"Mesenchymal" stem cells.

Two opposing descriptions of so-called mesenchymal stem cells (MSCs) exist at this time. One sees MSCs as the postnatal, self-renewing, and multipotent stem cells for the skeleton. This cell coincides with a specific type of bone marrow perivascular cell. In skeletal physiology, this skeletal stem cell is pivotal to the growth and lifelong turnover of bone and to its native regeneration capacity. In hematopoietic physiology, its role as a key player in maintaining hematopoietic stem cells in their niche and in regulating the hematopoietic microenvironment is emerging. In the alternative description, MSCs are ubiquitous in connective tissues and are defined by in vitro characteristics and by their use in therapy, which rests on their ability to modulate the function of host tissues rather than on stem cell properties. Here, I discuss how the two views developed, conceptually and experimentally, and attempt to clarify the confusion arising from their collision.

Epigenetic modifications underlie the differential adipogenic potential of preadipocytes derived from human subcutaneous fat tissue.

Ceiling culture-derived preadipocytes (ccdPAs) and adipose-derived stem cells (ASCs) can be harvested from human subcutaneous fat tissue using the specific gravity method. Both cell types possess a similar spindle shape without lipid droplets. We previously reported that ccdPAs have a higher adipogenic potential than ASCs, even after a 7-wk culture. We performed a genome-wide epigenetic analysis to examine the mechanisms contributing to the adipogenic potential differences between ccdPAs and ASCs. Methylation analysis of cytosines followed by guanine (CpG) using a 450-K BeadChip was performed on human ccdPAs and ASCs isolated from three metabolically healthy females. Chromatin immunoprecipitation sequencing was performed to evaluate trimethylation at lysine 4 of histone 3 (H3K4me3). Unsupervised machine learning using t-distributed stochastic neighbor embedding to interpret 450,000-dimensional methylation assay data showed that the cells were divided into ASC and ccdPA groups. In Kyoto Encyclopedia of Genes and Genomes pathway analysis of 1,543 genes with differential promoter CpG methylation, the peroxisome proliferator-activated receptor (PPAR) and adipocytokine signaling pathways ranked in the top 10 pathways. In the PPARγ gene, H3K4me3 peak levels were higher in ccdPAs than in ASCs, whereas promoter CpG methylation levels were significantly lower in ccdPAs than in ASCs. Similar differences in promoter CpG methylation were also seen in the fatty acid-binding protein 4 and leptin genes. In conclusion, we analyzed the epigenetic status of adipogenesis-related genes as a potential mechanism underlying the differences in adipogenic differentiation capability between ASCs and ccdPAs.

Human Umbilical Cord-Derived Mesenchymal Stem Cells for Acute Respiratory Distress Syndrome.

OBJECTIVES: To investigate the safety, feasibility, and possible adverse events of single-dose human umbilical cord-derived mesenchymal stem cells in patients with moderate-to-severe acute respiratory distress syndrome. DESIGN: Prospective phase I clinical trial. SETTING: Medical center in Kaohsiung, Taiwan. PATIENTS: Moderate-to-severe acute respiratory distress syndrome with a PaO2/FIO2 ratio less than 200. INTERVENTIONS: Scaling for doses was required by Taiwan Food and Drug Administration as follows: the first three patients received low-dose human umbilical cord-derived mesenchymal stem cells (1.0 × 10 cells/kg), the next three patients with intermediate dose (5.0 × 10 cells/kg), and the final three patients with high dose (1.0 × 10 cells/kg) between December 2017 and August 2019. MEASUREMENTS AND MAIN RESULTS: Nine consecutive patients were enrolled into the study. In-hospital mortality was 33.3% (3/9), including two with recurrent septic shock and one with ventilator-induced severe pneumomediastinum and subcutaneous emphysema. No serious prespecified cell infusion-associated or treatment-related adverse events was identified in any patient. Serial flow-cytometric analyses of circulating inflammatory biomarkers (CD14CD33/CD11b+CD16+/CD16+MPO+/CD11b+MPO+/CD14CD33+) and mesenchymal stem cell markers (CD26+CD45-/CD29+CD45-/CD34+CD45-/CD44+CD45-/CD73+CD45-/CD90+CD45-/CD105+CD45-/CD26+CD45-) were notably progressively reduced (p for trend < 0.001), whereas the immune cell markers (Helper-T-cell/Cytotoxity-T-cell/Regulatory-T-cell) were notably increased (p for trend < 0.001) after cell infusion. CONCLUSIONS: The result of this phase I clinical trial showed that a single-dose IV infusion of human umbilical cord-derived mesenchymal stem cells was safe with favorable outcome in nine acute respiratory distress syndrome patients.

Induced Pluripotent Stem Cell-Derived Mesenchymal Stromal Cells Are Functionally and Genetically Different From Bone Marrow-Derived Mesenchymal Stromal Cells.

There has been considerable interest in the generation of functional mesenchymal stromal cell (MSC) preparations from induced pluripotent stem cells (iPSCs) and this is now regarded as a potential source of unlimited, standardized, high-quality cells for therapeutic applications in regenerative medicine. Although iMSCs meet minimal criteria for defining MSCs in terms of marker expression, there are substantial differences in terms of trilineage potential, specifically a marked reduction in chondrogenic and adipogenic propensity in iMSCs compared with bone marrow-derived (BM) MSCs. To reveal the cellular basis underlying these differences, we conducted phenotypic, functional, and genetic comparisons between iMSCs and BM-MSCs. We found that iMSCs express very high levels of both KDR and MSX2 compared with BM-MSCs. In addition, BM-MSCs had significantly higher levels of PDGFRα. These distinct gene expression profiles were maintained during culture expansion, suggesting that prepared iMSCs are more closely related to vascular progenitor cells (VPCs). Although VPCs can differentiate along the chondrogenic, osteogenic, and adipogenic pathways, they require different inductive conditions compared with BM-MSCs. These observations suggest to us that iMSCs, based on current widely used preparation protocols, do not represent a true alternative to primary MSCs isolated from BM. Furthermore, this study highlights the fact that high levels of expression of typical MSC markers such as CD73, CD90, and CD105 are insufficient to distinguish MSCs from other mesodermal progenitors in differentiated induced pluripotent stem cell cultures. Stem Cells 2019;37:754-765.

Mesenchymal stem versus stromal cells: International Society for Cell & Gene Therapy (ISCT®) Mesenchymal Stromal Cell committee position statement on nomenclature.

The International Society for Cell & Gene Therapy (ISCT®) Mesenchymal Stromal Cell (ISCT MSC) committee offers a position statement to clarify the nomenclature of mesenchymal stromal cells (MSCs). The ISCT MSC committee continues to support the use of the acronym "MSCs" but recommends this be (i) supplemented by tissue-source origin of the cells, which would highlight tissue-specific properties; (ii) intended as MSCs unless rigorous evidence for stemness exists that can be supported by both in vitro and in vivo data; and (iii) associated with robust matrix of functional assays to demonstrate MSC properties, which are not generically defined but informed by the intended therapeutic mode of actions.

A comprehensive study on donor-matched comparisons of three types of mesenchymal stem cells-containing cells from human dental tissue.

BACKGROUND AND OBJECTIVE: Mesenchymal stem cells (MSCs) have been widely used in tissue engineering, such as for regenerating the supporting structures of teeth destroyed by periodontal diseases. In recent decades, dental tissue-derived MSCs have drawn much attention owing to their accessibility, plasticity and applicability. Dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs) and gingival MSCs (GMSCs) are the most readily available MSCs among all types of dental MSCs. The purpose of this study was to comprehensively compare the characteristics of MSCs from dental pulp (DP), periodontal ligament (PDL) and gingiva (G) in vitro and thus provide insight into optimizing the performance of cells and seed cell selection strategies for tissue regeneration. MATERIALS AND METHODS: In this study, patient-matched (n = 5) cells derived from DP, PDL and G which, respectively, contained DPSCs, PDLSCs and GMSCs were evaluated using multiple methods in terms of their proliferation, senescence, apoptosis, multilineage differentiation and stemness maintenance after long-term passage. RESULTS: Mesenchymal stem cells-containing cells from G (MSCs/GCs) showed superior proliferation capability, whereas patient-matched MSCs-containing cells from PDL (MSCs/PDLCs) exhibited excellent osteogenic and adipogenic differentiation ability; MSCs-containing cells from DP (MSCs/DPCs) achieved mediocre results in both aspects. In addition, MSCs/GCs were the least susceptible to senescence, while MSCs/PDLCs were the most prone to ageing. Furthermore, the biological properties of these three types of cells were all affected after long-term in vitro culture. CONCLUSION: These three types of dental MSCs showed different biological characteristics. MSCs/PDLCs are the best candidate cells for bone regeneration, but the application of MSCs/PDLCs might be limited to certain number of passages. Improving the differentiation of MSCs/GCs remains the key issue regarding their application in tissue engineering.

Identification of miRNA Reference Genes in Extracellular Vesicles from Adipose Derived Mesenchymal Stem Cells for Studying Osteoarthritis.

Osteoarthritis (OA) leads to chronic pain and disability, and traditional conservative treatments are not effective in the long term. The intra-articular injection of mesenchymal stem cells (MSCs) is considered a novel therapy for OA whose efficacy mainly relies on the adaptive release of paracrine molecules which are either soluble or extracellular vesicles (EVs) embedded. The correct quantification of EV-miRNAs using reliable reference genes (RGs) is a crucial step in optimizing this future therapeutic cell-free approach. The purpose of this study is to rate the stabilities of literature-selected proposed RGs for EV-miRNAs in adipose derived-MSCs (ASCs). EVs were isolated by ultracentrifugation from ASCs cultured with or without inflammatory priming mimicking OA synovial fluid condition. Expression of putative RGs (let-7a-5p, miR-16-5p, miR-23a-3p, miR-26a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p, U6 snRNA) was scored by using the algorithms geNorm, NormFinder, BestKeeper and ΔCt method. miR-16a-5p/miR-23a-3p yielded the most stable RGs, whereas let-7a-5p/miR-425-5p performed poorly. Outcomes were validated by qRT-PCR on miR-146a-5p, reported to be ASC-EVs enriched and involved in OA. Incorrect RG selection affected the evaluation of miR-146a-5p abundance and modulation by inflammation, with both values resulting strongly donor-dependent. Our findings demonstrated that an integrated approach of multiple algorithms is necessary to identify reliable, stable RGs for ASC-EVs miRNAs evaluation. A correct approach would increase the accuracy of embedded molecule assessments aimed to develop therapeutic strategies for the treatment of OA based on EVs.

Identification and characterization of human bone-derived cells.

This study was designed to identify and characterize primary bone-derived cells (BdCs) and investigate the potential role of osteoblast differentiation. Primary BdCs were isolated from surgical bone for comparative analysis with mesenchymal stem cells (MSCs) and fetal osteoblasts (FOBs) and for potential differentiation to mature osteoblasts. Using three different cells, we successfully cultivated human osteoblast differentiation and activity which were evaluated using microarray and biochemical methods. BdCs are more correlated to MSCs in bioinformatics result and similar with FOBs in gene expression. In particular, Osterix, osteoprogenitor marker, was high expressed in BdCs, while the expression in MSCs and FOBs were very low. Furthermore, BdCs exhibited a marked alkaline phosphatase (ALP) expression, early stage of osteogenic marker, and retained osteogenic properties and physiological changes into maturation as in FOBs. BdCs also showed an increase in bone morphogenic protein 2 (BMP2), osteopontin (OPN), and osteocalcin (OCN) mRNA expressions during differentiation. This study suggests that BdCs may be osteoprogenitor cells or undifferentiated preosteoblasts with strong capacity to differentiate toward mature osteoblasts.

Type 2 Diabetes Inhibited Human Mesenchymal Stem Cells Angiogenic Response by Over-Activity of the Autophagic Pathway.

The current study aimed to address the impact of serum from type 2 diabetes patients on the angiogenic properties of human bone marrow mesenchymal stem cells and its relationship to autophagy signaling. Human primary stem cells were enriched and incubated with serum from diabetic and normal subjects for 7 days. Compared to data from the control group, diabetic serum was found to induce a higher cellular death rate (P < 0.001) and apoptotic changes (P < 0.01). We also showed that diabetic condition significantly abolished angiogenesis tube formation on Matrigel substrate, decreased cell chemotaxis (P < 0.01) in response to SDF-1α, and inhibited endothelial differentiation rate (P < 0.0001). Western blotting showed autophagic status by high levels of P62 (P < 0.0001), beclin-1 (P < 0.0001), and increase in LC3II/I ratio (P < 0.001). In vivo Matrigel plug assay revealed that supernatant conditioned media prepared from cells exposed to diabetic serum caused a marked reduction in the recruitment of VE-cadherin- (P < 0.01) and α-SMA-positive (P < 0.0001) cells 7 days after subcutaneous injection. PCR expression array analysis confirmed the overexpression of autophagy and apoptosis genes in cultured cells in response to a diabetic condition (P < 0.05). Using bioinformatic analysis, we noted a crosstalk network between DM2, angiogenesis, and autophagy signaling. DM2 could potently modulate angiogenesis by the interaction of IL-1ß with downstream insulin receptor and upstream androgen receptor. Corroborating to data, diabetic serum led to abnormal regulation of P62 during the angiogenic response. These data demonstrate that diabetic serum decreased human mesenchymal stem cell angiogenic properties directly on angiogenesis pathways or by the induction of autophagy signaling. J. Cell. Biochem. 118: 1518-1530, 2017. © 2016 Wiley Periodicals, Inc.

A Conserved Role for VEGF Signaling in Specification of Homologous Mesenchymal Cell Types Positioned at Spatially Distinct Developmental Addresses in Early Development of Sea Urchins.

Comparative studies of early development in echinoderms are revealing the tempo and mode of alterations to developmental gene regulatory networks and to the cell types they specify. In euechinoid sea urchins, skeletogenic mesenchyme (SM) ingresses prior to gastrulation at the vegetal pole and aligns into a ring-like array with two bilateral pockets of cells, the sites where spiculogenesis will later occur. In cidaroid sea urchins, the anciently diverged sister clade to euechinoid sea urchins, a homologous SM cell type ingresses later in development, after gastrulation has commenced, and consequently at a distinct developmental address. Thus, a heterochronic shift of ingression of the SM cell type occurred in one of the echinoid lineages. In euechinoids, specification and migration of SM are facilitated by vascular endothelial growth factor (VEGF) signaling. We describe spatiotemporal expression of vegf and vegfr and experimental manipulations targeting VEGF signaling in the cidaroid Eucidaris tribuloides. Spatially, vegf and vegfr mRNA localizes similarly as in euechinoids, suggesting conserved deployment in echinoids despite their spatially distinct development addresses of ingression. Inhibition of VEGF signaling in E. tribuloides suggests its role in SM specification is conserved in echinoids. Temporal discrepancies between the onset of vegf expression and SM ingression likely result in previous observations of SM "random wandering" behavior. Our results indicate that, although the SM cell type in echinoids ingresses into distinct developmental landscapes, it retains a signaling mechanism that restricts their spatial localization to a conserved developmental address where spiculogenesis later occurs.

Comparative characterization of mesenchymal stromal cells from multiple abdominal adipose tissues and enrichment of angiogenic ability via CD146 molecule.

BACKGROUND: There are various types of adipose tissue in the human body, and their morphology is known to be closely related to cell function and metabolism. However, the functional differences among the mesenchymal stromal cells (MSCs) of different abdominal adipose tissues have not been clearly elucidated. METHODS: MSCs were isolated from different abdominal adipose tissues according to their regional distribution and included superficial subcutaneous, deep subcutaneous, omentum, mesentery and retroperitoneal MSCs. The immunophenotype, proliferative ability and angiogenic function of these MSCs were compared based on flow cytometry analysis, CCK-8 proliferation, in vitro differentiation, tubule formation and in vivo plug assay. RESULTS: The plastic adherence, cell morphology and general immunophenotype are similar among the MSCs. However, subcutaneous adipose tissue-derived MSCs have a faster growth rate and a higher level of CD146 expression than the other MSCs. Moreover, according to the fluorescence-activated cell sorting (FACS) enrichment procedure, the expression level of CD146 is positively related to the growth rate and angiogenic capability of MSCs. DISCUSSION: MSCs in adipose tissue showed slightly different characteristics depending on their location of origin, and they possessed different angiogenic abilities that were mediated by the expression of CD146. This study provides evidence that subcutaneous adipose tissue is the most appropriate source of MSCs for therapeutic cell transplantation in vascular disease.

Static magnetic field enhances the viability and proliferation rate of adipose tissue-derived mesenchymal stem cells potentially through activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) pathway.

The aim of this work was to investigate the effects of 0.5T static magnetic field (sMF) on the viability and proliferation rate of human adipose-derived mesenchymal stromal stem cells (hASCs) via activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) signaling pathway. In a 7-d culture we examined cell growth kinetic and population doubling time (PDT). We also examined cell morphology and the cellular senescence markers level. Exposure to sMF enhanced the viability of these cells. However, the effect was blocked by treating the cells with LY294002, a P13K inhibitor. We compared this effect by Western Blot analysis of Akt protein expression. We also examined whether the cell response on sMF stimulation is dependent on integrin engagement and we measured integrin gene expression. Our results suggest that stimulation using sMF is a viable method to improve hASC viability. sMF is involved in mechanisms associated with controlling cell proliferative potential signaling events.

Means of enhancing bone fracture healing: optimal cell source, isolation methods and acoustic stimulation.

BACKGROUND: The human body has an extensive capacity to regenerate bone tissue after trauma. However large defects such as long bone fractures of the lower limbs cannot be restored without intervention and often lead to nonunion. Therefore, the aim of the present study was to assess the pool and biological functions of human mesenchymal stromal cells (hMSCs) isolated from different bone marrow locations of the lower limbs and to identify novel strategies to prime the cells prior to their use in bone fracture healing. Following, bone marrow from the ilium, proximal femur, distal femur and proximal tibia was aspirated and the hMSCs isolated. Bone marrow type, volume, number of mononuclear cells/hMSCs and their self-renewal, multilineage potential, extracellular matrix (ECM) production and surface marker profiling were analyzed. Additionally, the cells were primed to accelerate bone fracture healing either by using acoustic stimulation or varying the initial hMSCs isolation conditions. RESULTS: We found that the more proximal the bone marrow aspiration location, the larger the bone marrow volume was, the higher the content in mononuclear cells/hMSCs and the higher the self-renewal and osteogenic differentiation potential of the isolated hMSCs were. Acoustic stimulation of bone marrow, as well as the isolation of hMSCs in the absence of fetal bovine serum, increased the osteogenic and ECM production potential of the cells, respectively. CONCLUSION: We showed that bone marrow properties change with the aspiration location, potentially explaining the differences in bone fracture healing between the tibia and the femur. Furthermore, we showed two new priming methods capable of enhancing bone fracture healing.

Mesenchymal stem cell treatment in hyperoxia-induced lung injury in newborn rats.

BACKGROUND: The aim of this study was to evaluate the effectiveness of tracheally delivered mesenchymal stem cells (MSC) on lung pathology in a hyperoxia-induced lung injury (HILI) model in neonatal rats. METHODS: For the HILI model, rat pups were exposed to 85-95% oxygen during the first 10 days of life. Rats were divided into six groups: room-air normoxia (n = 11); room air, sham (n = 11); hyperoxia exposed with normal saline as placebo (n = 9); hyperoxia exposed with culture medium of MSC (n = 10); hyperoxia exposed with medium remaining after harvesting of MSC (n = 8); and hyperoxia exposed with MSC (n = 17). Pathologic changes, number and diameter of alveoli, α-smooth muscle actin (α-SMA) expression and localization of MSC in the lungs were assessed. RESULTS: Number of alveoli increased and alveolar diameter decreased in the mesenchymal stem cell group so that there were no differences when compared with the normoxia group (P = 0.126 and P = 0.715, respectively). Expression of α-SMA decreased significantly in the mesenchymal stem cell group compared with the placebo group (P < 0001). Green fluorescent protein-positive cells were found in lung tissue from all rats given MSC. Some green fluorescent protein-positive MSC also expressed surfactant protein-C. CONCLUSION: Mesenchymal stem cells became localized in damaged lung tissue, and recovery approximated the room air control.

Bone Regeneration of the Maxillofacial Region Through the Use of Mesenchymal Cells Obtained by a Filtration Process of the Adipose Tissue.

In this work, the authors will discuss about a new protocol and rapid alternative to isolate the mesenchymal stromal cells from the stromal vascular fraction, without the use of collagenase, to regenerate the bone tissue in the maxillo-facial region. This method employs a device that allows the isolation and concentration of stromal vascular fraction by means of lipoaspirates, which separate the lipid component and fragments of the extracellular matrix. The innovative element consists in using a filtration device instead of a centrifuging device to separate the different components. The purpose of this work was to illustrate the results obtained with the above-mentioned method in a series of 8 patients suffering from cystic neoformations maxillary or mandibular.

Natural history of mesenchymal stem cells, from vessel walls to culture vessels.

Mesenchymal stem/stromal cells (MSCs) can regenerate tissues by direct differentiation or indirectly by stimulating angiogenesis, limiting inflammation, and recruiting tissue-specific progenitor cells. MSCs emerge and multiply in long-term cultures of total cells from the bone marrow or multiple other organs. Such a derivation in vitro is simple and convenient, hence popular, but has long precluded understanding of the native identity, tissue distribution, frequency, and natural role of MSCs, which have been defined and validated exclusively in terms of surface marker expression and developmental potential in culture into bone, cartilage, and fat. Such simple, widely accepted criteria uniformly typify MSCs, even though some differences in potential exist, depending on tissue sources. Combined immunohistochemistry, flow cytometry, and cell culture have allowed tracking the artifactual cultured mesenchymal stem/stromal cells back to perivascular anatomical regions. Presently, both pericytes enveloping microvessels and adventitial cells surrounding larger arteries and veins have been described as possible MSC forerunners. While such a vascular association would explain why MSCs have been isolated from virtually all tissues tested, the origin of the MSCs grown from umbilical cord blood remains unknown. In fact, most aspects of the biology of perivascular MSCs are still obscure, from the emergence of these cells in the embryo to the molecular control of their activity in adult tissues. Such dark areas have not compromised intents to use these cells in clinical settings though, in which purified perivascular cells already exhibit decisive advantages over conventional MSCs, including purity, thorough characterization and, principally, total independence from in vitro culture. A growing body of experimental data is currently paving the way to the medical usage of autologous sorted perivascular cells for indications in which MSCs have been previously contemplated or actually used, such as bone regeneration and cardiovascular tissue repair.

EphB2 isolates a human marrow stromal cell subpopulation with enhanced ability to contribute to the resident intestinal cellular pool.

To identify human bone marrow stromal cell (BMSC) subsets with enhanced ability to engraft/contribute to the resident intestinal cellular pool, we transplanted clonally derived BMSCs into fetal sheep. Analysis at 75 d post-transplantation showed 2 of the 6 clones engrafting the intestine at 4- to 5-fold higher levels (5.03±0.089 and 5.04±0.15%, respectively) than the other clones (P<0.01), correlating with the percentage of donor-derived Musashi-1(+) (12.01-14.17 vs. 1.2-3.8%; P<0.01) or leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5)(+) cells within the intestinal stem cell (ISC) region. Phenotypic and transcriptome analysis determined that the clones with enhanced intestinal contribution expressed high levels of Ephrin type B receptor 2 (EphB2). Intestinal explants demonstrated proliferation of the engrafted cells and ability to generate crypt-like structures in vitro still expressing EphB2. Additional transplants based on BMSC EphB2 expression demonstrated that, at 7 d post-transplant, the EphB2(high) BMSCs engrafted in the ISC region at levels of 2.1 ± 0.2%, while control EphB2(low) BMSCs engrafted at 0.3 ± 0.1% (P<0.01). Therefore we identified a marker for isolating and culturing an expandable subpopulation of BMSCs with enhanced intestinal homing and contribution to the ISC region.

Effect of bone marrow-derived mesenchymal stem cells on hepatic fibrosis in a thioacetamide-induced cirrhotic rat model.

BACKGROUND: Cirrhosis is a long-term consequence of chronic hepatic injury with fibrosis. No effective therapy is currently available for decompensated cirrhosis except liver transplantation. Hence, we investigated the effect of bone marrow-derived mesenchymal stem cells (BM-MSCs) on hepatic fibrosis in a thioacetamide (TAA)-induced cirrhotic rat model. METHODS: The BM-MSCs were injected directly into the right liver lobe twice, at 6 and 8 weeks during the 12-week TAA administration, in thioacetamide (TAA)-induced cirrhotic rats model, and hepatic fibrosis was evaluated. At 12 weeks, the effect of BM-MSCs on hepatic fibrosis was analyzed histomorphologically using the Laennec fibrosis scoring system, and the collagen proportionate area was quantified. Cirrhosis-related factors, such as transforming growth factor ß1 (TGF-ß1), type 1 collagen (collagen-1), α-smooth muscle actin (α-SMA), and P-Smad3/Smad3 expression levels, were evaluated using real-time polymerase chain reaction and western blot assays. RESULTS: According to the Laennec fibrosis scoring system, histological improvement was observed in hepatic fibrosis after BM-MSC treatment (P <0.01). The percentage of the collagen proportionate area decreased from 16.72 ± 5.51 to 5.06 ± 1.27 after BM-MSC treatment (P <0.01). The content of hepatic hydroxyproline was significantly lower in the BM-MSC treated group (46.25 ± 13.19) compared to the untreated cirrhotic group (85.81 ± 17.62; P <0.01). BM-MSC administration significantly decreased TGF-ß1, collagen-1, and α-SMA expression in TAA-induced cirrhotic rats (P <0.01). We also confirmed P-Smad3/Smad3, downstream effectors of the TGF-ß1 signaling pathway, and found that MSC transplantation inhibited Smad3 phosphorylation. CONCLUSIONS: BM-MSC treatment attenuated hepatic fibrosis in rats with TAA-induced cirrhosis, raising the possibility of the clinical use of BM-MSCs in the treatment of cirrhosis.

Mesenchymal stem cells and transplant tolerance.

Different strategies are being tried to induce transplant tolerance in clinical settings; however, none of them are both safe and effective. Mesenchymal stem cells have been found to be potent immunomodulators and immunosuppressants. We discuss in this review different sources of mesenchymal stem cells and the potent role of adipose tissue-derived mesenchymal stem cells in induction of transplant tolerance including when to use them and how to use them for achieving the Utopian dream of transplant tolerance.

Multiparameter flow cytometry for the characterisation of extracellular markers on human mesenchymal stem cells.

Extracellular surface proteins are used to identify fully-functional human mesenchymal stem cells (hMSCs) in a mixed population. Here, a multiparameter flow cytometry assay was developed to examine the expression of several bone marrow-derived hMSC markers simultaneously at the single cell level. The multiparameter approach demonstrates a depth of analysis that goes far beyond the conventional single or dual staining methods. CD73, CD90 and CD105 were chosen as positive markers as they are expressed on multipotent hMSCs, whilst CD34 and HLA-DR were chosen as negative indicators. Single colour analysis suggested a population purity of 100 %; in contrast, when analysed via the multiparameter method, the CD73(+ve)/CD105(+ve)/CD90(+ve)/HLA-DR(-ve)/CD34(-ve) phenotypes represented 94.5 ± 1.3 % of the total cell population. Also, although CD271 has been posited as a definite early stage hMSC marker, here we show it is not present on pre-passage cells, highlighting the need for careful marker selection.