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。     

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。     

Exosomes derived from human umbilical cord mesenchymal stem cells alleviate acute liver failure by reducing the activity of the NLRP3 inflammasome in macrophages

Human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-EXOs) play an important role in the regulation of the immune system and inflammatory responses; however, their role in acute liver failure (ALF) and related pathological conditions is unclear. In this study, we found that hUCMSC-EXOs can reduce the expression of the NLRP3 inflammasome and downstream inflammatory factors in acute liver failure. Western blot and ELISA results showed that hUCMSC-EXOs decreased the expression of NLRP3, caspase-1, IL-1β and IL-6 in LPS-stimulated RAW 264.7 macrophages. In vivo, the hUCMSC-EXOs repaired damaged liver tissue and decreased the expression of the NLRP3 inflammasome and the levels of ALT and AST in a mouse ALF model. The results of this study provide a new strategy for the application of human umbilical cord mesenchymal stem cell-derived exosomes in the treatment of ALF.     

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.     

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.     

脐带间充质干细胞对内毒素血症诱发的大鼠急性肝功能损伤的影响

目的评价脐带间充质干细胞（hUC-MSCs）对内毒素血症诱发的大鼠急性肝功能损伤的影响及其与凋亡机制的关系。 方法6周龄雄性SD大鼠18只,随机分为3组,分别是对照组（C组）、内毒素血症组（M组）和内毒素血症+hUC-MSCs治疗组（M+cells组）,每组6只。大鼠腹腔注射5 mg/kg脂多糖（LPS）诱导内毒素血症模型,并经尾静脉注射含20×10⁶个hUC- MSCs。4 h时检测血清谷草转氨酶（AST）和谷丙转氨酶（ALT）,ELISA方法检测肿瘤坏死因子（TNF-α）、白细胞介素6（IL-6）,HE常规染色鉴定肝脏组织病理,Western Blot法检测肝脏组织抗凋亡蛋白Bcl-2、促凋亡蛋白Bax、凋亡信号调节激酶1（ASK1）、应激活化蛋白激酶即JUN氨基末端激酶（JNK）蛋白的表达。多组间比较采用单因素方差分析,相关分析选用pearson。 结果（1）C组AST、ALT、TNF-α和IL-6浓度分别为（74.66±6.39）U/ L、（40.07±6.07）U/ L、（37.74±3.08）ng/L和（0.42±0.07）ng/L;与M组比较（310.75±9.13）U/L、（107.04±10.04）U/ L、（160.32±4.88）ng/L和（0.90±0.09）ng/L,差异具有统计学意义（P均 < 0.05）,M组AST、ALT、TNF-α、IL-6浓度分别为（310.75±9.13）U/L、（107.04±10.04）U/ L、（160.32±4.88）ng/ L和（0.90±0.09）ng/L,与M+cells组比较（204.49±15.36）U/L、（71.24± 7.34）U/ L、（117.61±9.37）ng/ L和（0.60±0.10）ng/L,差异具有统计学意义（P均 < 0.05）。（2）C组大鼠肝细胞形态正常,可见肝小叶结构清晰,肝汇管区无炎性细胞浸润,M组大鼠肝小叶散在点状坏死肝细胞伴炎性浸润,肝细胞间隙散布增生的Kuffer细胞,M+cells组大鼠肝小叶炎性细胞浸润及肝细胞间隙Kuffer细胞浸润改善。（3）与C组比较,M组大鼠肝脏组织JUN、ASK1和Bax蛋白表达均增高（P均 < 0.05）,Bcl-2蛋白表达降低（P < 0.05）;与M组比较,M+cells组大鼠肝脏组织JUN、ASK1和Bax蛋白表达降低（P均 < 0.05）,Bcl-2蛋白增加（P < 0.05）。（4）单因素相关分析显示大鼠血清ALT、AST与TNF-a指数呈正相关（r值分别为0.9580、0.9865,P均< 0.05）,大鼠血清ALT、AST与IL-6指数呈正相关（r值分别为0.9892、0.9630,P均 < 0.05）,大鼠血清ALT、AST分别与BAX、ASK1、JNK指数均呈正相关（r值分别为0.9993、0.9851、0.7901、0.9864、0.9557、0.7128,P均 < 0.05）,大鼠血清ALT、AST分别与BCL-2指数均呈负相关（r值分别为-0.8824、-0.9338,P均 < 0.05）,大鼠血清TNF-α分别与BAX、ASK1、JNK指数均呈正相关（r值分别为0.9466、0.8958、0.6025,P均< 0.05）,大鼠血清TNF-α与BCL-2指数呈负相关（r = -0.6025,P均 < 0.05）,大鼠血清IL-6分别与BAX、ASK1、JNK指数均呈正相关（r值分别为0.9941、0.9997、0.8679,P均< 0.05）,大鼠血清IL-6与BCL-2指数呈负相关（r = -0.8078,P均 < 0.05）。 结论hUC-MSCs具有减轻内毒素血症大鼠急性肝功能损伤的作用,其机制与抑制肝脏细胞凋亡相关。     

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。     

Human umbilical cord mesenchymal stem cells facilitate the up‐regulation of miR‐153‐3p,whereby attenuating MGO‐induced peritoneal fibrosis in rats

MiRNAs contribute greatly to epithelial to mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs), which is a crucial step in peritoneal fibrosis (PF). In this study, we tried to profile whether miRNA expression differences exist after human umbilical cord mesenchymal stem cells (hUCMSCs) treatment in PF rats and investigate the possible role of miR‐153‐3p involved in anti‐EMT process. We randomly assigned 34 rats into three groups: control group (Group Control), MGO‐induced PF rats (Group MGO) and hUCMSCs‐treated rats (Group MGO + hUCMSCs). MiRNA microarrays and real‐time PCR analyses were conducted in three groups. α‐SMA, Snail1 and E‐cadherin expression were detected by Western blot. Luciferase reporter assays were used to detect the effects of miR‐153‐3p overexpression on Snai1 in rat peritoneal mesothelial cells (RPMCs). We identified differentially expressed miRNAs related to EMT, in which miR‐153‐3p demonstrated the greatest increase in Group MGO + hUCMSCs. Transient cotransfection of miR‐153‐3p mimics with luciferase expression plasmids resulted in a significant repression of Snai1 3′‐untranslated region luciferase activity in RPMCs. These studies suggest that miR‐153‐3p is a critical molecule in anti‐EMT effects of hUCMSCs in MGO‐induced PF rats. MiR‐153‐3p might exert its beneficial effect through directly targeting Snai1.     

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 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.     

人脐带和胎盘不同层次间充质干细胞的生物学特性分析

间充质干细胞(mesenchymal stem cell,MSC)在再生医学中具有广阔的应用前景,其临床转化应用已成为研究热点,如何大量获取原代间充质干细胞以及针对不同疾病选择最为合适的细胞来源是关键。为了探讨不同来源间充质干细胞的异同,为临床治疗与研究选择合适的种子细胞提供参考,文中比较了人脐带和胎盘不同层次间充质干细胞(MSC)的生物学特性,包括细胞形态、表面标志物、分化能力及核型分析,并对4种胎儿来源的MSCs进行了转录组序列分析,针对转录组测序结果,从增殖能力和分泌细胞因子等方面对不同来源的MSCs进行了分析验证。结果显示,脐带(umbilical cord,UC)、羊膜(amniotic membrane,AM)、绒毛膜平滑肌层(chorionic membrane,CM)、绒毛膜滋养层(chorionicvilli,CV)和蜕膜(deciduae,DC)等5种不同来源的MSCs均符合2006年国际细胞治疗协会(international society for cell therapy,ISCT)的最低标准,符合干细胞的一般特性;核型分析表明除了DC来源MSCs来自母体,UC、AM、CM、CV来源MSCs均来自胎儿;转录组测序分析结果显示,来自脐带和胎盘的MSCs具有相似的基因表达模式,但也存在一些差异,这些特定基因涉及细胞周期、细胞分裂、细胞死亡、细胞生长和发育。这些基因还在转录调节、DNA修复、DNA复制和染色体稳定性中起作用,这是细胞或亚细胞组分运动、细胞通信、细胞组织突起、细胞因子分泌和激素代谢的重要组成部分。转录组测序分析的结果在一定程度上解释了不同来源的MSCs之间生物学特征的差异;基于测序结果的验证实验可知,不同来源的MSCs在增殖能力和细胞因子分泌能力存在显著差异。总的来说,UC-MSCs和CV-MSCs表现出更强的增殖能力,分泌更高水平的旁分泌因子,可能在未来疾病的治疗中有不同的优势。     

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.