Rev Diabet Stud. 2009 Winter; 6(4): 260270.

1Tissue Engineering and Banking Laboratory, National Center for Cell Science, Ganeshkhind Road, Pune MH 411007, India

2Division of Animal Sciences, Agharkar Research Institute, Agarkar Road, Pune, MH 411004, India

3Stem Cells and Diabetes Section, National Center for Cell Science, Ganeshkhind Road, Pune MH 411007, India

4Stempeutics Research Pvt. Ltd., 9th Floor, Manipal Hospital, HAL Airport Road, Bangalore 560017, India

Received October 2, 2009; Revised December 5, 2009; Accepted December 11, 2009.

Cellular microenvironment is known to play a critical role in the maintenance of human bone marrow-derived mesenchymal stem cells (BM-MSCs). It was uncertain whether BM-MSCs obtained from a 'diabetic milieu' (dBM-MSCs) offer the same regenerative potential as those obtained from healthy (non-diabetic) individuals (hBM-MSCs). To investigate the effect of diabetic microenvironment on human BM-MSCs, we isolated and characterized these cells from diabetic patients (dBM-MSCs). We found that dBM-MSCs expressed mesenchymal markers such as vimentin, smooth muscle actin, nestin, fibronectin, CD29, CD44, CD73, CD90, and CD105. These cells also exhibited multilineage differentiation potential, as evident from the generation of adipocytes, osteocytes, and chondrocytes when exposed to lineage specific differentiation media. Although the cells were similar to hBM-MSCs, 6% (3/54) of dBM-MSCs expressed proinsulin/C-peptide. Emanating from the diabetic microenvironmental milieu, we analyzed whether in vitro reprogramming could afford the maturation of the islet-like clusters (ICAs) derived from dBM-MSCs. Upon mimicking the diabetic hyperglycemic niche and the supplementation of fetal pancreatic extract, to differentiate dBM-MSCs into pancreatic lineage in vitro, we observed rapid differentiation and maturation of dBM-MSCs into islet-like cell aggregates. Thus, our study demonstrated that diabetic hyperglycemic microenvironmental milieu plays a major role in inducing the differentiation of human BM-MSCs in vivo and in vitro.

Keywords: diabetes, beta-cell, stem cell, differentiation, bone marrow, NGN3, NKX6.1, PAX6

Abbreviations: -MEM - -modified Eagle's medium (used for cell culture); AGE - advanced glycation end-product; ALL - acute lymphoblastic leukemia; ALS - amyotrophic lateral sclerosis; AML - acute myeloid leukemia; BM-MSC - bone marrow-derived mesenchymal stem cell; BRN4 - Brain 4 (transcription factor expressed in the brain and glucagon-expressing cells in the pancreas, also known as POU3F4); C-peptide - connecting peptide; Ct - cycle threshold; CXCR4 - alpha-chemokine receptor (also called fusin) specific for stromal-derived-factor-1 (SDF-1, also called CXCL12), a molecule endowed with potent chemotactic activity for lymphocytes; dBM-MSC - human diabetic BM-MSC; DME meduim - Dulbecco's modified Eagles medium; E-cadherin - epithelial cadherin (CDh1); EDTA - ethylenediaminetetraacetic acid (used as chelating agent that binds to calcium and prevents joining of cadher-ins between cells; it also prevents clumping of cells grown in liquid suspension, and is able to detach adherent cells for passaging); EGFP - enhanced green fluorescence protein; F(ab)2 - antigen-binding fragment of an antibody; FACS - fluorescence-activated cell sorting; GATA6 - binding protein that binds (A/T/C)GAT(A/T)(A) of the binding sequence; Glut2 - glucose transporter 2 (also known as solute carrier family 2 member 2 SLC2A2); GCG - glucagons gene; hBM-MSC - normal human BM-MSC; HD - Hodgkin disease; ICA - islet-like cell aggregate; ICAM-5 - intercellular adhesion molecule 5 (also known as telencephalin, CD# not yet assigned); ISL1 - insulin gene enhancer protein gene 1; NCAM-1 - neural cell adhesion molecule 1 (CD56); NDS - normal donkey serum; NGN-3 - neurogenin-3 (controls islet cell fate specification in pancreatic progenitor cells); NHL - non-Hodgkin lymphoma; NKX6-1 - NK6 homeobox 1 (transcription factor required for the development of beta-cells); Oil-Red-O - Solvent Red 27 (fat-soluble dye used for stain-ing of triglycerides and lipids); PBS - phosphate-buffered saline; PECAM-1 - platelet endothelial cell adhesion molecule-1 (CD31); PE - phycoerythrin (fluorescent dye for labeling antibodies); Pdx1 - pancreatic and duodenal homeobox 1 (transcription factor necessary for pancreatic development and beta-cell maturation); PFA - paraformaldehyde (used to fix cells); POU - class of genes that produce transcription factors; POU3F4 - POU class 3 homeobox 4 gene or gene product (also known as BRN4); RNA - ribonucleic acid; RPE - rat pancreatic extract; RT-PCR - reverse transcriptase polymerase chain reaction; TPVG - trypsin phosphate versene glucose; UCBS - human umbilical cord blood serum

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are able to differentiate into many cell types, and to proliferate ex vivo. These attributes makes them a potential therapeutic tool for cell replacement therapy in diabetes and other diseases. Stem cell differentiation is controlled by extracellular cues, the environment, and intrinsic genetic programs within stem cells [1, 2]. The fate of stem cell differentiation is influenced by both soluble and insoluble factors from the surrounding microenvironment. Several signaling cascades mediate the balance response of the stem cell to the need of the organism. Pathological conditions induced by dysregulation result in aberrant functions of stem cells or other targets [3-6].

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