Comparison of Gene Expression in Human Embryonic Stem Cells, hESC-Derived Mesenchymal Stem Cells and Human Mesenchymal Stem Cells

We present a strategy to identify developmental/differentiation and plasma membrane marker genes of the most primitive human Mesenchymal Stem Cells (hMSCs). Using sensitive and quantitative TaqMan Low Density Arrays (TLDA) methodology, we compared the expression of 381 genes in human Embryonic Stem Cells (hESCs), hESC-derived MSCs (hES-MSCs), and hMSCs. Analysis of differentiation genes indicated that hES-MSCs express the sarcomeric muscle lineage in addition to the classical mesenchymal lineages, suggesting they are more primitive than hMSCs. Transcript analysis of membrane antigens suggests that IL1R1low, BMPR1Blow, FLT4low, LRRC32low, and CD34 may be good candidates for the detection and isolation of the most primitive hMSCs. The expression in hMSCs of cytokine genes, such as IL6, IL8, or FLT3LG, without expression of the corresponding receptor, suggests a role for these cytokines in the paracrine control of stem cell niches. Our database may be shared with other laboratories in order to explore the considerable clinical potential of hES-MSCs, which appear to represent an intermediate developmental stage between hESCs and hMSCs

Osteogenic differentiation of human bone marrow mesenchymal stem cells seeded on melt based chitosan scaffolds for bone tissue engineering applications

The purpose of this study was to evaluate the growth patterns and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) when seeded onto new biodegradable chitosan/polyester scaffolds. Scaffolds were obtained by melt blending chitosan with poly(butylene succinate) in a proportion of 50% (wt) each and further used to produce a fiber mesh scaffold. hBMSCs were seeded on those structures and cultured for 3 weeks under osteogenic conditions. Cells were able to reduce MTS and demonstrated increasing metabolic rates over time. SEM observations showed cell colonization at the surface as well as within the scaffolds. The presence of mineralized extracellular matrix (ECM) was successfully demonstrated by peaks corresponding to calcium and phosphorus elements detected in the EDS analysis. A further confirmation was obtained when carbonate and phosphate group peaks were identified in Fourier Transformed Infrared (FTIR) spectra. Moreover, by reverse transcriptase (RT)-PCR analysis, it was observed the expression of osteogenic gene markers, namely, Runt related transcription factor 2 (Runx2), type 1 collagen, bone sialoprotein (BSP), and osteocalcin. Chitosan-PBS (Ch- PBS) biodegradable scaffolds support the proliferation and osteogenic differentiation of hBMSCs cultured at their surface in vitro, enabling future in vivo testing for the development of bone tissue engineering therapies.Ana Costa-Pinto was supported by a grant (SFRH/24735/2005) from the Portuguese Foundation for Science and Technology "Fundacao para a Ciencia e a Tecnologia" (FCT). This work was partially supported by the EU Integrated Project GENOSTEM (Adult Mesenchymal Stem Cells Engineering for connective tissue disorders: from the bench to the bedside, LSHB-CT-2003-5033161), and the European Network of Excellence EXPERTISSUES (NMP3-CT-2004-500283). The authors would like to acknowledge to the School of Health Sciences of the University of Minho for the opportunity of using its facilities

Microenvironmental changes during differentiation of mesenchymal stem cells towards chondrocytes

Chondrogenesis is a process involving stem-cell differentiation through the coordinated effects of growth/differentiation factors and extracellular matrix (ECM) components. Recently, mesenchymal stem cells (MSCs) were found within the cartilage, which constitutes a specific niche composed of ECM proteins with unique features. Therefore, we hypothesized that the induction of MSC differentiation towards chondrocytes might be induced and/or influenced by molecules from the microenvironment. Using microarray analysis, we previously identified genes that are regulated during MSC differentiation towards chondrocytes. In this study, we wanted to precisely assess the differential expression of genes associated with the microenvironment using a large-scale real-time PCR assay, according to the simultaneous detection of up to 384 mRNAs in one sample. Chondrogenesis of bone-marrow-derived human MSCs was induced by culture in micropellet for various periods of time. Total RNA was extracted and submitted to quantitative RT-PCR. We identified molecules already known to be involved in attachment and cell migration, including syndecans, glypicans, gelsolin, decorin, fibronectin, and type II, IX and XI collagens. Importantly, we detected the expression of molecules that were not previously associated with MSCs or chondrocytes, namely metalloproteases (MMP-7 and MMP-28), molecules of the connective tissue growth factor (CTGF); cef10/cyr61 and nov (CCN) family (CCN3 and CCN4), chemokines and their receptors chemokine CXC motif ligand (CXCL1), Fms-related tyrosine kinase 3 ligand (FlT3L), chemokine CC motif receptor (CCR3 and CCR4), molecules with A Disintegrin And Metalloproteinase domain (ADAM8, ADAM9, ADAM19, ADAM23, A Disintegrin And Metalloproteinase with thrombospondin type 1 motif ADAMTS-4 and ADAMTS-5), cadherins (4 and 13) and integrins (α4, α7 and β5). Our data suggest that crosstalk between ECM components of the microenvironment and MSCs within the cartilage is responsible for the differentiation of MSCs into chondrocytes

Adhesion, proliferation, and osteogenic differentiation of a mouse mesenchymal stem cell line (BMC9) seeded on novel melt-based chitosan/polyester 3D porous scaffolds

The aim of the present work was to study the biological behavior of a mouse mesenchymal stem cell line when seeded and cultured under osteogenic conditions onto novel processed melt-based chitosan scaffolds. Scaffolds were produced by compression molding, followed by salt leaching. Scanning electron microscopy (SEM) observations and lCT analysis showed the pore sizes ranging between 250 and 500 lm and the interconnectivity of the porous structure. The chitosan–poly(butylenes succinate) scaffolds presented high mechanical properties, similar to the ones of trabecular bone (E1%*75 MPa). Cytotoxicity assays were carried out using standard tests (accordingly to ISO/EN 10993 part 5 guidelines), namely, MTS test with a 24 h extraction period, revealing that L929 cells had similar metabolic activities to that obtained for the negative control. Cell culture studies were conducted using a mouse mesenchymal stem cell line (BMC9). Cells were seeded onto the scaffold and allowed to proliferate for 3 weeks under osteogenic conditions. SEM observations demonstrated that cells were able to proliferate and massively colonize the scaffolds structure. The cell viability assay MTS demonstrated that BMC9 cells were viable after 3 weeks of culture. The cells clearly evidenced a positive differentiation toward the osteogenic lineage, as confirmed by the high ALP activity levels. Moreover, energy dispersive spectroscopy (EDS) analysis revealed the presence of Ca and P in the elaborated extracellular matrix (ECM). These combined results indicate that the novel melt-based chitosan/polyester scaffolds support the adhesion, proliferation, and osteogenic differentiation of the mouse MSCs and shows adequate physicochemical and biological properties for being used as scaffolds in bone tissue engineering–related strategies

Assessment of the suitability of chitosan/polybutylene succinate scaffolds seeded with mouse mesenchymal progenitor cells for a cartilage tissue engineering approach

In this work, scaffolds derived from a new biomaterial originated from the combination of a natural material and a synthetic material were tested for assessing their suitability for cartilage tissue engineering applications. In order to obtain a better outcome result in terms of scaffolds’ overall properties, different blends of natural and synthetic materials were created. Chitosan and polybutylene succinate (CPBS) 50/50 (wt%) were melt blended using a twin-screw extruder and processed into 5 5 5mm scaffolds by compression moulding with salt leaching. Micro-computed tomography analysis calculated an average of 66.29% porosity and 92.78% interconnectivity degree for the presented scaffolds. The salt particles used ranged in size between 63 and 125 lm, retrieving an average pore size of 251.28 lm. Regarding the mechanical properties, the compressive modulus was of 1.73 ± 0.4MPa (Esec 1%). Cytotoxicity evaluation revealed that the leachables released by the developed porous structures were not harmful to the cells and hence were noncytotoxic. Direct contact assays were carried out using a mouse bone marrow–derived mesenchymal progenitor cell line (BMC9). Cells were seeded at a density of 5 105 cells/scaffold and allowed to grow for periods up to 3 weeks under chondrogenic differentiating conditions. Scanning electron microscopy analysis revealed that the cells were able to proliferate and colonize the scaffold structure, and MTS test demonstrated cell viability during the time of the experiment. Finally, Western blot performed for collagen type II, a natural cartilage extracellular matrix component, showed that this protein was being expressed by the end of 3 weeks, which seems to indicate that the BMC9 cells were being differentiated toward the chondrogenic pathway. These results indicate the adequacy of these newly developed C-PBS scaffolds for supporting cell growth and differentiation toward the chondrogenic pathway, suggesting that they should be considered for further studies in the cartilage tissue engineering field.J. T. Oliveira would like to acknowledge the grant (SFRH/ BD17135/2004) from Portuguese Foundation for Science and Technology (FCT). The authors would like to thank Fernanda Marques, at the Institute for Health and Life Sciences (ICVS), University of Minho, Braga, Portugal, for her help with the Western blot analysis, as well as the staff at ICVS for allowing to use their facilities. The monoclonal antibody for collagen type II was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283), and partially supported by the European Project GENOSTEM (LSHB-CT-2003-503161) and the FCT Project CartiScaff (POCTI/SAU-BMA/58991/2004)

Human bone marrow mesenchymal stem cells : a systematic reappraisal via the genostem experience

Genostem (acronym for “Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench to the bed side”) has been an European consortium of 30 teams working together on human bone marrow Mesenchymal Stem Cell (MSC) biological properties and repair capacity. Part of Genostem activity has been dedicated to the study of basic issues on undifferentiated MSCs properties and on signalling pathways leading to the differentiation into 3 of the connective tissue lineages, osteoblastic, chondrocytic and tenocytic. We have evidenced that native bone marrow MSCs and stromal cells, forming the niche of hematopoietic stem cells, were the same cellular entity located abluminally from marrow sinus endothelial cells. We have also shown that culture-amplified, clonogenic and highly-proliferative MSCs were bona fide stem cells, sharing with other stem cell types the major attributes of self-renewal and of multipotential priming to the lineages to which they can differentiate (osteoblasts, chondrocytes, adipocytes and vascular smooth muscle cells/pericytes). Extensive transcription profiling and in vitro and in vivo assays were applied to identify genes involved in differentiation. Thus we have described novel factors implicated in osteogenesis (FHL2, ITGA5, Fgf18), chondrogenesis (FOXO1A) and tenogenesis (Smad8). Another part of Genostem activity has been devoted to studies of the repair capacity of MSCs in animal models, a prerequisite for future clinical trials. We have developed novel scaffolds (chitosan, pharmacologically active microcarriers) useful for the repair of both bone and cartilage. Finally and most importantly, we have shown that locally implanted MSCs effectively repair bone, cartilage and tendonWork supported by the European Community (Key action 1.2.4-3 Integrated Project Genostem, contract No 503161)

Pericyte-Like Progenitors Show High Immaturity and Engraftment Potential as Compared with Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) and pericyte progenitors (PPs) are both perivascular cells with similar multipotential properties regardless of tissue of origin. We compared the phenotype and function of the 2 cell types derived from the same bone-marrow samples but expanded in their respective media – pericyte conditions (endothelial cell growth medium 2 [EGM-2]) for PPs and standard medium (mesenchymal stem cell medium [MSM]) for MSCs. After 3 weeks of culture, whatever the expansion medium, all cells showed similar characteristics (MSC markers and adipo-osteo-chondroblastic differentiation potential), although neuronal potential was greater in EGM-2– than MSM-cultured cells. As compared with MSM-cultured MSCs, EGM-2–cultured PPs showed higher expression of the pericyte-specific antigen 3G5 than a-smooth muscle actin. In addition, EGM-2–cultured PPs showed an immature phenotype, with upregulation of stemness OCT4 and SOX2 proteins and downregulation of markers of osteoblastic, chondroblastic, adipocytic and vascular smooth muscle lineages. Despite having less effective in vitro immunosuppression capacities than standard MSCs, EGM-2–cultured PPs had higher engraftment potentials when combined with biomaterials heterotopically-transplanted in Nude mice. Furthermore, these engrafted cells generated more collagen matrix and were preferentially perivascular or lined trabeculae as compared with MSM-cultured MSCs. In conclusion, EGM-2–cultured PPs are highly immature cells with increased plasticity and engraftment potential

Bone marrow mesenchymal stem cells: historical overview and concepts.: Bone marrow MSC history and concepts

International audienceThis review describes the historical emergence of the concept of bone marrow mesenchymal stem cells (MSCs), summarizing data on Wolf and Trentin's hematopoietic inductive microenvironment; Dexter's hematopoiesis-supportive stromal cells; Friedenstein's osteogenic cells; and Pittenger's trilineal osteoblastic, chondrocytic, and adipocytic precursors; to finally introduce the specific bone marrow mesenchymal stem cells with differentiation potential to four lineages (mesenchymal and vascular smooth muscle lineages), and stromal and immunomodulatory capacities. Two points are the object of detailed discussion. The first point envisions the stem cell attributes (multipotentiality, self-renewal, tissue regeneration, population heterogeneity, plasticity, and lineage priming) compared with that of the paradigmatic hematopoietic stem cell. In the second point, we discuss the possible existence of bone marrow cells with greater differentiation potential, eventually pluripotential cells. The latter point raises the issues of cell fusion, reprogramming, or selection under nonstandardized conditions of rare populations of neuroectodermal origin, or of cells that had undergone mesenchymal-to-epithelial transition. In the last section, we review data on MSC senescence and possible malignant transformation secondary to extensive culture, gene transfer of telomerase, or mutations such as leading to Ewing's sarcoma. The set of data leads to the conclusion that bone marrow MSCs constitute a specific adult tissue stem cell population. The multiple characteristics of this stem cell type account for the versatility of the mechanisms of injured tissue repair. Although MSC administration may be extremely useful in a number of clinical applications, their transplantation is not without risks that must not be overlooked when developing cell therapy protocols

Caractérisation des cellules souches mesenchymateuses multipotentes stromales des deux principaux sites de l'hématopoïese (foie foetal et moelle osseuse)

Using cell lines obtain from the fetal liver and the bone marrow, we established a molecular signature of these stromales multipotential mesenchymal stem cells (MSC). This signature is based on the characterization of the lines by a study of differentiation in the mesodermic lineage generally studied (osteoblastic, adipocygenic, chondrogenic and vascular smooth muscle) and by their capacity to support the hematopoiesis. Starting from these cellular studies it could be highlighted a molecular signature based on the common gene expression only in the MSC lines. This signature shows a possible implication of cytoskeleton and proteins related to the DNA, in the inhibition of the phenomena of differentiation and proliferation of cells. In the second time we carried out a preliminary study of the differentiation capacity of MSC at the clonal level, in the mesodermic and neurœctodermic lineageNous avons établis, à partir de lignés cellulaires originaires du foie fœtal et de la moelle osseuse, une signature moléculaire des cellules souches mésenchymateuses (CSM) stromales multipotentielle. Cette signature repose sur la caractérisation des lignées par une étude de différenciation dans les voies mésodermiques usuellement étudiées (ostéoblastique, adipocytaire, chondrocytaire et vasculaire musculaire lisse) et par leur capacité de soutien de l'hématopoïèse après 3 et 5 semaines. A partir de ces e tudes cellulaires il a put être mis en évidence une signature moléculaire réalisée à partir de la expression commune dans les lignées de types CSM, et montrant une possible relation entre le cytosquelette et les protéines liées à l'ADN, dans l'inhibition des phénomènes de différenciation et prolifération des cellules. Dans un second temps nous avons réalisé une étude préliminaire de la capacité de différenciation des CSM à l'échelon clonale, au travers des voies mésodermiques et neurœctodermique.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

La biologie des cellules souchesmésenchymateuses d’origine humaine

Cet article résume les principales caractéristiques des cellules souches mésenchymateuses (CSM) de la moelle osseuse et des adipose-derived stroma/stem cells (ASC) du tissu adipeux : méthodes d’obtention, phénotype, potentiel de différenciation, capacités de soutien de l’hématopoïèse (fonction stromale) et d’immunosuppression. Deux points font l’objet d’une discussion particulière : d’une part les critères permettant de qualifier les CSM comme une population de cellules souches : multipotence, autorenouvellement, plasticité, et d’autre part les mécanismes de réparation en jeu dans les différentes indications de thérapie cellulaire : reconstitution des compartiments tissulaires fonctionnels par repopulation après prolifération et différenciation ou reprogrammation, effets de type stromal par sécrétion de facteurs angiogéniques, antiapoptotiques, antifibrotiques, modulateurs de l’inflammation, etc