ABSTRACT
To establish a method for isolation, culture and identification of adipose-derived mesenchymal stem cells (ASCs) from the inbreed line miniature pig of Wuzhishan (ILMW). Methods: A total of 100 g adipose tissues were obtained from subcutaneous tissues of neck in six-month old healthy ILMW (3 samples, male). ASCs from ILMW (ILMW-ASCs) were isolated from adipose tissues through 0.1% collagenase digestion. The cells at the 3rd, 5th, 8th, 13th passages were collected. Cell morphology, size, phenotype, cell cycle, and apoptosis were monitored. Cell differentiation was induced and cell proliferation curve was drawn. Results: The ILMW-ASCs, fibroblast-like or whirlpool-like, began the adherence at 36 h and entered a logarithmic phase in the 5th day. Eighty percent of them were fused in the 7th day. The average diameter and volume of ILMW-ASCs were (17.00±0.54) µm and (2.58±0.24)×10-9 L, respectively. The expressions of CD29, CD44 and CD90 were positive, and there was no significant difference between the different passages (all P>0.05). The expressions of CD45, CD8a and HLA-DR were increased with the increase in passages after the 3th passage (all P<0.05). The adipogenic induction of ILMW-ASCs was observed by positive oil red O staining, and the osteogenic induction of ILMW-ASCs was determined by positive alizarin red staining. Apoptosis and senescence occurred in the 13 passage of ILMW-ASCs, and the proportion of S phase of cell cycle was lower than that in lower passages (all P<0.05). Conclusion: ILMW-ASCs are one of the best choice for porcine ASCs, which might provide a source of candidate stem cells for therapy of large animal disease models and tissue or organ repairment.
Subject(s)
Animals , Male , Adipose Tissue , Cell Differentiation , Cells, Cultured , Mesenchymal Stem Cells , Swine , Swine, MiniatureABSTRACT
BACKGROUND: Induced pluripotent stem cells have similar self-renewal, proliferation and differentiation abilities to embryonic stem cells. They have no source limitations, no ethical problems, and no current problems of cell xenogenesis, which are expected to be the source of cells for the treatment of liver diseases. OBJECTIVE: To observe the effect of induced pluripotent stem cell-derived hepatocyte-like cells on liver fibrosis. METHODS: The three-step method in vitro was used to induce the differentiation of induced pluripotent stem cells into hepatocyte-like cells. Glycogen staining, immunohistochemistry and low-density lipoprotein uptake assay were used to detect the ability of induced cells to synthesize glycogen, alpha-fetoprotein, albumin, CK18 protein and low-density lipoprotein uptake. Forty-five Sprague-Dawley rats (provided by the Experimental Animal Center, the Affiliated Haikou Hospital, Xiangya School of Medicine, Central South University) were randomized into three groups: normal control group, model group and cell transplantation group (n=15 per group). The rats in the latter two groups were intraperitoneally injected with carbon tetrachloride to establish liver fibrosis models. Cell transplantation group was given intravenous injectin of hepatocyte-like cells (induced for 21 days) , 0.5 mL, 2×109/L, at 3 days after modeling. Four weeks after cell transplantation, venous blood and liver tissue samples were taken to analyze the changes of liver function, liver fibrosis index and liver pathology. RESULTS AND CONCLUSION: (1) After 21 days of induction, human induced pluripotent stem cell clonal clusters became loose, mainly round or polygonal in shape, and presented with a dense paving stone-like arrangement. A large amount of pink glycogens could be seen in the cytoplasm, indicating that induced pluripotent stem cells have the ability to synthesize glycogen. Low-density lipoprotein uptake test showed that induced pluripotent stem cells had the ability to uptake low-density lipoprotein. Immunohistochemistry detection showed that the cells were positive for alpha fetoprotein, albumin and CK18. (2) At 4 weeks after cell transplantation, the level of albumin in the model group was significantly lower than that in the normal control group (P < 0.05) , while the levels of direct bilirubin, indirect bilirubin, aspartate aminotransferase, alanine aminotransferase, type IV collagen, serum hyaluronidase, serum type III procollagen in the model group were significantly higher than those in the normal control group (P < 0.05). Compared with the model group, the level of albumin in the cell transplantation group was significantly increased (P < 0.05) , while the levels of direct bilirubin, indirect bilirubin, aspartate aminotransferase, alanine aminotransferase, type IV collagen, serum hyaluronidase, serum type III procollagen in the cell transplantation group were significantly decreased (P < 0.05). (3) Four weeks after cell transplantation, inflammatory cell infiltration, hepatocyte degeneration and necrosis in the cell transplantation group were improved to different extents. Therefore, hepatocyte-like cells derived from induced pluripotent stem cells could significantly improve liver fibrosis in rats.
ABSTRACT
BACKGROUND: Induced pluripotent stem cells have similar self-renewal, proliferation and differentiation abilities to embryonic stem cells. They have no source limitations, no ethical problems, and no current problems of cell xenogenesis, which are expected to be the source of cells for the treatment of liver diseases. OBJECTIVE: To observe the effect of induced pluripotent stem cell-derived hepatocyte-like cells on liver fibrosis. METHODS: The three-step method in vitro was used to induce the differentiation of induced pluripotent stem cells into hepatocyte-like cells. Glycogen staining, immunohistochemistry and low-density lipoprotein uptake assay were used to detect the ability of induced cells to synthesize glycogen, alpha-fetoprotein, albumin, CK18 protein and low-density lipoprotein uptake. Forty-five Sprague-Dawley rats (provided by the Experimental Animal Center, the Affiliated Haikou Hospital, Xiangya School of Medicine, Central South University) were randomized into three groups: normal control group, model group and cell transplantation group (n=15 per group). The rats in the latter two groups were intraperitoneally injected with carbon tetrachloride to establish liver fibrosis models. Cell transplantation group was given intravenous injectin of hepatocyte-like cells (induced for 21 days), 0.5 mL, 2×109/L, at 3 days after modeling. Four weeks after cell transplantation, venous blood and liver tissue samples were taken to analyze the changes of liver function, liver fibrosis index and liver pathology. RESULTS AND CONCLUSION: (1) After 21 days of induction, human induced pluripotent stem cell clonal clusters became loose, mainly round or polygonal in shape, and presented with a dense paving stone-like arrangement. A large amount of pink glycogens could be seen in the cytoplasm, indicating that induced pluripotent stem cells have the ability to synthesize glycogen. Low-density lipoprotein uptake test showed that induced pluripotent stem cells had the ability to uptake low-density lipoprotein. Immunohistochemistry detection showed that the cells were positive for alpha fetoprotein, albumin and CK18. (2) At 4 weeks after cell transplantation, the level of albumin in the model group was significantly lower than that in the normal control group (P < 0.05), while the levels of direct bilirubin, indirect bilirubin, aspartate aminotransferase, alanine aminotransferase, type IV collagen, serum hyaluronidase, serum type III procollagen in the model group were significantly higher than those in the normal control group (P < 0.05). Compared with the model group, the level of albumin in the cell transplantation group was significantly increased (P < 0.05), while the levels of direct bilirubin, indirect bilirubin, aspartate aminotransferase, alanine aminotransferase, type IV collagen, serum hyaluronidase, serum type III procollagen in the cell transplantation group were significantly decreased (P < 0.05). (3) Four weeks after cell transplantation, inflammatory cell infiltration, hepatocyte degeneration and necrosis in the cell transplantation group were improved to different extents. Therefore, hepatocyte-like cells derived from induced pluripotent stem cells could significantly improve liver fibrosis in rats.
ABSTRACT
Objective To explore the pathogenic mechanism by detecting the expression of membrane glycoprotein in the platelets of nonmuscle myosin heavy chain 9 related disease (MYH9-RD)patients.Methods Periperal bloods were obtained from 11 MYH9-RD patients and 7 normal family members.Flow cytometry was used for detecting the expression of the platelet membrane glycoprotein including GP Ⅱ b/Ⅲa(CD41/61),GP Ⅰ a(CD49b),GP Ⅰ b/Ⅸ/Ⅴ (CD42a) GP Ⅰ b(CD42b) and GPⅣCD36).Results The expression fluorescence intensity of platelet membrane glycoprotein GP Ⅱ b/Ⅲ a CD41/61),GPⅠa(CD49b),GP Ⅰ b/Ⅸ/Ⅴ (CD42a) GP Ⅰ b(CD42b) and GPⅣ (CD36) are 653.7 ±192.7,420.0 ± 151.3,667.7 ± 371.3 and 236.4 ± 64.2 respectively,which are significantly higher than those in normal controls (406.7 ± 126.1,181.2 ± 29.3,271.4 ± 91.6 and 136.1 ± 23.5 ; P ＜ 0.01) ; The expression of GP Ⅰ a(CD49b) was lower in patients with MYH9-RD (139.1 ± 54.9) than that in normal controls (192.2 ± 143.4),but there was no significant difference (P ＞ 0.05).Conclusion In our study,the diverse clinical manifestations in patients with MYH9-RD is probably associated with the expression level of platelet membrane glycoprotein