小鼠骨髓间充质干细胞生物学特性和体外诱导分化

目的研究小鼠骨髓间充质干细胞的生物学性状和多系分化潜能。方法取Balb／c小鼠骨髓单个核细胞在低糖的培养液中培养出贴壁生长的细胞,进行形态学观察、细胞周期和免疫表型分析;在不同的因子作用下诱导向成骨细胞、软骨细胞,脂肪细胞分化,并检测诱导后细胞相应的基因表达。结果小鼠骨髓间充质干细胞贴壁生长后形态较均一,增殖能力随着传代逐渐增强,但从第8代后增殖能力明显减退。细胞表达CD29,CD38,CD44,CD106等标记,但CD34和H-2k表达阴性。在不同的诱导培养体系里间充质干细胞能分化为成骨细胞、软骨细胞和脂肪细胞,相应的骨钙蛋白基因,Ⅱ型胶原基因,脂蛋白脂酶基因表达都明显增强。结论从小鼠骨髓可以分离培养出间充质干细胞,在体外有效扩增和诱导分化。表明可以以小鼠为模型研究间充质干细胞在组织工程、细胞移植、基因治疗等领域的运用。     

Multiple differentiation capacity of STRO-1+/CD146+ PDL mesenchymal progenitor cells

Although mesenchymal progenitor cells can be isolated from periodontal ligament (PDL) tissues using stem cell markers STRO-1 and CD146, the proportion of these cells that have the capacity to differentiate into multiple cell lineages remains to be determined. This study was designed to quantify the proportions of primary human PDL cells that can undergo multilineage differentiation and to compare the magnitude of these capabilities relative to bone marrow-derived mesenchymal stem cells (MSCs) and parental PDL (PPDL) cells. PDL mesenchymal progenitor (PMP) cells were isolated from PPDL cells using the markers STRO-1 and CD146. The colony-forming efficiency and multilineage differentiation potential of PMP, PPDL, and MSCs under chondrogenic, osteogenic, and adipogenic conditions were determined. Flow cytometry revealed that on average 2.6% of PPDL cells were STRO-1(+)/CD146(+), whereas more than 63% were STRO-1(-)/CD146(-). Colony-forming efficiency of STRO-1(+)/CD146(+) PMP cells (19.3%) and MSCs (16.7%) was significantly higher than that of PPDL cells (6.8%). Cartilage-specific genes, early markers of osteoblastic differentiation, and adipogenic markers were significantly upregulated under appropriate conditions in PMP cells and MSCs compared to either their noninduced counterparts or induced PPDL cells. Consistent with these findings, immunohistochemistry revealed substantial accumulation of cartilaginous macromolecules, mineralized calcium nodules, and lipid vacuoles under chondrogenic, osteogenic, or adipogenic conditions in PMP and MSC cultures, respectively, compared to noninduced controls or induced PPDL cells. Thus STRO-1(+)/CD146(+) PMP cells demonstrate multilineage differentiation capacity comparable in magnitude to MSCs and could potentially be utilized for regeneration of the periodontium and other tissues.     

Multilineage cells from human adipose tissue: implications for cell-based therapies

Future cell-based therapies such as tissue engineering will benefit from a source of autologous pluripotent stem cells. For mesodermal tissue engineering, one such source of cells is the bone marrow stroma. The bone marrow compartment contains several cell populations, including mesenchymal stem cells (MSCs) that are capable of differentiating into adipogenic, osteogenic, chondrogenic, and myogenic cells. However, autologous bone marrow procurement has potential limitations. An alternate source of autologous adult stem cells that is obtainable in large quantities, under local anesthesia, with minimal discomfort would be advantageous. In this study, we determined if a population of stem cells could be isolated from human adipose tissue. Human adipose tissue, obtained by suction-assisted lipectomy (i.e., liposuction), was processed to obtain a fibroblast-like population of cells or a processed lipoaspirate (PLA). These PLA cells can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of PLA cells are of mesodermal or mesenchymal origin with low levels of contaminating pericytes, endothelial cells, and smooth muscle cells. Finally, PLA cells differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, the data support the hypothesis that a human lipoaspirate contains multipotent cells and may represent an alternative stem cell source to bone marrow-derived MSCs.     

体外扩增过程中人骨髓间充质干细胞的增殖与分化规律

目的：系统考察体外扩增过程中人骨髓间充质干细胞（MSC）的增殖与分化规律，为MSC任组织修复以及细胞治疗中的应用提供参考、方法：以全骨髓贴壁法分离成人肋骨骨髓MSC，在相同条件下分别考察各代细胞形态、生长、表面标记、细胞周期、成骨、成软骨及成脂肪能力的变化情况。结果：随代次增加，MSC增殖能力、成骨、成脂肪能力均有所下降，而成软骨能力无明显降低；成骨、成软骨及成脂肪能乃均保持到细胞衰老。存扩增过程中，MSC始终保持较高的纯度，CD29、CD44、CD105的阳性率均在90％以上，CD14、CD34和CD45的阳性率均在4％以下、结论：在体外培养过程中MSC干细胞特性逐渐丢失，其中向骨、脂肪方向的分化潜能较软骨方向更易失去；而多向分化能力的保持较之自我更新能力更为持久。MSC在7代以前可作为基础研究及临床应用的良好对象。     

软骨来源成体干细胞的分离及其鉴定

 目的 观察关节软骨中是否存在着软骨干细胞,并对软骨干细胞进行分离、培养和鉴定。方法 将软骨组织消化成单个软骨细胞,接种到预先用纤连蛋白处理过的平皿中,20mins后给予换液,培养2周时用克隆环挑取形成的单个细胞集落,在体外进行扩增。流式细胞仪检测扩增后细胞表面标志物,并对细胞的成脂肪、成骨、成软骨能力进行验证。结果 成功挑选出软骨细胞集落,培养至第3代,细胞呈均一的长梭形。细胞表面表达间充质干细胞相关抗原CD44、CD29、CD73、CD90和CD166,且几乎不表达CD45、CD34和CD133。当给与诱导培养基培养时,软骨干细胞能成功的向脂肪细胞、骨细胞和软骨细胞进行分化。结论 关节软骨中存在着软骨干细胞,并成功的对软骨干细胞进行了分离和鉴定。     

Bone marrow stromal cells as targets for gene therapy

The bone marrow (BM) is composed of the non-adherent hematopoietic and adherent stromal cell compartment. This adherent BM stromal cell fraction contains pluripotent mesenchymal stem cells (MSCs) and differentiated mesenchymal BM stromal cells. The MSCs self-renew by proliferation while maintaining their stem-cell phenotype and give rise to the differentiated stromal cells which belong to the osteogenic, chondrogenic, adipogenic, myogenic and fibroblastic lineages. A more primitive adherent stem cell was recently identified, the multipotent adult progenitor cell (MAPC) or mesodermal progenitor cell, which co-purifies with MSCs. These MAPCs differentiate into MSCs, endothelial, epithelial and even hematopoietic cells. BM stroma cells, including the primitive pluripotent MSCs and MAPCs, are attractive targets for cell and gene therapy. The BM stromal cell population and its multipotent stem cells can be engineered to secrete a series of different proteins in vitro and in vivo that could potentially treat a variety of serum protein deficiencies and other genetic or acquired diseases, including bone, cartilage and BM stromal disorders or even cancer.     

致敏小鼠骨髓间充质干细胞体外培养及多向分化功能的测定

目的：评价异基因脾细胞输注致敏的小鼠骨髓源性间充质干细胞(MSCs)的体外培养生长能力及其多向分化功能。方法：应用贴壁培养法体外培养间充质干细胞,流式检测其表面标志以及检测其成骨、成脂和成肌多向分化状况;结果：致敏小鼠骨髓源性MSCs与非致敏小鼠骨髓源性MSCs比较,形态学无差异且均表达CD29+、CD105+、CD44+和Sca-1+ ;CD34-、CD11b-;同时在相应的诱导条件下具有向成骨、成脂、成肌多向分化的能力。结论：异基因脾细胞输注致敏的小鼠,其骨髓源性MSCs的形态学和功能与正常小鼠的MSCs比较评估未见异常。     

人脐静脉来源的间质干细胞体外分离扩增及多向分化的研究

目的： 探讨人脐静脉来源的间质干细胞(MSCs) 的体外分离、纯化、扩增和多向分化条件。 方法： 无菌条件下取正常人脐静脉,1%胶原酶Ⅱ消化脐静脉细胞，以IMDM作为培养基进行培养和纯化细胞，瑞氏染色和电镜观察形态；FACS检测其免疫表型和细胞周期；体外诱导成骨细胞、脂肪细胞分化，von Kossa染色、油红O染色和RT-PCR检测骨钙蛋白、脂蛋白脂酶mRNA的表达以检测细胞向成骨、成脂肪细胞分化情况。 结果： 脐静脉来源的细胞呈纤维样贴壁生长，瑞氏染色和电镜观察具有MSCs特征；FACS检测结果显示, 表达MSCs相关的抗原CD29、CD44、CD105，而CD31、CD13、CD34、CD45、HLA-DR为阴性；体外诱导成骨细胞、脂肪细胞分化成功。 结论： 人脐静脉来源的MSCs的细胞形态、生长特性、免疫表型、多向分化能力与骨髓来源的MSCs相似，可作为满足实验和临床需要的MSCs来源。     

Isolation and characterization of chorionic mesenchymal stromal cells from human full term placenta

This study focused on the characterization of mesenchymal stromal cells (MSCs) from the chorion of human full term placenta from 15 donors. Chorionic MSCs revealed homologous fibroblast-like morphology and expressed CD73, CD29, CD105, and CD90. The hematopoietic stem cell markers including HLA DR, CD11b, CD34, CD79a, and CD45 were not expressed. The growth kinetics of their serial passage was steady at the later passages (passage 10). The multilineage capability of chorionic MSCs was demonstrated by successful adipogenic, osteogenic and chondrogenic differentiation and associated gene expression. Chorionic MSCs expressed genes associated with undifferentiated cells (NANOG, OCT4, REX1) and cardiogenic or neurogenic markers such as SOX2, FGF4, NES, MAP2, and NF. TERT was negative in all the samples. These findings suggest that chorionic MSCs undifferentiated stem cells and less likely to be transformed into cancer cells. A low HLA DR expression suggests that chorionic MSCs may serve as a great source of stem cells for transplantation because of their immune-privileged status and their immunosuppressive effect. Based on these unique properties, it is concluded that chorionic MSCs are pluripotent stem cells that are probably less differentiated than BM-MSCs, and they have considerable potential for use in cell-based therapies.     

小鼠骨髓来源Flk-1+间充质干细胞分离制备与生物学特点

背景：骨髓间充质干细胞具有广泛的应用前途,其三系分化的能力和免疫诱导特性使其具有再生医学的应用前景。 目的：从小鼠骨髓中研究分离制备Flk-1⁺间充质干细胞的新方法,检测了这类细胞的生物特性。 方法：以小鼠骨髓为研究对象,分离单个核细胞,体外培养并 扩增原始间充质干细胞,检测它们的细胞周期、表型和多系分化的能力。 结果与结论：小鼠来源的骨髓来源的原始间充质干细胞呈成纤维样生长,大部分细胞处于G₀/G₁期,并且高表达Flk1,CD13,CD29,CD44,小鼠骨髓来源Flk-1⁺间充质干细胞在光镜下均呈成纤维样或多角型贴壁生长,在体外相应的诱导液环境中均可以向三系分化。提示小鼠骨髓来源Flk-1⁺间充质干细胞具有多向分化潜能。     

Alkaline phosphatase, glutathione-S-transferase-P, and cofilin-1 distinguish multipotent mesenchymal stromal cell lines derived from the bone marrow versus peripheral blood

Multipotent mesenchymal stromal cells (MSCs) can be isolated from bone marrow or peripheral blood. To identify phenotypical and functional differences between MSCs derived from these sources, the human bone marrow-derived, fibroblast-like cell line L87/4 was compared with the peripheral blood-derived, fibroblast-like cell line V54/2. Both cell lines expressed similar levels of SH3+, CD45(-), CD68(-), CD133(-), and HLA-DR(-). The bone marrow-derived cells expressed higher surface levels of CD105, CD10, and CD117 and preferentially expressed alkaline phosphatase, glutathione S-transferase P, and cofilin-1. The peripheral blood-derived line showed a higher number of CD34+/CD105+ double-positive and side population (SP) cells. The results demonstrate the more multipotent, yet quiescent, stromal phenotype of bone marrow MSCs, whereas MSCs isolated from the circulation display more hematopoietic-lineage characteristics. Importantly, potential marker genes that distinguish the two stages of MSCs are defined.     

Adult Stem Cell Driven Genesis of Human-Shaped Articular Condyle

Uniform design of synovial articulations across mammalian species is challenged by their common susceptibility to joint degeneration. The present study was designed to investigate the possibility of creating human-shaped articular condyles by rat bone marrow-derived mesenchymal stem cells (MSCs) encapsulated in a biocompatible poly(ethylene glycol)-based hydrogel. Rat MSCs were harvested, expanded in culture, and treated with either chondrogenic or osteogenic supplements. Rat MSC-derived chondrogenic and osteogenic cells were loaded in hydrogel suspensions in two stratified and yet integrated hydrogel layers that were sequentially photopolymerized in a human condylar mold. Harvested articular condyles from 4-week in vivo implantation demonstrated stratified layers of chondrogenesis and osteogenesis. Parallel in vitro experiments using goat and rat MSCs corroborated in vivo data by demonstrating the expression of chondrogenic and osteogenic markers by biochemical and mRNA analyses. Ex vivo incubated goat MSC-derived chondral constructs contained cartilage-related glycosaminoglycans and collagen. By contrast, goat MSC-derived osteogenic constructs expressed alkaline phosphatase and osteonectin genes, and showed escalating calcium content over time. Rat MSC-derived osteogenic constructs were stiffer than rat MSC-derived chondrogenic constructs upon nanoindentation with atomic force microscopy. These findings may serve as a primitive proof of concept for ultimate tissue-engineered replacement of degenerated articular condyles via a single population of adult mesenchymal stem cells.     

Adipose tissue-derived stem cells for cell therapy of airway allergic diseases in mouse

The purpose of this study was to compare murine mesenchymal stem cells (MSCs) isolated from bone marrow (BM) and adipose tissue (AT) for the selection of suitable MSCs in cell therapy of an airway allergic animal model. We compared MSCs of BALB/c mice derived from BM and AT with respect to proliferation potential, immunophenotype, and multilineage differentiation capacity. In proliferation potential, MSCs from AT (ASCs) showed higher fibroblastoid colony-forming units frequencies and colony-forming efficiency than MSCs from BM (BMSCs). The flow cytometry analysis showed that both ASCs and BMSCs expressed MSCs-related antigens (CD90 and CD105), whereas they did not express hematopoiesis-related antigens (CD45 and CD11b). There was no significant difference in adipogenic, osteogenic, and chondrogenic differentiation between the murine ASCs and BMSCs. In conclusion, the present study has shown that ASCs had higher CFU-F frequencies and colony-forming efficiency than BMSCs. ASCs and BMSCs presented a similar surface immunophenotype and multilineage differentiation capacity. Therefore, ASCs in BALB/c mice might be a more useful material for cell therapy of the airway allergic experiment due to the abundance, relatively easy harvesting and high proliferation potential.     

Viability and osteogenic potential of cryopreserved human bone marrow-derived mesenchymal cells

Human bone marrow-derived mesenchymal cells contain mesenchymal stem cells (MSCs), which are well known for their osteo/chondrogenic potential and can be used for bone reconstruction. This article reports the viability of cryopreserved human mesenchymal cells and a comparison of the osteogenic potential between noncryopreserved and cryopreserved human mesenchymal cells with MSC-like characteristics, derived from the bone marrow of 28 subjects. The viability of cryopreserved mesenchymal cells was approximately 90% regardless of the storage term (0.3 to 37 months). It is clear by fluorescence-activated cell sorter analysis that the cell surface antigens of both noncryopreserved and cryopreserved mesenchymal cells were negative for hematopoietic cell markers such as CD14, CD34, CD45, and HLA-DR but positive for mesenchymal characteristics such as CD29 and CD105. To monitor the osteogenic potential of the cells, such as alkaline phosphatase (ALP) activity and in vitro mineralization, a subculture was conducted in the presence of dexamethasone, ascorbic acid, and glycerophosphate. No difference in osteogenic potential was found between cells with or without cryopreservation treatment. In addition, cells undergoing long-term cryopreservation (about 3 years) maintained high osteogenic potential. In conclusion, cryopreserved as well as noncryopreserved human mesenchymal cells could be applied for bone regeneration in orthopedics.     

Feridex labeling of mesenchymal stem cells inhibits chondrogenesis but not adipogenesis or osteogenesis

Magnetic resonance (MR) tracking of superparamagnetic iron oxide (SPIO)-labeled cells is a relatively new technique to non-invasively determine the biodistribution and migration of transplanted stem cells. A number of studies have recently reported encouraging results in the use of bone marrow-derived mesenchymal stem cells (MSCs) for repair of a variety of tissues. For MR tracking of SPIO-labeled MSCs, it is important to determine the effect that the magnetic labeling procedure may have on the differentiation capacity of labeled MSCs. Human MSCs were labeled with poly-L-lysine (PLL)-coated Feridex, with Feridex being an FDA-approved SPIO formulation in an off-label application, and assayed for cellular differentiation using five different assays. As compared with unlabeled controls, labeled MSCs exhibited an unaltered viability, proliferated similarly, and underwent normal adipogenic and osteogenic differentiation. However, there was a marked inhibition of chondrogenesis. The blocking of chondrogenic activity was mediated by the Feridex, rather than by the transfection agent (PLL). This is the first report showing Feridex blocking of cellular differentiation down a specific pathway (while not affecting viability and proliferation), and caution should thus be exercised when using Feridex-labeled MSCs for chondrogenic MR tracking studies. On the other hand, no detrimental effects of Feridex-labeling are anticipated for MR-guided osteogenic or adipogenic transplantation studies.     

Mesenchymal progenitor cells from canine fetal tissues: yolk sac, liver, and bone marrow

During fetal development, mesenchymal progenitor (MP) cells are co-localized in major hematopoietic territories, such as yolk sac (YS), bone marrow (BM), liver (LV), and others. Studies using mouse and human MP cells isolated from fetus have shown that these cells are very similar but not identical to adult mesenchymal stem cells (MSC). Their differentiation potential is usually restricted to production of highly committed osteogenic and chondrogenic precursors. Such properties of fetal MP cells can be very useful for tissue regeneration, when a great number of committed precursors are required. The objectives of this study were to isolate and characterize MP cells from canine YS, BM, and LV in early and late stages of fetal development. Gestational stage was identified, and cell culture conditions were evaluated for efficient isolation of canine MP cells. All canine fetal MP cells expressed vimentin, nestin, and CD44 proteins. Cytokeratin 18 expression was observed in BM- and LV-MP cells, and vascular endothelial (VE)-cadherin expression was observed only in YS-MP cells. A small number of MP cells (5%) from LV and YS expressed Oct3/4 protein. The differentiation potential of canine fetal MP cells varied significantly: YS- and BM-MP cells differentiated into bone and cartilage, whereas LV-MP cells differentiation was limited to osteogenic fate. None of the canine fetal MP cells were able to differentiate into adipose cells. Our data suggest that canine fetal MP cells are an appropriate in vitro model to study MP biology from hematopoietic territories and they are a source of committed osteogenic and chondrogenic precursors for regenerative medicine.