Mesenchymal stem cells (MSCs) have been investigated as promising candidates for use in new cell-based therapeutic strategies such as mesenchyme-derived tissue repair. MSCs are easily isolated from adult tissues and are not ethically restricted. MSC-related literature, however, is conflicting in relation to MSC differentiation potential and molecular markers. Here we compared MSCs isolated from bone marrow (BM), umbilical cord blood (UCB), and adipose tissue (AT). The isolation efficiency for both BM and AT was 100%, but that from UCB was only 30%. MSCs from these tissues are morphologically and immunophenotypically similar although their differentiation diverges. Differentiation to osteoblasts and chondroblasts was similar among MSCs from all sources, as analyzed by cytochemistry. Adipogenic differentiation showed that UCB-derived MSCs produced few and small lipid vacuoles in contrast to those of BM-derived MSCs and AT-derived stem cells (ADSCs) (arbitrary differentiation values of 245.57 +/- 943 and 243.89 +/- 145.52 mum(2) per nucleus, respectively). The mean area occupied by individual lipid droplets was 7.37 mum(2) for BM-derived MSCs and 2.36 mum(2) for ADSCs, a finding indicating more mature adipocytes in BM-derived MSCs than in treated cultures of ADSCs. We analyzed FAPB4, ALP, and type II collagen gene expression by quantitative polymerase chain reaction to confirm adipogenic, osteogenic, and chondrogenic differentiation, respectively. Results showed that all three sources presented a similar capacity for chondrogenic and osteogenic differentiation and they differed in their adipogenic potential. Therefore, it may be crucial to predetermine the most appropriate MSC source for future clinical applications.
Background-Cellular transplantation is emerging as a promising strategy for the treatment of postinfarction ventricular dysfunction. Whether its beneficial effects can be extended to other cardiomyopathies remains an unexplored question. We evaluated the histological and functional effects of simultaneous autologous transplantation of co-cultured stem cells and skeletal myoblasts in an experimental model of dilated cardiomyopathy caused by Chagas disease, characterized by diffuse fibrosis and impairment of microcirculation. Methods and Results-Wistar rats weighing 200 grams were infected intraperitoneally with 15ϫ10 4 trypomastigotes. After 8 months, 2-dimensional echocardiographic study was performed for baseline assessment of left ventricle (LV) ejection fraction (EF) (%), left ventricle end-diastolic volume (LVEDV) (mL), and left ventricle end-systolic volume (LVESV) (mL). Animals with LV dysfunction (EF Ͻ37%) were selected for the study. Autologous skeletal myoblasts were isolated from muscle biopsy and mesenchymal stem cells from bone marrow aspirates were co-cultured in vitro for 14 days, yielding a cell viability of Ͼ90%. Eleven animals received autologous transplant of 5.4ϫ10 6 Ϯ8.0ϫ10 6 cells (300 L) into the LV wall. The control group (nϭ10) received culture medium (300 L). Cell types were identified with vimentin and fast myosin. After 4 weeks, ventricular function was reassessed by echo. For histological analysis, heart tissue was stained with hematoxylin and eosin and immunostained for fast myosin.
Stem cells can either differentiate into more specialized cells or undergo self-renewal. Several lines of evidence from different organisms suggest that these processes depend on the post-transcriptional regulation of gene expression. The presence of the PUF [Pumilio/FBF (fem-3 binding factor)] domain defines a conserved family of RNA binding proteins involved in repressing gene expression. It has been suggested that a conserved function of PUF proteins is to repress differentiation and sustain the mitotic proliferation of stem cells. In humans, Pumilio-2 (PUM2) is expressed in embryonic stem cells and adult germ cells. Here we show that PUM2 is expressed in a subpopulation of adipose-derived stem cell (ASC) cultures, with a granular pattern of staining in the cytoplasm. Protein levels of PUM2 showed no changes during the differentiation of ASCs into adipocytes. Moreover, RNAi knockdown of pum2 did not alter the rate of adipogenic differentiation compared with wild-type control cells. A ribonomic approach was used to identify PUM2-associated mRNAs. Microarray analysis showed that PUM2-bound mRNAs are part of gene networks involved in cell proliferation and gene expression control. We studied pum2 expression in cell cultures with low or very high levels of proliferation and found that changes in pum2 production were dependent on the proliferation status of the cell. Transient knockdown of pum2 expression by RNAi impaired proliferation of ASCs in vitro. Our results suggest that PUM2 does not repress differentiation of ASCs but rather is involved in the positive control of ASCs division and proliferation.
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