Current issues and regulations in tendon regeneration and musculoskeletal repair with mesenchymal stem cells

Mesenchymal stem cells are multipotent stromal cells residing within the connective tissue of most organs. Their surface phenotype has been well described. Most commonly, mesenchymal stem cells demonstrate the ability to differentiate into mesenchymal tissues (bone, catailge, fat, etc...), however, under the proper conditions these cells can differentiate into epithelial cells and neuroectoderm derived lineages. Their developmental plasticity also depends on the ability of mesenchymal stem cells to alter the tissue microenvironment by secreting soluble factors, as well as their capacity for differentiation in tissue repair. It is the cell-matrix interaction which defines the tissue characteristics. The molecular and functional heterogeneity of this cell population may confound interpretation of their differentiation potential, but it is this heterogeneity that is believed to provide for their therapeutic efficacy. Stem cell therapies are an attractive therapeutic approach for soft tissues as they offer a vehicle for repair and regeneration at the end of a needle. The early introduction of stem cell treatments into the therapeutic armamentarium involves both commercial and non-commercial multidisciplinary partnerships and has occurred in a climate of regulatory reform, so not all the relevant information resides in the public domain, but early clinical studies have shown promising results. Against this backdrop, novel techniques and early results of a small series of tendon and musculotendinous junction interventions are being published and other ongoing studies are yet to report their results. The issue of ensuring governance of these novel technologies falls upon both the scientific community and the established licensing authorities.     

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 originating from ES cells show high telomerase activity and therapeutic benefits

We establish a novel method for the induction and collection of mesenchymal stem cells using a typical cell surface marker, CD105, through adipogenesis from mouse ES cells. ES cells were cultured in a medium for adipogenesis. Mesenchymal stem cells from mouse ES cells were easily identified by the expression of CD105, and were isolated and differentiated into multiple mesenchymal cell types. Mesenchymal stem cells showed remarkable telomerase activity and sustained their growth for a long time with a high potential for differentiation involving skeletal myogenesis in vitro. When mesenchymal stem cells were transplanted into the injured tibialis anterior muscles, they differentiated into skeletal muscle cells in vivo. In addition, they improved the vascular formation, but never formed teratoma for longer than 6 months. Gene expression profiles revealed that mesenchymal stem cells lost pluripotency, while they acquired high potential to differentiate into mesenchymal cell lines. They thus indicate a promising new source of cell-based therapy without teratoma formation.     

In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells

Cardiomyocyte loss in the ischemically injured human heart often leads to irreversible defects in cardiac function. Recently, cellular cardiomyoplasty with mesenchymal stem cells, which are multipotent cells with the ability to differentiate into specialized cells under appropriate stimuli, has emerged as a new approach for repairing damaged myocardium. In the present study, the potential of human umbilical cord-derived mesenchymal stem cells to differentiate into cells with characteristics of cardiomyocyte was investigated. Mesenchymal stem cells were isolated from endothelial/subendothelial layers of the human umbilical cords using a method similar to that of human umbilical vein endothelial cell isolation. Isolated cells were characterized by transdifferentiation ability to adipocytes and osteoblasts, and also with flow cytometry analysis. After treatment with 5-azacytidine, the human umbilical cord-derived mesenchymal stem cells were morphologically transformed into cardiomyocyte-like cells and expressed cardiac differentiation markers. During the differentiation, cells were monitored by a phase contrast microscope and their morphological changes were demonstrated. Immunostaining of the differentiated cells for sarcomeric myosin (MF20), desmin, cardiac troponin I, and sarcomeric alpha-actinin was positive. RT-PCR analysis showed that these differentiated cells express cardiac-specific genes. Transmission electron microscopy revealed a cardiomyocyte-like ultrastructure and typical sarcomers. These observations confirm that human umbilical cord-derived mesenchymal stem cells can be chemically transformed into cardiomyocytes and can be considered as a source of cells for cellular cardiomyoplasty.     

Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep

Mesenchymal stem cells are multipotent cells that can be isolated from adult bone marrow and can be induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle. Despite their potential clinical utility for cellular and gene therapy, the fate of mesenchymal stem cells after systemic administration is mostly unknown. To address this, we transplanted a well-characterized human mesenchymal stem cell population into fetal sheep early in gestation, before and after the expected development of immunologic competence. In this xenogeneic system, human mesenchymal stem cells engrafted and persisted in multiple tissues for as long as 13 months after transplantation. Transplanted human cells underwent site-specific differentiation into chondrocytes, adipocytes, myocytes and cardiomyocytes, bone marrow stromal cells and thymic stroma. Unexpectedly, there was long-term engraftment even when cells were transplanted after the expected development of immunocompetence. Thus, mesenchymal stem cells maintain their multipotential capacity after transplantation, and seem to have unique immunologic characteristics that allow persistence in a xenogeneic environment. Our data support the possibility of the transplantability of mesenchymal stem cells and their potential utility in tissue engineering, and cellular and gene therapy applications.     

Therapeutic Potential of Human Adipose-Derived Stem Cells (ADSCs) from Cancer Patients: A Pilot Study

Mesenchymal stem cells from adipose tissue (ADSCs) are an important source of cells for regenerative medicine. The therapeutic effect of culture-expanded adipose derived stem cells has been shown; however, optimal xeno-free culture conditions remain to be determined. Cancer patients, specifically those undergoing invasive surgery, constitute a subgroup of patients who could benefit from autologous stem cell transplantation. Although regenerative potential of their ADSCs could be affected by the disease and/or treatment, we are not aware of any study that has evaluated the therapeutic potential of ADSCs isolated from cancer patients in reference to that of ADSCs derived from healthy subjects. Here we report that ADSCs isolated from subabdominal adipose tissue of patients with urological neoplasms yielded similar growth kinetics, presented equivalent mesenchymal surface markers and showed similar differentiation potential into distinct mesodermal cell lineages: adipocytes, chondroblasts and osteoblasts than ADSCs isolated from adipose tissue of age-matched non-oncogenic participants, all under xeno-free growth culture conditions. Molecular karyotyping of patient expanded ADSCs genomes showed no disease-related alterations indicating their safety. In addition, vesicles <100 nm identified as exosomes (EXOs) which may be at least partly responsible for the attributed therapeutic paracrine effects of the ADSCs were effectively isolated from ADSCs and showed equivalent miRNA content regardless they were derived from cancer patients or non-oncogenic participants indicating that the repair capabilities of xeno-free expanded ADSCs are not compromised by patient condition and therefore their xeno-free culture expanded ADSCs should be suitable for autologous stem cell transplantation in a clinical setting.     

Mesenchymal stem cells: Sources, phenotype, and differentiation potential

Mesenchymal stem cells present in the bone marrow and some other organs are primitive pluripotent precursors of osseous, cartilaginous, adipose, and other mesenchymal tissues. The recently revealed capacity of these cells for differentiation into nonmesenchymal derivatives is of considerable theoretical and practical interest. However, many aspects of the biology of these cells remain obscure despite active research. This review considers possible sources and methods for the isolation of mesenchymal stem cells, their potential for proliferation and differentiation in different directions, and outlooks of their therapeutic application. A model of parent-progeny relationships of stromal cells is proposed, and the problems of regulation of proliferation and differentiation of mesenchymal precursors as well as their role in the maintenance of regeneration and tissue functioning are discussed.     

Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction

Mesenchymal stem cells are multipotent cells that can differentiate into cardiomyocytes and vascular endothelial cells. Here we show, using cell sheet technology, that monolayered mesenchymal stem cells have multipotent and self-propagating properties after transplantation into infarcted rat hearts. We cultured adipose tissue-derived mesenchymal stem cells characterized by flow cytometry using temperature-responsive culture dishes. Four weeks after coronary ligation, we transplanted the monolayered mesenchymal stem cells onto the scarred myocardium. After transplantation, the engrafted sheet gradually grew to form a thick stratum that included newly formed vessels, undifferentiated cells and few cardiomyocytes. The mesenchymal stem cell sheet also acted through paracrine pathways to trigger angiogenesis. Unlike a fibroblast cell sheet, the monolayered mesenchymal stem cells reversed wall thinning in the scar area and improved cardiac function in rats with myocardial infarction. Thus, transplantation of monolayered mesenchymal stem cells may be a new therapeutic strategy for cardiac tissue regeneration.     

Mesenchymal stem cells: a promising candidate in regenerative medicine

Mesenchymal stem cells were initially characterized as plastic adherent, fibroblastoid cells. In recent years, there has been an increasing focus on mesenchymal stem cells since they have great plasticity and are potential for therapeutic applications. Mesenchymal stem cells or mesenchymal stem cell-like cells have been shown to reside within the connective tissues of most organs. These cells can differentiate into osteogenic, adipogenic and chondrogenic lineages under appropriate conditions. A number of reports have also indicated that these cells possess the capacity to trans-differentiate into epithelial cells and lineages derived from the neuro-ectoderm, and in addition, mesenchymal stem cells can migrate to the sites of injury, inflammation, and to tumors. These properties of mesenchymal stem cells make them promising candidates for use in regenerative medicine and may also serve as efficient delivery vehicles in site-specific therapy.     

The role of stem cells in cardiac regeneration

After myocardial infarction, injured cardiomyocytes are replaced by fibrotic tissue promoting the development of heart failure. Cell transplantation has emerged as a potential therapy and stem cells may be an important and powerful cellular source. Embryonic stem cells can differentiate into true cardiomyocytes, making them in principle an unlimited source of transplantable cells for cardiac repair, although immunological and ethical constraints exist. Somatic stem cells are an attractive option to explore for transplantation as they are autologous, but their differentiation potential is more restricted than embryonic stem cells. Currently, the major sources of somatic cells used for basic research and in clinical trials originate from the bone marrow. The differentiation capacity of different populations of bone marrow-derived stem cells into cardiomyocytes has been studied intensively. The results are rather confusing and difficult to compare, since different isolation and identification methods have been used to determine the cell population studied. To date, only mesenchymal stem cells seem to form cardiomyocytes, and only a small percentage of this population will do so in vitro or in vivo. A newly identified cell population isolated from cardiac tissue, called cardiac progenitor cells, holds great potential for cardiac regeneration. Here we discuss the potential of the different cell populations and their usefulness in stem cell based therapy to repair the damaged heart.     

Comparative proteomic analysis of human mesenchymal and embryonic stem cells: Towards the definition of a mesenchymal stem cell proteomic signature

Mesenchymal stem cells (MSC) are adult multipotential progenitors which have a high potential in regenerative medicine. They can be isolated from different tissues throughout the body and their homogeneity in terms of phenotype and differentiation capacities is a real concern. To address this issue, we conducted a 2‐DE gel analysis of mesenchymal stem cells isolated from bone marrow (BM), adipose tissue, synovial membrane and umbilical vein wall. We confirmed that BM and adipose tissue derived cells were very similar, which argue for their interchangeable use for cell therapy. We also compared human mesenchymal to embryonic stem cells and showed that umbilical vein wall stem cells, a neo‐natal cell type, were closer to BM cells than to embryonic stem cells. Based on these proteomic data, we could propose a panel of proteins which were the basis for the definition of a mesenchymal stem cell proteomic signature.     

间充质干细胞的细胞成像技术比较与应用

间充质干细胞是一类具有强大增殖、多向分化潜能和免疫调节能力的多功能细胞,研究显示间充质干细胞移植可能治疗多种难治性疾病,例如帕金森病、脊髓损伤以及肿瘤等。但是,人们对移植后的细胞在宿主内的存活、分布、增殖、分化、免疫排斥反应以及成瘤特性等问题尚不清楚,所以许多疾病经过细胞移植治疗后的进展及转归情况仍难以获得确切的科学证据。而细胞成像技术(包括放射性核素成像、超声成像、磁共振成像以及光学成像)可以在体外或者体内实现对间充质干细胞实时、无创的示踪,在以间充质干细胞为研究基础的细胞移植治疗和细胞组织再生的医学领域里有着巨大的应用潜力。该文综述近十年来细胞成像技术应用于示踪间充质干细胞移植疗法的研究进展,旨在比较当下多种热门细胞成像技术的优劣,进而找寻更合适的干细胞示踪策略,为干细胞移植治疗的基础和临床研究提供进一步的理论证据支持和研究思路。     

Ascorbic acid induces in vitro proliferation of human subcutaneous adipose tissue derived mesenchymal stem cells with upregulation of embryonic stem cell pluripotency markers Oct4 and SOX 2

Mesenchymal stem cells (MSCs) have immense therapeutic potential because of their ability to self-renew and differentiate into various connective tissue lineages. The in vitro proliferation and expansion of these cells is necessary for their use in stem cell therapy. Recently our group has developed and characterized mesenchymal stem cells from subcutaneous and visceral adipose tissue. We observed that these cells show a slower growth rate at higher passages and therefore decided to develop a supplemented medium, which will induce proliferation. Choi et al. have recently shown that the use of ascorbic acid enhances the proliferation of bone marrow derived MSCs. We therefore studied the effect of ascorbic acid on the proliferation of MSCs and characterized their phenotypes using stem cell specific molecular markers. It was observed that the use of 250 μM ascorbic acid promoted the significant growth of MSCs without loss of phenotype and differentiation potential. There was no considerable change in gene expression of cell surface markers CD105, CD13, Nanog, leukemia inhibitory factor (LIF) and Keratin 18. Moreover, the MSCs maintained in the medium supplemented with ascorbic acid for a period of 4 weeks showed increase in pluripotency markers Oct4 and SOX 2. Also cells in the experimental group retained the typical spindle shaped morphology. Thus, this study emphasizes the development of suitable growth medium for expansion of MSCs and maintenance of their undifferentiated state for further therapeutic use.     

WJSC 6th Anniversary Special Issues（2）:Mesenchymal stem cells Enhancing the efficacy of mesenchymal stem cell therapy

Mesenchymal stem cell(MSC)therapy is entering a challenging phase after completion of many preclinical and clinical trials.Among the major hurdles encountered in MSC therapy are inconsistent stem cell potency,poor cell engraftment and survival,and age/disease-related host tissue impairment.The recognition that MSCs primarily mediate therapeutic benefits through paracrine mechanisms independent of cell differentiation provides a promising framework for enhancing stem cell potency and therapeutic benefits.Several MSC priming approaches are highlighted,which will likely allow us to harness the full potential of adult stem cells for their future routine clinical use.     

Multilineage differentiation potential of equine blood-derived fibroblast-like cells

Tissue engineering (TE) has emerged as a promising new therapy for the treatment of damaged tissues and organs. Adult stem cells are considered as an attractive candidate cell type for cell-based TE. Mesenchymal stem cells (MSC) have been isolated from a variety of tissues and tested for differentiation into different cell lineages. While clinical trials still await the use of human MSC, horse tendon injuries are already being treated with autologous bone marrow-derived MSC. Given that the bone marrow is not an optimal source for MSC due to the painful and risk-containing sampling procedure, isolation of stem cells from peripheral blood would bring an attractive alternative. Adherent fibroblast-like cells have been previously isolated from equine peripheral blood. However, their responses to the differentiation conditions, established for human bone marrow MSC, were insufficient to fully confirm their multilineage potential. In this study, differentiation conditions were optimized to better evaluate the multilineage capacities of equine peripheral blood-derived fibroblast-like cells (ePB-FLC) into adipogenic, osteogenic, and chondrogenic pathways. Adipogenic differentiation using rabbit serum resulted in a high number of large-size lipid droplets three days upon induction. Cells' expression of alkaline phosphatase and calcium deposition upon osteogenic induction confirmed their osteogenic differentiation capacities. Moreover, an increase of dexamethasone concentration resulted in faster osteogenic differentiation and matrix mineralization. Finally, induction of chondrogenesis in pellet cultures resulted in an increase in cartilage-specific gene expression, namely collagen II and aggrecan, followed by protein deposition after a longer induction period. This study therefore demonstrates that ePB-FLC have the potential to differentiate into adipogenic, osteogenic, and chondrogenic mesenchymal lineages. The presence of cells with confirmed multilineage capacities in peripheral blood has important clinical implications for cell-based TE therapies in horses.     

Abnormal osteogenesis in osteoporotic patients is reflected by altered mesenchymal stem cells dynamics.

Bone marrow contains a population of mesenchymal stem cells with the ability to differentiate into cells that form bone, cartilage, adipose, and other connective tissues. Stem cells can be isolated from bone marrow aspirates and expanded in vitro. Presently, most stem cells studies have been performed in cells obtained from "healthy" control subjects. The goal of this study was to compare the functional characteristics of mesenchymal stem cells derived from "healthy" control and osteoporotic postmenopausal women to better understand the mechanisms involved in the pathogenesis of this disease. Osteoporotic and control stem cells have similar morphology and size and express similar cell surface antigens as evidenced by their reactivity with cell specific monoclonal antibodies. Mesenchymal stem cells from osteoporotic women differ from controls in having a lower growth rate than control cells, being refractory to the mitogenic effect of IGF-1, and exhibiting a deficient ability to differentiate into the osteogenic linage as evidenced by the alkaline phosphatase activity and calcium phosphate deposition. We conclude that in osteoporosis stem cell growth, proliferative response and osteogenic differentiation are significantly affected. Also, the study of mesenchymal stem cells from osteoporotic postmenopausal women may provide a better understanding of the mechanisms involved in the pathogenesis of the osteoporosis. It may also serve to test in vitro in rapid manner novel new therapeutic strategies.     

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.     

Fat pad-derived mesenchymal stem cells as a potential source for cell-based adipose tissue repair strategies

Background: Mesenchymal stem cells are able to undergo adipogenic differentiation and present a possible alternative cell source for regeneration and replacement of adipose tissue. The human infrapatellar fat pad is a promising source of mesenchymal stem cells with many source advantages over from bone marrow. It is important to determine whether a potential mesenchymal stem‐cell exhibits tri‐lineage differentiation potential and is able to maintain its proliferation potential and cell‐surface characterization on expansion in tissue culture. We have previously shown that mesenchymal stem cells derived from the fat pad can undergo chondrogenic and osteogenic differentiation, and we characterized these cells at early passage. In the study described here, proliferation potential and characterization of fat pad‐derived mesenchymal stem cells were assessed at higher passages, and cells were allowed to undergo adipogenic differentiation. Materials and methods: Infrapatellar fat pad tissue was obtained from six patients undergoing total knee replacement. Cells isolated were expanded to passage 18 and proliferation rates were measured. Passage 10 and 18 cells were characterized for cell‐surface epitopes using a range of markers. Passage 2 cells were allowed to undergo differentiation in adipogenic medium. Results: The cells maintained their population doubling rates up to passage 18. Cells at passage 10 and passage 18 had cell‐surface epitope expression similar to other mesenchymal stem cells previously described. By staining it was revealed that they highly expressed CD13, CD29, CD44, CD90 and CD105, and did not express CD34 or CD56, they were also negative for LNGFR and STRO1. 3G5 positive cells were noted in cells from both passages. These fat pad‐derived cells had adipogenic differentiation when assessed using gene expression for peroxisome proliferator‐activated receptor γ2 and lipoprotein lipase, and oil red O staining. Discussion: These results indicate that the cells maintained their proliferation rate, and continued expressing mesenchymal stem‐cell markers and pericyte marker 3G5 at late passages. These results also show that the cells were capable of adipogenic differentiation and thus could be a promising source for regeneration and replacement of adipose tissue in reconstructive surgery.     

Mesenchymal stem cell therapy and acute graft-versus-host disease: a review

Mesenchymal stem cells (MSCs) are being widely studied as potential cell therapy agents due to their immunomodulatory properties, which have been established by in vitro studies and in several clinical trials. Within this context, mesenchymal stem cell therapy appears to hold substantial promise, particularly in the treatment of conditions involving autoimmune and inflammatory components. Nevertheless, many research findings are still contradictory, mostly due to difficulties in characterization of the effects of MSCs in vivo. The purpose of this review is to report the mechanisms underlying mesenchymal stem cell therapy for acute graft-versus-host disease, particularly with respect to immunomodulation, migration, and homing, as well as report clinical applications described in the literature.