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

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

Connexin 36 controls synchronization of Ca2+ oscillations and insulin secretion in MIN6 cells.

Cx36 is the predominant connexin isoform expressed by pancreatic beta-cells. However, little is known about the role of this protein in the functioning of insulin-secreting cells. To address this question, we searched for a cell line expressing Cx36 and having glucose-induced insulin secretion comparable to that of primary beta-cells. By evaluating Cx36 expression in MIN6, betaTC3, RIN2A, INS1, and HIT cell lines, which differ in their sensitivity to glucose, we found that wild-type MIN6 cells fit these requirements. Therefore, we stably transfected MIN6 cells with a cDNA coding for a Cx36 antisense sequence to study the role of Cx36 in these cells. Independent clones of MIN6 cells were obtained that had a markedly reduced Cx36 expression. Loss of Cx36 decreased functional gap junctional conductance in these clones. This alteration impaired the synchronization of glucose-induced [Ca(2+)](i) oscillations and insulin secretion in response to glucose, to secretagogues that increase [cAMP](i), and to depolarizing conditions. These data provide the first evidence that Cx36-made channels 1) mediate functional coupling in MIN6 cells, 2) provide for synchronous [Ca(2+)](i) oscillations, and 3) are necessary for proper insulin secretion in response to metabolizable and nonmetabolizable secretagogues.

Cell specific differences between human adipose-derived and mesenchymal-stromal cells despite similar differentiation potentials.

Stromal cells from bone marrow and adipose tissue are attractive sources of adult progenitors for cell-based therapy. However, whether those cell populations represent intrinsically different cell types is still largely under debate. The aim of this study was to systematically and quantitatively compare adipose-derived stromal cells (ADSC) and bone marrow-derived multipotent mesenchymal-stromal cells (BM-MSC). The quantitative comparison was realized using Taqman Low Density Array, 2D electrophoresis and differentiation functional assays in vitro. Furthermore, cells engineered to express TGFbeta1 were injected into the intra-articular space of mouse knee joints in order to determine whether they were able to form new differentiated tissues in vivo. Our data revealed cell specific differences at transcriptional and proteomic levels between both cell types according to their tissue origin as well as functional differences in their differentiation processes towards adipogenic, osteogenic and chondrogenic programs. Nevertheless, in vitro as well as in vivo ADSC displayed the same ability than MSC to differentiate towards chondrocytes/osteoblasts, comforting the status of both cell sources as promising regenerative cells. In summary, our observations indicate that ADSC and MSC are fundamentally different cell types and differently committed cells.

From heterogeneity to plasticity in adipose tissues: site-specific differences.

In mammals, two types of adipose tissues are present, brown (BAT) and white (WAT). WAT itself can be divided into subcutaneous and internal fat deposits. All these tissues have been shown to present a great tissue plasticity, and recent data emphasized on the multiple differentiation potentials obtained from subcutaneous WAT. However, no study has compared the heterogeneity of stroma-vascular fraction (SVF) cells and their differentiation potentials according to the localization of the fat pad. This study clearly demonstrates that WAT and BAT present different antigenic features and differentiation potentials. WAT by contrast to BAT contains a large population of hematopoietic cells composed essentially of macrophages and hematopoietic progenitor cells. In WAT, the non-hematopoietic population is mainly composed of mesenchymal stem cell (MSC)-like but contains also a significant proportion of immature cells, whereas in BAT, the stromal cells do not present the same phenotype. Internal and subcutaneous WAT present some discrete differences in the phenotype of their cell populations. WAT derived SVF cells give rise to osteoblasts, endothelial cells, adipocytes, hematopoietic cells, and cardiomyoblasts only from inguinal cells. By contrast, BAT derived SVF cells display a reduced plasticity. Adipose tissues thus appear as complex tissues composed of different cell subsets according to the location of fat pads. Inguinal WAT appears as the most plastic adipose tissue and represents a potential and suitable source of stem cell, considering its easy sampling as a major advantage for cell therapy.

Human subcutaneous adipose cells support complete differentiation but not self-renewal of hematopoietic progenitors.

Adipose tissue is now considered as an endocrine organ implicated in energy regulation, inflammation and immune response, and as a source of multipotent cells with a broad range of differentiation capacities. Some of these cells are of a mesenchymal type which can -- like their bone marrow (BM) counterpart -- support hematopoiesis, since in a previous study we were able to reconstitute lethally irradiated mice by cells isolated from adipose tissue. In the present study, we established that cells derived from the stroma-vascular fraction of human subcutaneous fat pads support the complete differentiation of hematopoietic progenitors into myeloid and B lymphoid cells. However, these cells are unable to maintain the survival and self-renewal of hematopoietic stem cells. These features, similar to those of BM adipocytes, are the opposite of those of other cell types derived from mesenchymal progenitors such as BM myofibroblasts or osteoblasts. Because it is abundant and accessible, adipose tissue could be a convenient source of cells for the short-term reconstitution of hematopoiesis in man.

Differentiating the effects of Cx36 and E-cadherin for proper insulin secretion of MIN6 cells.

Connexins have been implicated in many cell functions, even though in most cases it is still unclear whether these functions may actually be mediated by other proteins that are secondarily affected by connexin changes. Secretory systems provide useful models in which to tackle this central question. Primary pancreatic beta-cells and insulin-producing lines are connected by gap junction channels made of Cx36. Using stable transfection of an antisense Cx36 cDNA, we have previously obtained clones of MIN6 cells that featured a markedly reduced expression of Cx36 and impaired insulin secretion. Here, we first show that this change also resulted in loss of E-cadherin and occludin expression, thus preventing the attribution of the secretory defects to a specific type of membrane protein. To investigate this question, we have now restored the expression of either Cx36 or E-cadherin in the Cx36 antisense-transfected cells. We show that a lentivirus-mediated transduction efficiently restored Cx36 expression in MIN6 cells and allowed for expression of variable levels of this protein. We further document that adequate but not excessive levels of Cx36 allowed for recover of normal insulin secretion in response to various secretagogues. Finally, we demonstrate that restoration of normal E-cadherin expression was not able to achieve the same secretory correction. The data demonstrate that Cx36, but not E-cadherin, is necessary to control specific steps of beta-cell secretion.

Connexins and secretion.

Connexin channels clustered at gap junctions are obligatory attributes of all macroscopic endocrine and exocrine glands investigated so far and also connect most types of cells which produce secretory products in other tissues. Increasing evidence indicates that connexins, and the cell-to-cell communications that these proteins permit, contribute to control the growth of secretory cells, their expression of specific genes and their differentiated function, including their characteristic ability to biosynthetize and release secretory products in a regulated manner. Since the previous reviews which have been published on this topic, several lines of evidence have been added in support of multiple regulatory roles of gland connexins. Here, we review this novel evidence, point to the many questions which are still open and discuss some interesting perspectives of the field.

Lentivirus-mediated transduction of connexin cDNAs shows level- and isoform-specific alterations in insulin secretion of primary pancreatic beta-cells.

We have generated novel lentiviral vectors to integrate various connexin cDNAs into primary, non-dividing cells. We have used these vectors to test whether proper control of insulin secretion depends on a specific connexin isoform and/or on its level of expression. We have observed that transduced connexin32, connexin36 and connexin43 were expressed by primary adult beta-cells at membrane interfaces, were packed into typical gap junction plaques and formed functional channels that allowed a variable coupling, depending on the type and level of connexin expressed. The infected cells spontaneously reaggregated into three-dimensional pseudo-islet organs that could be maintained in culture. We have found that pseudo-islets made by cells transduced with either GFP- or connexin43-expressing lentivirus released insulin in response to various secretagogues similarly to controls. By contrast, pseudo-islets made by cells expressing connexin32, a connexin exogenous to pancreatic islets, or over-expressing connexin36, the endogenous islet connexin, featured a marked decrease in the secretory response to glucose. The data show: (1) that lentiviral vectors allow stable modulation of various connexin in primary, non-proliferating cells; (2) that specific connexin isoforms affect insulin secretion differently; and (3) that adequate levels of coupling via connexin36 channels are required for proper beta-cell function.