Mesenchymal stem cells promote the sustained expression of CD69 on activated T lymphocytes: roles of canonical and non-canonical NF-κB signalling

Mesenchymal stem cells (MSCs) are known to induce the conversion of activated T cells into regulatory T cells in vitro. The marker CD69 is a target of canonical nuclear factor kappa-B (NF-κB) signalling and is transiently expressed upon activation; however, stable CD69 expression defines cells with immunoregulatory properties. Given its enormous therapeutic potential, we explored the molecular mechanisms underlying the induction of regulatory cells by MSCs. Peripheral blood CD3(+) T cells were activated and cultured in the presence or absence of MSCs. CD4(+) cell mRNA expression was then characterized by microarray analysis. The drug BAY11-7082 (BAY) and a siRNA against v-rel reticuloendotheliosis viral oncogene homolog B (RELB) were used to explore the differential roles of canonical and non-canonical NF-κB signalling, respectively. Flow cytometry and real-time PCR were used for analyses. Genes with immunoregulatory functions, CD69 and non-canonical NF-κB subunits (RELB and NFKB2) were all expressed at higher levels in lymphocytes co-cultured with MSCs. The frequency of CD69(+) cells among lymphocytes cultured alone progressively decreased after activation. In contrast, the frequency of CD69(+) cells increased significantly following activation in lymphocytes co-cultured with MSCs. Inhibition of canonical NF-κB signalling by BAY immediately following activation blocked the induction of CD69; however, inhibition of canonical NF-κB signalling on the third day further induced the expression of CD69. Furthermore, late expression of CD69 was inhibited by RELB siRNA. These results indicate that the canonical NF-κB pathway controls the early expression of CD69 after activation; however, in an immunoregulatory context, late and sustained CD69 expression is promoted by the non-canonical pathway and is inhibited by canonical NF-κB signalling.     

Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34(+) hematopoietic progenitor cells

Megakaryocytopoiesis and thrombocytopoiesis result from the interactions between hematopoietic progenitor cells, humoral factors, and marrow stromal cells derived from mesenchymal stem cells (MSCs) or MSCs directly. MSCs are self-renewing marrow cells that provide progenitors for osteoblasts, adipocytes, chondrocytes, myocytes, and marrow stromal cells. MSCs are isolated from bone marrow aspirates and are expanded in adherent cell culture using an optimized media preparation. Culture-expanded human MSCs (hMSCs) express a variety of hematopoietic cytokines and growth factors and maintain long-term culture-initiating cells in long-term marrow culture with CD34(+) hematopoietic progenitor cells. Two lines of evidence suggest that hMSCs function in megakaryocyte development. First, hMSCs express messenger RNA for thrombopoietin, a primary regulator for megakaryocytopoiesis and thrombocytopoiesis. Second, adherent hMSC colonies in primary culture are often associated with hematopoietic cell clusters containing CD41(+) megakaryocytes. The physical association between hMSCs and megakaryocytes in marrow was confirmed by experiments in which hMSCs were copurified by immunoselection using an anti-CD41 antibody. To determine whether hMSCs can support megakaryocyte and platelet formation in vitro, we established a coculture system of hMSCs and CD34(+) cells in serum-free media without exogenous cytokines. These cocultures produced clusters of hematopoietic cells atop adherent MSCs. After 7 days, CD41(+) megakaryocyte clusters and pro-platelet networks were observed with pro-platelets increasing in the next 2 weeks. CD41(+) platelets were found in culture medium and expressed CD62P after thrombin treatment. These results suggest that MSCs residing within the megakaryocytic microenvironment in bone marrow provide key signals to stimulate megakaryocyte and platelet production from CD34(+) hematopoietic cells.     

Mesenchymal stem/stromal cells induce the generation of novel IL-10--dependent regulatory dendritic cells by SOCS3 activation

Suppression of immune response by mesenchymal stem/stromal cells (MSCs) is well documented. However, their regulatory effects on immune cells, especially regulatory dendritic cells, are not fully understood. We have identified a novel Sca-1(+)Lin(-)CD117(-) MSC population isolated from mouse embryonic fibroblasts (MEF) that suppressed lymphocyte proliferation in vitro. Moreover, the Sca-1(+)Lin(-)CD117(-) MEF-MSCs induced hematopoietic stem/progenitor cells to differentiate into novel regulatory dendritic cells (DCs) (Sca-1(+)Lin(-)CD117(-) MEF-MSC-induced DCs) when cocultured in the absence of exogenous cytokines. Small interfering RNA silencing showed that Sca-1(+)Lin(-)CD117(-) MEF-MSCs induced the generation of Sca-1(+)Lin(-)CD117(-) MEF-MSC-induced DCs via IL-10-activated SOCS3, whose expression was regulated by the JAK-STAT pathway. We observed a high degree of H3K4me3 modification mediated by MLL1 and a relatively low degree of H3K27me3 modification regulated by SUZ12 on the promoter of SOCS3 during SOCS3 activation. Importantly, infusion of Sca-1(+)CD117(-)Lin(-) MEF-MSCs suppressed the inflammatory response by increasing DCs with a regulatory phenotype. Thus, our results shed new light on the role of MSCs in modulating regulatory DC production and support the clinical application of MSCs to reduce the inflammatory response in numerous disease states.     

A comparison of DNA damage induced by aflatoxin B1 in hepatocyte-like cells, their progenitor mesenchymal stem cells and CD34(+) cells isolated from umbilical cord blood

This study compared the sensitivity of differentiated hepatocyte-like cells, their progenitor mesenchymal stem cells (MSCs) and CD34(+) stem cells to DNA damage and toxicity induced by aflatoxin B1 (AFB1). The hepatocyte-like cells and their progenitor cells (isolated from umbilical cord blood (UCB)) were each treated with AFB1 on day 15 of differentiation. Cell toxicity and genotoxicity effects were assessed using MTT and alkaline comet assays. AFB1 treatment resulted in a dose- and time-dependent inhibition of cell growth. The IC(50) values of AFB1 for hepatocytes differentiated from CD34(+) and MSCs were within the same range (44.7-46.8μM). The IC(50) calculated for non-differentiated MSCs and CD34(+) cells was slightly lower (42.0-43.4μM) than that calculated for their differentiated counterparts. However, the extent of DNA damage was different in differentiated and non-differentiated cells. The percentages of DNA (% DNA) in comet tails measured in hepatocytes differentiated from MSCs exposed to AFB1 (0, 2.5, 10 and 20μM) for 24h were ～15, 55, 65 and 70%, respectively. In comparison, hepatocytes from CD34(+) cells were more resistant to AFB1-induced DNA damage. Hepatocyte-MSCs were most sensitive to DNA damage, followed by UCB-CD34(+) cells, then UCB-MSCs and finally hepatocyte-CD34(+) cells. These results clearly showed that stem cells from different sources have different sensitivities to DNA damaging agents. These differences can be assigned to the expression levels of cytochrome P450 (CYP) particularly CYP3A4 in non-differentiated and differentiated cells. These data are useful in better understanding the susceptibility/resistance of stem cells in the process of differentiation to environmental toxicants.     

Fetal muscle-derived cells can repair dystrophic muscles in mdx mice

We have previously reported that CD34(+) cells purified from mouse fetal muscles can differentiate into skeletal muscle in vitro and in vivo when injected into muscle tissue of dystrophic mdx mice. In this study, we investigate the ability of such donor cells to restore dystrophin expression, and to improve the functional muscle capacity of the extensor digitorum longus muscle (EDL) of mdx mice. For this purpose green fluorescent-positive fetal GFP(+)/CD34(+) cells or desmin(+)/(-)LacZ/CD34(+) cells were transplanted into irradiated or non-irradiated mdx EDL muscle. Donor fetal muscle-derived cells predominantly fused with existing fibers. Indeed more than 50% of the myofibers of the host EDL contained donor nuclei delivering dystrophin along 80-90% of the length of their sarcolemma. The presence of significant amounts of dystrophin (about 60-70% of that found in a control wild-type mouse muscle) was confirmed by Western blot analyses. Dystrophin expression also outcompeted that of utrophin, as revealed by a spatial shift in the distribution of utrophin. At 1 month post-transplant, the recipient muscle appeared to have greater resistance to fatigue than control mdx EDL muscle during repeated maximal contractions.     

Identity and ranking of colonic mesenchymal stromal cells

Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146(+) MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions.     

Ex vivo glycan engineering of CD44 programs human multipotent mesenchymal stromal cell trafficking to bone

The capacity to direct migration ('homing') of blood-borne cells to a predetermined anatomic compartment is vital to stem cell-based tissue engineering and other adoptive cellular therapies. Although multipotent mesenchymal stromal cells (MSCs, also termed 'mesenchymal stem cells') hold the potential for curing generalized skeletal diseases, their clinical effectiveness is constrained by the poor osteotropism of infused MSCs (refs. 1-3). Cellular recruitment to bone occurs within specialized marrow vessels that constitutively express vascular E-selectin, a lectin that recognizes sialofucosylated determinants on its various ligands. We show here that human MSCs do not express E-selectin ligands, but express a CD44 glycoform bearing alpha-2,3-sialyl modifications. Using an alpha-1,3-fucosyltransferase preparation and enzymatic conditions specifically designed for treating live cells, we converted the native CD44 glycoform on MSCs into hematopoietic cell E-selectin/L-selectin ligand (HCELL), which conferred potent E-selectin binding without effects on cell viability or multipotency. Real-time intravital microscopy in immunocompromised (NOD/SCID) mice showed that intravenously infused HCELL(+) MSCs infiltrated marrow within hours of infusion, with ensuing rare foci of endosteally localized cells and human osteoid generation. These findings establish that the HCELL glycoform of CD44 confers tropism to bone and unveil a readily translatable roadmap for programming cellular trafficking by chemical engineering of glycans on a distinct membrane glycoprotein.     

A novel role for CD4+ T cells in the control of cachexia

Cachexia is the dramatic weight loss and muscle atrophy seen in chronic disease states, including autoimmunity, cancer, and infection, and is often associated with lymphopenia. We have previously shown that CD4(+) T cells that express the lowest density of CD44 (CD4(+)CD44(v.low)) are significantly reduced in diabetic NOD mice that are cachexic compared with diabetic mice that are not cachexic. Using this model, and a model of cancer cachexia, we test the hypothesis that CD4(+)CD44(v.low) cells play an active role in protecting the host from cachexia. CD4(+)CD44(v.low) cells, but not CD4(+) cells depleted of CD44(v.low) cells, delay the onset of wasting when infused into either diabetic or prediabetic NOD recipients. However, no significant effect on the severity of diabetes was detected. In a model of cancer cachexia, they significantly reduce muscle atrophy, and inhibit muscle protein loss and DNA loss, even when given after the onset of cachexia. Protection from wasting and muscle atrophy by CD4(+)CD44(v.low) cells is associated with protection from lymphopenia. These data suggest, for the first time, a role for an immune cell subset in protection from cachexia, and further suggest that the mechanism of protection is independent of protection from autoimmunity.     

Wnt signaling induces the myogenic specification of resident CD45+ adult stem cells during muscle regeneration

The observation that CD45(+) stem cells injected into the circulation participate in muscle regeneration raised the question of whether CD45(+) stem cells resident in muscle play a physiological role during regeneration. We found that CD45(+) cells cultured from uninjured muscle were uniformly nonmyogenic. However, CD45(+) cells purified from regenerating muscle readily gave rise to determined myoblasts. The number of CD45(+) cells in muscle rapidly expanded following injury, and a high proportion entered the cell cycle. Investigation of candidate pathways involved in embryonic myogenesis revealed that Wnt signaling was sufficient to induce the myogenic specification of muscle-derived CD45(+) stem cells. Moreover, injection of the Wnt antagonists sFRP2/3 into regenerating muscle markedly reduced CD45(+) stem cell proliferation and myogenic specification. Our data therefore suggest that mobilization of resident CD45(+) stem cells is an important factor in regeneration after injury and highlight the Wnt pathway as a potential therapeutic target for degenerative neuromuscular disease.     

一种新的人胚胎干细胞自身来源的滋养层支持其体外培养

摘要: 通过人胚胎干细胞(Human embryonic stem cells, hESCs)经体内分化获取间充质干细胞(Mesenchymal stem cells, MSCs)为人胚胎干细胞提供一种新的滋养层。将约5×106个hESCs注射入重症免疫联合缺陷小鼠形成畸胎瘤, 8周后再从畸胎瘤中分离MSCs并鉴定, 将MSCs作为hESCs的滋养层细胞, 并检测和观察hESCs的生长情况、细胞特性和分化能力。从畸胎瘤中获得了纯度较高的具有类似骨髓来源的MSC特性的细胞群, 其形态相似、表面抗原标志相似(CD34和CD45阴性, CD29、CD49b、CD105、CD73和CD90阳性), 经诱导可以向成骨细胞和成脂细胞分化。将hESCs在MSCs滋养层细胞上传代培养10代以上, hESCs依然具有正常的细胞形态, 反转录PCR证实其特异转录因子Oct4、Nanog的表达, 干细胞表面标记SSEA-1显示为阴性, SSEA-4、TRA-1-60、TRA-1-81显示为阳性, 碱性磷酸酶染色显示为阳性, 并且核型正常。体外EB形成和体内畸胎瘤形成证明了其全能性。因此来源于hESCs本身的MSCs可以被用来作为支持胚胎干细胞生长并维持其未分化状态的滋养层细胞。     

沉默Dnmt1基因表达诱导大白鼠骨髓间充质干细胞向心肌样细胞分化

骨髓间充质干细胞(MSCs)具有向心肌样细胞分化的潜能.本室前期研究发现,MSCs在体外经DNA甲基转移酶(Dnmt)抑制剂5-氮胞苷诱导可分化为心肌样细胞.本研究证明,沉默DNA甲基化转移酶1（Dnmt1）基因表达,可诱导大鼠MSCs向心肌样细胞分化.本文采用表达Dnmt1 siRNA 慢病毒感染MSCs,沉默Dnmt1表达.DNA甲基化分析显示,随着沉默Dnmt1时间延长（7-28 d）,Gata-4基因上游DNA调控序列的CpG甲基化水平明显降低,而Gata-4 mRNA的转录水平明显上调,说明敲减Dnmt1表达导致Gata-4基因激活.蛋白质印迹和/或免疫细胞化学揭示,与对照组比较,心肌相关基因MHC 和cTnT表达上调, 而骨髓干细胞标志物CD90和CD29随转染时间延长表达下调.同时,实时定量PCR显示,心肌早期发育调控基因Nkx2.5 mRNA水平与Gata-4 mRNA相同,随表达Dnmt1 siRNA的慢病毒感染而上调.上述结果提示,敲减Dnmt1可降低心肌发育调控基因Gata-4启动子CpG岛的甲基化水平,上调Gata-4基因的表达,诱导骨髓间充质干细胞向心肌样分化.     

Acute and Temporal Expression of Tumor Necrosis Factor (TNF)-α-stimulated Gene 6 Product,TSG6, in Mesenchymal Stem Cells Creates Microenvironments Required for Their Successful Transplantation into Muscle Tissue

Previously, we demonstrated that when mesenchymal stem cells (MSCs) from mouse ES cells were transplanted into skeletal muscle, more than 60% of them differentiated into muscles in the crush-injured tibialis anterior muscle in vivo, although MSCs neither differentiated nor settled in the intact muscle. Microenvironments, including the extracellular matrix between the injured and intact muscle, were quite different. In the injured muscle, hyaluronan (HA), heavy chains of inter-α-inhibitor (IαI), CD44, and TNF-α-stimulated gene 6 product (TSG-6) increased 24–48 h after injury, although basement membrane components of differentiated muscle such as perlecan, laminin, and type IV collagen increased gradually 4 days after the crush. We then investigated the microenvironments crucial for cell transplantation, using the lysate of C2C12 myotubules for mimicking injured circumstances in vivo. MSCs settled in the intact muscle when they were transplanted together with the C2C12 lysate or TSG6. MSCs produced and released TSG6 when they were cultured with C2C12 lysates in vitro. MSCs pretreated with the lysate also settled in the intact muscle. Furthermore, MSCs whose TSG6 was knocked down by shRNA, even if transplanted or pretreated with the lysate, could not settle in the muscle. Immunofluorescent staining showed that HA and IαI always co-localized or were distributed closely, suggesting formation of covalent complexes, i.e. the SHAP-HA complex in the presence of TSG6. Thus, TSG6, HA, and IαI were crucial factors for the settlement and probably the subsequent differentiation of MSCs.     

The efficiency of in vitro isolation and myogenic differentiation of MSCs derived from adipose connective tissue,bone marrow,and skeletal muscle tissue

The objective of the study is to evaluate efficiency of in vitro isolation and myogenic differentiation of mesenchymal stem cells (MSCs) derived from adipose connective tissue (AD-MSCs), bone marrow (BM-MSCs), and skeletal muscle tissue (MC-MSCs). MSCs were isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue of two adult 6-wk-old rats. Cultured MSCs were treated with 5-azacytidine (AZA) to induce myogenic differentiation. Isolated MSCs and differentiated cells were evaluated by immunocytochemistry (ICC), fluorescence-activated cell sorting (FACS), PCR, and RT-PCR. AD-MSCs showed the highest proliferation rate while BM-MSCs had the lowest one. In ICC, isolated MSCs had strong CD90- and CD44-positive expression and negative expression of CD45, CD31, and CD34, while AZA-treated MSCs had strong positive desmin expression. In FACS analysis, AD-MSCs had the highest percentage of CD90- and CD44-positive-expressing cells (99% and 96%) followed by BM-MSCs (97% and 94%) and MC-MSCs (92% and 91%).At 1 wk after incubation with AZA treatment, the peak of myogenin expression reached 93% in differentiated MC-MSCs, 83.3% in BM-MSCs, and 77% in AD-MSCs. MSCs isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue have the same morphology and phenotype, but AD-MSCs were the most easily accessible and had the highest rate of growth on cultivation and the highest percentage of stem cell marker expression. Moreover, although MC-MSCs showed the highest rate of myogenic differentiation potential and expression of myoblast markers, AD-MSCs and BM-MSCs still can be valuable alternatives. The differentiated myoblastic cells could be an available new choice for myoblastic auto-transplantation in regeneration medicine.     

Human mesenchymal stem cells isolated from the umbilical cord

Mesenchymal stem cells (MSCs) are known as a population of multi-potential cells able to proliferate and differentiate into multiple mesodermal tissues including bone, cartilage, muscle, ligament, tendon, fat and stroma. In this study human MSCs were successfully isolated from the umbilical cords. The research characteristics of these cells, e.g., morphologic appearance, surface antigens, growth curve, cytogenetic features, cell cycle, differentiation potential and gene expression were investigated. After 2weeks of incubation, fibroblast-like cells appeared to be dominant. During the second passage the cells presented a homogeneous population of spindle fibroblast-like cells. After more than 4months (approximately 26 passages), the cells continued to retain their characteristics. Flow cytometry analysis revealed that CD29, CD44, CD95, CD105 and HLA-I were expressed on the cell surface, but there was no expression of hematopoietic lineage markers, such as CD34, CD38, CD71 and HLA-DR. Chromosomal analysis showed the cells kept a normal karyotype. The cell cycle at the third passage showed the percentage of G(0)/G(1), G(2)/M and S phase were 88.86%, 5.69% and 5.45%, respectively. The assays in vitro demonstrated the cells exhibited multi-potential differentiation into osteogenic and adipogenic cells. Both BMI-1 and nucleostemin genes, expressed in adult MSCs from bone marrow, were also expressed in umbilical cord MSCs. Here we show that umbilical cords may be a novel alternative source of human MSCs for experimental and clinical applications.     

Enhanced neointima formation following arterial injury in immune deficient Rag-1-/- mice is attenuated by adoptive transfer of CD8 T cells

T cells modulate neointima formation after arterial injury but the specific T cell population that is activated in response to arterial injury remains unknown. The objective of the study was to identify the T cell populations that are activated and modulate neointimal thickening after arterial injury in mice. Arterial injury in wild type C57Bl6 mice resulted in T cell activation characterized by increased CD4(+)CD44(hi) and CD8(+)CD44(hi) T cells in the lymph nodes and spleens. Splenic CD8(+)CD25(+) T cells and CD8(+)CD28(+) T cells, but not CD4(+)CD25(+) and CD4(+)CD28(+) T cells, were also significantly increased. Adoptive cell transfer of CD4(+) or CD8(+) T cells from donor CD8-/- or CD4-/- mice, respectively, to immune-deficient Rag-1-/- mice was performed to determine the T cell subtype that inhibits neointima formation after arterial injury. Rag-1-/- mice that received CD8(+) T cells had significantly reduced neointima formation compared with Rag-1-/- mice without cell transfer. CD4(+) T cell transfer did not reduce neointima formation. CD8(+) T cells from CD4-/- mice had cytotoxic activity against syngeneic smooth muscle cells in vitro. The study shows that although both CD8(+) T cells and CD4(+) T cells are activated in response to arterial injury, adoptive cell transfer identifies CD8(+) T cells as the specific and selective cell type involved in inhibiting neointima formation.