Autologous Adipocyte Derived Stem Cells Favour Healing in a Minipig Model of Cutaneous Radiation Syndrome

Cutaneous radiation syndrome (CRS) is the delayed consequence of localized skin exposure to high doses of ionizing radiation. Here we examined for the first time in a large animal model the therapeutic potential of autologous adipose tissue-derived stroma cells (ASCs). For experiments, Göttingen minipigs were locally gamma irradiated using a 60Co source at the dose of 50 Gy and grafted (n = 5) or not (n = 8). ASCs were cultured in MEM-alpha with 10% fetal calf serum and basic fibroblast growth factor (2 ng.mL−1) and post irradiation were intradermally injected on days 25, 46, 67 and finally between days 95 and 115 (50×106 ASCs each time) into the exposed area. All controls exhibited a clinical evolution with final necrosis (day 91). In grafted pigs an ultimate wound healing was observed in four out of five grafted animals (day 130 +/− 28). Immunohistological analysis of cytokeratin expression showed a complete epidermis recovery. Grafted ASCs accumulated at the dermis/subcutis barrier in which they attracted numerous immune cells, and even an increased vasculature in one pig. Globally this study suggests that local injection of ASCs may represent a useful strategy to mitigate CRS

Genome-Wide Analysis of Histone H3 Lysine9 Modifications in Human Mesenchymal Stem Cell Osteogenic Differentiation

Mesenchymal stem cells (MSCs) possess self-renewal and multi-lineage differentiation potentials. It has been established that epigenetic mechanisms such as histone modifications could be critical for determining the fate of stem cells. In this study, full human genome promoter microarrays and expression microarrays were used to explore the roles of histone modifications (H3K9Ac and H3K9Me2) upon the induction of MSC osteogenic differentiation. Our results revealed that the enrichment of H3K9Ac was decreased globally at the gene promoters, whereas the number of promoters enriched with H3K9Me2 was increased evidently upon osteogenic induction. By a combined analysis of data from both ChIP-on-chip and expression microarrays, a number of differentially expressed genes regulated by H3K9Ac and/or H3K9Me2 were identified, implicating their roles in several biological events, such as cell cycle withdraw and cytoskeleton reconstruction that were essential to differentiation process. In addition, our results showed that the vitamin D receptor played a trans-repression role via alternations of H3K9Ac and H3K9Me2 upon MSC osteogenic differentiation. Data from this study suggested that gene activation and silencing controlled by changes of H3K9Ac and H3K9Me2, respectively, were crucial to MSC osteogenic differentiation

The Role of Paracrine and Autocrine Signaling in the Early Phase of Adipogenic Differentiation of Adipose-derived Stem Cells.

INTRODUCTION: High cell density is known to enhance adipogenic differentiation of mesenchymal stem cells, suggesting secretion of signaling factors or cell-contact-mediated signaling. By employing microfluidic biochip technology, we have been able to separate these two processes and study the secretion pathways. METHODS AND RESULTS: Adipogenic differentiation of human adipose-derived stem cells (ASCs) cultured in a microfluidic system was investigated under perfusion conditions with an adipogenic medium or an adipogenic medium supplemented with supernatant from differentiating ASCs (conditioned medium). Conditioned medium increased adipogenic differentiation compared to adipogenic medium with respect to accumulation of lipid-filled vacuoles and gene expression of key adipogenic markers (C/EBPα, C/EBPβ, C/EBPδ, PPARγ, LPL and adiponectin). The positive effects of conditioned medium were observed early in the differentiation process. CONCLUSIONS: Using different cell densities and microfluidic perfusion cell cultures to suppress the effects of cell-released factors, we have demonstrated the significant role played by auto- or paracrine signaling in adipocyte differentiation. The cell-released factor(s) were shown to act in the recruitment phase of the differentiation process

Identification and characterization of a rich population of CD34mesenchymal stem/stromal cells in human parotid, sublingual and submandibular glands

Mesenchymal stem/stromal cells (MSCs) play crucial roles in maintaining tissue homeostasis during physiological turnovers and injuries. Very little is known about the phenotype, distribution and molecular nature of MSCs in freshly isolated human salivary glands (SGs) as most reports have focused on the analysis of cultured MSCs. Our results demonstrate that the cell adhesion molecule CD34 was widely expressed by the MSCs of human major SGs, namely parotid (PAG), sublingual (SLG) and submandibular (SMG) glands. Further, gene expression analysis of CD34+ cells derived from fetal SMGs showed significant upregulation of genes involved in cellular adhesion, proliferation, branching, extracellular matrix remodeling and organ development. Moreover, CD34+ SMG cells exhibited elevated expression of genes encoding extracellular matrix, basement membrane proteins, and members of ERK, FGF and PDGF signaling pathways, which play key roles in glandular development, branching and homeostasis. In vitro CD34+ cell derived SG-MSCs revealed multilineage differentiation potential. Intraglandular transplantation of cultured MSCs in immunodeficient mice led to their engraftment in the injected and uninjected contralateral and ipsilateral glands. Engrafted cells could be localized to the stroma surrounding acini and ducts. In summary, our data show that CD34+ derived SG-MSCs could be a promising cell source for adoptive cell-based SG therapies, and bioengineering of artificial SGs

Native human adipose stromal cells: localization, morphology and phenotype

International audienceObjectives:Beside having roles in energy homeostasis and endocrine modulation, adipose tissue (AT) is now considered a promising source of mesenchymal stromal cells (adipose-derived stromal cells or ASCs) for regenerative medicine. Despite numerous studies on cultured ASCs, native human ASCs are rarely investigated. Indeed, the phenotype of ASCs in their native state, their localization within AT and comparison with bone marrow-derived mesenchymal stromal cells (BM-MSCs) has been poorly investigated.Design:To address these issues, the stroma vascular fraction (SVF) of human AT was extracted and native cell subtypes were isolated by immunoselection to study their clonogenic potential in culture. Immunohistology on samples of human AT in combination with reconstruction of confocal sections were performed in order to localize ASCs.Results:Compared with BM-MNCs, all native ASCs were found in the CD34(+) cell fraction of the AT-SVF. Native ASCs expressed classical mesenchymal markers described for BM-MSCs. Interestingly, CD34 expression decreased during ASC cell culture and was negatively correlated with cell proliferation rate. Immunohistological analysis revealed that native ASCs exhibited specific morphological features with protrusions. They were found scattered in AT stroma and did not express in vivo pericytic markers such as NG2, CD140b or alpha-smooth muscle actin, which appeared during the culture process. Finally, ASCs spontaneous commitment to adipocytic lineage was enhanced in AT from obese humans.Conclusions:The use of complementary methodological approaches to study native human ASCs revealed their immunophenotype, their specific morphology, their location within AT and their stemness. Furthermore, our data strongly suggest that human ASCs participate in adipogenesis during AT development.International Journal of Obesity advance online publication, 25 January 2011; doi:10.1038/ijo.2010.269

Genome-Wide Profile of Pleural Mesothelioma versus Parietal and Visceral Pleura: The Emerging Gene Portrait of the Mesothelioma Phenotype

Malignant pleural mesothelioma is considered an almost incurable tumour with increasing incidence worldwide. It usually develops in the parietal pleura, from mesothelial lining or submesothelial cells, subsequently invading the visceral pleura. Chromosomal and genomic aberrations of mesothelioma are diverse and heterogenous. Genome-wide profiling of mesothelioma versus parietal and visceral normal pleural tissue could thus reveal novel genes and pathways explaining its aggressive phenotype.Well-characterised tissue from five mesothelioma patients and normal parietal and visceral pleural samples from six non-cancer patients were profiled by Affymetrix oligoarray of 38 500 genes. The lists of differentially expressed genes tested for overrepresentation in KEGG PATHWAYS (Kyoto Encyclopedia of Genes and Genomes) and GO (gene ontology) terms revealed large differences of expression between visceral and parietal pleura, and both tissues differed from mesothelioma. Cell growth and intrinsic resistance in tumour versus parietal pleura was reflected in highly overexpressed cell cycle, mitosis, replication, DNA repair and anti-apoptosis genes. Several genes of the “salvage pathway” that recycle nucleobases were overexpressed, among them TYMS, encoding thymidylate synthase, the main target of the antifolate drug pemetrexed that is active in mesothelioma. Circadian rhythm genes were expressed in favour of tumour growth. The local invasive, non-metastatic phenotype of mesothelioma, could partly be due to overexpression of the known metastasis suppressors NME1 and NME2. Down-regulation of several tumour suppressor genes could contribute to mesothelioma progression. Genes involved in cell communication were down-regulated, indicating that mesothelioma may shield itself from the immune system. Similarly, in non-cancer parietal versus visceral pleura signal transduction, soluble transporter and adhesion genes were down-regulated. This could represent a genetical platform of the parietal pleura propensity to develop mesothelioma.Genome-wide microarray approach using complex human tissue samples revealed novel expression patterns, reflecting some important features of mesothelioma biology that should be further explored

Defective proliferation and osteogenic potential with altered immunoregulatory phenotype of native bone marrow-multipotential stromal cells in atrophic fracture non-union

Bone marrow-Multipotential stromal cells (BM-MSCs) are increasingly used to treat complicated fracture healing e.g., non-union. Though, the quality of these autologous cells is not well characterized. We aimed to evaluate bone healing-related capacities of non-union BM-MSCs. Iliac crest-BM was aspirated from long-bone fracture patients with normal healing (U) or non-united (NU). Uncultured (native) CD271highCD45low cells or passage-zero cultured BM-MSCs were analyzed for gene expression levels, and functional assays were conducted using culture-expanded BM-MSCs. Blood samples were analyzed for serum cytokine levels. Uncultured NU-CD271highCD45low cells significantly expressed fewer transcripts of growth factor receptors, EGFR, FGFR1, and FGRF2 than U cells. Significant fewer transcripts of alkaline phosphatase (ALPL), osteocalcin (BGLAP), osteonectin (SPARC) and osteopontin (SPP1) were detected in NU-CD271highCD45low cells. Additionally, immunoregulation-related markers were differentially expressed between NU- and U-CD271highCD45low cells. Interestingly, passage-zero NU BM-MSCs showed low expression of immunosuppressive mediators. However, culture-expanded NU and U BM-MSCs exhibited comparable proliferation, osteogenesis, and immunosuppression. Serum cytokine levels were found similar for NU and U groups. Collectively, native NU-BM-MSCs seemed to have low proliferative and osteogenic capacities; therefore, enhancing their quality should be considered for regenerative therapies. Further research on distorted immunoregulatory molecules expression in BM-MSCs could potentially benefit the prediction of complicated fracture healing