Direct Differentiation of Bone Marrow Mononucleated Cells Into Insulin-Producing Cells Using 4 Specific Soluble Factors

Abstract

Bone marrow-derived stem cells are self-renewing and multipotent adult stem cells that differentiate into several types of cells. Here, we investigated a unique combination of 4 differentiation-inducing factors (DIFs), including putrescine (Put), glucosamine (GlcN), nicotinamide, and BP-1-102, to develop a differentiation method for inducing mature insulin-producing cells (IPCs) and apply this method to bone marrow mononucleated cells (BMNCs) isolated from mice. BMNCs, primed with the 4 soluble DIFs, were differentiated into functional IPCs. BMNCs cultured under the defined conditions synergistically expressed multiple genes, including those for PDX1, NKX6.1, MAFA, NEUROG3, GLUT2, and insulin, related to pancreatic beta cell development and function. They produced insulin/C-peptide and PDX1, as assessed using immunofluorescence and flow cytometry. The induced cells secreted insulin in a glucose-responsive manner, similar to normal pancreatic beta cells. Grafting BMNC-derived IPCs under kidney capsules of mice with streptozotocin (STZ)-induced diabetes alleviated hyperglycemia by lowering blood glucose levels, enhancing glucose tolerance, and improving glucose-stimulated insulin secretion. Insulin- and PDX1-expressing cells were observed in the IPC-bearing graft sections of nephrectomized mice. Therefore, this study provides a simple protocol for BMNC differentiation, which can be a novel approach for cell-based therapy in diabetes mellitus.

Keywords: a unique combination of differentiation-inducing factors; bone marrow mononucleated cells; differentiation; insulin-producing cells.

Graphical Abstract

Figure 1.

Synergistic effect of 4 DIFs in BMNC-derived IPCs. Beta cell-specific gene expression in IPCs on day 6 of differentiation from BMNCs treated with Put, GlcN, nicotinamide, and BP-1-102 was analyzed using qPCR. Results are presented as mean ± SEM from at least 3 independent experiments. Put, putrescine; GlcN, glucosamine. *Mean values are significantly different from that of non-treated BMNCs at *P < .05; **P < .01; ***P < .001. ^#Mean values are significantly different from that of BMNCs primed with Put + GlcN + nicotinamide at ^#P < .05.

Figure 2.

Characterization of IPCs following in vitro culture of murine BMNCs with 4 DIFs. (A) Expression of pancreatic developmental markers during differentiation was determined using PCR with reverse transcription. Data are representative of 3 independent experiments. (B) Immunofluorescence staining of cells using anti-insulin and anti-PDX1 antibodies, followed by DAPI staining for nuclei and observation using a confocal fluorescence microscope. Scale bar = 50 µm; original magnification, 1200×. (C) Representative FACS plots illustrating the expression of C-peptide and PDX1 in BMNCs-derived IPCs. The numbers mark the percentage of cells in each quadrant. Ins, insulin; Gcg, glucagon; Sst, somatostatin.

Figure 3.

The functionality of BMNC-derived IPCs. (A) Ultrasensitive ELISA measured insulin secretion from differentiated IPCs on day 6 at low (2 mM) and high (20 mM) glucose concentrations. The results presented are those of 5 separate experiments. *Mean values significantly differ from that of non-treated control BMNCs at *P < .05 and ***P < .001. ^#Mean values significantly differ from that of primed cells at low glucose concentrations at ^#P < .001. (B) mRNA expression of Glut2, Gck, and Syt in differentiated IPCs was measured using qPCR. The results presented are those of at least 3 separate experiments. Data are mean ± SEM. *P < .05; **P < .01. Glut2, glucose transporter 2; Gck, glucokinase; Syt, synaptotagmin.

Figure 4.

Transplantation of primed BMNCs improves hyperglycemia in STZ-induced diabetic mice. (A) Summarized scheme of animal experiments. Male C57BL/6 mice were intraperitoneally injected with STZ. After 7 days, primed or non-primed BMNCs (1 × 10⁶ cells/mouse) were transplanted under the kidney capsule, followed by monitoring for 42 days. (B) Food (left panel) and water (right panel) intake was measured every 3-4 days. Changes in random feeding blood glucose levels (C) and body weight (D) of mice transplanted with primed BMNCs (filled circle, straight line) or comparable non-primed BMNCs (empty circle, dotted line) were measured on the indicated days. (E) Blood glucose levels from the intraperitoneal glucose tolerance test (ipGTT). Glucose (1 g/kg body weight) was injected after overnight fasting for 27 days post-grafting. AUC (right panel) was measured. (F) Plasma insulin levels at the indicated time points post-glucose injection (2 g/kg, body weight) following overnight fasting was measured using ultrasensitive ELISA for mouse insulin. Data are presented as mean ± SEM. *P < .05; **P < .01 for non-primed vs. primed BMNC-transplanted mice. STZ, streptozotocin.

Figure 5.

Analysis of grafts following kidney excision using immunofluorescence staining for insulin and PDX1 expression. (A) Representative immunofluorescent images of nephrectomized grafts stained with anti-insulin and anti-PDX1 antibodies, followed by DAPI staining for nuclei and observation under a confocal fluorescence microscope. Scale bar = 50 µm; original magnification, 1200×. (B) Representative immunohistochemical images of the pancreas for insulin harvested 42 days after transplantation. Images were acquired with an ECLIPSE Ci-L microscope. Scale bar = 100 µm; original magnification, 200×. (C) Pancreatic insulin content in mice was determined using high-range ELISA for mouse insulin from pancreatic tissues harvested 42 days after transplantation. All data are presented as mean ± SEM from 5-6 mice from each group.