Identification of islet-like structures differentiated from bone marrow mesenchymal stem cells induced by pancreatic tissue lysate

doi:10.3969/j.issn.2095-4344.1696

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells have been induced into islet-like cell mass in vitro. However,little researches reported on the morphological changes of cells, the types of endocrine cells in the islet-like cell mass and their relationships.
OBJECTIVE: To investigate the morphological changes of cells in the differentiation of bone marrow mesenchymal stem cells into islet-like structure and to explore the composition and distribution of endocrine cells.
METHODS: Passage 3 bone marrow mesenchymal stem cells growing well were cultured and expanded until the cell colonies occupies 80% of the bottom of the culture bottle, and the pancreatic tissue lysate was added for continuous induction. Dithizone staining was used to screen the islet-like cell mass directly differentiated from bone marrow mesenchymal stem cells. The types and distribution of endocrine cells were identified by Mallory staining. Expressions of insulin, C-peptide, glucagon and somatostatin protein were detected by immunofluorescence cytochemical staining.
RESULTS AND CONCLUSION: (1) Dithizone staining showed that the number of positive cells was increased over the induction time. (2) Mallory staining showed the red α-like cells were located in the periphery of the islet-like cell mass, the yellow β-like cells located in the center and periphery, and the light blue fibroblasts were distributed around the cell mass. (3) Immunofluorescence staining showed insulin, C-peptide, glucagon and somatostatin positive cells in the islet-like cell mass. To conclude, under certain microenvironment, bone marrow mesenchymal stem cells can differentiate into islet-like structures which containing α, β, δ-like cells, and are surrounded by fibroblast-like cells.

Key words: Bone Marrow, Mesenchymal Stem Cells, Cell Differentiation, Islets of Langerhans, Endocrine Cells, Tissue Engineering

CLC Number:

Liu Hongjing, Wang Huifeng, Wei Yashu, Zhao Wenjing, Chen Weiping. Identification of islet-like structures differentiated from bone marrow mesenchymal stem cells induced by pancreatic tissue lysate[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(13): 2088-2093.

Figures/Tables 1

2.1 骨髓间充质干细胞多向分化潜能及细胞表面标志物鉴定结果成骨诱导培养2周，细胞呈多角形，成骨诱导培养3周，细胞逐渐聚集，茜素红染色可见橘红色钙盐；成脂诱导培养1周，细胞内出现脂滴，持续诱导，脂滴数量增多，油红O染色细胞内可见红色脂滴。骨髓间充质干细胞表面抗原标志物CD90、CD29的阳性率分别为98.2%、98.9%，造血干细胞表面抗原标志物CD34、CD45的阳性率分别为0.254%、0.214%。 2.2 骨髓间充质干细胞经胰腺组织裂解液定向诱导分化过程中细胞的形态变化诱导前，倒置显微镜下观察骨髓间充质干细胞以梭形为主，细胞质折光性好，见图1A；经胰腺组织裂解液定向诱导后细胞形态变化有明显的规律：诱导约1周，细胞界限清晰，形态大小均一，呈串珠样或“品”字形排列，细胞逐渐聚集，见图1B；诱导约3周，细胞团逐渐变大、贴壁、边界清晰，见图1C；诱导约5周，细胞团边缘清晰、规则平整，中央细胞轮廓模糊，不透光，见图1D。 2.3 胰岛样结构的鉴定结果 2.3.1 双硫腙染色诱导约1周，早期聚集形成的细胞团内隐约可见被染成浅红色的细胞，见图2A；诱导约3周，逐渐增大的细胞团细胞界限清晰，中央细胞增多、密集、呈深红色，见图2B；诱导约5周，较大的细胞团边缘规则、平整，中央细胞轮廓不清，呈棕红色。细胞团周围贴壁细胞呈梭形、未着色，见图2C。 2.3.2 Mallory染色诱导约5周，选取边缘规则、平整、较大的细胞团进行Mallory染色，中央及边缘呈黄色，周边大部分呈红色，周围贴壁的长梭形细胞呈淡蓝色，见图2D。 2.3.3 免疫荧光染色诱导约5周，选取边缘规则、平整、较大的细胞团进行免疫细胞化学染色，结果显示：胰岛素阳性细胞和C肽阳性细胞数多，分布在中央及边缘，见图3A，B；胰高血糖素阳性细胞较多，分布在周边，见图3C；生长抑素阳性细胞少，散在分布，见图3D。阳性细胞的胞质被激发出黄绿色荧光。"

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