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.     

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.     

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。     

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。     

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.     

Low immunogenicity of allogeneic human umbilical cord blood-derived mesenchymal stem cells in vitro and in vivo

Evaluation of the immunogenicity of human mesenchymal stem cells (MSCs) in an allogeneic setting during therapy has been hampered by lack of suitable models due to technical and ethical limitations. Here, we show that allogeneic human umbilical cord blood derived-MSCs (hUCB-MSCs) maintained low immunogenicity even after immune challenge in vitro. To confirm these properties in vivo, a humanized mouse model was established by injecting isolated hUCB-derived CD34+ cells intravenously into immunocompromised NOD/SCID IL2γnull (NSG) mice. After repeated intravenous injection of human peripheral blood mononuclear cells (hPBMCs) or MRC5 cells into these mice, immunological alterations including T cell proliferation and increased IFN-γ, TNF-α, and human IgG levels, were observed. In contrast, hUCB-MSC injection did not elicit these responses. While lymphocyte infiltration in the lung and small intestine and reduced survival rates were observed after hPBMC or MRC5 transplantation, no adverse events were observed following hUCB-MSC introduction. In conclusion, our data suggest that allogeneic hUCB-MSCs have low immunogenicity in vitro and in vivo, and are therefore “immunologically safe” for use in allogeneic clinical applications.     

人脐带间充质干细胞静脉输注小鼠的安全性

目的探讨C57／BL6J小鼠重复多次尾静脉输注人脐带间充质干细胞后的免疫反应和毒性。方法将SPF级别的32只C57／BL6J小鼠随机分为阴性对照组、细胞移植组,每组16只,雌雄各半,细胞移植组小鼠尾静脉注射分离培养的第5代人脐带间充质干细胞,一次5×10。／只,每周注射一次,连续注射4周;阴性对照组每次注射相同容积的PBS。注射后后观察小鼠的一般症状,末次注射后i周、4周进行血细胞计数、血生化、免疫反应指标、脏器质量测定和组织病理学检查。结果细胞移植组小鼠血细胞计数、血生化、脏器重量和脏器系数与对照组无显著性差异（P〉0．05）,脏器组织病理学在光镜下检查结果与对照组无形态学差别,以及免疫结果测定（T细胞亚群CD3＋、CD4＋、CD8＋及CD4＋／CD8＋）与对照组无显著性差异（P〉0．05）。结论人脐带间充质干细胞重复多次尾静脉输注C57／BL6J小鼠是安全可行的,对受者无明显免疫反应和毒副作用。     

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.     

Extracellular vesicles from human umbilical cord mesenchymal stem cells improve nerve regeneration after sciatic nerve transection in rats

Peripheral nerve injury results in limited nerve regeneration and severe functional impairment. Mesenchymal stem cells (MSCs) are a remarkable tool for peripheral nerve regeneration. The involvement of human umbilical cord MSC‐derived extracellular vesicles (hUCMSC‐EVs) in peripheral nerve regeneration, however, remains unknown. In this study, we evaluated functional recovery and nerve regeneration in rats that received hUCMSC‐EV treatment after nerve transection. We observed that hUCMSC‐EV treatment promoted the recovery of motor function and the regeneration of axons; increased the sciatic functional index; resulted in the generation of numerous axons and of several Schwann cells that surrounded individual axons; and attenuated the atrophy of the gastrocnemius muscle. hUCMSC‐EVs aggregated to rat nerve defects, down‐regulated interleukin (IL)‐6 and IL‐1β, up‐regulated IL‐10 and modulated inflammation in the injured nerve. These effects likely contributed to the promotion of nerve regeneration. Our findings indicate that hUCMSC‐EVs can improve functional recovery and nerve regeneration by providing a favourable microenvironment for nerve regeneration. Thus, hUCMSC‐EVs have considerable potential for application in the treatment of peripheral nerve injury.     

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     

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。     

Human umbilical cord mesenchymal stem cell transplantation restores damaged ovaries

Ovarian injury because of chemotherapy can decrease the levels of sexual hormones and potentia generandi of patients, thereby greatly reducing quality of life. The goal of this study was to investigate which transplantation method for human umbilical cord mesenchymal stem cells (HUMSCs) can recover ovarian function that has been damaged by chemotherapy. A rat model of ovarian injury was established using an intraperitoneal injection of cyclophosphamide. Membrane‐labelled HUMSCs were subsequently injected directly into ovary tissue or tail vein. The distribution of fluorescently labelled HUMSCs, estrous cycle, sexual hormone levels, and potentia generandi of treated and control rats were then examined. HUMSCs injected into the ovary only distributed to the ovary and uterus, while HUMSCs injected via tail vein were detected in the ovary, uterus, kidney, liver and lung. The estrous cycle, levels of sex hormones and potentia generandi of the treated rats were also recovered to a certain degree. Moreover, in some transplanted rats, fertility was restored and their offspring developed normally. While ovary injection could recover ovarian function faster, both methods produced similar results in the later stages of observation. Therefore, our results suggest that transplantation of HUMSCs by tail vein injection represents a minimally invasive and effective treatment method for ovarian injury.     

CXC chemokine receptor 1 enhances the ability of human umbilical cord blood-derived mesenchymal stem cells to migrate toward gliomas

In this study, we showed that knocking-down interleukin-8 (IL-8) in glioma cells, or its receptor, CXC chemokine receptor 1 (CXCR1) in hUCB-MSCs reduced hUCB-MSC migration toward glioma cells in a Transwell chamber. In contrast, CXCR1-transfected hUCB-MSCs (CXCR1-MSCs) showed a superior capacity to migrate toward glioma cells in a Transwell chamber compared to primary hUCB-MSCs. Furthermore, these transfected cells also demonstrated the same ability to migrate toward tumors in mice bearing intracranial human gliomas as shown by histological and in vivo imaging analysis. Our findings indicate that overexpression of CXCR1 could be a useful tool for MSC-based gene therapy to achieve a sufficient quantity of therapeutic MSCs that are localized within tumors.     

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.     

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.