Human mesenchymal stem cells - current trends and future prospective

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton''s jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.     

Mesenchymal stem cells and their use in therapy: What has been achieved?

The considerable therapeutic potential of human multipotent mesenchymal stromal cells or mesenchymal stem cells (MSCs) has generated increasing interest in a wide variety of biomedical disciplines. Nevertheless, researchers report studies on MSCs using different methods of isolation and expansion, as well as different approaches to characterize them; therefore, it is increasingly difficult to compare and contrast study outcomes. To begin to address this issue, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposed minimal criteria to define human MSCs. First, MSCs must be plastic-adherent when maintained in standard culture conditions (α minimal essential medium plus 20% fetal bovine serum). Second, MSCs must express CD105, CD73 and CD90, and MSCs must lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules. Third, MSCs must differentiate into osteoblasts, adipocytes and chondroblasts in vitro. MSCs are isolated from many adult tissues, in particular from bone marrow and adipose tissue. Along with their capacity to differentiate and transdifferentiate into cells of different lineages, these cells have also generated great interest for their ability to display immunomodulatory capacities. Indeed, a major breakthrough was the finding that MSCs are able to induce peripheral tolerance, suggesting that they may be used as therapeutic tools in immune-mediated disorders. Although no significant adverse events have been reported in clinical trials to date, all interventional therapies have some inherent risks. Potential risks for undesirable events, such as tumor development, that might occur while using these stem cells for therapy must be taken into account and contrasted against the potential benefits to patients.     

Multipotent mesenchymal stem cells of desquamated endometrium: Isolation,characterization, and application as a feeder layer for maintenance of human embryonic stem cells

In this study, we characterize new multipotent human mesenchymal stem cell lines (MSCs) derived from desquamated (shedding) endometrium of menstrual blood. The isolated endometrial MSC (eMSC) is an adhesive to plastic heterogeneous population composed mainly of endometrial glandular and stromal cells. The established cell lines meet the criteria of the International Society for Cellular Therapy for defining multipotent human MSCs of any origin. The eMSCs have positive expression of CD13, CD29, CD44, CD73, CD90, and CD105 markers and lack hematopoietic cell surface antigens CD19, CD34, CD45, CD117, CD130, and HLA-DR (class II). Multipotency of the established eMSCs is confirmed by their ability to differentiate into other mesodermal lineages, such as osteocytes and adipocytes. In addition, the isolated eMSCs partially (over 50%) express the pluripotency marker SSEA-4. However, they do not express Oct-4. Immunofluorescent analysis of the derived cells revealed the expression of the neural precursor markers nestin and β-III-tubulin. This suggests a neural predisposition of the established eMSCs. These cells are characterized by a high proliferation rate (doubling time 22–23 h) and a high colony-forming efficiency (about 60%). In vitro, the eMSCs undergo more than 45 population doublings without karyotypic abnormalities. We demonstrate that mitotically inactivated eMSCs are perfect feeder cells for maintenance of human embryonic stem cell lines (hESCs) C612 and C910. The eMSCs, being a feeder culture, sustain the hESC pluripotent status that verified by expression of Oct-4, alkaline phosphatase and SSEA-4 markers. The hESCs cocultured with the eMSCs retain their morphology and proliferative rate for more than 40 passages and exhibit the capability for spontaneous differentiation into embryoid bodies comprising three embryonic germ layers. Thus, an easy and noninvasive isolation of the eMSCs from menstrual blood, their multipotency and high proliferative activity in vitro without karyotypic abnormalities demonstrate the potential of use of these stem cells in regenerative medicine. Using the derived eMSCs as the feeder culture eliminates the risks associated with animal cells while transferring hESCs to clinical setting.     

Isolation of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells

The presence within bone marrow of a population of mesenchymal stem cells (MSCs) able to differentiate into a number of different mesenchymal tissues, including bone and cartilage, was first suggested by Friedenstein nearly 40 years ago. Since then MSCs have been demonstrated in a variety of fetal and adult tissues, including bone marrow, fetal blood and liver, cord blood, amniotic fluid and, in some circumstances, in adult peripheral blood. MSCs from all of these sources can be extensively expanded in vitro and when cultured under specific permissive conditions retain their ability to differentiate into multiple lineages including bone, cartilage, fat, muscle, nerve, glial and stromal cells. There has been great interest in these cells both because of their value as a model for studying the molecular basis of differentiation and because of their therapeutic potential for tissue repair and immune modulation. However, MSCs are a rare population in these tissues. Here we tried to identify cells with MSC-like potency in human placenta. We isolated adherent cells from trypsin-digested term placentas and examined these cells for morphology, surface markers, and differentiation potential and found that they expressed several stem cell markers. They also showed endothelial and neurogenic differentiation potentials under appropriate conditions. We suggest that placenta-derived cells have multilineage differentiation potential similar to MSCs in terms of morphology and cell-surface antigen expression. The placenta may prove to be a useful source of MSCs.     

An efficient method for isolation of murine bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) have been isolated based on the ability of adherence to plastic surfaces. The potential of these cells to differentiate along multiple lineages is the key to identifying stem cell populations in the absence of molecular markers. Here we describe a homogenous population of MSCs from mouse bone marrow isolated using a relatively straightforward and novel approach. This method is based on the combination of frequent medium change (FMC) and treatment of the primary cultures with trypsin. Cells isolated using this method demonstrated the MSCs characteristics including their ability to differentiate into mesenchymal lineages. MSCs retained the differentiation potentials in expanded cultures up to 10 passages. Isolated MSCs were reactive to the CD44, Sca-1, and CD90 cell surface markers. MSCs were negative for the hematopoietic surface markers such as CD34, CD11b, CD45, CD31, CD106, CD117 and CD135. The data presented in this report indicated that this method can result in efficient isolation of homogenous populations of MSCs from mouse bone marrow.     

Derivation,characterization and gene modification of cynomolgus monkey mesenchymal stem cells

Mesenchymal stem cells (MSCs) have received considerable attention in recent years. Particularly exciting is the prospect that MSCs could be differentiated into specialized cells of interest, which could then be used for cell therapy and tissue engineering. MSCs derived from nonhuman primates could be a powerful tool for investigating the differentiation potential in vitro and in vivo for preclinical research. The purpose of this study was to isolate cynomolgus mesenchymal stem cells (cMSCs) from adult bone marrow and characterize their growth properties and multipotency. Mononuclear cells were isolated from cynomolgus monkey bone marrow by density-gradient centrifugation, and adherent fibroblast-like cells grew well in the complete growth medium with 10 μM Tenofovir. cMSCs expressed mesenchymal markers, such as CD29, CD105, CD166 and were negative for hematopoietic markers such as CD34, CD45. Furthermore, the cells were capable of differentiating into osteogenic, chondrogenic, and adipogenic lineages under certain conditions, maintaining normal karyotype throughout extended culture. We also compared different methods (lipofection, nucleofection and lentivirus) for genetic modification of cMSCs and found lentivirus proved to be the most effective method with transduction efficiency of up to 44.6% and lowest level of cell death. The cells after transduction stably expressed green fluorescence protein (GFP) and maintained the abilities to differentiate down osteogenic and adipogenic lineages. In conclusion, these data showed that cMSCs isolated from cynomolgus bone marrow shared similar characteristics with human MSCs and might provide an attractive cell type for cell-based therapy in higher-order mammalian species disorder models.     

Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow

Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). MSCs possess self-renewal capacity and pluripotency defined by their ability to differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells. MSCs are also known to differentiate into neurons and glial cells in vitro, and in vivo following transplantation into the brain of animal models of neurological disorders including ischemia and intracerebral hemorrhage (ICH) stroke. In order to obtain sufficient number and homogeneous population of human MSCs, we have clonally isolated permanent and stable human MSC lines by transfecting primary cell cultures of fetal human bone marrow MSCs with a retroviral vector encoding v-myc gene. One of the cell lines, HM3.B10 (B10), was found to differentiate into neural cell types including neural stem cells, neurons, astrocytes and oligodendrocytes in vitro as shown by expression of genetic markers for neural stem cells (nestin and Musashi1), neurons (neurofilament protein, synapsin and MAP2), astrocytes (glial fibrillary acidic protein, GFAP) and oligodendrocytes (myelin basic protein, MBP) as determined by RT-PCR assay. In addition, B10 cells were found to differentiate into neural cell types as shown by immunocytochical demonstration of nestin (for neural stem cells), neurofilament protein and beta-tubulin III (neurons) GFAP (astrocytes), and galactocerebroside (oligodendrocytes). Following brain transplantation in mouse ICH stroke model, B10 human MSCs integrate into host brain, survive, differentiate into neurons and astrocytes and induce behavioral improvement in the ICH animals. B10 human MSC cell line is not only a useful tool for the studies of organogenesis and specifically for the neurogenesis, but also provides a valuable source of cells for cell therapy studies in animal models of stroke and other neurological disorders.     

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.     

Ovarian stem cells: From basic to clinical applications

The field of reproductive biology has undergone significant developments in the last decade. The notion that there is a fixed reserve pool of oocytes before birth was established by Zuckerman in 1951. However, in 2004, an article published in nature challenged this central dogma of mammalian reproductive biology. Tilly’s group reported the existence of ovarian germline stem cells (GSCs) in postnatal ovaries of mice and suggested that the bone marrow could be an extragonadal source of ovarian GSCs. These findings were strongly criticized; however, several independent groups have since successfully isolated and characterized ovarian GSCs in postnatal mice. The ovarian GSCs are located in the ovarian surface epithelium and express markers of undifferentiated GSCs. When transplanted into mouse ovaries, mouse ovarian GSCs could differentiate and produce embryos and offspring. Similarly, in a recent study, ovarian GSCs were found to be present in the ovaries of women of reproductive age. Conversely, there is increasing evidence that stem cells responsible for maintaining a healthy state in normal tissue may be a source of some cancers, including ovarian cancer. Cancer stem cells (CSCs) have been found in many tissues, including ovaries. Some researchers have suggested that ovarian cancer may be a result of the transformation and dysfunction of ovarian GSCs with self-renewal properties. Drug resistant and metastasis-generating CSCs are responsible for many important problems affecting ovarian cancer patients. Therefore, the identification of CSCs will provide opportunities for the development of new therapeutic strategies for treatments for infertility and ovarian cancer. In this article, we summarize the current understanding of ovarian GSCs in adult mammals, and we also discuss whether there is a relationship between GSCs and CSCs.     

Human adipose tissue is a source of multipotent stem cells

Much of the work conducted on adult stem cells has focused on mesenchymal stem cells (MSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.     

Fetal mesenchymal stem cells derived from human umbilical cord sustain primitive characteristics during extensive expansion

Stem cells of fetal origin lie between embryonic and adult stem cells in terms of potentiality. Because of the ethical controversy surrounding embryonic stem cells and the relatively inferior quality of adult stem cells, the use of fetal stem cells would be an attractive option in future therapeutic applications. Here, we have investigated primitive characteristics of human umbilical-cord-derived fetal mesenchymal stem cells (UC fMSCs) during extensive expansion. We have successfully isolated and cultured UC fMSCs from all UC samples, but with two early fungal contaminations. UC fMSCs proliferated without significant evidence of morphological changes, and the average cumulative population-doubling level was over 25 for about 3 months. UC fMSCs showed the positive expression of several CD markers, known to be related to MSCs, including CD73 (SH-3, 4), CD90 (Thy-1), CD105 (SH-2), CD117 (c-kit), and CD166 (ALCAM). They demonstrated primitive properties throughout the expansion period: multilineage differentiation potentials examined by functional assays, a variety of pluripotent stem cell markers including Nanog, Oct-4, Sox-2, Rex-1, SSEA-3, SSEA-4, Tra-1–60, and Tra-1–81, minimal evidence of senescence as shown by β-galactosidase staining, and the consistent expression of telomerase activity. These results suggest that UC fMSCs have more primitive properties than adult MSCs, which might make them a useful source of MSCs for clinical applications. This work was supported by the Seoul R&BD Program (10548).     

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.Download video file.^{(94M, mp4)}     

The adult human brain harbors multipotent perivascular mesenchymal stem cells

Blood vessels and adjacent cells form perivascular stem cell niches in adult tissues. In this perivascular niche, a stem cell with mesenchymal characteristics was recently identified in some adult somatic tissues. These cells are pericytes that line the microvasculature, express mesenchymal markers and differentiate into mesodermal lineages but might even have the capacity to generate tissue-specific cell types. Here, we isolated, purified and characterized a previously unrecognized progenitor population from two different regions in the adult human brain, the ventricular wall and the neocortex. We show that these cells co-express markers for mesenchymal stem cells and pericytes in vivo and in vitro, but do not express glial, neuronal progenitor, hematopoietic, endothelial or microglial markers in their native state. Furthermore, we demonstrate at a clonal level that these progenitors have true multilineage potential towards both, the mesodermal and neuroectodermal phenotype. They can be epigenetically induced in vitro into adipocytes, chondroblasts and osteoblasts but also into glial cells and immature neurons. This progenitor population exhibits long-term proliferation, karyotype stability and retention of phenotype and multipotency following extensive propagation. Thus, we provide evidence that the vascular niche in the adult human brain harbors a novel progenitor with multilineage capacity that appears to represent mesenchymal stem cells and is different from any previously described human neural stem cell. Future studies will elucidate whether these cells may play a role for disease or may represent a reservoir that can be exploited in efforts to repair the diseased human brain.     

Comparison of the Neural Differentiation Potential of Human Mesenchymal Stem Cells from Amniotic Fluid and Adult Bone Marrow

Human mesenchymal stem cells (MSCs) are considered a promising tool for cell-based therapies of nervous system diseases. Bone marrow (BM) has been the traditional source of MSCs (BM-MSCs). However, there are some limitations for their clinical use, such as the decline in cell number and differentiation potential with age. Recently, amniotic fluid (AF)-derived MSCs (AF-MSCs) have been shown to express embryonic and adult stem cell markers, and can differentiate into cells of all three germ layers. In this study, we isolated AF-MSCs from second-trimester AF by limiting dilution and compared their proliferative capacity, multipotency, neural differentiation ability, and secretion of neurotrophins to those of BM-MSCs. AF-MSCs showed a higher proliferative capacity and more rapidly formed and expanded neurospheres compared to those of BM-MSCs. Both immunocytochemical and quantitative real-time PCR analyses demonstrated that AF-MSCs showed higher expression of neural stemness markers than those of BM-MSCs following neural stem cell (NSC) differentiation. Furthermore, the levels of brain-derived growth factor and nerve growth factor secreted by AF-MSCs in the culture medium were higher than those of BM-MSCs. In addition, AF-MSCs maintained a normal karyotype in long-term cultures after NSC differentiation and were not tumorigenic in vivo. Our findings suggest that AF-MSCs are a promising and safe alternative to BM-MSCs for therapy of nervous system diseases.     

Growth factor-defined culture medium for human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) are potential cellular sources of therapeutic stem cells as they have the ability to proliferate and differentiate into a wide array of mesenchymal cell types such as osteoblasts, chondroblasts and adipocytes. hMSCs have been used clinically to treat patients with graft vs. host disease, osteogenesis imperfect, or alveolar cleft, suggesting that transplantation of hMSCs is comparatively safe as a stem cell-based therapy. However, conventional culture medium for hMSCs contains fetal bovine serum (FBS). In the present study, we developed a growth factor-defined, serum-free medium for culturing hMSCs. Under these conditions, TGF-beta1 promoted proliferation of hMSCs. The expanded hMSC population expressed the human pluripotency markers SSEA-3, -4, NANOG, OCT3/4 and SOX2. Furthermore, double positive cells for SSEA-3 and a mesenchymal cell marker, CD105, were detected in the population. The potential to differentiate into osteoblasts and adipocytes was confirmed. This work provides a useful tool to understand the basic biological properties of hMSCs in culture.     

Mesenchymal stromal cells from unconventional model organisms

Mesenchymal stromal cells (MSCs) are multipotent, plastic, adherent cells able to differentiate into osteoblasts, chondroblasts and adipocytes. MSCs can be isolated from many different body compartments of adult and fetal individuals. The most commonly studied MSCs are isolated from humans, mice and rats. However, studies are also being conducted with the use of MSCs that originate from different model organisms, such as cats, dogs, guinea pigs, ducks, chickens, buffalo, cattle, sheep, goats, horses, rabbits and pigs. MSCs derived from unconventional model organisms all present classic fibroblast-like morphology, the expression of MSC-associated cell surface markers such as CD44, CD73, CD90 and CD105 and the absence of CD34 and CD45. Moreover, these MSCs have the ability to differentiate into osteoblasts, chondroblasts and adipocytes. The MSCs isolated from unconventional model organisms are being studied for their potential to heal different tissue defects and injuries and for the development of scaffold compositions that improve the proliferation and differentiation of MSCs for tissue engineering.     

Jak-STAT regulation of male germline stem cell establishment during Drosophila embryogenesis

Germline stem cells (GSCs) in Drosophila are descendants of primordial germ cells (PGCs) specified during embryogenesis. The precise timing of GSC establishment in the testis has not been determined, nor is it known whether mechanisms that control GSC maintenance in the adult are involved in GSC establishment. Here, we determine that PGCs in the developing male gonad first become GSCs at the embryo to larval transition. This coincides with formation of the embryonic hub; the critical signaling center that regulates adult GSC behavior within the stem cell microenvironment (niche). We find that the Jak-STAT signaling pathway is activated in a subset of PGCs that associate with the newly-formed embryonic hub. These PGCs express GSC markers and function like GSCs, while PGCs that do not associate with the hub begin to differentiate. In the absence of Jak-STAT activation, PGCs adjacent to the hub fail to exhibit the characteristics of GSCs, while ectopic activation of the Jak-STAT pathway prevents differentiation. These findings show that stem cell formation is closely linked to development of the stem cell niche, and suggest that Jak-STAT signaling is required for initial establishment of the GSC population in developing testes.