Trans-differentiation of Bone Marrow-derived EGFP-mMSCs and EGFP-pMSCs into Insulin-Producing Cells
Date Issued
2008
Date
2008
Author(s)
Wu, Cheng-Yu
Abstract
Type I diabetes is an autoimmune disorder characterized by the progressing destructively loss of pancreatic β- cell. Efforts toward routine islet cell transplantation as a means for reversing type I diabetes have been hampered by islet availability as well as allograft rejection. Several studies have confirmed mesenchymal stem cells (MSCs) could trans-differentiate into insulin producing cells (IPCs), however, it took a long period (4 months) at a low frequency (10%). It was also reported that cells migration in three-dimensional extra-cellular matrix (ECM) plays a key role of cluster formation. Therefore, using ECM and cell mass structure to generate an ameliorated protocol for preparing a renewable and abundant transplantable islet clusters is demanded. Marrow-derived MSCs isolating from the femur of transgenic porcine and mice (EGFP-pMSCs/EGFP-mMSCs) harboring with β-actin-EGFP (enhanced green fluorescent protein) gene were used in the studies. The first experiment, to generate an appropriate induction conditions that EGFP-pMSCs and EGFP-mMSCs differentiate into islet cells, different concentration (17.5, 23.0, 28.5, 33, 39.5 mM) glucose was added to induction medium to determine the effect of dose dependent. The result shows EGFP-pMSCs inducted with 28.5 mM glucose for 18 days, the formation ratio of islet-like cluster (0.25 cluster/cm2) is extremely higher than other groups (P<0.01). In the result of EGFP-mMSCs inducted to islet-like cluster (0.14 cluster/cm2) is similar to the tendency of EGFP-pMSCs induction by using 33 mM glucose. The second experiment, poly-D-lysine with three different molecular weight (MW) (MW > 120,000, MW > 300,000, MW > 500,000) were allocated to treatment groups (A, B and C) as ECM, respectively. The results indicated that islet-like cluster formation ratio (5.56 clusters/cm2) of IPCs in group B was significantly higher than other treated groups (P<0.05). Furthermore, EGFP-mMSCs/pMSCs with colonies and single cells were simultaneously seeded and cultured in induction medium to determine whether three-dimensional structures would effect IPCs induction. Experimental results revealed EGFP-mMSCs/pMSCs seeded by colonies exhibiting the largest number of IPCs (6.54 clusters/cm2) within 9 days (P<0.05), clarified the importance of colony formation. In addition, multiple genes include: Insulin, Glucagon, Somatostatin, Glucokinase, Pax6, Nkx6.1, Glut2, expression on inducted islet-like cells were determined by RT-PCR assay. Genes (Insulin, Islet1, Nkx6.1, Glut2) expression profile of time dependent (day0、day3、day6) were determined by Real-Time PCR. Immunocytochemistry was also used to confirm that EGFP-mIPCs/pIPCs are able to synthesize C-peptide and insulin. Insulin secretion level (8.94±0.9 ng/ml) in high glucose (28.5 mM) conditioned medium was quantitated by EIA. Further study regarding the hyperglycemia in NOD (non-obesity diabetes) mice were alleviated significantly after subcutaneous xenograft of 6×106 EGFP-pIPCs and the survival rate of transplanted mice within 20 days was 100%, but NOD mice treated with PBS, EGFP-pMSCs and control group survive up to 18 day. According to above results indicate glucose concentration containing in induction medium and ECM play a key role of cluster formation in EGFP-mMSCs/pMSCs induction, and improve their efficiency trans-differentiating into functional islet cell clusters even secret insulin to reverse the hyperglycemia of NOD mice via subcutaneous xenograft of EGFP-pIPCs. These observations propose that MSCs expressing GFP is capable of trans-differentiating into functional insulin-producing cells and is possible to extend to the studies of pig models of humans diabetes as a reference means to alleviate limitations of surrounding islet cell transplantation.
Subjects
diabetes
insulin producing cells
stem cells
Type
thesis
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