, butylated hydroxyanisole, FGF-4 and HGF. Differentiating characterization of UCB-derived MSCs were detected by cytochemistry, immunocytochemistry, radioimmunoassay, RT-PCR and urea assay. The results showed UCB-derived MSCs could differentiate into osteoblasts, adipocytes and neuron-like cells. When MSCs were cultured with FGF-4 and HGF, approximately 63.6% of cells became small, round and epithelioid on day 28 by morphology. Compared with the control, levels of AFP in the supernatant liquid increased significantly from day 12 and were higher on day 28 (P<0.01). Albumin increased significantly from day 16 (P<0.01). Urea was first detected on day 20 (P<0.01), and continued to increase on day 28 (P<0.01). Cells first expressed CK-18 on day 16 through immunocytochemistry analysis. RT-PCR analysis showed that differentiated cells could express a number of hepatocyte-specific genes in a time-dependent manner. Glycogen storage was first seen on day 24. Our results suggest that UCB-derived MSCs can differentiate not only into osteoblasts, adipocytes and neuron-like cells, but also into hepatocytes. Human UCB-derived MSCs are a new source of cell types for cell transplantation and therapy.     

Application of human umbilical cord blood-derived mesenchymal stem cells in disease models

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) are regarded as an alternative source of bone marrow-derived mesenchymal stem cells because collection of cord blood is less invasive than that of bone marrow. hUCB-MSCs have recently been studied for evaluation of their potential as a source of cell therapy. In this review, the general characteristics of hUCB-MSCs and their therapeutic effects on various diseases in vitro and in vivo will be discussed.     

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.     

In vitro mesengenic potential of human umbilical cord blood-derived mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) have been paid a great deal of attention because of their unprecedented therapeutic merits endowed by powerful ex vivo expansion and multilineage differentiation potential. Umbilical cord blood (UCB) is a convenient but not fully proven source for hMSCs, and hence, greater experience is required to establish UCB as a reliable source of hMSCs. To this end, we attempted to isolate hMSC-like adherent cells from human UCB. The isolated cells were highly proliferative and exhibited an immunophenotype of CD13+ CD14- CD29+ CD31- CD34- CD44+ CD45- CD49e+ CD54+ CD90+ CD106- ASMA+ SH2+ SH3+ HLA-ABC+ HLA-DR-. More importantly, these cells, under appropriate conditions, could differentiate into a variety of mesenchymal lineage cells such as osteoblasts, chondrocytes, adipocytes, and skeletal myoblasts. This mesengenic potential assures that the UCB-derived cells are multipotent hMSCs and further implicates that UCB can be a legitimate source of hMSCs.     

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.     

诱导人脐带间充质干细胞分化为视网膜色素上皮样细胞的研究

目的：探讨用猪视网膜色素上皮细胞（RPE）条件培养基诱导hUCMSCs分化为RPE样细胞的方法。方法通过流式细胞技术鉴定hUCMSCs的表面标记分子;通过诱导hUCMSCs分化成为脂肪、骨和软骨细胞确定其多系分化能力;将hUCMSCs培养在猪RPE的条件培养液中并添加诱导因子来诱导hUCMSCs向RPE细胞分化。对照组与处理组Q-PCR结果采用t检验比较。结果 hUCMSCs表达CD105、CD90、CD73、CD44和CD29,但不表达CD34,CD45和MHCII等分子标记,在成脂、成骨、成软骨分化培养基中可分化为脂肪、骨和软骨细胞。单独使用猪RPE条件培养液不能有效诱导hUCMSCs向RPE细胞分化,但联合应用猪RPE条件培养液和诱导因子（视黄酸,activin-A和人重组骨形成蛋白-7）可有效诱导hUCMSCs分化为RPE样细胞,RPE细胞标记分子RPE65、Mitf和Ck8/18的基因表达量分别提高了2.1±0.4、6.8±1.3和2.5±0.3倍（P〈0.05）。诱导产生的RPE样细胞呈多边形,但不含色素颗粒。结论猪RPE条件培养液联合诱导因子可有效诱导hUCMSCs分化为RPE样细胞,可能会为治疗视网膜变性疾病提供合适的种子细胞。     

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.     

A slowly proliferating subpopulation in human umbilical cord mesenchymal stem cells in culture

In this report, a slow-growing subpopulation of human umbilical cord mesenchymal stromal cells (MSCs) was identified. These cells were around 5 μm in diameter and their relative gravity was between 1.031 and 1.043 g/ml. In sharp contrast to the parent MSCs, they expressed highly CD271 and poorly the receptor for platelet-derived growth factor. Quantitative PCR with the identification of the products by DNA sequencing proved that these cells expressed Nanog at a higher level than cells from the other subpopulation (approximately 30-fold), which was further confirmed by western blotting. Furthermore, they did not grow at clonal density and depletion of these cells from the population had little effect on the colony formation of the parent MSCs. The results here indicate that a subpopulation of cells with special biological features exist in human cord MSCs in culture.     

人脐带间充质干细胞在组织工程中的研究进展

人脐带间充质干细胞是一类具有自我更新、增殖和多向分化潜能的干细胞,具有来源广泛、易于采集、保存和运输、无异体排斥、避免伦理争议等诸多优点.流式细胞仪分析发现人脐带间充质干细胞高表达间质细胞标志(CD44、CD105)、整合素受体(CD29、CD49b、CD49c、CD51),不表达造血系标志(CD34、CD45)人白细胞抗原HLA-DR和内皮细胞标志CD31.人脐带间充质干细胞在体内外可以分化为骨细胞、软骨细胞、肝细胞、心肌细胞、骨骼肌细胞以及神经元细胞等.目前人脐带间充质干细胞在组织工程骨、人工血管以及基因治疗等临床应用研究中已逐渐深入,并已显示出广阔的应用前景.本文就人脐带间充质干细胞的生物学特性及其在组织工程中的研究作一综述.     

Immunomodulatory effect of human umbilical cord Wharton's jelly-derived mesenchymal stem cells on lymphocytes

Studies have shown that mesenchymal stem cells (MSCs) have low immunogenicity and immune regulation. Human umbilical cord Wharton’s jelly provides a new source for MSCs that are highly proliferative and have multi-differentiation potential. To investigate immunomodulatory effects of human Wharton’s jelly cells (WJCs) on lymphocytes, we successfully isolated MSCs from human umbilical cord Wharton’s jelly. WJCs expressed MSC markers but low levels of human leukocyte antigen (HLA)-ABC and no HLA-DR. These results indicate that WJCs have low immunogenicity. Both WJCs and their culture supernatant could inhibit the proliferation of phytohemagglutinin-stimulated human peripheral blood lymphocytes and mouse splenocytes. Additionally, WJCs suppressed secretion of transforming growth factor-β1 and interferon-γ by human peripheral blood lymphocytes. We conclude that the immunomodulatory effect of WJCs may be related to direct cell contact and inhibition of cytokine secretion by human peripheral blood lymphocytes.