Status Of Mesenchymal Stem Cells Research Articles

Elevated TRAF4 expression impaired LPS-induced autophagy in mesenchymal stem cells from ankylosing spondylitis patients

Ankylosing spondylitis (AS) is a type of autoimmune disease that predominantly affects the spine and sacroiliac joints. However, the pathogenesis of AS remains unclear. Some evidence indicates that infection with bacteria, especially Gram-negative bacteria, may have an important role in the onset and progression of AS. Recently, many studies have demonstrated that mesenchymal stem cells (MSCs) dysfunction may contribute to the pathogenesis of many rheumatic diseases. We previously demonstrated that MSCs from AS patients exhibited markedly enhanced osteogenic differentiation capacity in vitro under non-inflammatory conditions. However, the properties of MSCs from AS patients in an inflammatory environment have never been explored. Lipopolysaccharide (LPS), a proinflammatory substance derived from the outer membrane of Gram-negative bacteria, can alter the status and function of MSCs. However, whether MSCs from AS patients exhibit abnormal responses to LPS stimulation has not been reported. Autophagy is a lysosome-mediated catabolic process that participates in many physiological and pathological processes. The link between autophagy and AS remains largely unknown. The level of autophagy in ASMSCs after LPS stimulation remains to be addressed. In this study, we demonstrated that although the basal level of autophagy did not differ between MSCs from healthy donors (HDMSCs) and ASMSCs, LPS-induced autophagy was weaker in ASMSCs than in HDMSCs. Specifically, increased TRAF4 expression in ASMSCs impaired LPS-induced autophagy, potentially by inhibiting the phosphorylation of Beclin-1. These data may provide further insight into ASMSC dysfunction and the precise mechanism underlying the pathogenesis of AS.

Two-photon FLIM of NAD(P)H and FAD in mesenchymal stem cells undergoing either osteogenic or chondrogenic differentiation

BackgroundMetabolic plasticity and the versatility of different lineages of stem cells as they satisfy their energy demands are not completely understood. In this study we investigated the metabolic changes in mesenchymal stem cells (MSCs) undergoing differentiation in two directions, osteogenic and chondrogenic, using two-photon fluorescence microscopy combined with FLIM.MethodsDifferentiation was induced by incubating the human bone marrow MSCs in osteogenic or chondrogenic mediums. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. The cells were imaged before the induction of differentiation (day 0) and on days 7, 14, and 21 of osteogenic and chondrogenic differentiation.ResultsBased on the data for the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound NAD(P)H, we registered a metabolic shift toward a more glycolytic status in the process of MSC differentiation. The difference was that, in osteogenic differentiation, an increase in oxidative phosphorylation preceded the shift to the glycolytic status in the process of such MSC differentiation. The fluorescence lifetime characteristics of FAD indicated the stimulation of an unknown metabolic pathway, where protein-bound FAD participates.ConclusionsIn this study, probing of the metabolic status of MSCs during osteogenic and chondrogenic differentiation was implemented for the first time with the use of optical metabolic imaging of the two cofactors - NAD(P)H and FAD. Our data suggest that biosynthetic processes, associated, presumably, with the synthesis of collagen, drive energy metabolism in differentiating cells, and promote a metabolic shift from a more oxidative to a more glycolytic state.

Human adipose mesenchymal stem cell-derived exosomal-miRNAs are critical factors for inducing anti-proliferation signalling to A2780 and SKOV-3 ovarian cancer cells.

An enigmatic question exists concerning the pro- or anti-cancer status of mesenchymal stem cells (MSCs). Despite growing interest, this question remains unanswered, and the debate became intensified with new evidences backing each side. Here, we showed that human adipose MSC (hAMSC)-derived conditioned medium (CM) exhibited inhibitory effects on A2780 human ovarian cancer cells by blocking the cell cycle, and activating mitochondria-mediated apoptosis signalling. Explicitly, we demonstrated that exosomes, an important biological component of hAMSC-CM, could restrain proliferation, wound-repair and colony formation ability of A2780 and SKOV-3 cancer cells. Furthermore, hAMSC-CM-derived exosomes induced apoptosis signalling by upregulating different pro-apoptotic signalling molecules, such as BAX, CASP9, and CASP3, as well as downregulating the anti-apoptotic protein BCL2. More specifically, cancer cells exhibited reduced viability following fresh or protease-digested exosome treatment; however, treatment with RNase-digested exosomes could not inhibit the proliferation of cancer cells. Additionally, sequencing of exosomal RNAs revealed a rich population of microRNAs (miRNAs), which exhibit anti-cancer activities by targeting different molecules associated with cancer survival. Our findings indicated that exosomal miRNAs are important players involved in the inhibitory influence of hAMSC-CM towards ovarian cancer cells. Therefore, we believe that these comprehensive results will provide advances concerning ovarian cancer research and treatment.

Mesenchymal Stem Cells and Cell Therapy for Bone Repair.

Mesenchymal stem cells (MSCs) represent a new therapeutic paradigm for a number of diseases because they possess unique biological characteristics such as multipotency, immunomodulation and production of cytokines. Currently, 425 MSC based clinical trials have been conducted for at least 12 kinds of pathological conditions, with many completed trials demonstrating the safety and efficacy of MSCs. Here, we provide an overview of the clinical status of MSCs by searching the public clinical trials database http://clinicaltrials.gov. Particularly, the role of MSCs in clinical trials to treat bone defects and injuries is highlighted.

Alteration of the immune status of umbilical cord mesenchymal stem cells stimulated by TLR1/2 agonist, Pam3Csk.

Mesenchymal stem cells (MSCs) have been widely used in clinical trials due to their multiple differentiation ability, low immunogenicity and immunosuppressant effects on immune response. However, accumulating evidence has indicated that MSCs may stimulate invivo immune responses and result in the disappearance of MSCs following engrafting. Toll‑like receptors (TLRs) are important in immune response induction against invaded pathogens, however, the function of TLRs in regulating the immune status of MSCs has been seldom reported. The present stimulated umbilical cord (UC) MSCs by treatment with the TLR1/2 agonist, Pam3Csk, the to determine whether activation of TLR1/2 signaling alters the immune status of UCMSCs. The results indicated that activation of TLR1/2 increased the proliferation of peripheral blood mononuclear cells (PBMCs) and the production of lactate dehydrogenase in a PBMC‑MSC co‑culture system. The study also demonstrated that Pam3Csk induced the secretion of pro‑inflammatory molecules, and increased the expression levels of cytokine and chemokines in UCMSCs. Flow cytometry analysis indicated that the levels of surface co‑stimulators, CD80 and CD86, were increased on UCMSCs in the presence of Pam3Csk, whereas activation of TLR1/2 exerted no observable effect on the differentiation abilities of UCMSCs. The results of the current study indicated that activation of TLR1/2 signaling may alter the immune status of UCMSCs, however, further mechanistic research is required in future studies.

Cell type dependent morphological adaptation in polyelectrolyte hydrogels governs chondrogenic fate

Repair of critical-size articular cartilage defects typically involves delivery of cells in biodegradable, 3D matrices. Differences in the developmental status of mesenchymal stem cells (MSCs) and terminally differentiated mature chondrocytes might be a critical factor in engineering appropriate 3D matrices for articular cartilage tissue engineering. This study examined the relationship between material-driven early cell morphological adaptations and chondrogenic outcomes, by studying the influence of aligned collagen type I (Col I) presentation on chondrocytes and MSC in interfacial polyelectrolyte complexation (IPC)-based hydrogels. In the absence of Col I, both chondrocytes and MSCs adopted rounded cell morphology and formed clusters, with chondrocyte clusters favoring the maintenance of hyaline phenotype, while MSC clusters differentiated to fibro-superficial zone-like chondrocytes. Encapsulated chondrocytes in IPC-Col I hydrogel adopted a fibroblastic morphology forming fibro-superficial zone-like phenotype, which could be reversed by inhibiting actin polymerization using cytochalasin D (CytD). In contrast, adoption of fibroblastic morphology by encapsulated MSCs in IPC-Col I facilitated superior chondrogenesis, generating a mature, hyaline neocartilage tissue. CytD treatment abrogated the elongation of MSCs and brought about a single cell-like state, resulting in insignificant chondrogenic differentiation, underscoring the essential requirement of providing matrix environments that are amenable to cell–cell interactions for robust MSC chondrogenic differentiation. Our study demonstrates that MSCs and culture-expanded chondrocytes favour differential microenvironmental niches and emphasizes the importance of designing biomaterials that meet cell type-specific requirements, in adopting chondrocyte or MSC-based approaches for regenerating hyaline, articular cartilage.

IIB or not IIB: development of a screening method by flow cytometry to evaluate correct chondrogenic conversion of human mesenchymal stem cells in hydrogel

Purpose: The use of mesenchymal stem cells (MSCs) represents a promising alternative cellular model to chondrocytes for articular cartilage repair but satisfactory protocols allowing proper chondrogenic conversion together with sufficient cartilage matrix production are still missing. The purpose of this study was to undertake an extensive characterization of human MSCs isolated from Wharton’s jelly (WJ), adipose tissue (AT), dental pulp (DP) and bone marrow (BM), and to confront their chondrogenic potential in serum-free culture conditions. Methods: MSCs were isolated and expanded in serum-free conditions. Their proliferation kinetics were evaluated. An extensive immunophenotyping analysis by flow cytometry was undertaken at the end of their first passage (P1). We then explored their potential to engage into adipogenic, osteogenic and chondrogenic lineages, the latter being assessed in serum-free conditions with a view of clinical application. This chondrogenic conversion was performed in micromass pellets and in another cellular model using MSCs embedded in agarose hydrogel. This model offers two advantages: 1) it mimicks a cartilage tissue engineering protocol where MSCs converted into chondrocytes and embedded in hydrogel form a cartilage gel that could be used to treat cartilage lesions and 2) the agarose nature of the hydrogel allows the release of the cells from the gel (by enzymatic digestion) at the end of their chondrogenic conversion, giving therefore the possibility to analyze them directly by flow cytometry. Recently, we developed and characterized the first antibody capable of detecting the IIB isoform of human type II procollagen, the only isoform of this collagen type that is expressed by well-differentiated chondrocytes. Here, we used this antibody by flow cytometry to look for intracellular IIB procollagen expression in MSCs undergoing chondrocyte differentiation in hydrogel. Results: The mean population doubling times between P1 and P5 ranged between 30 h and 55 h, depending on the tissue origin of MSCs. The doubling times remained constant from P1 to P5 and a minimal reservoir of 300 × 10ˆ6 cells (e.g., BM-MSCs) could be obtained after 5 passages, starting with 1 × 10ˆ6 cells harvested at the end of P1. Importantly, no karyotype abnormalities were detected after 5 passages. In brief, all cell sources yielded clinical-scale amounts of MSCs after expansion in serum-free culture conditions. By using an original panel of 27 surface markers that were analyzed by 8-color flow cytometry, our results revealed that all MSC sources were positive for the expression of the ISCT-validated markers (CD73, CD90, CD105) as well as for other recognized MSC markers (CD44). Nevertheless, differences in expression for other markers were found between the sources. BM-MSCs were the only cells to include subpopulations positive for all the putative skeletal markers analyzed (CD29, CD56, MSCA-1, Stro-1, CD106, CD146, CD271). All cell sources showed signs of adipogenic, osteogenic and chondrogenic conversion in monolayer cultures, after amplification in serum-free conditions. However, only BM-MSCs were able to clearly convert into true chondrocytes in micromass pellets or hydrogel cultures, as attested in particular by the expression of type IIB procollagen, monitored by immunohistochemistry or flow cytometry. Conclusions: It is possible to drive MSCs to the chondrogenic lineage in hydrogel after expansion and differentiation in serum-free conditions. BM-MSCs appear to be the best candidates for chondrogenic conversion in the conditions used in this study and this is in concordance with the largest panel of putative skeletal markers revealed by flow cytometry analysis for this category of MSCs. In this context, we have introduced an innovative quality control of the chondrocytic status of MSCs embedded in hydrogel, via measurement by flow cytometry of intracellular IIB procollagen, a marker of true chondrocytes.

Establishment of mesenchymal stem cell lines derived from the bone marrow of green fluorescent protein-transgenic mice exhibiting a diversity in intracellular transforming growth factor-β and bone morphogenetic protein signaling.

Cytokines and their intercellular signals regulate the multipotency of mesenchymal stem cells (MSCs). The present study established the MSC lines SG-2, -3, and -5 from the bone marrow of green fluorescent protein (GFP)-transgenic mice. These cell lines clearly expressed mouse MSC markers Sca-1 and CD44, and SG-2 and -5 cells retained the potential for osteogenic and adipogenic differentiation in the absence of members of the transforming growth factor (TGF)-β superfamily. By contrast, SG-3 cells only retained adipogenic differentiation potential. Analysis of cytokine and cytokine receptor expression in these SG cell lines showed that bone morphogenetic protein (BMP) receptor 1B was most highly expressed in the SG-3 cells, which underwent osteogenesis in response to BMP, while TGF-β receptor II was most highly expressed in SG-3 and -5 cells. However, it was unexpectedly noted that phosphorylation of Smad 2, a major transcription factor, was induced by TGF-β1 in SG-2 cells but not in SG-3 or -5 cells. Furthermore, TGF-β1 clearly induced the expression of Smad-interacting transcription factor CCAAT/enhancer binding protein-β in SG-2 but not in SG-3 or -5 cells. These results demonstrated the establishment of TGF-β-responsive SG-2 MSCs, BMP-responsive SG-3 MSCs and TGF-β/BMP-unresponsive SG-5 MSCs, each of which was able to be traced by GFP fluorescence after transplantation into in vivo experimental models. In conclusion, the present study suggested that these cell lines may be used to explore how the TGF-β superfamily affects the proliferation and differentiation status of MSCs in vivo.

Human bone marrow- and adipose-mesenchymal stem cells secrete exosomes enriched in distinctive miRNA and tRNA species.

IntroductionAdministration of mesenchymal stem cells (MSCs) represents a promising treatment option for patients suffering from immunological and degenerative disorders. Accumulating evidence indicates that the healing effects of MSCs are mainly related to unique paracrine properties, opening opportunities for secretome-based therapies. Apart from soluble factors, MSCs release functional small RNAs via extracellular vesicles (EVs) that seem to convey essential features of MSCs. Here we set out to characterize the full small RNAome of MSC-produced exosomes.MethodsWe set up a protocol for isolating exosomes released by early passage adipose- (ASC) and bone marrow-MSCs (BMSC) and characterized them via electron microscopy, protein analysis and small RNA-sequencing. We developed a bioinformatics pipeline to define the exosome-enclosed RNA species and performed the first complete small RNA characterization of BMSCs and ASCs and their corresponding exosomes in biological replicates.ResultsOur analysis revealed that primary ASCs and BMSCs have highly similar small RNA expression profiles dominated by miRNAs and snoRNAs (together 64-71 %), of which 150–200 miRNAs are present at physiological levels. In contrast, the miRNA pool in MSC exosomes is only 2-5 % of the total small RNAome and is dominated by a minor subset of miRNAs. Nevertheless, the miRNAs in exosomes do not merely reflect the cellular content and a defined set of miRNAs are overrepresented in exosomes compared to the cell of origin. Moreover, multiple highly expressed miRNAs are precluded from exosomal sorting, consistent with the notion that these miRNAs are involved in functional repression of RNA targets. While ASC and BMSC exosomes are similar in RNA class distribution and composition, we observed striking differences in the sorting of evolutionary conserved tRNA species that seems associated with the differentiation status of MSCs, as defined by Sox2, POU5F1A/B and Nanog expression.ConclusionsWe demonstrate that primary MSCs release small RNAs via exosomes, which are increasingly implicated in intercellular communications. tRNAs species, and in particular tRNA halves, are preferentially released and their specific sorting into exosomes is related to MSC tissue origin and stemness. These findings may help to understand how MSCs impact neighboring or distant cells with possible consequences for their therapeutic usage.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0116-z) contains supplementary material, which is available to authorized users.

I–2 Low Intensity Pulsed Ultrasound (LIPUS) Helps to Maintain the Undifferentiated Status of Mesenchymal Stem Cells

Mesenchymal Stem Cells (MSCs) are pluripotent cells with the capability of self-renewal. It has been established that MSCs can transform into osteoblasts, adipocytes or chondrocytes depending on the culture condition. Regenerative medicine is considered to become an essential therapy in the field of

Large-Scale Expansion and Differentiation of Mesenchymal Stem Cells in Microcarrier-Based Stirred Bioreactors.

Mesenchymal stem cells (MSCs) have emerged as an important tool for tissue engineering, thanks to their differentiation potential and their broad trophic activities. However, for clinical purposes or for relevant in vitro applications, large quantities of MSCs are required, which could hardly be reached using conventional cultivation in plastic dishes. Microcarriers have high surface to volume ratio, which enables the easy scale-up of the expansion and differentiation of MSCs. In addition, the agitation in stirred tank bioreactors limits the diffusion gradient of nutrients or morphogens, thus providing a physiologically relevant environment to favor MSC production at large scale. This work describes a simple method for the mass expansion and differentiation of MSCs, including the procedures to monitor the proliferation, metabolic status and phenotype of MSCs during suspension culture. Moreover, this work proposes suitable materials for cGMP compliant culture conditions enabling the clinical grade production of MSCs.

Modulation of the stemness and osteogenic differentiation of human mesenchymal stem cells by controlling RGD concentrations of poly(carboxybetaine) hydrogel.

In vitro modulation of the differentiation status of mesenchymal stem cells (MSCs) is important for their application to regenerative medicine. We suggested that the morphology and differentiation states of MSCs could be modulated by controlling the cell affinity of a substrate. The objective of this study was to investigate the effects of surface bio-adhesive signals on self-renewal and osteogenic differentiation of MSCs using a low-fouling platform. Cell-resistant poly(carboxybetaine) hydrogel was conjugated with 5 μM or 5 mM of cell-adhesive arginine-glycine-aspartic acid (RGD) peptides in order to control the cells' affinity to the substrate. Human mesenchymal stem cells (hMSCs) were cultured on the RGD-modified poly(carboxybetaine) hydrogel and then the cells' states of stemness and osteogenic differentiation were evaluated using reverse-transcriptase polymerase chain reaction. The hMSCs formed three-dimensional spheroids on the 5 μM RGD substrate, while cells on the 5 mM RGD substrate exhibited spreading morphology. Furthermore, cells on the 5 μM RGD hydrogel maintained a better stemness phenotype, while the hMSCs on the 5 mM RGD hydrogel proliferated faster and underwent osteogenic differentiation. In conclusion, the stemness of hMSCs was best maintained on a low RGD surface, while osteogenic differentiation of hMSCs was enhanced on a high RGD surface.

Promoting effect of thrombin on proliferation of bone marrow-derived mesenchymal stem cells and its mechanisms

This study was aimed to investigate the growth-promoting activity of thrombin on mesenchymal stem cells (MSC) and its mechanisms. Human bone marrow MSC were cultured in serum-free medium supplemented with graded concentrations of thrombin, and the proliferation status of MSC was detected by MTT test. The expression levels of protease-activated receptors (PAR) and c-MYC gene were detected by PCR. Activated Akt signaling pathway was revealed by Western blot, and specific inhibitors of the signaling pathways were used to confirm the effects. The results showed that thrombin stimulated MSC proliferation in a dose-dependent manner; the minimal concentration of thrombin for stimulating MSC growth was 0.5 U/ml, and the promoting effect reached its maximum when thrombin at a dose of 8 U/ml was employed. PCR results showed that MSC expressed the two types of PAR1 and PAR2. After PAR1 was blocked with a specific inhibitor SCH79797, the growth-promoting effect of thrombin was inhibited, while this phenomenon was not observed when MSC were exposed to FSLLRY-NH2, a specific inhibitor for PAR2. Further experiments showed that after exposure to thrombin, the AKT signaling pathway in MSC was promptly activated, and c-MYC expression was greatly up-regulated. Meanwhile, when LY294002, a specific AKT inhibitor, was added into the culture medium, the up-regulation of c-MYC expression was reduced, accompanied by the low rate of MSC growth. It is concluded that thrombin can stimulate MSC proliferation by eliciting PAR1-mediated AKT activation and subsequent up-regulation of c-MYC expression.

Concise Review: The Immune Status of Mesenchymal Stem Cells and Its Relevance for Therapeutic Application

Multipotentiality and anti-inflammatory activity, the two main properties of mesenchymal stem cells (MSCs), underlie their therapeutic prospective. During the past decade, numerous studies in animal models and clinical trials explored the potential of MSCs in the treatment of diseases associated with tissue regeneration and inflammatory control. Other qualities of MSCs: ready accessibility in bone marrow and fat tissue and rapid expansion in culture make the therapeutic use of patients' own cells feasible. The prevailing belief that MSCs are nonimmunogenic encouraged the use of unrelated donor cells in immune-competent recipients. The data emerging from studies performed with immune-incompatible cells in animal models for a wide-range of human diseases show, however, conflicting results and cast doubt on the immune privileged status of MSCs. Our analysis of the preclinical literature in this review is aimed to gain a better understanding of the therapeutic potential of immune-incompatible MSCs. Emphasis was laid on applications for enhancement of tissue repair in the absence of immune-suppressive therapy.

Mesenchymal stem cell-collagen microspheres for articular cartilage repair: Cell density and differentiation status

Mesenchymal stem cells (MSC) hold promise for cartilage repair. A microencapsulation technique was previously established to entrap MSC in collagen microspheres, and the collagen fibrous meshwork was found to be an excellent scaffold for supporting MSC survival, growth and differentiation. This study investigates the importance of cell density and differentiation status of MSC–collagen microspheres in cartilage repair. MSC were isolated from rabbit bone marrow and encapsulated in collagen microspheres. The effects of pre-differentiating the encapsulated MSC into chondrogenic lineages and different cell densities on cartilage repair were investigated in rabbits. Implantation of undifferentiated collagen–MSC microspheres formed hyaline-like cartilage rich in type II collagen and glycosaminoglycans (GAG) at 1month post-implantation. By 6months, hyaline cartilage rich in type II collagen and GAG, but negative for type I collagen, and partial zonal organization were found in both undifferentiated and chondrogenically differentiated groups in the high cell density group. The undifferentiated group and high cell density group significantly improved the O’Driscoll histological score. Moreover, the undifferentiated group significantly increased the GAG content. The mechanically differentiated group showed stiffer but thinner cartilage, while the undifferentiated group showed thicker but softer cartilage compared with their respective contra-lateral controls. This work suggests that a higher local cell density favors cartilage regeneration, regardless of the differentiation status of MSC, while the differentiation status of MSC does significantly affect regeneration outcomes.

Mesenchymal stem cells as a vector for the inflammatory prostate microenvironment

Mesenchymal stem cells (MSCs) have an inherent tropism for sites of inflammation, which are frequently present in sites of cancer, including prostatic lesions. MSCs have been defined as CD73/CD90/CD105 triple-positive cells in the absence of hematopoietic lineage markers with the ability to differentiate into multiple mesodermal lineages, including osteoblasts, adipocytes, and chondrocytes. Our group has previously demonstrated that MSCs represent between 0.01 and 1.1% of the total cells present in human prostatectomy tissue. In addition to their multi-lineage differentiation potential, MSCs are immunoprivileged in nature and have a range of immunomodulatory effects on both the innate and adaptive arms of the immune system. MSCs have been detected in an increasing array of tissues, and evidence suggests that they are likely present in perivascular niches throughout the body. These observations suggest that MSCs represent critical mediators of the overall immune response during physiological homeostasis and likely contribute to pathophysiological conditions as well. Chronic inflammation has been suggested as an initiating event and progression factor in prostate carcinogenesis, a process in which the immunosuppressive properties of MSCs may play a role. MSCs have also been shown to influence malignant progression through a variety of other mechanisms, including effects on tumor proliferation, angiogenesis, survival, and metastasis. Additionally, human bone marrow-derived MSCs have been shown to traffic to human prostate cancer xenografts in immunocompromised murine hosts. The trafficking properties and immunoprivileged status of MSCs suggest that they can be exploited as an allogeneic cell-based vector to deliver cytotoxic or diagnostic agents for therapy.

Comparative characteristics of new lines of mesenchymal stem cells derived from human embryonic stem cells, bone marrow, and foreskin

New human nonimmortalized fibroblast-like cell lines were derived from various sources: from embryonic stem cells (ESCs) (the SC5-MSC and SC3a-MSC lines), from bone marrow of a 5- to 6-week-old fetus (the FetMSC line), and from foreskin of a 3-year-old child (the FRSN line). All the lines are successfully used as feeders during cultivation of human ESCs. The mean doubling time of the cell populations fluctuates depending on the line from 25.5 h in the SC5-MSC line to 38.8 h in the SC3a-MSC line. The growth curves indicate active cell proliferation of all lines. Numerical and structural karyotypical analysis has shown these lines to have a normal karyotype: 46, XX (SC5-MSC and SC3a-MSC) and 46, XY (FetMSC and FRSN). To determine the status of these lines, comparative analysis of surface markers was performed with the aid of flow cytofluorimetry, and expression of the c antigens characteristic of human mesenchymal stem cells (MSCs) was revealed: CD44, CD73, CD90, CD105, and HLA-ABC and the absence of expression of CD34 and HLA-DR. Interlinear differences in the expression level of the marker CD117 (c-kit) were revealed. Immunofluorescent and cytofluorimetric analysis of expression of surface markers and transcription factor Oct-4 that are characteristic of human ESCs has shown that, in all four lines, expression of TRA-1-60 and Oct-4 is absent, whereas in expression of SSEA-4 there are observed the interlinear differences not depending on the origin of cells. At present it is not yet clear whether the revealed interlinear differences affect essentially the functional status of mesenchymal stem cells. Immunofluorescent analysis in cells of all lines showed expression of markers of early differentiation into derivatives of three germ layers characterizing ESCs, which might possibly provide wide MSC possibilities during reparation of different tissue damages, depending on the corresponding microenvironment. The capability of cells of all lines for directed differentiation into the adipogenic and osteogenic directions was revealed.

Therapeutic potential of adult bone marrow‐derived mesenchymal stem cells in diseases of the skeleton

Mesenchymal stem cells (MSCs) are the most popular among the adult stem cells in tissue engineering and regenerative medicine. Since their discovery and functional characterization in the late 1960s and early 1970s, MSCs or MSC-like cells have been obtained from various mesodermal and non-mesodermal tissues, although majority of the therapeutic applications involved bone marrow-derived MSCs. Based on its mesenchymal origin, it was predicted earlier that MSCs only can differentiate into mesengenic lineages like bone, cartilage, fat or muscle. However, varied isolation and cell culturing methods identified subsets of MSCs in the bone marrow which not only differentiated into mesenchymal lineages, but also into ectodermal and endodermal derivatives. Although, true pluripotent status is yet to be established, MSCs have been successfully used in bone and cartilage regeneration in osteoporotic fracture and arthritis, respectively, and in the repair of cardiac tissue following myocardial infarction. Immunosuppressive properties of MSCs extend utility of MSCs to reduce complications of graft versus host disease and rheumatoid arthritis. Homing of MSCs to sites of tissue injury, including tumor, is well established. In addition to their ability in tissue regeneration, MSCs can be genetically engineered ex vivo for delivery of therapeutic molecule(s) to the sites of injury or tumorigenesis as cell therapy vehicles. MSCs tend to lose surface receptors for trafficking and have been reported to develop sarcoma in long-term culture. In this article, we reviewed the current status of MSCs with special emphasis to therapeutic application in bone-related diseases.

Fate of mesenchymal stem cells transplanted to osteonecrosis of femoral head

The purpose of this study was to investigate the survival and differentiation status of MSCs transplanted to ONFH. Traumatic ONFH was surgically produced in skeletally mature mongrel dogs. Osteonecrosis was treated with either saline (control) or autologous mesenchymal stem cells (MSCs) transplantation after decompression. Green fluorescent protein (GFP) was used to track the transplanted MSCs, the differentiation of MSCs were evaluated by fluorescent double-labeling with GFP between osteocalcin or von Willebrand factor (vWF) at 2nd, 8th, and 12th week after the transplantation. It was demonstrated that GFP-positive cells were present in the necrotic area up to 12 weeks after the transplantation, their number increased from 15% at 2nd week to 38% at 12th week (p < 0.05). Neither osteocalcin nor vWF was detected by immunocytochemistry in GFP-labeled MSCs in vitro, but osteocalcin was immunohistochemically positive in 90% of the GFP-labeled MSCs in vivo, while vWF was still negative. The vWF expression was of no significant difference between the control group and MSCs-transplanted group. The percentages of trabeculae bone volume were 9.36% and 8.42% at 2nd week (p > 0.05), 22.82% and 14.72% at 8th week, and 31.08% and 20.66% at 12th week (p < 0.05) in MSCs-transplanted group and control group, respectively; new trabeculae bone in MSCs-transplanted group was significantly increased as compared to that of control group at 8th and 12th week. The results demonstrated that the transplanted MSCs could survive, proliferate, and differentiate into osteoblasts directly, which contributed to the accelerated repair process. The possible mechanism is site-dependant differentiation.

Astrocytes facilitate the growth and differentiation of co-cultured mesenchymal stem cells

The effect of astrocytes on the growth and differentiation of co-cultured mesenchymal stem cells (MSCs) was studied. MSCs of Wistar rats were isolated, cultured and labeled with Hoechst33342. The labeled MSCs were co-cultured with astrocytes in DMEM/F12/10%FBS. The growth status and morphologic change of MSCs were observed. The expression of specific protein NeuN, GFAP and CNP was detected by using immunofluorescence staining. The results showed that the two kinds of co-cultured cells grew well. Some labeled MSCs stretched out long processes with spin, triangle and star shapes. Immunofluorescence stain showed that these cells expressed NeuN, GFAP or CNP. It was suggested that astrocytes could promote the proliferation of co-cultured MSCs and induce them to differentiate into neural like cells.