Pancreatic line Cell Differentiation of bone marrow Mesenchymal Stromal Cells in Acellular Pancreatic Scaffolds

Abstract

Background: To evaluate the potential differentiation ability of bone mesenchymal stromal cells(BMSCs) to pancreatic line Cells on rat acellular pancreatic bioscaffold (APB). Methods: Fresh pancreata from 20 Adult Sprague Dawley rats (between 6 and 7 weeks old) were soaked and perfused using Easy-Load Digital Drive peristaltic pumps. After BMSCs were isolated and identified, they were dynamic cultured on the APB and static cultured in tissue culture flask(TCF). Based on whether the differentiation was induced by the growth factors (GF) in the culture system, our study was divided into 4 groups: BMSCs cultured in TCF without any GF(TCF-GF(-)), BMSCs cultured in TCF with GF(TCF-GF(+)), BMSCs cultured on APB without GF(APB-GF(-)), BMSCs cultured on APB with GF (APB-GF(+)).The cytological behavior such as the proliferation and differentiation of BMSCs in all the above kinds of culture system with or without GF were assessed by morphological observation, flow cytometry, ELASA analysis, qRT-PCR assay and western blot analysis. Results: 4ml/min was the most appropriate flow rate for the dynamic culture of BMSCs. Under culture conditions, BMSCs populations could attach to and proliferate within the APB. APB could promote the proliferation and viability of BMSCs significantly better in dynamic culture with optimal flow rate 4ml/min, when compared to the static culture system. Also, the proliferation rate of BMSCs in the APB groups were significantly increased compared to TCF system. During the pancreatic line cell differentiation process, APB could induce BMSCs into pancreatic-like cells which expressed markers such as pancreatic duodenal homeodomain containing transcription factor (PDX-1) and pancreatic exocrine transcription factor (PTF-1) higher at mRNA levels compared to TCF system. In contrast, the marker Oct4 (octamer-binding transcription factor 4) was expressed at a lower level in APB group. For the pancreatic functional cytoketatins including α-Amylase(α-Amy), cytokeratin 7(CK7), fetal liver kinase-1 (Flk-1),and C-peptide, they were all expressed at higher level in APB group than in the TCF group. And metabolic enzymes secretion such as amylase and insulin were promoted significantly in APB system. By scanning electron microscope(SEM) and transmission electron microscopy(TEM), the ultrastructure of BMSCs in the APB group could further demonstrated the morphological characteristics of pancreatic-like cells. In addition, in both the dynamic and static system, GF could significantly facilitate the function of proliferation, differentiation and cell engraftment . Conclusion: Together our data show the capacity of APB , 3D pancreatic biomatrix, promoting BMSCs differentiate toward pancreatic line phenotypes, and the considerable potential of using these cells for pancreatic cell therapies and tissue engineering.