Differentiation of Mesenchymal Stem Cells Derived from Pancreatic Islets and Bone Marrow into Islet-Like Cell Phenotype

BACKGROUND:Regarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs) for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself. METHODOLOGY/PRINCIPAL FINDINGS:In the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs) and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs) were cultured in custom-made serum-free medium, using suitable conditions in order to induce differentiation into Islet-like Cells (ILCs). HI-MSCs and BM-MSCs were positive for the MSC markers CD105, CD73, CD90, CD29. Following this induction, HI-MSC and BM-MSC formed evident islet-like structures in the culture flasks. To investigate functional modifications after induction to ILCs, ultrastructural analysis and immunofluorescence were performed. PDX1 (pancreatic duodenal homeobox gene-1), insulin, C peptide and Glut-2 were detected in HI-ILCs whereas BM-ILCs only expressed Glut-2 and insulin. Insulin was also detected in the culture medium following glucose stimulation, confirming an initial differentiation that resulted in glucose-sensitive endocrine secretion. In order to identify proteins that were modified following differentiation from basal MSC (HI-MSCs and BM-MSCs) to their HI-ILCs and BM-ILCs counterparts, proteomic analysis was performed. Three new proteins (APOA1, ATL2 and SODM) were present in both ILC types, while other detected proteins were verified to be unique to the single individual differentiated cells lines. Hierarchical analysis underscored the limited similarities between HI-MSCs and BM-MSCs after induction of differentiation, and the persistence of relevant differences related to cells of different origin. CONCLUSIONS/SIGNIFICANCE:Proteomic analysis highlighted differences in the MSCs according to site of origin, reflecting spontaneous differentiation and commitment. A more detailed understanding of protein assets may provide insights required to master the differentiation process of HI-MSCs to functional beta cells based only upon culture conditioning. These findings may open new strategies for the clinical use of BM-MSCs in diabetes

Analysis of Islet expression by Flow cytometry and measurement of insulin secretion by ELISA.

<p>(<b>A</b>) Representative FACS analyses shows that HI-MSCs were positive for markers characteristic of ILCs (Insulin, PDX-1, C-peptide and Glut-2) and that (<b>B</b>) BM-MSCs were only positive for Glut-2; (<b>C</b>) ELISA test shows insulin levels of supernatants from MSCs at basal level (BM-MSCs and HI-MSCs) and after differentiation to ILCs (BM-ILCs and HI-ILCs).</p

Proteins shared by MSCs and ILCs, as identified by MALDI-TOF MS.

a<p>MW, molecular weight.</p>b<p>pI, isoelectric point.</p>c<p>Number of matched mass values on number of total mass values searched.</p>d<p>The sequence coverage, which is calculated as the percentage of identified sequence to the complete sequence of the matched protein.</p>e<p>Mascot Score.</p>f<p>Ratio between level of spot avarage expression indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028175#pone-0028175-g005" target="_blank">Fig. 5</a> Standard deviation is indicated. For the significance two-sided Student's <i>t</i> test was used (*p<0.05,**p<0.01).</p

Proteins differentially expressed in ILCs as identified by MALDI-TOF MS.

a<p>MW, molecular weight.</p>b<p>pI, isoelectric point.</p>c<p>Number of matched mass values on number of total mass values searched.</p>d<p>The sequence coverage, which is calculated as the percentage of identified sequence to the complete sequence of the matched protein.</p>e<p>Mascot Score.</p>f<p>Ratio between level of spot avarage expression indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028175#pone-0028175-g005" target="_blank">Fig. 5</a> Standard deviation is indicated. For the significance two-sided Student's <i>t</i> test was used (*p<0.05,**p>0.01).</p

Multilineage differentiation of BM and HI-MSCs.

<p>(<b>A</b>) cultured BM-MSCs and (<b>B</b>) HI-MSCs before differentiation. (<b>C–D</b>) Representative micrographs of osteogenic differentiation: calcium depositions were detected by Alizarin Red after culturing BM-MSCs (<b>C</b>) and HI-MSCs (<b>D</b>) for 21 days in specific osteogenic medium (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028175#s4" target="_blank">Material and Methods</a>). (<b>E–F</b>) Representative micrographs of adipogenic differentiation showing the presence of lipid droplets after 21 days in adipogenic differentiating medium in of BM-MSCs (<b>E</b>) and HI-MSCs (<b>F</b>) respectively. (magnification ×200). (<b>G and H</b>) Representative micrographs of chondrogenic differentiation shown by formation of a pellet positive for safranin O (<b>G</b>) and alcian blue (<b>H</b>) after culturing HI-MSC in chondrogenic medium for 28 days (magnification ×100). Five different lines were studied with similar results.</p

Hierarchical Cluster Analysis.

<p>This figure shows a cluster map of the proteins expressed in the different cell lines. The tree on the left represents the hierarchical cluster analysis on the 72 proteins. In the heat map, black squares show the expression of the corresponding protein in the correspondent cell lines, while white squares show no expression.</p

Proteins differentially expressed in MSCs as identified by MALDI-TOF MS.

a<p>MW, molecular weight.</p>b<p>pI, isoelectric point.</p>c<p>Number of matched mass values on number of total mass values searched.</p>d<p>The sequence coverage, which is calculated as the percentage of identified sequence to the complete sequence of the matched protein.</p>e<p>Mascot Score.</p>f<p>Ratio between level of spot avarage expression indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028175#pone-0028175-g005" target="_blank">Fig. 5</a> Standard deviation is indicated. For the significance two-sided Student's <i>t</i> test was used (*p<0.05,**p<0.01).</p

Morphological changes and ultrastructural analysis of BM and HI-MSCs during differentiation.

<p>(<b>A</b>) Representative HI-MSCs undifferentiated cells shows a fibroblast-like cell morphology (<b>B</b>) after 7 days in the complete differentiative medium HI-MSCs started to organize into small aggregates (<b>C</b>) finally, during maturation, HI-ILCs formed islet-like spatial structures. <b>Electron microscopy</b>: (<b>D</b>) HI-MSCs before differentiation showed stem cell features with undifferentiated cytoplasm and bean-shaped nucleus, (<b>E</b>) High magnification of a cytoplasm from a BM-ILCs derived cell showing numerous RER cisternae associated with some electron-dense core insulin-like granules. (<b>F</b>) HI- ILCs shows few secretory granules with a central electron-dense core and a peripheral halo resembling immature insulin-secreting granules.</p