The effect of diabetes mellitus on differentiation of mesenchymal stem cells into insulin-producing cells

Abstract

Background: Diabetes mellitus (DM) is a global epidemic with increasing incidences. DM is a metabolic disease associated with chronic hyperglycemia. Aside from conventional treatments, there is no clinically approved cure for DM up till now. Differentiating mesenchymal stem cells (MSCs) into insulin-producing cells (IPCs) is a promising approach for curing DM. Our study was conducted to investigate the effect of DM on MSCs differentiation into IPCs in vivo and in vitro.

Methods: We isolated adipose-derived mesenchymal stem cells (Ad-MSCs) from the epididymal fat of normal and STZ-induced diabetic Sprague-Dawley male rats. Afterwards, the in vitro differentiation of normal-Ad-MSCs (N-Ad-MSCs) and diabetic-Ad-MSCs (DM-Ad-MSCs) into IPCs was compared morphologically then through determining the gene expression of β-cell markers including neurogenin-3 (Ngn-3), homeobox protein (Nkx6.1), musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), and insulin-1 (Ins-1) and eventually, through performing glucose-stimulated insulin secretion test (GSIS). Finally, the therapeutic potential of N-Ad-MSCs and DM-Ad-MSCs transplantation was compared in vivo in STZ-induced diabetic animals.

Results: Our results showed no significant difference in the characteristics of N-Ad-MSCs and DM-Ad-MSCs. However, we demonstrated a significant difference in their abilities to differentiate into IPCs in vitro morphologically in addition to β-cell markers expression, and functional assessment via GSIS test. Furthermore, the abilities of both Ad-MSCs to control hyperglycemia in diabetic rats in vivo was assessed through measuring fasting blood glucose (FBGs), body weight (BW), histopathological examination of both pancreas and liver and immunoexpression of insulin in pancreata of study groups.

Conclusion: Our findings reveal the effectiveness of N-Ad-MSCs in differentiating into IPCs in vitro and controlling the hyperglycemia of STZ-induced diabetic rats in vivo compared to DM-Ad-MSCs.

Keywords: Adipose tissue; Diabetes mellitus; Differentiation; Insulin-producing cells; Mesenchymal stem cells.

Fig. 1

A Phase contrast images of cultured Ad-MSCs; either freshly isolated N- Ad-MSCs (Day7, P0) showing a homogenous population of fibroblast-like shaped cells (left), or freshly isolated DM-Ad-MSCs (Day 7, P0) showing a homogenous population (right). B Immunophenotyping of N-Ad-MSCs (upper) showing positive expression of mesenchymal markers (CD105-FITC: 99.20%, CD90-FITC: 100%) and negative expression of hematopoietic markers (CD34-PE: 9.50%, the blue peak represents positive PE-stained cell). While the immunophenotyping of DM-Ad-MSCs (lower panel) showed positive expression of mesenchymal markers (CD105-FITC 100%, CD90-FITC 89.89%) and almost negative expression of hematopoietic markers (CD34-PE 1.45%). C and D Multilineage differentiation of N- and DM-Ad-MSCs: C N-Ad-MSCs differentiated into adipocytes; showing oil red staining, osteocytes; showing alizarin red staining and chondrocytes; showing alcain blue staining (left) as compared to control cells (right). D DM-Ad-MSCs differentiated into adipocytes, osteocytes, and chondrocytes (right) as compared to control cells (left). N-Ad-MSCs normal adipose MSCs, DM-Ad-MSCs diabetic adipose MSCs, FITC Fluorescein isothiocyanate, PE phycoerythrin

Fig. 2

Morphological assessment of IPCs generated from N-Ad-MSCs and DM-Ad-MSCs. A Schematic presentation of the differentiation protocol (Phase I, II, III). B Phase contrast images of unstained differentiated N-MSCs and DM-MSCs into IPCs; upon differentiation, both N-Ad-MSCs (upper) and DM-Ad-MSCs (lower panel) aggregate to form clusters in contrast to control N-Ad-MSCs and DM-Ad-MSCs which retain fibroblast-like morphology (at magnification 10×). C Phase contrast images of DTZ-stained differentiated IPCs of N-Ad-MSCs and DM-Ad-MSCs; DTZ-stained IPCs of N-Ad-MSCs (upper) showing crimson red large clusters with different masses as compared to stained IPCs of DM-Ad-MSCs (lower). Both control N-Ad-MSCs and DM-Ad-MSCs retained the unstained fibroblast-like morphology (at magnification 20x)

Fig. 3

Gene expression of β-cell markers and GSIS upon differentiation of N-Ad-MSC or DM-Ad-MSCs into IPCs: Relative mRNA fold change expression of A Ngn-3, B NKX6.1, C MafA and D Ins-1, throughout differentiation protocol of N-Ad-MSCs and DM-Ad-MSCs into IPCs. Insulin release of differentiated IPCs derived from E N-Ad-MSCs and F in response to LG (2mM) and HG (25mM). a significant different from Control N-Ad-MSCs at p-value < 0.05. b significant different from D3 IPCs N-Ad-MSCs at p-value < 0.05. c significant different from Final IPCs N-Ad-MSCs at p-value < 0.05. * means the significant difference from LG (control) at p-value < 0.05. N-Ad-MSCs Normal Adipose-MSCs, DM-Ad-MSCs Diabetic Adipose-MSCs, control Undifferentiated cells, D3 Day 3 differentiation, Final Final generated IPCs

Fig. 4

Transplantation of N- and DM- Ad-MSCs in STZ induced diabetic rats: A FBG levels of STZ-induced diabetic rats for 42 days post-transplantation of N-Ad-MSCs and DM-Ad-MSCs. B Body weights of STZ-induced diabetic rats for 42 days post-transplantation of N-Ad-MSCs and DM-Ad-MSCs. C Histopathological examination of pancreas of different study groups (at magnification 10× and 40×). D Histopathological examination of liver of different study groups (at magnification 10 × and 40×). Control: non injected group; STZ + PBS, n = 6: group injected with STZ and PBS, n = 6; N-Ad-MSCs: animals injected with STZ and transplanted with N-Ad-MSCs, n = 6; DM-Ad-MSCs: animals injected with STZ and transplanted with DM-Ad-MSCs, n = 6. *: significant different from Control at p-value < 0.05. #: significant different from STZ + PBS at p-value < 0.05. $: significant different from STZ + DM-Ad-MSCs at p-value < 0.05

Fig. 5

Immunostaining of insulin in pancreata of STZ-induced diabetic rats. A Immunostaining of insulin in control non injected group (upper most), STZ-induced diabetic rats (second from top), STZ-induced rats treated with N-Ad-MSCs (third from top) or DM-Ad-MSCs (lowest). B Quantification of insulin positive cells/high power field (HPF). Control: non injected group; STZ + PBS, n = 3: group injected with STZ and PBS, n = 3; N-Ad-MSCs: animals injected with STZ and transplanted with N-Ad-MSCs, n = 3; DM-Ad-MSCs: animals injected with STZ and transplanted with DM-Ad-MSCs, n = 3. a: significant different from Control, at p-value < 0.05. b: significant different from STZ + PBS, at p-value < 0.05