Human adipose stromal cells expanded in human serum promote engraftment of human peripheral blood hematopoietic stem cells in NOD/SCID mice

Human mesenchymal stem cells (hMSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles, and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle, and neuron. Therefore, hMSC are attractive candidates for cell and gene therapy. The optimal conditions for hMSC expansion require medium supplemented with fetal bovine serum (FBS). Some forms of cell therapy will involve multiple doses, raising a concern over immunological reactions caused by medium-derived FBS proteins. In this study, we cultured human adipose stromal cells (hADSC) and bone marrow stroma cells (HBMSC) in human serum (HS) during their isolation and expansion, and demonstrated that they maintain their proliferative capacity and ability for multilineage differentiation and promote engraftment of peripheral blood-derived CD34(+) cells mobilized from bone marrow in NOD/SCID mice. Our results indicate that hADSC and hBMSC cultured in HS can be used for clinical trials of cell and gene therapies, including promotion of engraftment after allogeneic HSC transplantation.     

Cell therapy for type-1 diabetes

A promising approach to treating type-1 diabetes mellitus is cell therapy. Nowadays, in laboratory experiments and clinical studies, allogeneic and xenogeneic cells of Langergance islets, bone marrow cells, hematopoietic stem cells, mesenchymal stem cells, and cord blood stem cells are used for transplantation. Any type of these cells correct the patient status to a certain extent; however, full recovery after cell therapy has not yet been achieved.     

间充质干细胞体外分化为多巴胺能神经元的研究进展

骨髓间充质干细胞（MSCs）有来源广泛、易于分离培养、不易引起免疫排斥等特点,使其成为细胞治疗和基因治疗的种子细胞,具有广泛的科研和临床应用价值。骨髓MSCs具有多向分化潜能,在特定条件下能诱导分化成神经元甚至是更为特异的多巴胺能神经元,为帕金森病进行细胞移植疗法提供了理想的细胞来源。本文就近年来体外诱导MSCs向多巴胺能神经元定向分化所涉及到的常用诱导因素和诱导方法及途径予以综述。     

Adult mesenchymal stem cells: a pluripotent population with multiple applications

Mesenchymal stem cells (MSCs) have been isolated not only from bone marrow, but also from many other tissues such as adipose tissue, skeletal muscle, liver, brain and pancreas. Because MSC were found to have the ability to differentiate into cells of multiple organs and systems such as bone, fat, cartilage, muscle, neurons, hepatocytes and insulin-producing cells, MSCs have generated a great deal of interest for their potential use in regenerative medicine and tissue engineering. Furthermore, given the ease of their isolation and their extensive expansion rate and differentiation potential, mesenchymal stem cells are among the first stem cell types that have a great potential to be introduced in the clinic. Finally, mesenchymal stem cells seem to be not only hypoimmunogenic and thus be suitable for allogeneic transplantation, but they are also able to produce immunosuppression upon transplantation. In this review we summarize the latest research in the use of mesenchymal stem cells in transplantation for generalized diseases, local implantation for local tissue defects, and as a vehicle for genes in gene therapy protocols.     

Mesenchymal stem cells

Within the bone marrow stroma there exists a subset of nonhematopoietic cells referred to as mesenchymal stem or mesenchymal progenitor cells. These cells can be ex vivo expanded and induced, either in vitro or in vivo, to terminally differentiate into osteoblasts, chondrocytes, adipocytes, tenocytes, myotubes, neural cells, and hematopoietic-supporting stroma. The multipotential of these cells, their easy isolation and culture, as well as their high ex vivo expansive potential make these cells an attractive therapeutic tool. In this work we will review the information dealing with the biology of mesenchymal progenitors as it has been revealed mainly by ex vivo studies performed with bone marrow-derived cells. The discussed topics include, among others, characteristics of mesenchymal progenitors, evidence for the existence of a vast repertoire of uncommitted and committed progenitors both in the bone marrow and in mesenchymal tissues, a diagram for their proliferative hierarchy, and comments on mobilization, microenvironment, and clinical use of mesenchymal progenitors. Despite the enormous data available at molecular and cellular levels, it is evident that a number of fundamental questions still need to be resolved before mesenchymal progenitors can be used for safe and effective clinical applications in the context of both cell and gene therapies.     

Homeostasis of adult stem cells and carcinogenesis

Treatment of malignant tumors using radiotherapy, chemotherapy, immunosuppressive drugs required to recover bone marrow transplant by the donor bone marrow or purified adult stem cells. During the next 1-15 years of follow up of these patients compared with healthy individuals of the same age increases the risk of multiple malignancies. It used to be attributed to the influence of therapeutic effects. However, it is revealed that some of the cells and the stroma of the secondary tumors are composed of descendants of transplanted stem cells. This indicates the important role of stem cells in tumor growth. This is also evidenced by numerous studies showing that adult stem cells from both mice and humans multiplied in vitro, after transplantation into the body give the sarcoma, cancer foci and other types of malignant growth. In this, malignant growth is most intense in the presence of focal chronic inflammation. No less than the experimental data, including those obtained in humans suggest that the transplanted stem cells actively colonize stroma of tumor tissue, stimulating the growth of the tumor and its metastasis. The human condition of survival is the presence of rigid homeostatic control mechanisms of low numbers of stem cells in the body and the limit their division, even in areas of regeneration. After the transplantation of stem cells their number in the bloodstream and, consequently, in the pathological foci of regeneration, increases in many dozens of times--this level can not be achieved by the organism itself. This leads to a sharp increase in the rate of regeneration of tissues, which creates conditions for amplification of malignant growth.     

Effects of autologous stem cell transplantation on ventricular electrophysiology in doxorubicin-induced heart failure

To investigate whether stem cell transplantation affects ventricular electrophysiology in vivo, either autologous bone marrow mesenchymal stem cells or skeletal myoblast cells were transplanted via a catheter into a doxorubicin-treated failing heart. Four weeks after transplantation, electrophysiological investigation showed that transplantation of either cell type prolonged the local activation time and increased the activation time dispersion. In the stem cell transplantation groups, a positive correlation was demonstrated between activation time dispersion and the number of stem cell-derived cells in the pacing site. It is concluded that transplantation of either mesenchymal stem cells or skeletal myoblast cells might exacerbate abnormalities of local ventricular conduction in the doxorubicin-treated failing heart.     

Homeostasis of adult human stem cells and carcinogenesis

Treatment of malignant diseases with radiation therapy, chemotherapy, and immunodepressants requires subsequent restoration of bone marrow by the use of transplantation of donor bone marrow or separated adult stem cells to the body. During the next 1–15 years, in these patients, the risk of malignant neoplasia substantially increases as compared to healthy persons. This was previously considered as the effect of treatment. However, it has been found that part of cells and the stroma of a secondary tumor consist of progenies of transplanted stem cells. This demonstrates an important role of stem cells in tumorigenesis. Numerous studies also show that adult mouse or human stem cells cultured in vitro can form foci of sarcoma, cancer or other types of malignant growth. Malignant growth is more intense when chronic inflammation is present in the body. A lot of experimental data including studies in humans demonstrated that, after transplantation, stem cells actively occupy the tumor stroma, stimulate tumorigenesis and its metastasis. An important condition of human life is the presence of strong homeostatic mechanisms that control the number of stem cells in the body and limit their division even in regeneration foci. After transplantation of stem cells, their number in the blood and, correspndingly, in a pathological regeneration location increases by the dozens. This level of cells in the body cannot be reached spontaneously. This significantly enhances the rate of tissue regeneration, which creates conditions for malignant growth.     

人鼠肝组织嵌合体模型的制备

目的研究制备人鼠肝组织嵌合小鼠模型。方法将人骨髓干细胞直接注射到一定日龄胎鼠肝组织,每只注射移植约1×109人骨髓干细胞。用免疫组化对出生一定日龄移植小鼠肝脏进行甲胎蛋白免疫组织化学检测,检定分析人肝细胞在小鼠体内嵌合生长情况。结果移植人骨髓干细胞胎鼠出生2月龄、12月龄可检测到甲胎蛋白。结论将人骨髓干细胞移植小鼠肝脏内能够存活并分化成人肝细胞并能够长期存活。     

Functional expression of human CD8 in fully reconstituted mice after retroviral-mediated gene transfer of hemopoietic stem cells.

Retroviral-mediated gene transfer has been used in an attempt to efficiently and stably express functional cell-surface molecules in lymphoid and myeloid cells. The human CD8 molecule is a T cell-specific surface receptor that is intimately involved in class I MHC-restricted Ag recognition and subsequent T cell activation. After infection with a recombinant, replication-defective retrovirus containing the human CD8 alpha cDNA, bone marrow cells were transplanted into lethally irradiated recipients. The majority of lymphoid and myeloid cells of reconstituted animals expressed high levels of human CD8 for at least 8 months after transplantation. Transfer of bone marrow and spleen cells from these recipients 100 days after transplantation into secondary recipients also resulted in long term expression of CD8 in lymphoid and myeloid cells. CD8 expressed in splenic T cells associated with the lymphoid-specific tyrosine protein kinase p56lck, participated in T cell activation and conferred an increased xenogeneic response to human MHC class I Ag. Thus, retroviral-mediated gene transfer allows the long term, functional expression of cell-surface molecules in normal murine lymphoid and myeloid cells.     

Adult mesenchymal stem cells and cell-based tissue engineering

The identification of multipotential mesenchymal stem cells (MSCs) derived from adult human tissues, including bone marrow stroma and a number of connective tissues, has provided exciting prospects for cell-based tissue engineering and regeneration. This review focuses on the biology of MSCs, including their differentiation potentials in vitro and in vivo, and the application of MSCs in tissue engineering. Our current understanding of MSCs lags behind that of other stem cell types, such as hematopoietic stem cells. Future research should aim to define the cellular and molecular fingerprints of MSCs and elucidate their endogenous role(s) in normal and abnormal tissue functions.     

Human fetal mesenchymal stem cells

Stem cells have been isolated at all stages of development from the early developing embryo to the post-reproductive adult organism. However, the fetal environment is unique as it is the only time in ontogeny that there is migration of stem cells in large numbers into different organ compartments. While fetal neural and haemopoietic stem cells (HSC) have been well characterised, only recently have mesenchymal stem cells from the human fetus been isolated and evaluated. Our group have characterised in human fetal blood, liver and bone marrow a population of non-haemopoietic, non-endothelial cells with an immunophenotype similar to adult bone marrow-derived mesenchymal stem cells (MSC). These cells, human fetal mesenchymal stem cells (hfMSC), are true multipotent stem cells with greater self-renewal and differentiation capacity than their adult counterparts. They circulate in first trimester fetal blood and have been found to traffic into the maternal circulation, engrafting in bone marrow, where they remain microchimeric for decades after pregnancy. Though fetal microchimerism has been implicated in the pathogenesis of autoimmune disease, the biological role of hfMSC microchimerism is unknown. Potential downstream applications of hfMSC include their use as a target cell for non-invasive pre-natal diagnosis from maternal blood, and for fetal cellular and gene therapy. Using hfMSC in fetal therapy offers the theoretical advantages of avoidance of immune rejection, increased engraftment, and treatment before disease pathology sets in. Aside from allogeneic hfMSC in utero transplantation, the use of autologous hfMSC has been brought a step forward with the development of early blood sampling techniques, efficient viral transduction and clonal expansion. Work is ongoing to determine hfMSC fate post-transplantation in murine models of genetic disease. In this review we will examine what is known about hfMSC biology, as well as discussing areas for future research. The implications of hfMSC trafficking in pregnancy will be explored and the potential clinical applications of hfMSC in prenatal diagnosis and fetal therapy discussed.     

Adult mesenchymal stem cells and cell-based tissue engineering

The identification of multipotential mesenchymal stem cells (MSCs) derived from adult human tissues, including bone marrow stroma and a number of connective tissues, has provided exciting prospects for cell-based tissue engineering and regeneration. This review focuses on the biology of MSCs, including their differentiation potentials in vitro and in vivo, and the application of MSCs in tissue engineering. Our current understanding of MSCs lags behind that of other stem cell types, such as hematopoietic stem cells. Future research should aim to define the cellular and molecular fingerprints of MSCs and elucidate their endogenous role(s) in normal and abnormal tissue functions.     

Behavior of transplanted bone marrow-derived GFP mesenchymal cells in osteochondral defect as a simulation of autologous transplantation.

To elucidate the behavior of autologously transplanted mesenchymal cells in osteochondral defects, we followed transplanted cells using green fluorescent protein (GFP) transgenic rats, in which all cells express GFP signals in their cytoplasm and nuclei as transplantation donors. Bone marrow-derived mesenchymal cells, which contain mesenchymal stem cells (MSCs), were obtained from transgenic rats. Then, dense mesenchymal cell masses created by hanging-drop culture were transplanted and fixed with fibrin glue into osteochondral defects of wild-type rats. At 24 weeks after surgery, the defects were repaired with hyaline-like cartilage and subchondral bone. GFP positive cells, indicating transplanted mesenchymal-derived cells, were observed in the regenerated tissues for 24 weeks although GFP positive cells decreased in number with time. Because GFP causes no immunological rejection and requires no chemicals for visualization, transplantation between transgenic and wild-type rats can be regarded as a simulation of autologous transplantation, and the survivability of transplanted cells are able to be followed easily and reliably. Thus, the behavior of transplanted mesenchymal cells was able to be elucidated in vivo by this strategy, and the results could be essential in future tissue engineering for the regeneration of osteochondral defects with original hyaline cartilage and subchondral bone.     

From bone marrow to therapeutic applications: different behaviour and genetic/epigenetic stability during mesenchymal stem cell expansion in autologous and foetal bovine sera?

Bone marrow-derived mesenchymal stem cells are a multipotent adult cellular population endowed with broad differentiation potential. Their regeneration capability, ease to undergo gene modifications, and immuno-suppressive capacity makes them optimal tools for tissue engineering, gene- and immuno-therapy. Due to the ever-increasing number of studies on the clinical applications of mesenchymal stem cells in regenerative medicine, these cells have become attractive targets in clinical transplantation. However, the identification and definition of mesenchymal stem cell culture media for their clinical application in cell therapy is currently a matter of strong discussion. Up to now, clinical studies have been conducted with mesenchymal stem cells cultured in foetal calf serum, and the chance of contamination or immunological reaction towards xenogeneic compounds must be taken into consideration. On the other hand, a serum-free medium without the addition of growth factors is not able to expand these cells in vitro; so the evaluation of which is best, among foetal calf serum, human serum (whether autologous or allogeneic) and platelet-rich plasma, is a hot topic urgently needing further research efforts. The need for the establishment of standardized protocols for mesenchymal stem cell preparations, in order not to interfere with their self-renewal and differentiation processes, assuring durable engraftment and long-term therapeutic effects, is evidently crucial. Therefore, the search for optimal culture conditions for the effective clinical-scale production of vast numbers of mesenchymal stem cells for cellular therapy is of paramount importance and the need for a robust passage from basic to translational research is fundamental.     

Impact of bone marrow-derived mesenchymal stromal cells on experimental xenogeneic graft-versus-host disease

Background aimsGraft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous studies have suggested that mesenchymal stromal cells (MSCs) could exert potent immunosuppressive effects.MethodsThe ability of human bone marrow derived MSCs to prevent xenogeneic GVHD in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and in NOD/SCID/interleukin-2Rγ(null) (NSG) mice transplanted with human peripheral blood mononuclear cells (PBMCs) was assessed.ResultsInjection of 200 × 10⁶ human PBMCs intraperitoneally (IP) into sub-lethally (3.0 Gy) irradiated NOD/SCID mice also given anti-asialo GM₁ antibodies IP 1 day prior and 8 days after transplantation induced lethal xenogeneic GVHD in all tested mice. Co-injection of 2 × 10⁶ MSCs IP on day 0 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Similarly, injection of 30 × 10⁶ human PBMCs IP into sub-lethally (2.5 Gy) irradiated NSG mice induced a lethal xenogeneic GVHD in all tested mice. Injection of 3 × 10⁶ MSCs IP on days 0, 7, 14 and 21 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs.ConclusionsInjection of MSCs did not prevent xenogeneic GVHD in these two humanized mice models.     

CD271 as a marker to identify mesenchymal stem cells from diverse sources before culture

Mesenchymal stem cells, due to their characteristics are ideal candidates for cellular therapy. Currently, in culture these cells are defined by their adherence to plastic, specific surface antigen expression and multipotent differentiation potential. However, the in vivo identification of mesenchymal stem cells, before culture, is not so well established. Pre-culture identification markers would ensure higher purity than that obtained with selection based on adherence to plastic. Up until now, CD271 has been described as the most specific marker for the characterization andpurification of human bone marrow mesenchymal stem cells. This marker has been shown to be specifically expressed by these cells. Thus, CD271 has been proposed as a versatile marker to selectively isolated and expand multipotent mesenchymal stem cells with both immunosuppressive and lymphohematopoietic engraftment-promoting properties. This review focuses on this marker, specifically on identification of mesenchymal stem cells from different tissues. Literature revision suggests that CD271 should not be defined as a universal marker to identify mesenchymal stem cells before culture from different sources. In the case of bone marrow or adipose tissue, CD271 could be considered a quite suitable marker; however this marker seems to be inadequate for the isolation of mesenchymal stem cells from other tissues such as umbilical cord blood or wharton’s jelly among others.     

骨髓间充质干细胞在大鼠实验中的移植途径及比较

骨髓间充质干细胞是具有自我更新能力和分化潜能的一类成体干细胞,经过局部微环境的诱导,可在体内外进行扩展,到晚期可分化成为多种细胞系。当组织受损伤时,可迅速到达损伤部位,分化为特异的组织细胞,参与组织修复。骨髓间充质干细胞这种惊人的分化及组织修复能力,为治疗退行性疾病和器官损伤性疾病提供广阔前景,故成为科研热点。国内外相关实验研究多以大鼠为动物模型,而骨髓间充质干细胞如何进入大鼠体内并定植,是实验成功的重要前提。因此如何找到最合适、最安全的移植途径将骨髓间充质干细胞有效地移植进入大鼠疾病模型体内的受损区域,是研究者关心的重点。本文就目前骨髓间充质干细胞在大鼠实验中不同移植途径进行综述,并比较各种途径的优缺点,希望能对临床科研工作提供参考,并期待能有更成熟的移植手段来推动骨髓间充质干细胞实验研究的进展。     

Induction of decidual differentiation in endometrial mesenchymal stem cells

In this study, we compared the ability of human mesenchymal stem cells (eMSCs) derived from menstrual blood and mesenchymal stem cells (MSCs) from other tissues to differentiate into decidual cells in vitro. It was demonstrated that, during differentiation, secretion of prolactin and insulin-like growth factor binding protein-1 (key decidualization markers) markedly increased in eMSCs slightly augmented in bone marrow MSC (BM-MSCs) and did not change in MSCs from adipose tissue (AT-MSCs). Thus, eMSCs exhibited higher capacity for differentiation into decidual cells than BM-MSCs or AT-MSCs. This makes eMSCs promising for application in cellular therapy of infertility associated with insufficient decidualization of endometrium.