Expression of vascular cell adhesion molecule-1 indicates the differentiation potential of human bone marrow stromal cells

Bone marrow stromal cells (BMSCs) are a mixture of cells differing in differentiation potential including mesenchymal stem cells, and so far no CD antigens were found to be predictable for the differentiation property of each BMSC. Here we attempted to isolate differentiation-associated CD antigens using 100 immortalized human BMSC (ihBMSC) clones. Among 13 CD antigens analyzed, only CD106/Vascular cell adhesion molecule-1 (VCAM-1) showed a clear correlation with the differentiation potential of each clone; CD106-positive ihBMSC clones were less osteogenic and more adipogenic than CD106-negative clones. This association was confirmed in primary BMSCs sorted by CD106, showing that the CD106-positive fraction contained less osteogenic and more adipogenic cells than the CD106-positive fraction. The evaluation of CD106 fraction of BMSC strains in early passages predicted clearly the osteogenic and adipogenic potential after in vitro induction of differentiation, indicating the usefulness of CD106 as a differentiation-predicting marker of BMSC.     

外源性Wnt3a持续作用对小鼠胚胎干细胞Wnt／β-catenin信号通路的调控作用

目的观察Wnt/β-catenin信号通路是否在体外以外源性Wnt3a持续作用小鼠胚胎干细胞后被激活,并进一步调控该通路下游基因的表达。方法应用外源性Wnt3a持续作用ES-E14TG2a小鼠胚胎干细胞21d,通过细胞免疫荧光及Western Blotting检测细胞内β-catenin蛋白,以观察该蛋白的胞内积聚情况;同时QRT-PCR检测WNT下游靶标基因的表达量,采用完全随机F检验并用LSD法进行两两比较,来确定经典WNT／β-catenin信号通路是否被激活。结果ES-E14TG2a小鼠胚胎干细胞经Wnt3a连续培养21d后,β-catenin蛋白的细胞荧光明显较强,而对照组中的荧光强度较弱,说明细胞内β-catenin蛋白没有被降解而是在胞内大量积累;Western Blotting检测结果显示Wnt3a连续培养21d后ES-E14TG2a细胞内β-catenin蛋白条带明显比空白对照的蛋白条带粗;ES—E14TG2a细胞经wnt3a培养后Pitx2、Frizzled、Sox17的表达量均持续上升,Pitx2在培养7d、14d、21d分别为4．17±0．20、7．27±0．35、8．59±0．21（F=222．757,P=0．000）;Frizzled在培养7d、14d、21d分别为1．01±0．06、2．93±0．22、5．44±0．30（F=302．703,P=0．000）;Sox17在培养7d、14d、21d分别为8．45±0．41、18．35±0．17、34．93±0．16（F=7217．083,P=0．000）;Oct4培养到7d、14d的表达量持续增加分别为1．22±0．21、1．56±0．04,而连续培养21d后Oct4基因的表达量下降为1．15±0．07（F=8．827,P=0．016）。结论Wnt3a持续作用可激活Wnt／β-catenin信号通路,并调控下游基因的表达。     

Tissue-engineered bone formation with cryopreserved human bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) have become the main cell source for bone tissue engineering. It has been reported that cryopreserved human MSCs can maintain their potential for proliferation and osteogenic differentiation in vitro. There are, however, no reports on osteogenesis with cryopreserved human MSCs in vivo. The aim of this study was to determine whether cryopreservation had an effect on the proliferation capability and osteogenic differentiation of human MSCs on scaffolds in vitro and in vivo. MSCs were isolated from human bone marrow, cultured in vitro until passage 2, and then frozen and stored at −196 °C in liquid nitrogen with 10% Me₂SO as cryoprotectant for 24 h. The cryopreserved MSCs were then thawed rapidly, seeded onto partially demineralized bone matrix (pDBM) scaffolds and cultured in osteogenic media containing 10 mM sodium β-glycerophosphate, 50 μM l-ascorbic acid, and 10 nM dexamethasone. Non-cryopreserved MSCs seeded onto the pDBM scaffolds were used as control groups. Scanning electronic microscopy (SEM) observation, DNA content assays, and measurements of alkaline phosphatase (ALP) activity and osteocalcin (OCN) content were applied, and the results showed that the proliferation potential and osteogenic differentiation of MSCs on pDBM in vitro were not affected by cryopreservation. After 2 weeks of subculture, the MSCs/pDBM composites were subcutaneously implanted into the athymic mice. The constructs were harvested at 4 and 8 weeks postimplantation, and histological examination showed tissue-engineered bone formation in the pDBM pores in both groups. Based on these results, it can be concluded that cryopreservation allows human MSCs to be available for potential therapeutic use to tissue-engineer bone.     

Novel Daple-like protein positively regulates both the Wnt/beta-catenin pathway and the Wnt/JNK pathway in Xenopus

Wnt signaling pathways are essential in various developmental processes including differentiation, proliferation, cell migration, and cell polarity. Wnt proteins execute their multiple functions by activating distinct intracellular signaling cascades, although the mechanisms underlying this activation are not fully understood. We identified a novel Daple-like protein in Xenopus and named it xDal (Xenopus Daple-like). As with Daple, xDal contains several leucine zipper-like regions (LZLs) and a putative PDZ domain-binding motif, and can interact directly with the dishevelled protein. In contrast to mDaple, injection of xDal mRNA into the dorso-vegetal blastomere does not induce ventralization and acted synergistically with xdsh in secondary axis induction. XDal also induced expression of siamois and xnr-3, suggesting that XDal functions as a positive regulator of the Wnt/beta-catenin pathway. Injection of xDal mRNA into the dorso-animal blastomere, however, induced gastrulation-defective phenotypes in a dose-dependent manner. In addition, xDal inhibited activin-induced elongation of animal caps and enhanced c-jun phosphorylation. Based on these findings, xDal is also thought to function in the Wnt/JNK pathway. Moreover, functional domain analysis with several deletion mutants indicated that xDal requires both a putative PDZ domain-binding motif and at least one LZL for its activity. These findings with xDal will provide new information on the Wnt signaling pathways.     

Xeno-free proliferation of human bone marrow mesenchymal stem cells

The proliferation of human bone marrow mesenchymal stem cells (MSCs) employing xeno-free materials not containing fetal calf serum (FCS) and porcine trypsin was investigated for the regenerative medicine of cartilage using MSCs. Four sequential subcultivations of MSCs using a medium containing 10% FCS and recombinant trypsin (TrypLESelect™) resulted in cell growth comparable to that with porcine trypsin. There was no apparent difference in the cell growth and morphology between two kinds of MSC stored in liquid nitrogen using 10% FCS plus DMSO or serum-free TC protector™. MSCs were isolated from human bone marrow cells, stored in liquid nitrogen, and sequentially subcultivated four times employing conventional materials that included FCS, porcine trypsin, and DMSO, or xeno-free materials that included serum-free medium (MesenCult-XF™), TC protector™ and TrypLESelect™. Cells in the culture using the xeno-free materials maintained typical fibroblast-like morphology and grew more rapidly than the cells in the culture using the conventional materials, while the cell surface markers of MSCs (CD90 and CD166) were well maintained in both cultures. Chondrogenic pellet cultures were carried out using these subcultivated cells and a medium containing TGFβ3 and IGF1. The pellet culture using cells grown with the xeno-free materials showed an apparently higher gene expression of aggrecan, a chondrocyte marker, than the pellet culture using cells grown with the conventional materials. Consequently, MSCs that are isolated, stored, and grown using the xeno-free materials including the serum-free medium (MesenCult-XF™), TC protector™, and recombinant trypsin (TrypLESelect™) might be applicable for regenerative medicine of cartilage.     

Down-regulation of Wnt signaling could promote bone marrow-derived mesenchymal stem cells to differentiate into hepatocytes

Bone marrow-derived mesenchymal stem cells (BMSCs) have been demonstrated to be able to differentiate into hepatocytes, but the precise mechanisms controlling this process are unclear. Our aim is try to explore the role of Wnt signaling on the differentiation of BMSCs into hepatocytes. Our study demonstrated that BMSCs could successfully differentiate into hepatocytes under in vitro induction of the tissue extract of damaged liver. The mRNA level of Wnt-1, Wnt-5a, Frizzled1, DSH (disheveled), GSK-3β (glycogen synthase kinase 3 beta) and β-catenin on day 21 when the differentiation direction was determined, was lower than that on days 0, 7, and 11. Furthermore, blocking Wnt-1 signaling by treating BMSCs with Dkk1 could induce BMSCs to express albumin earlier and up-regulation of Wnt signaling by treating BMSCs with Wnt-1 could inhibit BMSCs to differentiate into hepatocytes. Above results indicated that inhibition on Wnt signaling can promote BMSCs to differentiate into hepatocytes.     

Arterial-venous endothelial cell fate is related to vascular endothelial growth factor and Notch status during human bone mesenchymal stem cell differentiation

Human bone mesenchymal stem cells (hMSCs) can differentiate into endothelial cells (ECs), so we aimed to investigate whether hMSCs could also differentiate into a specific arterial or venous ECs. hMSCs were induced to differentiate into ECs using vascular endothelial growth factor (VEGF). Low VEGF concentration (50ng/ml) upregulated the venous marker gene EphB4, however high concentration (100ng/ml) upregulated the arterial marker genes ephrinB2, Dll4 and Notch4, and downregulated the venous marker genes EphB4 and COUP-TFll. This VEGF dose-dependent induction was largely blocked by inhibition of the Notch pathway in hMSCs treated with gamma-secretase inhibitor. Therefore, differentiation of hMSCs into arterial- or venous-specific ECs depends on VEGF and is regulated by the Notch pathway.     

Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow

The future use of adult mesenchymal stem cells (MSCs) for human therapies depends on the establishment of preclinical studies with other mammals such as mouse. Surprisingly, purification and characterisation of murine MSCs were only poorly documented. The aim of this study was to purify mouse MSCs from adult bone marrow and to functionally characterise their abilities to differentiate along diverse lineages. Adherent cells from adult C57Bl/6J mouse bone marrow were depleted of granulo-monocytic cells and subsequently allowed to grow on fibronectin-coated dishes in presence of fetal bovine serum and growth factors. The growing fibroblastoid cell population primarily consisted of spindle- and star-shaped cells with significant renewal capacity as they were cultured until 30 passages (about 60 doubling population). We fully demonstrated the MSC phenotype of these cells by inducing them to differentiate along osteoblastic, adipocytic, and chondrocytic pathways. Mouse MSCs (mMSCs) sharing the same morphological and functional characteristics as human MSCs can be successfully isolated from adult bone marrow without previous mouse or bone marrow treatment. Therefore, mMSCs will be an important tool to study the in vivo behaviour and fate of this cell type after grafting in mouse pathology models.     

Lithium chloride promotes proliferation of neural stem cells in vitro,possibly by triggering the Wnt signaling pathway

The objective of this study was to clarify the relationship between the effect and associated mechanisms of lithium chloride on neural stem cells (NSCs) and the Wnt signaling pathway. The expression of key molecules proteins related to the Wnt signaling pathway in the proliferation and differentiation of control NSCs and lithium chloride-treated NSCs was detected by Western blot analysis. Flow cytometry analysis was applied to study the cell cycle dynamics of control NSCs and NSCs treated with lithium chloride. The therapeutic concentrations of lithium chloride stimulated NSC proliferation. β-catenin expression gradually decreased, while Gsk-3β expression gradually increased (P?P?in vitro and preventing the cells from differentiating, which is potentially mediated by activation of the Wnt signaling pathway.     

Gene delivery to human bone marrow mesenchymal stem cells by microporation

Electroporation has been considered one of the most efficient non-viral based methods to deliver genes regardless of frequently observed high cell mortality. In this study we used a microporation technique to optimise the delivery of plasmid DNA encoding green fluorescence protein (GFP) to human bone marrow mesenchymal stem cells (BM-MSC). Using resuspension buffer (RB) and as low as 1.5 × 10⁵ cells and 1 μg of DNA, we achieved 40% of cells expressing the transgene, with cell recovery and cell viabilities of 85% and 90%, respectively. An increase in DNA amount did not significantly increase the number of transfected cells but clearly reduced cell recovery. A face-centered composite design was used to unveil the conditions giving rise to optimal plasmid delivery efficiencies when using a sucrose based microporation buffer (SBB). The BM-MSC proliferation kinetics were mainly affected by the presence of plasmid and not due to the microporation process itself although no effect was observed on their immunophenotypic characteristics and differentiative potential. Based on the data shown herein microporation demonstrated to be a reliable and efficient method to genetically modify hard-to-transfect cells giving rise to the highest levels of cell survival reported so far along with superior gene delivery efficiencies.     

The immunobiological development of human bone marrow mesenchymal stem cells in the course of neuronal differentiation

The central nervous system (CNS) has been referred to as the "immunological privileged site". However, it is now clear that the privileged status of the CNS is a result of a balance between immune privilege and effective response. In vitro, human bone marrow mesenchymal stem cells (MSCs) have the ability to differentiate into neurons. Based on this biological attribute we gain the possibility by means of using MSCs as the donors to develop a future cell therapy in clinical application. But using MSCs as donor cells inevitably raises the question as to whether these donor cells would be immunogenic, and if so, would they be rejected after transplantation. To investigate this, human MSCs were cultured in vitro and induced to differentiate along neuronal lineage. The expression of human leukocyte antigen (HLA) class I and class II molecules and the co-stimulatory protein CD80 were increased on the surface of MSCs in the course of neuronal differentiation. But neither of the co-stimulatory proteins, CD40 or CD86, was expressed. After IFN-gamma exposure, the expression of the HLA molecules was further enhanced, but the co-stimulatory proteins were unaffected. MSCs that had been differentiated along neuronal lineage were not capable of inducing the proliferation of peripheral blood lymphocytes (PBLs). Even after IFN-gamma exposure, PBLs remained unresponsive. Furthermore, MSCs differentiated along neuronal lineage suppressed the proliferation of PBLs induced by allogeneic PBLs and mitogens. The mechanisms involved in the immunosuppression may be related to the effect of soluble factors and cell-cell interactions of neuronal differentiated MSCs and PBLs. From the above data we suggested that the low immunogenicity and immunomodulatory function of MSCs in the course of neuronal differentiation in vitro, which will be helpful to further investigation in order to establish the new way for future medical application.     

Forced expression of Sox2 or Nanog in human bone marrow derived mesenchymal stem cells maintains their expansion and differentiation capabilities

Mesenchymal stem cells (MSCs) derived from human bone marrow have capability to differentiate into cells of mesenchymal lineage. The cells have already been applied in various clinical situations because of their expansion and differentiation capabilities. The cells lose their capabilities after several passages, however. With the aim of conferring higher capability on human bone marrow MSCs, we introduced the Sox2 or Nanog gene into the cells. Sox2 and Nanog are not only essential for pluripotency and self-renewal of embryonic stem cells, but also expressed in somatic stem cells that have superior expansion and differentiation potentials. We found that Sox2-expressing MSCs showed consistent proliferation and osteogenic capability in culture media containing basic fibroblast growth factor (bFGF) compared to control cells. Significantly, in the presence of bFGF in culture media, most of the Sox2-expressing cells were small, whereas the control cells were elongated in shape. We also found that Nanog-expressing cells even in the absence of bFGF had much higher capabilities for expansion and osteogenesis than control cells. These results demonstrate not only an effective way to maintain proliferation and differentiation potentials of MSCs but also an important implication about the function of bFGF for self-renewal of stem cells including MSCs.     

JNK、ERK信号通路在NaAsO_2诱导骨髓间充质干细胞增殖中的作用

目的：研究c-jnk氨基末端激酶（JNK）、细胞外信号调节激酶（ERK）在亚砷酸钠（NaAs02）诱导骨髓间充质干细胞（BMSC）增殖中的作用。方法：体外培养骨髓间充质干细胞,四甲基偶氮唑盐比色法（MTT法）检测细胞增殖,Western-blot检测磷酸化JNK、ERK表达水平。结果：低浓度1、2μmol/LNaAs02对BMSC有明显的促进增殖作用;高浓度16、32μmol/LNaAs02则对细胞生长产生抑制作用,具有一定剂量-效应关系;2、4、8μmol/LNaAs02处理BMSC24h后,JNK磷酸化表达水平明显增加,ERK磷酸化表达水平明显降低;JNK抑制剂SP600125可明显降低高浓度16、32μmol/LNaAs02的生长抑制作用;ERK抑制剂PD98059可抑制低浓度1、2μmol/LNaAs02对BMSC的促增殖作用。结论：低浓度NaAs02激活ERK信号通路,提高细胞增殖率,可被抑制剂PD98059阻断;高浓度NaAs02激活JNK信号通路,提高细胞凋亡率,可被抑制剂SP600125阻断。NaAs02致癌机制可能与JNK、ERK信号通路作用相关。     

Impact of stromal cell composition on BMP-induced chondrogenic differentiation of mouse bone marrow derived mesenchymal cells

Chondrogenic differentiation in mesenchymal stromal cells (MSCs) has been actively studied due to their potential use in mesenchymal tissue repair. Our goal was to develop a simple isolation protocol for adherent mouse MSCs to simultaneously clear off hematopoietic cells and expand to obtain enough starting material for differentiation studies. CD34 and CD45 expressing cells were rapidly removed by inhibiting growth of hematopoietic cells to yield short-term selected (STS) cells. Further passaging enriched more primitive, uniformly Sca-1 expressing, long-term selected (LTS) cells. The efficacy of several BMPs to induce chondrogenesis in pellet culture was compared in STS and LTS cells. In STS cells, chondrogenesis progressed rapidly to terminal differentiation while LTS cells differentiated at a slower rate with no hypertrophy. In LTS cells, rhBMP homodimers -2, -4, -6 and rhBMP2/7 heterodimer were effective enhancers of chondrogenesis over that of rhBMP-5 and -7. In STS cells, rhBMP-2 and rhBMP-7 supported rapid chondrogenesis and terminal differentiation over that of rhBMP-6. These data indicate the impact of stromal cell composition on the chondrogenic differentiation profile, which is an important aspect to be considered when standardizing differentiation assay conditions as well as developing MSC based cartilage repair technologies.     

Cell cycle dependent telomere regulation by telomerase in human bone marrow mesenchymal stem cells

Human bone marrow mesenchymal stem cells (hMSCs) are a promising source for clinical stem cell transplantation. However, telomere regulation mechanisms, as one of the possible major mechanisms by which hMSCs sustain their stem cell characteristics, remain unknown. We isolated hMSCs by plastic adhesion and characterized these cells by morphology, immune phenotype and differentiation capacity. Telomerase was found negative in hMSCs, but slightly up-regulated in hMSC-derived adipocytes by the Telomeric Repeat Amplification Protocol (TRAP) assay. Moreover, hMSCs lack the alternative lengthening of telomeres (ALT) mechanism, because the hallmarks of ALT, such as very long and heterogeneous telomeres, extra-chromosome telomere repeat DNA (ECTR), and ALT-associated promyelocytic leukemia bodies (APBs), were not evident. However, when hMSCs were arrested in S phase with a combination of serum deprivation and aphidicolin, previously undetectable telomerase activity became predominantly positive. Meanwhile, the expression level of hTERT protein and mRNA increased, paralleled with the appearance of a large cohort of synchronized hMSCs at S phase. These findings provide a profile of telomere regulation by cell cycle dependent expression of telomerase in hMSCs and may lead to a better understanding of the stem cell nature of these cells.     

Characterization of mesenchymal stem cells (MSCs) from human fetal lung: Potential differentiation of germ cells

Pluripotent mesenchymal stem-like cell lines were established from lungs of 3–4 months old aborted fetus. The cells present the high ex vivo expansion potential of MSC, a typical fibroblast-like morphology and proliferate up to 15 passages without displaying clear changes in morphology. Immunological localization and flow cytometry analyses showed that these cells are positive for OCT4, c-Kit, CD11, CD29, CD44, telomerase, CD106, CD105, CD166, and SSEA1, weakly expression or negative for SSEA1, SSEA3, SSEA4, CD34, CD105 and CD106. These cells can give rise to the adipogenic as evidenced by accumulation of lipid-rich vacuoles within cells identified by Oil-red O when they were induced with 0.5 mM isobutylmethylxanthine, 200 μM indomethacin, 10⁻⁶ M dexamethasone, and 10 μg/ml of insulin in high-glucose DMEM. Osteogenic lineage cells were generated in 0.1 μM dexamethasone, 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate, which are shaped as the osteoblastic morphology, expression of alkaline phosphatase (AP), and the formation of a mineralized extracellular matrix identified by Alizarin Red staining. Neural cells are observed when the cultures were induced with 2-mercapometal, which are positive for nestin, NF-100, MBP and GFAP. Additionally, embryoid bodies (EBs) and sperm like cells are obtained in vitro differentiation of these lung MSCs induced with 10⁻⁵ M retinoic acid (RA). These results demonstrated that these MSCs are pluripotent and may provide an in vitro model to study germ-cell formation and also as a potential source of sperms for male infertility.