Under a nonadherent state, bone marrow mesenchymal stem cells can be efficiently induced into functional islet-like cell clusters to normalize hyperglycemia in mice: a control study

Abstract

Introduction: Bone marrow mesenchymal stem cells (BMSCs) possess low immunogenicity and immunosuppression as an allograft, can differentiate into insulin-producing cells (IPCs) by in vitro induction, and may be a valuable cell source to regenerate pancreatic islets. However, the very low differentiation efficiency of BMSCs towards IPCs under adherent induction has thus far hindered the clinical exploitation of these cells. The aim of this study is to explore a new way to efficiently induce BMSCs into IPCs and lay the groundwork for their clinical exploitation.

Methods: In comparison with adherent induction, BMSCs of human first-trimester abortus (hfBMSCs) under a nonadherent state were induced towards IPCs in noncoated plastic dishes using a three-stage induction procedure developed by the authors. Induction effects were evaluated by statistics of the cell clustering rate of induced cells, and ultrastructural observation, dithizone staining, quantitative polymerase chain reaction and immunofluorescence assay, insulin and c-peptide release under glucose stimulus of cell clusters, as well as transplantation test of the cell clusters in diabetic model mice.

Results: With (6.175 ± 0.263) × 105 cells in 508.5 ± 24.5 cell clusters, (3.303 ± 0.331) × 105 single cells and (9.478 ± 0.208) × 105 total cell count on average, 65.08 ± 2.98% hfBMSCs differentiated into pancreatic islet-like cell clusters after nonadherent induction. With (3.993 ± 0.344) × 105 cells in 332.3 ± 41.6 cell clusters, (5.437 ± 0.434) × 105 single cells and (9.430 ± 0.340) × 105 total cell count on average, 42.37 ± 3.70% hfBMSCs differentiated into pancreatic islet-like cell clusters after adherent induction (P < 0.01, n = 10). The former is significantly higher than the latter. Calculated according to the cell clustering rate and IPC percentage in the cell clusters, 29.80 ± 3.95% hfBMSCs differentiated into IPCs after nonadherent induction and 18.40 ± 2.08% hfBMSCs differentiated into IPCs after adherent induction (P < 0.01, n = 10), the former significantly higher than the latter. The cell clusters expressed a broad gene profile related to pancreatic islet cells, released insulin and c-peptide in a glucose concentration-dependent manner, and normalized hyperglycemia of streptozocin-induced mice for at least 80 days following xenograft. Blood glucose of grafted mice rose again after their graft removed. A series of examination of the grafts showed that transplanted cells produced human insulin in recipients.

Conclusions: Our studies demonstrate that nonadherent induction can greatly promote BMSCs to form pancreatic islet-like cell clusters, thereby improving the differentiation efficiency of BMSCs towards IPCs.

Figure 1

Isolation and identification of bone marrow mesenchymal stem cells of human first-trimester abortus. Adhered spindle cells appeared in culture of whole bone marrow (A) and they clonally grew and spread at the bottom of a culture dish within the 12th day (B). Flow cytometric analysis showed that passage 6 of the isolated cells strongly expressed the surface markers of mesenchymal stem cells, such as CD29, CD44 and CD166, but almost no markers of Hematopoietic Stem Cells, such as CD11a, CD14 and CD34 (C). FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Figure 2

Differentiation of bone marrow mesenchymal stem cells of human first-trimester abortus into islet-like cell clusters. Many irregular cell clusters formed in the first-stage induction (A), became compact in the second-stage induction (B), and became spherical with blurred boundaries among adjacent cells in the third-stage induction, similar to the morphology of the islets of Langerhans, whereas some of the nonclustered cells adhered to the dish bottom and became similar to bone marrow mesenchymal stem cells of human first-trimester abortus in shape in the nonadherent induction group (C), (D). Irregular cell clusters after the first-stage induction (E) became hemispherical in the second-stage induction (F), uplifted with blurred boundaries among adjacent cells in the third-stage induction in the adherent induction group (G), (H), and always adhered to the dish bottom. Transmission electron microscopy showing a large number of endocrine cells (I) and secretion granules in cytoplasma of the cells in the cell clusters from the nonadherent induction group (J) and the adherent induction group (K) after the three-stage induction, histologically similar to those in fetal pancreatic islets as the positive control (L).

Figure 3

Dithizone staining and quantitative polymerase chain reaction analysis of islet-like cell clusters from bone marrow mesenchymal stem cells of human first-trimester abortus. (A) Islet-like cell clusters were positive for dithizone staining in the nonadherent induction group (a) and the adherent induction group (b), similar to those of fetal pancreatic islets (c), whereas non-induced bone marrow mesenchymal stem cells of human first-trimester abortus were negative for dithizone (d). (B) Expression levels of Pdx1, Ngn3, Pax4, NeuroD1, Nkx2.2, Nkx6.1, PCSK1, insulin, glucagon, SST and PP genes in fluorescent quantitative reverse transcriptase-polymerase chain reaction test. The islet-like cell clusters expressed significantly more Ngn3 and Pax4, and significantly less insulin, glucagon, SST and PP than fetal pancreatic islets (P < 0.01, n = 10). The expressions of all the genes were not different between the nonadherent induction group and the adherent induction group (P > 0.05, n = 10).

Figure 4

Immunofluorescence assay of whole islet-like cell clusters. The strongest expression of nestin is in islet-like cell clusters after the first-stage induction in the nonadherent induction group (A), (B), (C) and the adherent induction group (D), (E), (F). The strongest expression of insulin and c-peptide is in islet-like cell clusters after the third-stage induction in the nonadherent induction group (G), (H), (I), (J) and the adherent induction group (K), (L), (M), (N). Non-induced bone marrow mesenchymal stem cells of human first-trimester abortus were negative for insulin and c-peptide (O), (P), (Q), (R).

Figure 5

Immunofluorescence assay of single cells from islet-like cell clusters. On enzymatic dispersion and reculture in lysine-coated plastic dishes, the single cells from islet-like cell clusters adhered and expressed insulin (A), (B), (C), (D), glucagon (E), (F), (G), (H), SST (I), (J), (K), (L) and PP (M), (N), (O), (P) respectively.

Figure 6

Insulin and C-peptide release by islet-like cell clusters. Radioimmunoassay results show that the release of insulin (A) and c-peptide (B) is significantly different between the nonadherent induction group and the non-induction control group, between the nonadherent induction group and the positive control group, and between the different glucose levels in the nonadherent induction group (all at P < 0.01, n = 10), but were not different between the nonadherent induction group and the adherent induction group (P > 0.05, n = 10).

Figure 7

Effects of xenograft with islet-like cell clusters on the recovery of streptozocin-induced diabetic mice. In the nonadherent induction group, the blood glucose levels of 10 diabetic mice fell to normal within 2 weeks following the transplantation, but three of the 10 mice regained hyperglycemia when their xenograft was removed 28 days post transplantation and all died within 45 days after the removal; the remaining seven mice without the removal of xenograft maintained normal levels of blood glucose for at least 80 days (A) and gained body weight slightly (B). The intraperitoneal glucose tolerance test indicated that the islet-like cell clusters had a normal glucose clearance rate after transplantation, but were not as effective as native pancreatic beta-cells (C). The effects of xenograft in the adherent induction group are similar to those in the nonadherent induction group. In the non-induction control group, six diabetic mice maintained high blood glucose levels (>18 mmol/l) after bone marrow mesenchymal stem cell of human first-trimester abortus transplantation, lost their body weight continuously and died within 45 days after the transplantation, and so did the mice with removal of their testes 28 days post transplantation.

Figure 8

Immunofluorescence examination of dewaxed sections of the grafts. Red CM-DiI-labeled cells in a graft section (A) in the nonadherent induction group were visualized only in the interstitial tissue among the seminiferous tubules (B) and showed green fluorophore (C) (merged in (D)) after staining with human insulin antibody under a fluoroscope. A similar phenomenon occurred in a graft section in the adherent induction group (E), (F), (G), (H), whereas CM-DiI-labeled cells in the non-induction control group did not express insulin (I), (J), (K), (L).