Expression of epithelial markers by human umbilical cord stem cells. A topographical analysis

IntroductionHuman umbilical cord stem cells have inherent differentiation capabilities and potential usefulness in regenerative medicine. However, the epithelial differentiation capability and the heterogeneity of these cells have not been fully explored to the date.MethodsWe analyzed the expression of several undifferentiation and epithelial markers in cells located in situ in different zones of the umbilical cord –in situ analysis– and in primary ex vivo cell cultures of Wharton's jelly stem cells by microarray and immunofluorescence.ResultsOur results demonstrated that umbilical cord cells were heterogeneous and had intrinsic capability to express in situ stem cell markers, CD90 and CD105 and the epithelial markers cytokeratins 3, 4, 7, 8, 12, 13, 19, desmoplakin and zonula occludens 1 as determined by microarray and immunofluorescence, and most of these markers remained expressed after transferring the cells from the in situ to the ex vivo cell culture conditions. However, important differences were detected among some cell types in the umbilical cord, with subvascular zone cells showing less expression of stem cell markers and cells in Wharton's jelly and the amnioblastic zones showing the highest expression of stem cells and epithelial markers.ConclusionsThese results suggest that umbilical cord mesenchymal cells have intrinsic potential to express relevant epithelial markers, and support the idea that they could be used as alternative cell sources for epithelial tissue engineering.     

The protective effects of human umbilical cord mesenchymal stem cell-derived extracellular vesicles on cisplatin-damaged granulosa cells

ObjectiveLong term exposure to gonadotoxic chemotherapy is becoming a major cause of premature ovarian failure/insufficiency (POF/POI) with the increasing cancer incidence among young women. The present study was designed to investigate the protective effects of human cord mesenchymal stem cells (HUCMSCs)-derived extracellular vesicles (EVs) on cisplatin (CDDP)-damaged granulosa cells (GCs) in vitro.Materials and methodsEVs were obtained from supernatant of cultured HUCMSCs by ultracentrifugation method, purified by Sucrose density gradient centrifugation, and then were co-cultured with cisplatin-damaged GCs of 3-weeks female Sprague–Dawley (SD) rats. PKH26 labeled EVs could be observed in normal and CDDP-damaged GCs after 6 h co-culture.ResultsThe surviving GCs were significantly higher and apoptotic GCs were significantly lower in EVs + CDDP group compared with CDDP group. Meanwhile, the levels of E2 and StAR (the key gene related to synthesis of steroid hormone) were significantly higher in EVs + CDDP group compared with CDDP group. Furthermore, the mRNA expression of Caspase 3 was down-regulated significantly and the ratio of Bcl-2/Bax was up-regulated significantly in EVs + CDDP group. Moreover, the protective effect of EVs on CDDP-damaged GCs showed a dose-dependent effect.ConclusionHUCMSCs-derived EVs could become incorporated to CDDP-damaged GCs, and increase the number of living cells, therefore playing important roles in promoting resistance to cisplatin-induced GCs apoptosis and restoring synthesis and secretion of steroid hormone in GCs. This study might provide a theoretical and experimental basis for use of mesenchymal stem cells (MSCs) derived EVs instead of MSCs as a cell-free therapeutic strategy for the patients with POI induced by chemotherapeutic agents.     

小鼠非黏附骨髓间充质干细胞分化为神经元样细胞的实验研究

目的 探讨小鼠非黏附骨髓间充质干细胞(non-adherent bone marrow-derived mesen-chymal stem cell,NA-BM-MSC)在体外、体内尤其是缺血损伤的脑内向神经元样细胞分化的能力.方法 传代纯化后的NA-BM-MSC经表皮生长因子和碱性成纤维细胞生长因子诱导后,通过甲苯胺蓝染色和免疫细胞化学染色检测神经元特异性核蛋白和神经中间丝蛋白的表达情况;将来自β-半乳糖苷酶转基因小鼠的NA-BM-MSC在立体定位仪下移植到小鼠缺血损伤的脑内,8周后通过LacZ组织化学染色和免疫组织化学染色检测供体细胞在缺血脑内微环境中存活以及神经元特异性核蛋白的表达情况. 结果 NA-BM-MSC经过诱导能够表达神经元特异性核蛋白和神经中间丝蛋白并有尼氏体形成;移植到损伤脑内后,LacZ组织化学染色发现在缺血侧供体细胞能够存活,免疫组织化学单、双染显示在缺血坏死区及周围都检测到了β-半乳糖苷酶阳性的供体细胞,其中的部分阳性细胞还同时表达神经元特异性核蛋白. 结论 NA-BM-MSC在体外能够分化成神经元样细胞,在小鼠缺血损伤的脑组织内也能够存活、迁移,部分细胞还能分化为神经元样细胞.     

A genome-wide comparison of mesenchymal stem cells derived from human placenta and umbilical cord

Objective

The human umbilical cord and placenta have been considered as attractive alternative sources for noninvasive isolation of human mesenchymal stem cells (hMSCs). Different sources of MSC may have individual differentiation potential and phenotype. In this study, we compared the genome-wide expression data of umbilical cord and placenta derived hMSCs to identify specific differential expression genes (DEGs) and corresponding functions.

人脐带间充质干细胞移植治疗大鼠压力性尿失禁的研究

目的 探讨人脐带间充质干细胞(HUCMSC)移植治疗雌鼠压力性尿失禁(SUI)的可行性和有效性.方法 14只SD大鼠分娩后立即采用气囊压迫大鼠阴道壁4h模拟产伤,2周后行双侧卵巢切除,常规饲养2个月后建成12只SD大鼠SUI模型.建模2个月后,将转染pEGFP-N1质粒的HUCMSC注射于6只SUI大鼠尿道周围(移植治疗组),另6只相同部位注射等量生理盐水(实验对照组),比较两组尿动力学指标后,获取泌尿生殖道组织,观察尿道及其周围组织和膀胱的形态学变化;荧光显微镜观察SUI模型大鼠泌尿生殖道组织标本荧光细胞的分布.结果 成功建模的12只大鼠漏尿点压力值为(23.8±4.2)mm Hg(1 mm Hg=0.133 kPa).移植治疗组喷嚏试验阳性率为1/6,实验对照组为5/6,两组比较,差异有统计学意义(P＜0.05).移植治疗组漏尿点压力值为(30.6±2.8) mm Hg,实验对照组为(21.4±7.0) mmHg,两组比较,差异有统计学意义(P＜0.05).HUCMSC移植治疗后大鼠尿道壁肌层增厚,肌层和筋膜间结缔组织紧密,尿道及周围组织切片可见到荧光细胞分布.结论 采用气囊压迫分娩大鼠阴道壁模拟产伤并行双侧卵巢切除能成功获得稳定的SUI动物模型.HUCMSC可以在损伤的大鼠尿道周围组织中存活、增殖,改善尿流动力学指标和喷嚏试验的阳性率;形态学上观察可修复尿道周围的支持组织.     

EphA2-positive human umbilical cord-derived mesenchymal stem cells exert anti-fibrosis and immunomodulatory activities via secretion of prostaglandin E2

Objective

Previous study has demonstrated that EphA2 is a biomarker of mesenchymal stem cells (MSCs) from human placenta or umbilical cord and is able to distinguish MSCs from fibroblasts. In this study, we further examine the potential efficacy of EphA2⁺ human umbilical cord-derived MSCs (hUC-MSCs).

人脐带间充质干细胞移植对围绝经期大鼠INHB和AMH的影响

目的:探讨人脐带间充质干细胞(h UCMSCs)移植对围绝经期大鼠AMH、INHB的影响。方法:阴道脱落细胞涂片筛选30只13~15月龄的SPF级SD围绝经期大鼠模型,随机分为A、B、C组(每组10只)。A组不予处理;B组经尾静脉移植浓度1×106细胞/ml h UCMSCs 1ml,隔日1次,共2次;C组移植等量的细胞培养液。于成模时和末次移植后7天和14天,检测各组大鼠的AMH、INHB水平。结果:3组成模时及末次移植后7天的AMH、INHB水平比较,差异均无统计学意义(P0.05)。末次移植后14天,B组的AMH、INHB水平高于A、C组,差异有统计学意义(P0.05);移植后,A、C组的AMH、INHB水平逐渐下降,移植后第14天时下降显著,与成模时比较差异有统计学意义(P0.05)。结论:h UCMSCs移植可延缓围绝经期SD大鼠AMH、INHB下降水平,提示对延缓围绝经期大鼠卵巢储备功能下降有积极意义。     

子痫前期重度患者血清损伤后血管内皮细胞上清对脐带间充质干细胞的趋化作用

目的探讨子痫前期重度患者血清培育后的血管内皮细胞上清是否对人脐带间充质干细胞(UC-MSCs)有趋化作用。方法 2009年10月至11月在中国医科大学附属盛京医院将内皮细胞以1×105个/mL的浓度接种于6孔培养板,进行分组干预:每组用含15%子痫前期重度患者血清(P组)、健康孕妇血清(H组)、不含血清的DMEM培养液(N组)培养24h,取各组上清液于无菌EP管中,离心20min后加入Transwell小室下室,取第5代UC-MSCs用含5%胎牛血清的DF-12调整细胞浓度为1×105个/mL,加入预处理后的Transwell小室上室,将Tran-swell小室置于37℃、5%CO2孵箱孵育24h。取出后4%多聚甲醛固定、伊红染色后于20倍镜下随机记数5个无重叠细胞视野的透膜细胞数,取其平均数代表该组细胞的侵袭力。结果 N组越膜细胞数:40.87±6.33;H组越膜细胞数:45.47±6.89;P组越膜细胞数:56.07±6.76。P组越膜细胞最多,差异有统计学意义(P<0.01,F=20.53)。结论 UC-MSCs具有向子痫前期重度患者血清培育后血管内皮细胞趋化的生理特性。     

Tissue-specific engraftment after in utero transplantation of allogeneic mesenchymal stem cells into sheep fetuses

OBJECTIVE: Mesenchymal stem cells (MSC) have multiorgan differentiation capacity, providing the potential for prenatal treatment of genetic disorders. We address the question if in utero transplantation of MSC results in short-term organ-specific engraftment in the fetal sheep. STUDY DESIGN: Sheep fetal liver-derived MSC selected by adherence culture (passage 1) were transplantated into the fetal peritoneal cavity with ultrasound-guidance (mean gestational age, 59 days). After 14 days recipient fetuses were analyzed by fluorescence-activated cell sorting (FACS), real-time polymerase chain reaction (PCR), and immunohistochemistry. RESULTS: Fetuses (n = 11) were transplanted with 7.7 x 10(6) MSCs (mean). All surviving fetuses (n = 5) showed engraftment with mean levels of 3.2% (lung), 0.8% (spleen), 0.6% (liver, brain), 0.4% (bone marrow), 0.1% (blood, thymus), and <0.1% (kidneys) by flow cytometry. Immunohistochemistry showed organ-specific distribution. CONCLUSION: In utero transplantation of allogeneic MSC results in low level, multiorgan engraftment at 14 days post transplant. This supports the potential of in utero MSC transplantation for the treatment of nonhematopoietic genetic disorders of the fetus.     

Fetal and perinatal stem cells in cardiac regeneration: Moving forward to the paracrine era

Cardiovascular disease (CD) is a major burden for Western society. Regenerative medicine has provided encouraging results, yet it has not addressed the focal defects causing CD and mainly related to the inefficient repair programme of the heart. In this scenario, stem cells have been broadly investigated and their paracrine effect proposed as a possible working strategy to boost endogenous mechanisms of repair and regeneration from within the cardiac tissue.The scientific community is now focusing on identifying the most effective stem cell secretome, as the whole of bioactive factors and extracellular vesicles secreted by stem cells and endowed with regenerative potential. Indeed, the adult stem cell-paracrine potential for cardiac regeneration have been widely analyzed with positive outcome. Nevertheless, low yield, invasive sampling and controversial self-renewal may limit adult stem cell application. On the contrary, fetal and perinatal stem cells, which can be easily isolated from leftover sample via prenatal screening during gestation or as clinical waste material after birth, can offer an ideal alternative. These broadly multipotent immature progenitors share features with both adult and embryonic stem cells, show high self-renewal, but they are not tumorigenic neither cause any ethical concern. While fetal and perinatal stem cells demonstrated to improve cardiac function when injected in the injured heart, the comprehensive characterization of their secretome for future applications is still at its infancy.In this review, we will discuss the paracrine potential of the fetal and perinatal stem cell secretome to provide cardiac repair and resurge the dormant mechanisms of cardiac regeneration for future therapy.     

模拟子宫内膜微环境体外诱导兔骨髓间充质干细胞向子宫内膜上皮细胞分化的实验研究

目的:探讨模拟子宫内膜微环境体外诱导兔骨髓间充质干细胞(BMSCs)向子宫内膜上皮细胞方向分化的可行性。方法:原代培养兔BMSCs并鉴定,提取子宫内膜条件培养液,用子宫内膜条件培养液和雌激素(β-雌二醇,1×10-7mol/L)体外诱导分化BMSCs,用细胞免疫荧光法和Western blot检测分化的BMSCs是否表达上皮细胞特异性标记蛋白。结果:兔BMSCs具有较强的增殖潜能,呈克隆性生长,表达CD44和CD90,表达率分别为99.91%、98.64%;不表达CD45。子宫内膜条件培养液和雌激素培养BMSCs5天后,大部分细胞形态由间质细胞向上皮样细胞分化;细胞免疫荧光鉴定诱导分化后的BMSCs表达角蛋白;Western blot检测实验组角蛋白表达量明显增加,与对照组相比有统计学差异(P<0.05)。结论:子宫内膜条件培养液联合雌激素可以诱导兔BMSCs向子宫内膜上皮细胞方向分化。体外模拟的子宫内膜微环境在BMSCs向子宫内膜上皮细胞分化中起重要作用。     

干细胞条件培养液对小鼠体外成熟卵母细胞发育潜能的影响

目的:探讨骨髓间充质干细胞(MSCs)条件培养液对小鼠体外成熟卵母细胞发育潜能的影响。方法:分离、培养小鼠MSCs,获得MSCs条件培养液;收集4~6周小鼠生发泡期(GV)卵母细胞,在MSCs条件培养液中体外成熟(IVM);同时部分小鼠促排处理,收集体内成熟卵母细胞;体外受精(IVF)后,比较受精率、4-细胞形成率及囊胚形成率差异;Hoechst33342染色后观察内细胞团(ICM)和滋养层细胞形成情况,计数内细胞团数。结果:IVM卵母细胞IVF后的受精率、4-细胞形成率和囊胚形成率及囊胚内细胞总数与体内成熟组比较,无显著差异(P0.05);IVM组各时期的胚胎形态正常,囊胚内细胞染色后的荧光图像与体内成熟组基本一致。结论:MSCs条件培养液能保持小鼠IVM卵母细胞良好的质量和发育潜能,是一种较理想的IVM培养体系。     

Therapeutic benefit of mesenchymal stem cells in pregnant rats with angiotensin receptor agonistic autoantibody-induced hypertension: Implications for immunomodulation and cytoprotection

Immunomodulation by mesenchymal stem cells (MSCs) is potentially important for maintaining peripheral tolerance. Preeclampsia may be due to maternal immune rejection of the genetically foreign fetus. This study aimed to investigate the biological function of human umbilical cord–derived mesenchymal stem cells (HU-MSCs) for the treatment of angiotensin receptor agonistic autoantibody (AT1-AA)-induced hypertension during pregnancy. HU-MSCs were isolated, cultured, and labeled in vitro. AT1-AA and HU-MSCs were administered to pregnant rats. Green fluorescent protein (GFP)–positive HU-MSCs infused in vivo were identified by immunofluorescence. Systolic blood pressure (SBP) was evaluated. The effects of HU-MSCs on fetal weight, kidney burden, and spiral artery remodeling, as well as on the expression of tumor necrosis factor α (TNF-α), interleukin 10 (IL-10), and heme oxygenase 1 (HO-1), were investigated. The SBP levels in the HU-MSC-treated pregnant hypertension rats decreased by gestational day 19. The reduction in fetal weight was largely ameliorated after HU-MSC treatment. Lesion burden in the kidney was attenuated and spiral artery remodeling was improved in HU-MSC-treated pregnant hypertension rats. However, green fluorescent protein (GFP)–labeled cells were sparingly observed in the kidney and placenta. Intravenous infusion of HU-MSCs into AT1-AA-induced rats significantly downregulated serum TNF-α levels and upregulated IL-10 levels, concomitant with increased placenta and mesometrial triangle (MT) HO-1 expression. Taken together, intravenous infusion of HU-MSCs ameliorates AT1-AA-induced pregnancy hypertension, intrauterine growth retardation, kidney impairment, and spiral artery remodeling impairment. Moreover, the potential benefits of HU-MSCs may be attributable to both an interference with the pathogenic immune response and a paracrine cytoprotective action.     

Trial evaluation of bone marrow derived mesenchymal stem cells (MSCs) transplantation in revival of spermatogenesis in testicular torsion

Defective spermatogenesis due to the failure in germ cell proliferation and differentiation is the major sign of male infertility pathogenesis; and male factor is involved in up to half of all infertile couples. Testicular torsion is an acute vascular event causing the rotation of the vascular pedicle of the testis, thereby impeding the blood flow to the testis and the scrotal contents. Azoospermia caused by torsion in testis is a common source of impotency, which has not been touched by this approach, yet. Here, we use the capacity of mesenchymal stem cells (MSCs), as multipotent adult stem cells, to revive spermatogenesis in torsion-induced azoospermia. For this purpose, MSCs were extracted from rat bone marrow, cultured and transplanted into torsioned testis. Germ cell specific markers (Oct4, Vasa and c-Kit) were monitored for the differentiation of MSCs after transplantation into the torsion azoospermed testis. This study is a trial evaluation of mesenchymal stem cells in rat torsion testis to follow up the regenerative capacity of stem cells in spermatogenesis revival. These approaches can provide the powerful tools to investigate the basic biology of stem cells for reproductive engineering and infertility treatment.     

Transendothelial migration of human umbilical mesenchymal stem cells across uterine endothelial monolayers: Junctional dynamics and putative mechanisms

IntroductionDuring pregnancy, fetal stem cells can transfer to the maternal circulation and participate in tissue repair. How they transmigrate across maternal endothelial barriers and whether they can subsequently influence maternal endothelial integrity is not known.MethodsMesenchymal stem cells (WJ-MSC) were isolated from Wharton's jelly and their interactions with human uterine microvascular endothelial cell (HUtMEC) monolayers, junctional occupancy and expression/phosphorylation of vascular endothelial (VE)- cadherin and vascular endothelial growth factor (VEGF-A) secretion was studied over 48h by real time, confocal microscopy, immunoblotting and ELISA.ResultsWJ-MSC displayed exploratory behaviour with interrogation of paracellular openings and spreading into the resultant increased gaps followed by closing of the endothelium over the WJ-MSC. 62% of added cells crossed within 22h to sub-endothelial niches. There was a concomitant loss of junctional VE-cadherin in HUtMEC followed by a full return and increased VE-cadherin expression after 22h. During early hours, VE-cadherin showed a transient phosphorylation at Tyrosine (Tyr)-685 when VEGF-A secretion were high. From 16 to 22h, there was increased de-phosphorylation of Tyr-731. Anti-VEGF-A blocked Tyr-685 phosphorylation but not the decrease in P-Tyr731; this partially inhibited WJ-MSC transmigration.DiscussionFetal WJ-MSC can traverse uterine endothelial monolayers by mediating a non-destructive paracellular pathway. They can promote junctional stability of uterine endothelium from the sub-endothelial niche. Mechanistically, WJ-MSC induces VEGF-dependent phosphorylation events linked with paracellular permeability and VEGF-independent de-phosphorylation events associated with leukocyte extravasation. Our data also allows consideration of a possible role of fetal MSC in mature functioning of the uterine vasculature needed for optimal utero-placental perfusion.