Pressure and Distortion Regulate Human Mesenchymal Stem Cell Gene Expression

While the concept that physical forces such as tension and compression are involved in mature tissue modeling is widely accepted, the role of these specific types of mechanical loading in the differentiation and maturation of uncommitted cell types like human mesenchymal stem cells (hMSCs) is currently unknown. We observed that hMSCs have the fundamental ability to distinguish between dynamic tensile and compressive loading by regulating distinct gene expression patterns and that these differences in gene expression can be related to conformational changes in cell shape and volume. Dynamic tension was found to regulate both fibroblastic and osteogenic associated genes while dynamic compression up-regulated genes associated with chondrogenesis. Identifying genes involved in the mechanotransduction of different modes of physical loading in hMSC may greatly enhance the ability to rationally design tissue regeneration systems to restore proper tissue function.     

Neuropotency of Human Mesenchymal Stem Cell Cultures: Clonal Studies Reveal the Contribution of Cell Plasticity and Cell Contamination

Various studies have shown neuropotency of bone marrow-derived human mesenchymal stem cells (hMSC) based on the appearance of cells with neural phenotype before or after neural induction protocols. However, to date, it is unclear which mechanisms account for this observation. We hypothesized that neural phenotypes observed in hMSC cultures can be because of both intrinsic cell plasticity and contamination by cells of neural origin. Therefore, we characterized 38 clones from hMSC cultures by assessing their adipogenic/osteogenic potential with specific mesenchymal differentiation protocols, and their molecular neural phenotype by RT-PCR analysis before and after exposure to a defined neural stem cell (NSC) medium for 8 days (neural protocol). We found 33 clones with mesenchymal potential and 15 of them also showed a neural phenotype. As neural phenotypes were maintained during the neural protocol, this suggested neural cell plasticity in 39% of all clones through pluripotency. Importantly, we were able to induce neural phenotypes in 11 of mesenchymal clones applying the neural protocol, demonstrating neural cell plasticity in 29% of all clones through the mechanism of transdifferentiation. Finally, 2 of 5 nonmesenchymal clones (5% of all clones) displayed a neural phenotype indicating neural cell contamination of hMSC cultures. In conclusion, we found 2 different ways of neuropotency of hMSC cultures: cell plasticity and cell contamination.     

人脐带、胎盘间充质干细胞样细胞中“干”性蛋白表达的研究

检测原代分离培养的人脐带、胎盘间充质干细胞样细胞中CD29、CD34、CD44、CD133、HLA-DR、vWF、MRP1、ABCG2 、P75NTR及Nestin的表达.采用组织块贴壁培养法分离培养人脐带、胎盘间充质干细胞样细胞,通过培养扩增后用流式细胞仪检测CD29、CD34、CD44、CD133、HLA-DR及vWF的表达,应用免疫荧光技术检测人脐带、胎盘间充质干细胞样细胞中MRP1、ABCG2、P75 NTR、Nestin的表达.在培养的人脐带、胎盘间充质干细胞样细胞中,流式细胞仪检测示CD29、CD44表达阳性,CD34、CD133、HLA-DR及vWF表达阴性;免疫荧光示MRP1、ABCG2 、P75NTR及Nestin在人脐带、胎盘间充质干细胞样细胞中表达阳性且荧光定位在细胞的胞浆.人脐带、胎盘间充质干细胞样细胞表达间充质干细胞的免疫表型;MRP1、ABCG2、P75NTR、Nestin在人脐带、胎盘间充质干细胞样细胞中表达阳性,提示人脐带与胎盘来源的间充质干细胞样细胞“干”性蛋白的表达情况相似.     

Specific Interaction of Cultured Human Mesenchymal and Hemopoietic Stem Cells under Conditions of Reduced Oxygen Content

We studied the effect of reduced oxygen content (5%) on the phenotype and functional activity of cultured human mesenchymal stem cells. The expression of main immunophenotypic markers for mesenchymal stem cells (CD13, CD29, CD44, CD73, CD90, CD105, and HLA-I) remained practically unchanged under conditions of hypoxia. The expression of cell adhesion molecules (CD54 and CD106) increased during coculturing of mesenchymal stem cells and hemopoietic stem cells. These changes were accompanied by increased production of hemopoietins (interleukin-6 and interleukin-8) and enhanced colony-forming capacity of hemopoietic stem cells. Coculturing of mesenchymal stem cells and hemopoietic stem cells during hypoxia was followed by increased formation of hemopoietic islets and intensive production of interleukin-6, interleukin-8, and vascular endothelial growth factor (compared to cultures under normoxic conditions).     

CD146 Expression Influences Periapical Cyst Mesenchymal Stem Cell Properties

Recent studies have identified a new human dental derived progenitor cell population with multi-lineage differentiation potential referred to as human periapical cyst mesenchymal stem cells (hPCy-MSCs). In the present study, we compared two subpopulations of hPCy-MSCs characterised by the low or high expression of CD146 to establish whether this expression can regulate their stem cell properties. Using flow cytometry, we evaluated the stem cell marker profile of hPCy-MSCs during passaging. Furthermore, CD146^Low and CD146^High cells were sorted by magnetic beads and subsequently both cell populations were evaluated for differences in their proliferation, self-renewal, stem cell surface markers, stemness genes expression and osteogenic differentiation potential.We found that hPCy-MSCs possessed a stable expression of several mesenchymal stem cell surface markers, whereas CD146 expression declined during passaging.In addition, sorted CD146^Low cells proliferated significantly faster, displayed higher colony-forming unit-fibroblast capacity and showed higher expression of Klf4 when compared to the CD146^High subset. Significantly, the osteogenic potential of hPCy-MSCs was greater in the CD146^Low than in CD146^High population. These results demonstrate that CD146 is spontaneously downregulated with passaging at both mRNA and protein levels and that the high expression of CD146 reduces the proliferative, self-renewal and osteogenic differentiation potential of hPCy-MSCs. In conclusion, our study demonstrates that changes in the expression of CD146 can influence the stem cell properties of hPCy-MSCs.     

Substrates for Human Pluripotent Stem Cell Cultures in Conditioned Medium of Mesenchymal Stem Cells

We aimed to establish a culture system of human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), free from xenogeneic proteins, Matrigel^? and conditioned medium of mouse embryonic fibroblasts. The conditioned culture medium consisted of mesenchymal stem cells derived from human bone marrow. We examined surface properties suitable for hPSC culture by using self-assembled monolayers (SAMs) of alkanethiols with four different functional groups: CH₃, OH, COOH and NH₂. hPSCs neither adhered nor proliferated on surfaces with a water contact angle higher than 40°. Based on this finding, the contact angle of a polystyrene (PSt) culture dish was reduced to less than 40°, and COOH and OH groups were introduced to its surface by oxygen plasma treatment, making the PSt dish suitable for hPSC culture. This combination of a PSt dish treated with oxygen plasma treatment and conditioned medium of mesenchymal stem cells achieved a long-term maintenance of hPSCs without differentiation.     

人脐血间充质干细胞培养及其生物学特性的研究

目的建立人脐血间充质干细胞体外分离培养的最佳条件,并观察其生物学特性。方法在无菌条件下抽取人脐血,用密度梯度离心的方法获得脐血单个核细胞,接种含10%胎牛血清的IMDM培养基中。对单个核细胞行贴壁培养后,进行细胞形态学观察,细胞生长、细胞周期分析及细胞凋亡检测,免疫细胞化学鉴定。结果采用Percoll(1.073g/ml)分离的脐血间充质干细胞(mesenchymal stem cells,MSCs)大小较为均匀,呈梭形或星形的上皮样细胞,传代培养后的细胞体积较大,成纤维样细胞逐渐增多。细胞生长曲线测定表明接种后第5d细胞进入指数增生期,至第9d进入平台期;流式细胞术证明G2+S期细胞为14.8%;免疫细胞化学染色结果表明42.0%细胞为CD45阳性。结论体外分离培养脐血间充质干细胞生长稳定,可作为组织工程的种子细胞。     

Embryonic Stem Cell Marker Expression Pattern in Human Mesenchymal Stem Cells Derived from Bone Marrow, Adipose Tissue, Heart and Dermis

Mesenchymal stem cells (MSCs) have been isolated from a variety of human tissues, e.g., bone marrow, adipose tissue, dermis, hair follicles, heart, liver, spleen, dental pulp. Due to their immunomodulatory and regenerative potential MSCs have shown promising results in preclinical and clinical studies for a variety of conditions, such as graft versus host disease (GvHD), Crohn’s disease, osteogenesis imperfecta, cartilage damage and myocardial infarction. MSC cultures are composed of heterogeneous cell populations. Complications in defining MSC arise from the fact that different laboratories have employed different tissue sources, extraction, and cultivation methods. Although cell-surface antigens of MSCs have been extensively explored, there is no conclusive evidence that unique stem cells markers are associated with these adult cells. Therefore the aim of this study was to examine expression of embryonic stem cell markers Oct4, Nanog, SOX2, alkaline phosphatase and SSEA-4 in adult mesenchymal stem cell populations derived from bone marrow, adipose tissue, dermis and heart. Furthermore, we tested whether human mesenchymal stem cells preserve tissue-specific differences under in vitro culture conditions. We found that bone marrow MSCs express embryonic stem cell markers Oct4, Nanog, alkaline phosphatase and SSEA-4, adipose tissue and dermis MSCs express Oct4, Nanog, SOX2, alkaline phosphatase and SSEA-4, whereas heart MSCs express Oct4, Nanog, SOX2 and SSEA-4. Our results also indicate that human adult mesenchymal stem cells preserve tissue-specific differences under in vitro culture conditions during early passages, as shown by distinct germ layer and embryonic stem cell marker expression patterns. Studies are now needed to determine the functional role of embryonic stem cell markers Oct4, Nanog and SOX2 in adult human MSCs.