Differentiation Potential of Nestin (+) and Nestin (-) Cells Derived from Human Bone Marrow Mesenchymal Stem Cells into Functional Insulin Producing Cells

Abstract

The feasibility of isolating and manipulating mesenchymal stem cells (MSCs) from human patients provides hope for curing numerous diseases and disorders. Recent phenotypic analysis has shown heterogeneity of MSCs. Nestin progenitor cell is a subpopulation within MSCs which plays a role in pancreas regeneration during embryogenesis. This study aimed to separate nestin ⁽⁺⁾ cells from human bone marrow MSCs, and differentiate these cells into functional insulin producing cells (IPCs) compared with nestin ^(-) cells. Manual magnetic separation was performed to obtain nestin ⁽⁺⁾ cells from MSCs. Approximately 91±3.3% of nestin ⁽⁺⁾ cells were positive for anti-nestin antibody. Pluripotent genes were overexpressed in nestin ⁽⁺⁾ cells compared with nestin ^(-) cells as revealed by quantitative real time-PCR (qRT-PCR). Following in vitro differentiation, flow cytometric analysis showed that 2.7±0.5% of differentiated nestin ⁽⁺⁾ cells were positive for anti-insulin antibody in comparison with 0.08±0.02% of nestin ^(-) cells. QRT-PCR showed higher expression of insulin and other endocrine genes in comparison with nestin ^(-) cells. While immunofluorescence technique showed the presence of insulin and C-peptide granules in nestin ⁽⁺⁾ cells. Therefore, our results introduced nestin ⁽⁺⁾ cells as a pluripotent subpopulation within human MSCs which is capable to differentiate and produce functional IPCs.

Keywords: Human bone marrow derived mesenchymal stem cells; diabetes mellitus; insulin producing cells; mesenchymal stem cells; real time-quantitative PCR.

Fig. 1

Morphological features of nestin (+) and nestin (-) cells during expansion. A: cultured nestin (+) cells; B: cultured nestin (-) cells

Fig. 2

Multilineage differentiation of MSCs. A: adipocyte cells stained with oil red; B: chondrocyte cells stained with alcian blue; C: osteocyte cells stained with alizarin red

Fig. 3

Immunofluorescence staining of nestin (+) and nestin (-) cells with counterstaning for DAPI (blue). A: nestin (+) cells stained with anti-nestin (red); B: nestin (-) cells stained with anti-nestin; C: IPCs derived from nestin (+) cells stained with anti-insulin (green); D: IPCs derived from nestin (+) cells stained with anti-C-peptide (red); E: co-expression of insulin and C-peptide by the same IPCs derived from nestin (+) cells by electron merge (yellow); F: nestin (-) cells stained with anti-insulin

Fig. 4

QRT-PCR of nestin ⁽⁺⁾ and nestin ^(-) cells. A: relative gene expression of pluripotent markers, nestin and PDX1 of the undifferentiated nestin ⁽⁺⁾ and nestin ^(-) cells relative to MSCs; B: endocrine gene expression of IPCs derived from nestin ⁽⁺⁾ and nestin ^(-) cells relative to human islets gene expression

Fig. 5

Human insulin and C-peptide release by IPCs derived from nestin ⁽⁺⁾ and nestin ^(-) cells by ELISA technique in response to glucose concentration challenge. A: insulin release; B: C-peptide release