Mesenchymal precursor cells in the blood of normal individuals

INTRODUCTION: Adult human bone marrow contains a minority population of MSCs that contribute to the regeneration of tissues such as bone, cartilage, muscle, ligaments, tendons, fat, and stroma. Evidence that these MSCs are pluripotent, rather than being a mixture of committed progenitor cells each with a restricted potential, includes their rapid proliferation in culture, a characteristic morphology, the presence of typical marker proteins, and their consistent differentiation into various mesenchymal lineages. These attributes are maintained through multiple passages and are identifiable in individual stem cells. AIMS: Since stem cells are present in both the bone marrow and other tissues, we thought it possible that cells with a similar appearance and pluripotent mesenchymal potential would be present in the blood. We applied techniques used successfully with marrow MSCs to identify similar cells in elutriation fractions of normal human blood. METHODS: BMPCs were elutriated by diluting the buffy coats from 500 ml of anticoagulant-treated, platelet-depleted blood 1:4 in RPMI-1640 medium (RPMI) and layering 25-ml portions over 20 ml of Lymphoprep(™). These samples were centrifuged at 2000 rpm for 20 min. The leukocyte-rich interface cells were collected, made up to 20 ml in RPMI, and separated by density-gradient centrifugation. The interface cells, now depleted of red blood cells, were collected, resuspended in 50 ml of sterile RMPI and 5% heat-inactivated FCS, and introduced into the sample line of the flow system of a Beckman JE-50 cell elutriator charged with elutriation buffer. The chamber was centrifuged at 25 000 rpm at 10°C and the flow rate adjusted to 12 ml/min. After about 150 ml had been collected, the flow rate was increased by 1 ml/min. Fractions nos. 1-6 (flow rates of 12-16 ml/min) contained most of the lymphocytes. Monocytes usually appeared in fractions 6 or 7 (as determined by flow cytometric analysis in a fluorescence-activated cell sorter (FACS). BMPCs were concentrated in fractions 7 and 8, along with monocytes and lymphocytes. Elutriation fractions with more than 50% and less than 75% monocytes were collected and concentrated by centrifugation at 1200 rpm for 5 min, and the cell pellets were combined, reconstituted in DMEM plus 20% sterile heat-inactivated FCS, counted, washed in medium, repelleted, and then resuspended in DMEM to 5 × 10(6)/ml and dispensed into either tissue-culture plastic slides or glass chamber slides. Cells thus obtained were observed in time-lapse cinematography, assayed for proliferation, and examined immunohistologically and histochemically, and their ability to become fibroblasts, osteoclasts, osteoblasts, and adipocytes was documented. RESULTS: BMPCs were found in elutriation fractions containing less than 30% T cells and more than 60% monocytes from the blood of more than 100 normal persons. BMPCs adhered to plastic and glass and proliferated logarithmically in DMEM-20% FCS without added growth factors. The initial elutriate had only small, round, mononuclear cells; upon culture, these were replaced by fibroblast-like cells and large, round, stromal cells. The formation of cells with fibroblast-like and stromal morphology was not affected by eliminating CD34, CD3, or CD14 cells from the elutriation fraction. Osteogenic supplements (dexamethasone, ascorbic acid, and β-glycero-phosphate) added to the culture inhibited fibroblast formation, and BMPCs assumed the cuboidal shape of osteoblasts. After 5 days in supplemented medium, the elutriated cells displayed AP and its production was doubled by the addition of BMP2 (1 ng) (P < 0.04). Two weeks later, 30% of the cells were very large and reacted with anti-osteocalcin antibody. The same cultures contained two other types of cell: sudanophlic adipocytes and multinucleated giant cells, which stain for TRAP and vitronectin receptors (attributes of osteoclasts). Cultured BMPCs were immunostained by antibodies to vimentin, type I collagen, and BMP receptors (heterodimeric structures expressed on mesenchymal lineage cells). The cultured cells also stained strongly for the SH-2 (endoglin) antigen, a putative marker for marrow MSCs. BMPCs express the gene for SDF-1, a potent stroma-derived CXCα chemokine. DISCUSSION: In the circulation of normal individuals is a small population of CD34(-) mononuclear cells that proliferate rapidly in culture as an adherent population with a variable morphology, display cytoskeletal, cytoplasmic, and surface markers of mesenchymal precursors, and differentiate into several lineages (fibroblasts, osteoblasts, and adipocytes). These are all features found in bone-marrow-derived MSCs. Therefore, autologous blood could provide cells useful for tissue engineering and gene therapy. In addition, the demonstration of similar cells in the inflammatory joint fluids and synovium of patients with rheumatoid arthritis (RA) suggests that these cells may play a role in the pathogenesis of RA.

引言：成人骨髓中存在少量间充质干细胞（Mesenchymal Stem Cells, MSCs），这类细胞可参与骨、软骨、肌肉、韧带、肌腱、脂肪及基质等多种组织的再生修复。多项证据表明这类MSCs具有多向分化潜能，而非仅由谱系受限的定向祖细胞混合组成——这些证据包括其在体外培养时的快速增殖能力、典型的细胞形态、标志性蛋白的表达，以及可稳定分化为多种间充质细胞谱系的特性。上述特性可在多次传代培养中保持稳定，且可在单个干细胞中被检测到。

研究目的：鉴于干细胞不仅存在于骨髓，也分布于其他组织中，我们推测血液中可能存在具有相似形态特征及多向间充质分化潜能的同类细胞。本研究将此前成功用于骨髓MSCs分离培养的技术，应用于正常人血液的淘洗分级组分中，以鉴定此类相似细胞。

实验方法：血液间充质祖细胞（Blood Mesenchymal Progenitor Cells, BMPCs）的分离流程如下：将500ml经抗凝处理并去除血小板的血液所分离得到的棕黄层以1:4的比例用RPMI-1640培养基（简称RPMI）稀释，取25ml该稀释液铺于20ml Lymphoprep(™)分层液上层。将上述样本以2000转/分钟离心20分钟，收集富含白细胞的界面层细胞，用RPMI培养基定容至20ml后，再次进行密度梯度离心分离。收集去除红细胞后的界面层细胞，重悬于50ml无菌RPMI培养基及5%热灭活胎牛血清（Fetal Calf Serum, FCS）中，随后注入装有淘洗缓冲液的贝克曼JE-50细胞淘洗仪的流式进样管路。将淘洗舱以25000转/分钟、10℃条件下离心，并将流速调整为12ml/min。收集约150ml洗脱液后，将流速提升1ml/min。第1-6号洗脱组分（流速12-16ml/min）中包含大部分淋巴细胞。单核细胞通常出现在第6或7号组分中（该结果通过荧光激活细胞分选仪（Fluorescence-Activated Cell Sorter, FACS）的流式细胞分析得以确认）。BMPCs与单核细胞、淋巴细胞共同富集于第7、8号洗脱组分中。收集单核细胞占比50%-75%的淘洗组分，以1200转/分钟离心5分钟进行浓缩，合并细胞沉淀后，用含20%无菌热灭活FCS的DMEM培养基重悬，计数后用培养基洗涤、再次离心沉淀，最终用DMEM培养基重悬至浓度为5×10^6个/ml，随后接种至组织培养用塑料玻片或玻璃腔室玻片上。对获取的细胞进行延时摄影观测、增殖能力检测，以及免疫组织化学和组织化学分析，并记录其向成纤维细胞、破骨细胞、成骨细胞及脂肪细胞的分化潜能。

实验结果：在100余名健康志愿者的血液样本中，BMPCs均存在于T细胞占比<30%、单核细胞占比>60%的淘洗组分中。BMPCs可黏附于塑料及玻璃表面，在添加20% FCS的DMEM培养基中无需额外添加生长因子即可呈对数增殖。初始淘洗得到的细胞仅为小型圆形单核细胞；体外培养后，此类细胞逐渐被成纤维样细胞及大型圆形基质细胞取代。从淘洗组分中去除CD34+、CD3+或CD14+细胞后，并不会影响成纤维样及基质样细胞的形成。向培养基中添加成骨诱导添加剂（地塞米松、抗坏血酸及β-甘油磷酸钠）后，成纤维细胞的形成受到抑制，BMPCs会转变为成骨细胞特有的立方形形态。在添加诱导剂的培养基中培养5天后，洗脱得到的细胞可表达碱性磷酸酶（Alkaline Phosphatase, AP）；加入1ng骨形态发生蛋白2（Bone Morphogenetic Protein 2, BMP2）后，AP的表达量提升了一倍（P<0.04）。培养两周后，30%的细胞体积显著增大，且可与抗骨钙素抗体发生特异性结合反应。同一培养体系中还存在另外两类细胞：苏丹染色阳性的脂肪细胞，以及可被抗酒石酸酸性磷酸酶（Tartrate-Resistant Acid Phosphatase, TRAP）和玻连蛋白受体抗体染色的多核巨细胞（此类细胞符合破骨细胞的特征）。体外培养的BMPCs可被波形蛋白、I型胶原及骨形态发生蛋白受体（在间充质细胞谱系中表达的异二聚体结构）的抗体特异性染色。培养后的细胞还可强表达SH-2（内皮糖蛋白，endoglin）抗原，该抗原是骨髓MSCs的公认标志物之一。BMPCs可表达基质细胞衍生因子1（Stromal Cell-Derived Factor 1, SDF-1）的编码基因，该因子是一种强效的基质源性CXCα趋化因子。

讨论：健康个体的循环血液中存在少量CD34阴性的单核细胞，此类细胞在体外培养时可黏附于培养表面，形态具有一定异质性且增殖迅速，同时表达间充质前体细胞特有的细胞骨架、胞质及表面标志物，并可分化为多种细胞谱系（成纤维细胞、成骨细胞及脂肪细胞）。上述特征均与骨髓来源的MSCs一致。因此，自体血液可作为组织工程及基因治疗的细胞来源。此外，在类风湿关节炎（Rheumatoid Arthritis, RA）患者的炎性关节液及滑膜组织中也已检测到此类相似细胞，提示这类细胞可能参与RA的发病过程。