Assessment of DNA Damage in Human Bone Marrow Cells and Multipotent Mesenchymal Stromal Cells

We carried out a comparative analysis of DNA damage (percentage of DNA in comet tail) and frequencies of comets in apoptotic cells in BM samples and cultures of BM multipotent mesenchymal stromal cells at different terms of culturing (passages 3–11). The levels of DNA damage in mesenchymal stromal cells remained unchanged during culturing (3.5 ± 0.9 and 4.4 ± 1.2%) and did not differ from those in BM cells (3.6 ± 0.8%). In BM samples, 10-28% atypical cells with high level of DNA damage were detected. In mesenchymal stromal cells, 2.8 ± 0.9 and 3.6 ± 1.8% apoptotic cells were detected at early and late passages, respectively.     

Characteristics of Mesenchymal Stromal Precursor Cells Labeled with Lentiviral Vector in Long-Term Bone Marrow Culture

Mouse mesenchymal stromal precursor cells were labeled with lentiviral vector in long-term bone marrow culture. We studied the fate of labeled cells in the stromal sublayer of the longterm bone marrow culture and in ectopic hemopoiesis foci formed from the labeled cultures. The incidence of labeled polypotent fibroblast CFU in sublayers of long-term bone marrow culture and in ectopic hemopoiesis foci formed from these sublayers under the renal capsule of syngeneic mice was also analyzed. It was shown that the marker gene was present in about 40% cells of the stromal sublayer and 30% fibroblast CFU and that effective gene transfer did not affect the total production of hemopoietic cells. The size of ectopic hemopoietic foci formed after implantation of labeled sublayers of the long-term bone marrow culture under the renal capsule did not differ from the control. Differentiated cells of the osseous shell in these foci carried the marker gene in 40% cases. Analysis of fibroblast CFU in these foci showed that despite the total concentration of fibroblast CFU was comparable to that in the bone marrow, the concentration of labeled fibroblast CFU was about 6%, which suggests that one more class of precursors probably exists in the hierarchy of stromal cells presumably between mesenchymal stem cells and fibroblast CFU. Our findings demonstrate the capacities of mesenchymal stem cells to self-maintenance and differentiation without loosing the marker gene integrated into the genome.     

Resistance of Rat Bone Marrow Mesenchymal Stromal Precursor Cells to Anoxia In Vitro

We studied the effect of significantly reduced oxygen content (0% O₂ in gaseous phase) in culture medium on rat bone marrow mesenchymal stromal precursor cells. The cells retained their morphology and expression of characteristic markers during several days under these conditions; they were viable, proliferated, and some cultured cells were even capable of realizing the initial stages of osteogenic and adipogenic differentiation. Further culturing of precursor cells under conditions of anoxia led to activation of apoptosis in cultures and to progressive necrosis.     

In Vivo Effects of Human Bone Marrow Mesenchymal Stromal Cells on the Development of Experimental B16 Melanoma in Mice

Bulletin of Experimental Biology and Medicine - Experiments on F1(CBA×C57BL/6) mice with experimental metastatic melanoma B16 F10 showed that single intravenous injection of xenogeneic bone...     

Co‐culture of Spleen Stromal Cells with Bone Marrow Mononuclear Cells Leads to the Generation of A Novel Macrophage Subset

Macrophages adopt diverse activation states depending on the microenvironment. Recently, stromal cells have been demonstrated to be organizers of the microenvironment. Here, using splenic stromal cells to mimic the splenic microenvironment in vivo, we show that spleen stromal cells can programme bone marrow‐derived mononuclear cells to differentiate and polarize into a novel macrophage subset. These differentiated macrophages (Diff‐Mφ) exhibited pronounced production of IL‐10, IL‐6 and TNF‐α, but diminished the production of IL‐12 in response to LPS. The generation of Diff‐Mφ depended on cell–cell contact as well as on soluble factors. Diff‐Mφ directly suppressed the antigen‐non‐specific (CD3/CD28) CD4⁺ T cell proliferative response and induced cell death of activated CD4⁺ T cells. As for cytokine production in CD4⁺ T cells, Diff‐Mφ promoted IL‐10 and IL‐17 production, whereas inhibited IL‐4 production and did not alter IFN‐γ production. Besides, Diff‐Mφ also expressed iNOS, CD16/CD32, CD54, CD43, CCR7, CD44, PD‐L1 and FasL, which might be involved in the function of Diff‐Mφ. These results suggest that splenic microenvironment may physiologically induce a novel type of macrophages differentiation.     

Treatment Efficacy with Bone Marrow Derived Mesenchymal Stem Cells and Minocycline in Rats After Cerebral Ischemic Injury

Objective

We aimed to investigate the effects of bone marrow derived mesenchymal stem cells (MSCs), minocycline, and these two therapies combined on functional and histological improvement in cerebral ischemic injury created rats.

Materials and methods

Twenty-eight Sprague Dawley female rats, weighing 250–300 g, were included in the study. Two male rats with similar properties were sacrificed for bone marrow derived MSC production. Group 1 was established as the control group. Group 2 was the group of only minocycline administered rats. Group 3 was the one of only MSCs administered rats. Group 4 was composed of the rats given the combination of MSCs and minocycline. Hematoxylin and eosin staining was done to assess the degeneration of the cells. Immunohistochemical staining was performed to evaluate the regeneration. Motor functions were examined by using Bederson’s score.

Results

Cell degeneration was the least in group 4. The cells stained with GFAP were observed mostly in group 4. The cells stained with Neu N in group 1 were statistically lower than in other groups. When the groups were ordered in terms of functional improvement at the end of the second week, group 4 had the most and group 1 had the least.

Conclusions

Bone marrow derived MSCs can lead to more histological and functional improvement when administered with minocycline, which is a neuroprotective agent as early as 24 h following the ischemic injury in a rat model. Minocycline therapy alone can be as effective as bone marrow derived MSCs therapy alone in ischemic cerebral rat model.     

PKH26标记大鼠骨髓间质干细胞的实验研究

目的建立PKH26标记大鼠骨髓间质干细胞（MSCs）的方法，并探讨标记MSCs的基本生物学活性。方法大鼠MSCs按PKH26标记程序进行标记后培养，采用激光共聚焦显微镜、流式细胞分析仪等观察细胞生长状态和荧光标记活性变化；应用RT-PCR检测标记后MSCs的GAPDH、nucleostemin及Bmi-1基因的表达；选用碱性磷酸酶染色、Von Kossa染色及骨形成蛋白3（BMP3）基因表达分析等技术，观察标记后MSCs体外分化成骨细胞的特性。结果PKH26标记后细胞呈红色荧光，荧光的强度随着PKH26浓度的增加而递增，并与传代时间和代数相关；标记后细胞生长状态良好，基本生长特性如传代培养和生长曲线无明显改变；细胞GAPDH、nucleostemin及Bmi-1基因表达未见改变；标记MSCs诱导后ALP、Von Kossa染色阳性，呈现典型的成骨细胞形态和生物学特征，并表达BMP3基因。结论PKH26可稳定标记大鼠骨髓MSCs并能传代培养，标记后细胞形态、生长活力及多向分化潜能等无明显影响，该技术可用于追踪MSCs转归、可塑性及干细胞移植方面的实验研究。     

骨髓间质干细胞体外分化为成骨细胞的实验研究

建立猪骨髓间质干细胞 (mesenchymalstemcells ,MSCs)体外分离培养方法。对猪MSCs体外分化为成骨细胞的能力进行研究。抽取猪骨髓 ,体外培养MSCs。取第二代MSCs ,以含有不同浓度的抗坏血酸、β -磷酸甘油、地塞米松及碱性成纤维细胞生长因子等条件培养基进行成骨细胞诱导分化。通过细胞形态变化 ,碱性磷酸酶染色及钙盐沉积对成骨细胞进行鉴定。结果表明MSC细胞形态由长梭形向多边形转变 ,ALP染色阳性 ,VonKossa染色阳性 ,经体外诱导分化后呈典型的成骨细胞样改变。猪骨髓MSCs可在体外长期、稳定培养 ,具有向成骨细胞分化的潜能 ,可以为骨组织工程研究提供较理想的细胞来源和动物模型。     

骨髓间充质干细胞自体移植促进缺血肢体血管生成的实验研究

目的探索骨髓间充质干细胞在血管生成中的作用,为临床建立一种简便安全的下肢动脉缺血性疾病的治疗途径。方法24只新西兰兔分为实验组(12只)和对照组(12只),分离培养实验组兔骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),Brdu标记,建立兔后肢缺血动物模型,将取于自体的骨髓间充质干细胞制成悬液注射于实验组兔缺血部位,对照组注射等量生理盐水。2周后行实验组和对照组兔股动脉的二维及彩色多普勒超声检测,观察移植前后的血管内径,血流峰值速度,血流加速时间等;取缺血部位肌肉组织,免疫荧光染色观察移植细胞分布及血管生成情况。结果细胞移植后2周,实验组的股动脉内径,血流峰值速度,加速时间均大于对照组,有显著性差异(P<0.01);免疫荧光染色结果显示移植部位有移植细胞存在,移植组血管生成情况明显优于未移植组。结论骨髓间充质干细胞具有促进血管生成的作用,自体移植可望成为一种简单的、有效的治疗下肢缺血的方法。     

鼠骨髓基质干细胞的特性及向成骨细胞的分化

目的分离纯化成年小鼠骨髓基质干细胞并进行定向诱导。方法利用Percoll液分离成年小鼠骨髓基质干细胞,Ter119、CD45磁珠纯化细胞,培养2-3代的细胞用矿化液进行定向诱导。对诱导前后的细胞进行免疫组化染色。结果 镜下见原代细胞呈多种形态。Ter119、CD45磁珠筛选可获得纯度达57.95％以上骨髓基质干细胞。碱性磷酸酶、Von Kossa染色阳性,油红染色弱阳性,矿化液诱导2周后,可形成钙结节。结论利用Percoll液结合磁珠分离法是离体条件下获得骨髓基质干细胞的一个简便有效的方法,纯化后定向诱导可得到较纯的成骨细胞。     

Adipogenic Mesenchymal Stromal Cells from Bone Marrow and Their Hematopoietic Supportive Role: Towards Understanding the Permissive Marrow Microenvironment in Acute Myeloid Leukemia

Purpose

The role of bone marrow-derived mesenchymal stem/stromal cells (MSCs) in creating a permissive microenvironment that supports the emergence and progression of acute myeloid leukemia (AML) is not well established. We investigated the extent to which adipogenic differentiation in normal MSCs alters hematopoietic supportive capacity and we undertook an in-depth comparative study of human bone marrow MSCs derived from newly diagnosed AML patients and healthy donors, including an assessment of adipogenic differentiation capacity.

Findings

MSCs from healthy controls with partial induction of adipogenic differentiation, in comparison to MSCs undergoing partial osteogenic differentiation, expressed increased levels of hematopoietic factors and induced greater proliferation, decreased quiescence and reduced in vitro hematopoietic colony forming capacity of CD34⁺ hematopoietic stem and progenitor cells (HSPCs). Moreover, we observed that AML-derived MSCs had markedly increased adipogenic potential and delayed osteogenic differentiation, while maintaining normal morphology and viability. AML-derived MSCs, however, possessed reduced proliferative capacity and decreased frequency of subendothelial quiescent MSCs compared to controls.

Conclusion

Our results support the notion of a bone marrow microenvironment characterized by increased propensity toward adipogenesis in AML, which may negatively impact normal hematopoiesis. Larger confirmatory studies are needed to understand the impact of various clinical factors. Novel leukemia treatments aimed at normalizing bone marrow niches may enhance the competitive advantage of normal hematopoietic progenitors over leukemia cells.

     

Bone Marrow Multipotent Mesenchymal Stromal Cells from Patients with Aplastic Anemia Retain Their Ability to Support Hematopoietic Precursors despite Pronounced Changes in Gene Expression

Bulletin of Experimental Biology and Medicine - The properties of bone marrow-derived multipotent mesenchymal stromal cells (MSC) of patients with aplastic anemia at the onset of the disease are...     

人骨髓间充质干细胞结合多孔β-TCP构建生物相容性人工骨

研究人骨髓间充质干细胞(HBMSCs)结合多孔β磷酸三钙(β-TCP)的生物相容性与体内成骨作用. 密度梯度离心结合差异贴壁法分离HBMSCs,常规扩增传代,相差显微镜形态学观察和流式细胞仪检测细胞表面标记CD13、CD29、HLA-2、CD34、CD45和HLA-DR;分别在成骨、成软骨和成脂肪细胞培养基中定向诱导分化,验证其多向分化能力.将HBMSCs在低压下载入多孔β-TCP立方块,形成MSCs/β-TCP复合物,电镜观察材料内部与细胞结合情况.继续成骨诱导培养2周后植入裸鼠背部皮下,于植入4周和8周后取出复合物做组织学检查.设非成骨诱导培养复合物为对照. 原代和传代细胞呈梭形外观,生长增殖能力良好;流式细胞仪检测间充质细胞来源表面标记物CD13、CD29,HLA-2阳性,造血细胞来源表面标记物CD34、CD45 和HLA-DR阴性;能成功高效定向分化为成骨细胞、软骨细胞、脂肪细胞;细胞在β-TCP材料表面贴附、增殖良好;MSCs/β-TCP复合物植入皮下4周后即有少量新骨生成,至8周时更明显;对照组新骨生成量较少.本方法快速、高效分离扩增HBMSCs;HRMSCs与多孔可降解β-TCP材料生物相容性良好;二者结合能显著提高体内新骨生成,提示其可用于临床作为骨移植替代物,可提高骨移植修复骨缺损的疗效.     

Treatment of Graft versus Host Disease with Mesenchymal Stromal Cells: A Phase I Study on 40 Adult and Pediatric Patients

This phase I multicenter study was aimed at assessing the feasibility and safety of intravenous administration of third party bone marrow–derived mesenchymal stromal cells (MSC) expanded in platelet lysate in 40 patients (15 children and 25 adults), experiencing steroid-resistant grade II to IV graft-versus-host disease (GVHD). Patients received a median of 3 MSC infusions after having failed conventional immunosuppressive therapy. A median cell dose of 1.5 × 10⁶/kg per infusion was administered. No acute toxicity was reported. Overall, 86 adverse events and serious adverse events were reported in the study, most of which (72.1%) were of infectious nature. Overall response rate, measured at 28 days after the last MSC injection, was 67.5%, with 27.5% complete response. The latter was significantly more frequent in patients exhibiting grade II GVHD as compared with higher grades (61.5% versus 11.1%, P = .002) and was borderline significant in children as compared with adults (46.7 versus 16.0%, P = .065). Overall survival at 1 and 2 years from the first MSC administration was 50.0% and 38.6%, with a median survival time of 1.1 years. In conclusion, MSC can be safely administered on top of conventional immunosuppression for steroid resistant GVHD treatment. Eudract Number 2008-007869-23, NCT01764100.     

Third‐party Tolerogenic Dendritic Cells Reduce Allo‐reactivity In vitro and Ameliorate the Severity of Acute graft‐Versus‐host Disease in Allo‐bone Marrow Transplantation

Tolerogenic dendritic cells (tDCs) potently induce and maintain tolerance based on their distinct characteristics compared with conventional DCs. Recent reports show that donor or host tDCs promote allograft survival in mice. In this study, the efficacy of third‐party tDCs in the prevention of acute graft‐versus‐host disease (aGVHD) was evaluated. In vitro, tDCs derived from the bone marrow (BM) of D1 mice were induced by GM‐CSF, IL‐10 and TGF‐β1. The phenotypes, expression of cytokines and suppression of tDCs were analysed. In vivo, the effects of adoptive transfer of third‐party‐tDCs were evaluated in an MHC‐mismatched aGVHD mouse model. Survival, body weight, GVHD scoring, histopathological specimens and serum cytokines were analysed in tDC‐treated mice and untreated controls. Tolerogenic DCs had low expression of MHC and co‐stimulatory molecules, expressed high levels of ‘immunosuppressive’ cytokines and suppressed allo‐CD4⁺T cell proliferation. In the B6→D2 mouse model, all aGVHD mice died within 18 days. Fortunately, third‐party tDCs transferred at low doses (10⁴) effectively prolonged survival after allo‐BMT. Furthermore, in the mice treated with 10⁴ tDCs, serum levels of IL‐10/TGF‐β were significantly higher and the percentage of Foxp3⁺ cells continually increased compared with the mice treated with other doses of tDCs. Third‐party tDCs play a crucial role in reducing the severity of aGVHD by modulating the secretion of various cytokines and expanding Foxp3⁺ regulatory T cells, which suggests the possibility of using third‐party tDCs for therapeutic applications. Furthermore, special attention should be paid to the optimal range of tDCs for preventing allograft rejection.     

Long-Term Donor Chimerism after MHC (RT1) Mismatched Bone Marrow Transplantation in the Rat: the Role of Host Alloreactive NK Cells

We have investigated the role of major histocompatibility complex (MHC) (RT1) disparities in the engraftment of bone marrow (BM) cells after whole body irradiation of rats. Mononuclear BM cells from PVG.RT7.2 (RT1c) rats were injected i.v. into sublethally (10Gy) whole body irradiated PVG (RT1c) rats and RT1 congenic and recombinant PVG rats. Repopulation of the BM, spleen, and blood with donor cells was assessed by FACS analysis of cells labelled with the fluorescein isothiocyanate (FITC)-labelled HIS41 monoclonal antibody (MoAb) against the RT7.2 marker. In RT1 matched (PVG.RT7.2 --> PVG) and RT1-mismatched combinations (PVG.RT7.2 --> PVG.1AV1), where radioresistant host natural killer (NK) cells could not recognize the BM inoculum as foreign, a donor chimerism close to 100% was observed after 6-8 weeks. However, in rat strain combinations where host NK cells could recognize an RT1 mismatch, almost no donor cells survived, and the rats were repopulated with leukocytes of host origin. In intra-MHC recombinant rat strains the element determining rejection or acceptance of the allograft mapped to the RT1-B/D-C/E/M region in PVG.R8 and PVG.R23 rats, in accordance with the patterns of NK alloreactivity in these strain combinations. NK cells may therefore be a primary obstacle to successful allogeneic BM engraftment in this model.