不同来源成体骨髓间充质干细胞调控免疫功能的比较研究

目的 比较霍奇金淋巴瘤(HL)、非霍奇金淋巴瘤(NHL)患者与正常人骨髓间充质干细胞(MSC)的免疫调节能力.方法 获取正常人、HL和NHL患者的骨髓MSC,用低血清培养液进行培养.采用流式细胞仪检测骨髓MSC的免疫表型;应用酶联免疫吸附试验检测骨髓MSC培养上清液中转化生长因子β1(TGF-β1)的水平;采用Transwell培养体系检测骨髓MSC抑制T淋巴细胞增殖的能力;应用混合淋巴细胞反应检测骨髓MSC抑制异体T淋巴细胞增殖的能力.结果 正常人、HL和NHL患者骨髓间充质干细胞具有相似的细胞形态和免疫表型,均具有分泌TGF-β1的能力,均不表达HLA-DR和共刺激分子CD80、CD86和CD40.HL和NHL患者骨髓MSC具有抑制异体T淋巴细胞增殖的能力,这种抑制能力随着MSC细胞数量的增加而增强,并可以被抗TGF-β1抗体所逆转.体外诱导分化后的骨髓MSC仍然具有抑制异体T淋巴细胞增殖的能力.结论 HL和NHL患者骨髓MSC具有和正常成人骨髓MSC相同的免疫调控能力,这种抑制T淋巴细胞增殖的能力不随着其体外诱导分化而改变.     

同种异体共培养大鼠骨髓间充质干细胞的神经分化诱导鉴定

目的：探讨同种异体大鼠骨髓间充质干细胞（BMSC）在体外的共培养方法，并行神经分化诱导。方法：分别通过全骨髓直接培养法及密度梯度法分离培养大鼠骨髓间充质干细胞，各传至第3代后。取等量细胞混合后共培养，共培养的细胞再次传代后进行神经方向诱导，并行Nestin、NSE、GFAP免疫细胞化学染色鉴定。结果：共培养的大鼠骨髓间充质干细胞经诱导分化后出现神经元和胶质细胞形态，经Nestin、NSE、GFAP免疫细胞化学染色证实为神经细胞。结论：同种异体共培养的骨髓间充质干细胞可在体外定向诱导为神经元和胶质细胞等神经细胞。     

多发性骨髓瘤患者间充质干细胞的免疫原性及其对免疫功能的调控作用

目的 探讨多发性骨髓瘤(MM)患者的骨髓间充质干细胞(MSC)的免疫原性及其调控免疫的功能和机制.方法 获取MM患者的骨髓MSC,在低血清培养液中培养和扩增.流式细胞仪检测免疫表型;酶联免疫吸附试验检测MSC培养上清液中细胞因子的分泌水平;混合淋巴细胞反应检测骨髓MSC抑制异体T淋巴细胞增殖的能力;流式细胞仪检测MSC对树突状细胞(DC)吞噬功能的影响;混合淋巴细胞反应检测MM骨髓MSC抑制DC介导的异体T淋巴细胞增殖能力.结果 MM患者的MSC不表达HLA-DR和共刺激分子CD80、CD83、CD86和CD40.MM患者的MSC通过分泌转化生长因子β1 (TGF-β1)和肝细胞生长因子(HGF)抑制异体T淋巴细胞的增殖.MM患者的MSC具有抑制DC吞噬作用的功能;MM骨髓MSC抑制DC分泌白细胞介素12(IL-12),并可以抑制DC介导的异体T淋巴细胞增殖.结论 MM骨髓MSC具有低免疫原性及体外调节免疫的功能,该免疫调节功能与其分泌细胞因子有关.     

成骨诱导后的骨髓间充质干细胞对外周血单个核细胞增殖的免疫调节作用

目的:探索骨髓间充质干细胞(BMSCs)及成骨诱导后的骨髓间充质干细胞(OM-BMSCs)对同种异体的外周血单个核细胞(PBMCs)的免疫调节作用。方法:分离比格犬BMSCs,成骨诱导培养基(ost eogenic medium,OM)进行成骨诱导,建立BMSCs、OM-BMSCs与同种异体PBMCs的共培养体系,分三种实验条件,共9组。A组:5×104受体PBMCs加入5×104供体PBMCs共培养;B组:A组组分加入已铺被1×104/孔BMSCs的96孔板;C组:A组组分加入已铺被1×104/孔OM-BMSCs的96孔板;D组:5×104受体PBMCs加入植物血凝素A(PHA)刺激物;E组、F组:将D组组分按B、C组方式处理;G组:5×104受体PBMCs加入IL-2炎症因子;H组、I组:G组组分按B、C组方式处理。A、D、G组分别为三个实验条件的对照组,单独接种1×104/孔BMSCs、OM-BMSCs作为空白对照组Bl ank1组、Bl ank2组。以上各组分别培养120h后,应用Cell Titer 96 Aqueous检测各组PBMCs的增殖情况。结果:在各实验条件下与相应对照组相比较,BMSCs与OM-BMSCs均能抑制双向混合淋巴细胞反应(MLR)中PBMCs的增殖,均能抑制经PHA刺激后PBMCs的增殖,均能抑制经IL-2刺激后PBMCs的增殖(P<0.01)。结论:BMSCs及成骨分化的BMSCs对刺激细胞、PHA、IL-2刺激的同种异体PBMSs的增殖均表现为抑制效应,成骨分化后的骨髓间充质干细胞对同种异体免疫反应仍具有免疫调节作用。     

骨髓间充质干细胞定向分化肝细胞及肝内移植研究

目的 观察体外诱导骨髓问充质干细胞分化及肝纤维化形成环境中移植情况。方法 首先行骨髓间充质干细胞提取、分离和培养，加入肝细胞生长因子（HGF，20μg／L）和表皮生长因子（EGF，1．5mg／L）诱导定向分化。肝纤维化形成的大鼠随机分成2组，每组10只。使用5-溴脱氧尿苷（BrdU）标记诱导的骨髓间充质干细胞，经门静脉向肝纤维化形成的SD大鼠肝脏移植，对照组用BrdU标记未经诱导的骨髓间充质干细胞。2周后通过免疫组织化学方法检测大鼠肝脏标记细胞的分布及BrdU^＋／ALB^＋细胞数量。结果 体外诱导骨髓间充质干细胞定向分化的细胞CK8及ALB表达阳性。移植2周后大鼠肝脏均可检测到BrdU标记细胞，与对照组相比诱导后骨髓问充质干细胞组BrdU^＋／ALB^＋细胞数较多，差异有统计学意义（P〈0．05）。结论 经体外诱导骨髓间充质干细胞能分化为肝细胞，移植在大鼠肝纤维化形成环境中，白蛋白表达细胞数更多。     

hIL-10基因修饰骨髓间充质干细胞对异种T淋巴细胞增殖的影响及机制研究

目的 研究hIL-10基因修饰的MSCs(骨髓间充质干细胞)对异种混合T淋巴细胞增殖的影响及机制.方法 RT-PCR克隆hIL-10基因并将构建慢病毒hIL-10/pLOX-cwGFPs,然后将慢病毒转导hIL-10入豚鼠MSCs;ELISA测定hIL-10-MSCs培养上清hIL-10含量;CCK-8法检测hIL-10基因修饰的骨髓间充质干细胞对体外混合淋巴细胞培养的影响;ELISA测定其培养上清对外周血单个核细胞分泌IFN-γ的影响.结果 成功构建慢病毒hIL-10/pLOX-cwGFP,hIL-10-MSCs培养上清的IL-10水平明显高于未转染(P<0.05);hIL-10-MSCs能抑制异种T淋巴细胞混合培养的增殖反应(P<0.05);加入IL-2能逆转MSCs的抑制作用(P<0.05);hIL-10-MSCs培养上清组能抑制PHA刺激的PBMC分泌IFN-γ(P<0.05).结论 hIL-10-MSCs对混合异种淋巴细胞培养有显著的抑制作用,能抑制PHA刺激的PBMC分泌IFN-γ.     

不同年龄小鼠骨髓间充质干细胞中miR203的差异及 其对细胞增殖的调控

目的探索不同年龄骨髓间充质干细胞中miR203的表达变化,及其对骨髓间充质干细胞自我更新的作用机制。方法分别分离培养4周龄和18~24月龄的Balb/c小鼠BMSCs,对比不同年龄小鼠BMSCs增殖潜能的差异,并检测不同年龄小鼠BMSCs中miR-203的表达变化差异,从而探讨miR-203在骨髓间充质干细胞增殖调节中的作用机制。结果根据干细胞贴壁特性获得了稳定的骨髓间充质干细胞,其在分化诱导条件下可获得经茜素红染色呈红色结节及油红O染色显示有脂质沉淀,且成骨诱导后Ⅰ型胶原蛋白显著表达。在增殖条件下,与年轻BMSCs相比,老年BMSCs增殖(传代)能力明显下降。年轻小鼠(4周龄)BMSCs中miR-203远低于老年小鼠(18~24月龄)BMSCs中miR-203表达(P0.05)。结论年轻骨髓间充质干细胞增殖能力优于老年骨髓间充质干细胞,可能与miR-203表达较低有关。     

骨髓间充质干细胞向淋巴管内皮细胞的分化潜能

目的：观察骨髓间充质干细胞在体外分化成淋巴管内皮细胞的可能性。方法：采用密度梯度离心法分离成人骨髓间充质干细胞,体外扩增,流式细胞术检测细胞表面标志。用内皮细胞培养基EGM-2培养,加入VEGF-C（156s）诱导后,相差显微镜下观察细胞形态变化,流式细胞术检测淋巴管内皮细胞特异性标志物Podoplanin和VEGFR-3,RT-PCR检测相应基因的表达。成管实验检测分化的淋巴管内皮细胞的功能。结果：分离培养的骨髓间充质细胞形态上为纤维样,经流式细胞术测定表达CD90、CD105,其阳性表达率分别为90.03%、97.86%,而CD14、CD34、HLA-DR的阳性阳性率分别为2.00%、2.18%、1.56%。经诱导刺激后,间充质干细胞呈鹅卵石样外观,Podoplanin和VEGFR-3的表达率分别为26.25%和12.70%。RT-PCR结果显示特异性的条带,成管实验显示诱导后的淋巴管内皮细胞具有成管的功能。结论：从骨髓中可以提取到骨髓间充质干细胞,在内皮细胞培养基和VEGF-C（156s）的诱导下骨髓间充质干细胞可以分化为淋巴管内皮细胞。     

大鼠睾丸支持细胞对骨髓问充质干细胞免疫抑制作用的影响

目的探索睾丸支持细胞（Sertoli细胞）对骨髓间充质干细胞（BMSC）免疫抑制作用的影响,为二者在移植免疫中的联合应用提供思路。方法二步酶解法处理大鼠睾丸,分离Sertoli细胞;Percoll法分离大鼠BMSC;Ficoll法分离淋巴细胞;刀豆蛋白A（ConA）刺激进行T细胞转化试验;将Sertoli细胞、BMSC和Sertoli细胞＋BMSC分别加入未经ConA处理的静止淋巴细胞培养体系和经ConA处理后的T细胞转化体系,MTT法测定淋巴细胞增殖情况,观察BMSC、Sertoli细胞或二者共培养对T细胞活化、增殖的影响。结果 BMSC、Sertoli细胞以及二者共培养对静止的淋巴细胞无明显作用。BMSC、Sertoli细胞及二者共培养对T细胞的活化、增殖均有明显的抑制作用,且Sertoli细胞与BMSC共培养时抑制作用呈现一定的协同性。结论 BMSC和Sertoli细胞均具有负性免疫调节作用,二者共培养可以进一步增强BMSC的免疫抑制效应。     

扁桃体间充质干细胞免疫学特性的初步研究

目的 探讨扁桃体间充质干细胞(tonsil mesenchymal stem cells,TMSCs)的免疫学特性及机制.方法 取慢性扁桃体炎患儿的扁桃体组织,分离、培养TMSCs,通过流式细胞术检测HLA-Ⅰ、HLA-Ⅱ、CD80、CD86等免疫分子的表达情况.以牙周膜干细胞作为对照,观察TMSCs能否引起同种异体外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)的增殖,以及TMSCs对混合淋巴细胞反应(mixed lymphocyte reaction,MLR)和植物血凝素(phytohemagglutinin,PHA)引起的淋巴细胞增殖的影响.建立TMSCs+PHA+异体PBMCs、TMSCs+MLR的培养体系,测定细胞上清液中的犬尿氨酸浓度.在上述反应体系进行中和实验,观察被TMSCs抑制了的淋巴细胞重新发生增殖的情况.每个实验重复3次,每组6个佯本.统计学方法采用方差分析,P ＜0.05为差异有统计学意义.结果 TMSCs表达HLA-Ⅰ,但不表达HLA-Ⅱ和共刺激分子CD80、CD86.TMSCs与异体PBMCs共培养5d后,刺激指数为1.38±0.26,而单纯PBMCs培养5d后的刺激指数为1.22±0.28,2组差异无统计学意义(P＞0.05),证实TMSCs不会引起异体PBMCs增殖.TMSCs与异体PBMCs、PHA共培养5d后,刺激指数分别为1.49±0.29(TMSCs∶ PBMCs为0.5∶1)和1.23±0.22(TMSCs∶ PBMCs为1∶1),而PBMCs+PHA组培养5d后的刺激指数为4.60±0.81,2组之间的差异均有统计学意义(P＜0.05),说明TMSCs能够抑制PHA引起的淋巴细胞增殖.TMSCs与MLR共培养5d后,刺激指数分别为1.29±0.23(TMSCs∶ PBMCs为0.5∶1)和1.26±0.27(TMSCs∶ PBMCs为1∶1),而MLR培养5d后的刺激指数为3.04±0.66,2组之间的差异均有统计学意义(P＜0.05),说明TMSCs能够抑制MLR引起的淋巴细胞增殖.在TMSCs+PHA+异体PBMCs、TMSCs+MLR的培养体系中,犬尿氨酸浓度显著升高,分别为(26.0±2.3) μmol/L和(23.5±4.5)μmol/L.中和实验发现,1-甲基-L-色氨酸基本恢复了被TMSCs抑制的淋巴细胞增殖.结论 TMSCs具有低免疫原性和免疫抑制特性,有望进行同种异体移植.     

Risk of viral transmission via bone marrow progenitor cells versus umbilical cord blood hematopoietic stem cells in bone marrow transplantation

Hematopoietic stem cell transplantation (HSCT) is the treatment of choice for children and certain adults with malignant and nonmalignant hematologic disease. Since viral infections are the major problem, this study examined those that might potentially be transmitted to HSCT recipients via bone marrow (BM) versus umbilical cord blood (UCB). BM progenitor cells, peripheral blood leukocytes, and plasma samples were collected from 30 allogenic BM donors. Umbilical cord blood hematopoietic stem cells and plasma samples were also collected from 34 UCB donors. Viral DNA extracted and purified from collected specimens was processed using nested polymerase chain reactions (PCR) to detect human parvovirus B19 (HPV B19), human herpesvirus-6 (HHV-6), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV). The prevalences of HCMV DNA in collected BM progenitor cells versus UCB hematopoietic stem cells were 73% versus 23%, respectively. Conversely, HHV-6 DNA was not detected in any collected specimen by simple PCR. Distribution of the other investigated virus DNAs except EBV DNA was similar in specimens collected from both groups. EBV DNA was not determined in UCB hematopoietic stem cells. The results indicate that the risk of viral transmission to BM transplant recipients via UCB hematopoietic stem cells is less than that with BM progenitor cells.     

Gene delivery using human cord blood-derived CD34+cells into inflamed glomeruli in NOD/SCID mice

BACKGROUND: Bone marrow reconstitution using genetically-modified hematopoietic stem cells has been reported to confer resistance to inflammation and prevent renal injury in glomerulonephritis. Although this strategy has potentials for clinical use, taking hematopoietic stem cells from bone marrow is highly stressful for patients. In this regard, umbilical cord blood may be a useful alternative and, therefore, we focused on their suitability as a source of hematopoietic stem cells for transplantation-based therapy for glomerulonephritis. METHODS: CD34+ cells were obtained from human umbilical cord blood, retrovirally transduced with human beta-glucuronidase (HBG) gene, and transplanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. After confirming the successful chimerism, these mice were treated with lipopolysaccharide (LPS), and local HBG expression in glomeruli was examined using immunohistochemical analysis, HBG bioassay, and Western blot analysis. RESULTS: Clonogenic assay showed that 88.4 +/- 5.9% burst-forming unit-erythroid (BFU-E), 79.7 +/- 11.4% in colony-forming unit-macrophage (CFU-M), and 81.1 +/- 14.1% in colony-forming unit-granulocyte (CFU-G), respectively, possessed the transgene after transfection, suggesting that precommited cells were susceptible to retroviral infection. Flow cytometric analysis revealed that 24.1 +/- 14.5% of bone marrow cells in these chimera mice expressed human lymphocyte antigen (HLA) 8 weeks after transplantation. Also, clonogenic assay showed that a sustained engraftment of human hematopoietic cells expressed HBG. CD14-positive cells were recruited into the glomeruli upon LPS treatment and they secreted bioactive HBG, suggesting that cord blood-derived CD34+cells may differentiate into monocyte lineage while maintaining the expression of the transgene. CONCLUSION: These data indicate that umbilical cord blood cells can be utilized as a source of hematopoietic stem cells for the transplantation-based therapy of glomerulonephritis.     

Allogenic peripheral blood derived mesenchymal stem cells (MSCs) enhance bone regeneration in rabbit ulna critical-sized bone defect model.

Mesenchymal stem cells (MSCs) were demonstrated to exist within peripheral blood (PB) of several mammalian species including human, guinea pig, mice, rat, and rabbit. Whether or not the PB derived MSCs (PBMSCs) could enhance the regeneration of large bone defects have not been reported. In this study, rabbit MSCs were obtained from mononuclear cells (MNCs) cultures of both the PB and bone marrow (BM) origin. The number of PBMSCs was relatively lower, with the colony forming efficiency (CFE) ranging from 1.2 to 13 per million MNCs. Under specific inductive conditions, PBMSCs differentiated into osteoblasts, chondrocytes, and adipocytes, showing multidifferentiation ability similar to BMMSCs. Bilateral 20 mm critical-sized bone defects were created in the ulnae of 12 6-month-old New Zealand white rabbits. The defects were treated with allogenic PBMSCs/Skelite (porous calcium phosphate resorbable substitute), BMMSCs/Skelite, PBMNCs/Skelite, Skelite alone, and left empty for 12 weeks. Bone regeneration was evaluated by serial radiography, peripheral quantitative computed tomography (pQCT), and histological examinations. The X-ray scores and the pQCT total bone mineral density in the PBMSCs/Skelite and BMMSCs/Skelite treated groups were significantly greater than those of the PBMNCs/Skelite and Skelite alone groups ( p < 0.05), respectively. Histologically, newly formed bone was evident in the PBMSCs/Skelite and BMMSCs/Skelite treated groups. The findings demonstrated that the rabbit PBMSCs possessed multidifferentiation potential comparable with BMMSCs, allogenic PBMSCs seeded onto porous calcium phosphate resorbable substitutes enhanced bone regeneration in the rabbit ulna critical-sized bone defect model, suggesting allogenic PBMSCs may be a new source of circulating osteogenic stem cells for bone regeneration and tissue engineering.     

Differential expression of Galalpha1,3Gal epitopes on fetal and adult porcine hematopoietic cells

Galα1-3Gal (Gal) is the major epitope on pig tissues bound by human natural antibodies. Xenogeneic hematopoietic cell transplantation is being investigated to induce immunological tolerance to xenografts. We have investigated the level of Gal expression on pig hematopoietic cells. Cells were collected from pig fetal liver and bone marrow (BM), and also from adult BM and peripheral blood, before and after treatment with pig-specific hematopoietic growth factors. Fluorescent activated cell sorting (FACS) analysis was performed with the M86 monoclonal antibody (specific for Gal), lineage markers, and biotinylated stem cell factor (SCF) to detect c-kit expression. In fetal pig BM and liver, there was no significant difference in Gal expression between monocytes/macrophages (myeloid cells) and lymphocytes. In adult hematopoietic cells from all sources, Gal-positive subpopulations in T cells showed weak expression of Gal, whereas B cells demonstrated higher expression, and myeloid cells showed highest expression. Adult BM and mobilized peripheral blood progenitor cells contained small populations with very low or negligible expression of Gal. A very small population of c-kit-positive cells, indicating progenitor cells, were Gal-negative. The small Gal-negative population that exists in progenitor cells might explain why some pig colony forming units (CFU) can be resistant to human serum.     

由脐血单个核细胞和富集的CD34+细胞扩增的造血干/祖细胞的生理功能比较

目的 探讨由脐血单个核细胞(MNC)和富集的CD34+细胞起始扩增所得的造血干/祖细胞在体内植入及造血重建的能力.方法 从人脐血中分离出MNC和CD34+细胞,在体外扩增7 d.将非肥胖糖尿病型重症联合免疫缺陷型(NOD/SCID)小鼠分为四组,在接受亚致死剂量铯源照射后,进行细胞移植,实验A组接受由MNC培养得到的CD34+细胞和CD34-细胞;实验B组接受由富集的CD34+细胞培养得到的CD34+细胞和CD34-细胞;阳性对照组接受从脐血新鲜分离的CD34+细胞和CD34-细胞;阴性对照组不接受细胞移植,仅输注相同体积的IMDM培养基.6周后处死存活的小鼠,取其骨髓、脾脏和外周血细胞,分别进行细胞表型分析、集落和人特异性基因的检测.结果 经过体外扩增,以富集的CD34+细胞起始培养者的细胞总扩增倍数为39.8倍,远高于以MNC为起始细胞者的1.88倍.移植6周后,所有接受细胞移植的小鼠均存活,存活小鼠的骨髓和脾脏细胞中均能检测到人源细胞(CD45+细胞)及人源的各系血细胞,实验A组各类细胞的含量稍高于实验B组,且接受细胞移植小鼠的骨髓和脾脏细胞中可检测出人特异的Alu序列.结论 与从脐血中新鲜分离的细胞相比,扩增后的造血干/祖细胞的体内植入能力有所下降,以MNC起始扩增的造血干/祖细胞体内植入能力优于以富集的CD34+细胞起始扩增者,但二者体内造血重建能力的差异不显著.     

Xenoreactivity and engraftment of human mesenchymal stem cells transplanted into infarcted rat myocardium

OBJECTIVE: It is thought that adult human mesenchymal stem cells do not induce immunoreactivity even to xenografts. We wanted to study whether adult human mesenchymal stem cells survive and engraft in experimentally induced ischemic rat myocardium. METHODS: Bone marrow-derived adult human mesenchymal stem cells (2.5 x 10(6)) were injected into the myocardium of 4 Sprague-Dawley rats. One week after injection, peripheral blood rat lymphocytes were added to adult human mesenchymal stem cells in a mixed lymphocyte reaction. Furthermore, an infarction was created by left anterior descending artery ligation of 8 Sprague-Dawley rats, 4 of which were immunosuppressed with tacrolimus (0.1 mg/kg/d) and 4 RNU athymic rats. One week after left anterior descending artery ligation, 2.5 to 3.5 x 10(6) adult human mesenchymal stem cells were injected around the infarcted area. The adult human mesenchymal stem cells were identified with fluorescence in situ hybridization technique and myocardial antigens by immunohistochemistry. The immune response was studied by hematoxylin and eosin staining and by antibodies directed toward macrophages. RESULTS: Significant rat lymphocyte proliferation was observed when adult human mesenchymal stem cells were added to peripheral blood from Sprague-Dawley rats previously exposed to adult human mesenchymal stem cells. No reactivity was seen in lymphocytes from untreated Sprague-Dawley rats and athymic rats. Adult human mesenchymal stem cells could only be identified in the myocardium of athymic rats. Further, in normal Sprague-Dawley rats, there was a significant myocardial infiltration of round cells, mostly macrophages, in the area of injection of adult human mesenchymal stem cells. In RNU rats, this reaction was less intense. CONCLUSION: Adult human mesenchymal stem cells did not induce xenoreactivity in vitro in previously unexposed immunocompetent Sprague-Dawley rats. However, although mesenchymal stem cells are transplantable across allogeneic barriers, transplant rejection can occur in a xenogenic model. When transplanted into an immunoincompetent host, adult human mesenchymal stem cells showed persistent engraftment.     

Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and osteogenesis in rabbit femoral head osteonecrosis

PurposeOsteonecrosis of the femoral head may be a disease resulting from abnormal proliferation or differentiation of mesenchymal stem cells. The present investigation explored the novel strategy of hypoxia-preconditioned BMMSCs to reverse the impairment of osteonecrosis BMMSCs and enhance the therapeutic potential of hypoxia-treated BMMSC transplantation.MethodsBMMSCs from the anterior superior iliac spine region of osteonecrosis rabbit were cultured under 20% O₂ or 2% O₂ conditions. Normal BMMSCs were cultured under 20% O₂ condition as control. Growth factors secreted were examined by enzyme-linked immunosorbent assay. 20% O₂ or 2% O₂ BMMSCs were injected into the femoral head of rabbits after core decompression. Cell viability and apoptosis were assessed in vitro, and TUNEL staining of the femoral head was analyzed after transplantation. Angiogenesis (capillary-like structure formation, CD31 immunohistochemical staining and ink infusion angiography) and osteogenesis (Alizarin red-S staining, micro-CT scanning and OCN immunohistochemical staining) tests were conducted as well.Results2% O₂ exposure up-regulated growth factor secretion in BMMSCs. Apoptosis in 2% O₂ group was lower when compared with that in 20% O₂ osteonecrosis group. Cell viability in 2% O₂ was significantly higher when compared with that in 20% O₂ osteonecrosis group. Growth factor secretion, cell viability, apoptosis, capillary-like structure formation, Alizarin red-S staining, and ALP staining showed no difference between the 2% O₂ BMMSC and normal BMMSC groups. Transplantation of 2% O₂ versus 20% O₂ mesenchymal stem cells after core decompression resulted in an increase in angiogenesis function and a decrease in local tissue apoptosis. Our study also found that osteogenesis function was improved after hypoxic stem cell transplantation.ConclusionHypoxic preconditioning of BMMSCs is an effective means of reversing the impairment of osteonecrosis BMMSCs, promoting their regenerative capability and therapeutic potential for the treatment of osteonecrosis.     

Human cordonal stem cell intraperitoneal injection can represent a rescue therapy after an acute hepatic damage in immunocompetent rats

BACKGROUND AND AIM: Tissue homeostasis and turnover require reserve stem proliferating cells. Several studies performed on immunodeficient animals have suggested a degree of plasticity by the hematopoietic stem cell compartment that may represent source for liver regeneration. We sought to explore the hepatic differentiation potential of hematopoietic stem cells from human cord blood, after toxic liver damage induced by allyl-alcohol in immunocompetent rats. MATERIALS AND METHODS: Wistar rats were divided into groups (A) allyl-alcohol intraperitoneal injection with hematopoietic stem cell intraperitoneal infusion at 1 day and sacrifice 3 days later; (B) stem cell injection and sacrifice 3 days later; (C) allyl-alcohol infusion and sacrifice 4 days later; and (D) sacrifice without any treatment. Livers, spleens, and bone marrows were analysed for human stem cells using flow-cytometry; livers were also tested by histology and immunohistochemistry to study the pattern of hepatic regeneration after damage and human stem cell conversion into hepatocyte-like cells, respectively. RESULTS: Flow-cytometry revealed selective recruitment of human hematopoietic stem cells by damaged livers (group A) compared with control group B. In addition, liver damage was reduced in animals treated with stem cells. Immunohistochemistry demonstrated that human stem cells could convert hepatic cells. CONCLUSIONS: Our study demonstrated that hematopoietic stem cells selectively recruited by injured livers can contribute to hepatic regeneration after acute toxic damage in immunocompetent recipients.