Human pluripotent stem cells represent a potentially unlimited source of functional pancreatic endocrine lineage cells. Here we report a highly efficient approach to induce human embryonic stem （ES） cells and induce...Human pluripotent stem cells represent a potentially unlimited source of functional pancreatic endocrine lineage cells. Here we report a highly efficient approach to induce human embryonic stem （ES） cells and induced pluripo- tent stem （iPS） cells to differentiate into mature insulin-producing cells in a chemical-defined culture system. The differentiated human ES cells obtained by this approach comprised nearly 25% insulin-positive cells as assayed by flow cytometry analysis, which released insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets. Most of these insulin-producing cells co-expressed mature β cell-specific markers such as NKX6-1 and PDX1, indicating a similar gene expression pattern to adult islet β cells in vivo. In this study, we also demonstrated that EGF facilitates the expansion of PDXl-positive pancreatic progenitors. Moreover, our protocol also succeeded in efficiently inducing human iPS cells to differentiate into insuIin-producing ceils. Therefore, this work not only provides a new model to study the mechanism of human pancreatic specialization and maturation in vitro, but also enhances the possibility of utilizing patient-specific iPS cells for the treatment of diabetes.展开更多
The capacity for self-renewal and differentiation of human embryonic stem （ES） cells makes them a potential source for generation of pancreatic beta cells for treating type I diabetes mellitus. Here, we report a new...The capacity for self-renewal and differentiation of human embryonic stem （ES） cells makes them a potential source for generation of pancreatic beta cells for treating type I diabetes mellitus. Here, we report a newly developed and effective method, carried out in a serum-free system, which induced human ES cells to differentiate into insulin-producing cells. Activin A was used in the initial stage to induce definitive endoderm differentiation from human ES cells, as detected by the expression of the definitive endoderm markers Sox17 and Brachyury. Further, all-trans retinoic acid （RA） was used to promote pancreatic differentiation, as indicated by the expression of the early pancreatic transcription factors pdxl and hlxb9. After maturation in DMEM/F12 serum-free medium with bFGF and nicotinamide, the differentiated cells expressed islet specific markers such as C-peptide, insulin, glucagon and glut2. The percentage of C-peptide-positive cells exceeded 15%. The secretion of insulin and C-peptide by these cells corresponded to the variations in glucose levels. When transplanted into renal capsules of Streptozotocin （STZ）-treated nude mice, these differentiated human ES cells survived and maintained the expression of beta cell marker genes, including C-peptide, pdxl, glucokinase, nkx6.1, lAPP, pax6 and Tcfl. Thirty percent of the transplanted nude mice exhibited apparent restoration of stable euglycemia; and the corrected phenotype was sustained for more than six weeks. Our new method provides a promising in vitro differentiation model for studying the mechanisms of human pancreas development and illustrates the potential of using human ES cells for the treatment of type I diabetes mellitus.展开更多
BACKGROUND: Mesenchymal stem cells derived from human umbilical cord blood (UCB-MSCs) have good research and application prospects in the treatment of diabetes. We once induced UCB-MSCs to differentiate into insulin-p...BACKGROUND: Mesenchymal stem cells derived from human umbilical cord blood (UCB-MSCs) have good research and application prospects in the treatment of diabetes. We once induced UCB-MSCs to differentiate into insulin-producing cells (IPCs) in vitro, but we did not know the functions of these cells in vivo. The aim of this study was to assess the functional effects of IPCs on insulin secretion and their role in the treatment of diabetes in vivo. METHODS: UCB-MSCs were induced to IPCs by an inducing protocol with extracellular matrix gel. BALB/C nude mice were made hyperglycemic by intraperitoneal injection of streptozotocin. The diabetic mice were transplanted with 1x10(7) IPCs under the renal capsule or with phosphate-buffered saline as a control. After transplantation, the grafts were analyzed by immunocytochemistry for the expression of human insulin; the serum human insulin levels were measured; and blood glucose and body weight status were monitored. RESULTS: Immunofluorescence showed that numerous IPCs under the kidney capsule were insulin-positive. On day 14 after transplantation, the serum human insulin level of the treatment group (n=9) averaged 0.44 +/- 0.12 mU/L, which was higher than that of the control group (n=9) that did not express insulin (t=10.842, P<0.05). The diabetic mice remained hyperglycemic and kept losing body weight after IPC transplantation, and there was no significant difference in the control group. CONCLUSION: IPCs differentiated from UCB-MSCs generate human insulin in diabetic mice, but more research is needed to make further use of them to regulate hyperglycemia and body weight in vivo. (Hepatobiliary Pancreat Dis Int 2009; 8: 255-260)展开更多
目的比较超顺磁化氧化铁颗粒-多聚左旋赖氨酸(SPIO-PLL)标记与未标记诱导后大鼠胰岛素分泌细胞的生物活性,探讨两者MRI成像表现。方法分离培养大鼠骨髓间质干细胞(BMSC),经二期方案诱导成胰岛素分泌细胞,SPIO-PLL标记细胞,普鲁士蓝染色...目的比较超顺磁化氧化铁颗粒-多聚左旋赖氨酸(SPIO-PLL)标记与未标记诱导后大鼠胰岛素分泌细胞的生物活性,探讨两者MRI成像表现。方法分离培养大鼠骨髓间质干细胞(BMSC),经二期方案诱导成胰岛素分泌细胞,SPIO-PLL标记细胞,普鲁士蓝染色显示细胞内铁;放射免疫分析法测定标记及未标记细胞的胰岛素分泌情况;同时采用临床应用型1．5 T MR仪对两组细胞群进行T_1WI、T_2WI、T_2*WI 3个序列成像。结果普鲁士蓝染色显示标记细胞蓝色铁颗粒位于细胞内。标记后的细胞能分泌胰岛素,经统计学分析分泌量与未标记细胞无显著性差异。标记后的细胞在以T_2*WI序列信号降低最明显,信号强度变化率最大。结论SPIO-PLL可以有效标记诱导后大鼠胰岛素分泌细胞,且对其生物学活性无明显影响,临床应用型1．5 T MR仪可对标记细胞群进行体外成像。展开更多
Background: Insulitis is defined by the presence of immune cells infiltrating in the pancreatic islets that might progress into the complete β-cell loss. The immunomodulatory properties of bone marrow-derived mesench...Background: Insulitis is defined by the presence of immune cells infiltrating in the pancreatic islets that might progress into the complete β-cell loss. The immunomodulatory properties of bone marrow-derived mesenchymal stem cells(BM-MSCs) have attracted much attention. This study aimed to evaluate the possible immunomodulatory effects of rat BM-MSCs and MSCs-derived insulin-producing cells(IPCs) in a mouse model of pancreatic insulitis. Methods: Insulitis was induced in BALB/c mice using five consecuti ve doses of streptozotocin. MSCs or IPCs were directly injected into the pancreas of mice and their effects on the expression of Th subsetsrelated genes were evaluated. Results: Both BM-MSCs and IPCs significantly reduced the expression of pancreatic Th1-related IFN-γ( P < 0.001 and P < 0.05, respectively) and T-bet genes(both P < 0.001). Moreover, the expression of IL-10 gene was significantly increased in IPC-treated compared to BM-MSC-or PBS-treated mice( P < 0.001 both comparisons). Conclusions: BM-MSCs and IPCs could successfully suppress pathologic Th1 immune responses in the mouse model of insulitis. However, the marked increase in IL-10 gene expression by IPCs compared to BM-MSCs suggests that their simultaneous use at the initial phase of autoimmune diabetes might be a better option to reduce inflammation but these results need to be verified by further experiments.展开更多
BACKGROUND Despite the availability of current therapies,including oral antidiabetic drugs and insulin,for controlling the symptoms caused by high blood glucose,it is difficult to cure diabetes mellitus,especially typ...BACKGROUND Despite the availability of current therapies,including oral antidiabetic drugs and insulin,for controlling the symptoms caused by high blood glucose,it is difficult to cure diabetes mellitus,especially type 1 diabetes mellitus.AIM Cell therapies using mesenchymal stem cells(MSCs)may be a promising option.However,the therapeutic mechanisms by which MSCs exert their effects,such as whether they can differentiate into insulin-producing cells (IPCs) beforetransplantation, are uncertain.METHODSIn this study, we used three types of differentiation media over 10 d to generateIPCs from human Wharton’s jelly MSCs (hWJ-MSCs). We further transplantedthe undifferentiated hWJ-MSCs and differentiated IPCs derived from them intothe portal vein of rats with streptozotocin-induced diabetes, and recorded thephysiological and pathological changes.RESULTSUsing fluorescent staining and C-peptide enzyme-linked immunoassay, we wereable to successfully induce the differentiation of hWJ-MSCs into IPCs.Transplantation of both IPCs derived from hWJ-MSCs and undifferentiated hWJMSCshad the therapeutic effect of ameliorating blood glucose levels andimproving intraperitoneal glucose tolerance tests. The transplanted IPCs homedto the pancreas and functionally survived for at least 8 wk after transplantation,whereas the undifferentiated hWJ-MSCs were able to improve the insulitis andameliorate the serum inflammatory cytokine in streptozotocin-induced diabeticrats.CONCLUSIONDifferentiated IPCs can significantly improve blood glucose levels in diabetic ratsdue to the continuous secretion of insulin by transplanted cells that survive in theislets of diabetic rats. Transplantation of undifferentiated hWJ-MSCs cansignificantly improve insulitis and re-balance the inflammatory condition indiabetic rats with only a slight improvement in blood glucose levels.展开更多
文摘Human pluripotent stem cells represent a potentially unlimited source of functional pancreatic endocrine lineage cells. Here we report a highly efficient approach to induce human embryonic stem （ES） cells and induced pluripo- tent stem （iPS） cells to differentiate into mature insulin-producing cells in a chemical-defined culture system. The differentiated human ES cells obtained by this approach comprised nearly 25% insulin-positive cells as assayed by flow cytometry analysis, which released insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets. Most of these insulin-producing cells co-expressed mature β cell-specific markers such as NKX6-1 and PDX1, indicating a similar gene expression pattern to adult islet β cells in vivo. In this study, we also demonstrated that EGF facilitates the expansion of PDXl-positive pancreatic progenitors. Moreover, our protocol also succeeded in efficiently inducing human iPS cells to differentiate into insuIin-producing ceils. Therefore, this work not only provides a new model to study the mechanism of human pancreatic specialization and maturation in vitro, but also enhances the possibility of utilizing patient-specific iPS cells for the treatment of diabetes.
基金This research was supported by the Ministry of Science and Technology Grant （2001CB510106）；Science and Technology Plan of Beijing Municipal Government （H020220050290）；National Natural Science Foundation of China Awards for 0utstanding Young Scientists （30125022）；for Creative Research Groups （30421004）；Bill ＆ Melinda Gates Foundation Grant （37871） to H Deng.
文摘The capacity for self-renewal and differentiation of human embryonic stem （ES） cells makes them a potential source for generation of pancreatic beta cells for treating type I diabetes mellitus. Here, we report a newly developed and effective method, carried out in a serum-free system, which induced human ES cells to differentiate into insulin-producing cells. Activin A was used in the initial stage to induce definitive endoderm differentiation from human ES cells, as detected by the expression of the definitive endoderm markers Sox17 and Brachyury. Further, all-trans retinoic acid （RA） was used to promote pancreatic differentiation, as indicated by the expression of the early pancreatic transcription factors pdxl and hlxb9. After maturation in DMEM/F12 serum-free medium with bFGF and nicotinamide, the differentiated cells expressed islet specific markers such as C-peptide, insulin, glucagon and glut2. The percentage of C-peptide-positive cells exceeded 15%. The secretion of insulin and C-peptide by these cells corresponded to the variations in glucose levels. When transplanted into renal capsules of Streptozotocin （STZ）-treated nude mice, these differentiated human ES cells survived and maintained the expression of beta cell marker genes, including C-peptide, pdxl, glucokinase, nkx6.1, lAPP, pax6 and Tcfl. Thirty percent of the transplanted nude mice exhibited apparent restoration of stable euglycemia; and the corrected phenotype was sustained for more than six weeks. Our new method provides a promising in vitro differentiation model for studying the mechanisms of human pancreas development and illustrates the potential of using human ES cells for the treatment of type I diabetes mellitus.
基金supported by grants from the Natural Science Foundation of Heilongjiang Province(No.ZJY0505)the Innovation of Foundation of Outstanding Teachers of Heilongjiang Provincial University(No.1054G026)
文摘BACKGROUND: Mesenchymal stem cells derived from human umbilical cord blood (UCB-MSCs) have good research and application prospects in the treatment of diabetes. We once induced UCB-MSCs to differentiate into insulin-producing cells (IPCs) in vitro, but we did not know the functions of these cells in vivo. The aim of this study was to assess the functional effects of IPCs on insulin secretion and their role in the treatment of diabetes in vivo. METHODS: UCB-MSCs were induced to IPCs by an inducing protocol with extracellular matrix gel. BALB/C nude mice were made hyperglycemic by intraperitoneal injection of streptozotocin. The diabetic mice were transplanted with 1x10(7) IPCs under the renal capsule or with phosphate-buffered saline as a control. After transplantation, the grafts were analyzed by immunocytochemistry for the expression of human insulin; the serum human insulin levels were measured; and blood glucose and body weight status were monitored. RESULTS: Immunofluorescence showed that numerous IPCs under the kidney capsule were insulin-positive. On day 14 after transplantation, the serum human insulin level of the treatment group (n=9) averaged 0.44 +/- 0.12 mU/L, which was higher than that of the control group (n=9) that did not express insulin (t=10.842, P<0.05). The diabetic mice remained hyperglycemic and kept losing body weight after IPC transplantation, and there was no significant difference in the control group. CONCLUSION: IPCs differentiated from UCB-MSCs generate human insulin in diabetic mice, but more research is needed to make further use of them to regulate hyperglycemia and body weight in vivo. (Hepatobiliary Pancreat Dis Int 2009; 8: 255-260)
文摘目的比较超顺磁化氧化铁颗粒-多聚左旋赖氨酸(SPIO-PLL)标记与未标记诱导后大鼠胰岛素分泌细胞的生物活性,探讨两者MRI成像表现。方法分离培养大鼠骨髓间质干细胞(BMSC),经二期方案诱导成胰岛素分泌细胞,SPIO-PLL标记细胞,普鲁士蓝染色显示细胞内铁;放射免疫分析法测定标记及未标记细胞的胰岛素分泌情况;同时采用临床应用型1．5 T MR仪对两组细胞群进行T_1WI、T_2WI、T_2*WI 3个序列成像。结果普鲁士蓝染色显示标记细胞蓝色铁颗粒位于细胞内。标记后的细胞能分泌胰岛素,经统计学分析分泌量与未标记细胞无显著性差异。标记后的细胞在以T_2*WI序列信号降低最明显,信号强度变化率最大。结论SPIO-PLL可以有效标记诱导后大鼠胰岛素分泌细胞,且对其生物学活性无明显影响,临床应用型1．5 T MR仪可对标记细胞群进行体外成像。
基金This study was supported by a grant from Shiraz University of Medical Sciences(No.94-7616).
文摘Background: Insulitis is defined by the presence of immune cells infiltrating in the pancreatic islets that might progress into the complete β-cell loss. The immunomodulatory properties of bone marrow-derived mesenchymal stem cells(BM-MSCs) have attracted much attention. This study aimed to evaluate the possible immunomodulatory effects of rat BM-MSCs and MSCs-derived insulin-producing cells(IPCs) in a mouse model of pancreatic insulitis. Methods: Insulitis was induced in BALB/c mice using five consecuti ve doses of streptozotocin. MSCs or IPCs were directly injected into the pancreas of mice and their effects on the expression of Th subsetsrelated genes were evaluated. Results: Both BM-MSCs and IPCs significantly reduced the expression of pancreatic Th1-related IFN-γ( P < 0.001 and P < 0.05, respectively) and T-bet genes(both P < 0.001). Moreover, the expression of IL-10 gene was significantly increased in IPC-treated compared to BM-MSC-or PBS-treated mice( P < 0.001 both comparisons). Conclusions: BM-MSCs and IPCs could successfully suppress pathologic Th1 immune responses in the mouse model of insulitis. However, the marked increase in IL-10 gene expression by IPCs compared to BM-MSCs suggests that their simultaneous use at the initial phase of autoimmune diabetes might be a better option to reduce inflammation but these results need to be verified by further experiments.
基金Taipei Veterans General Hospital,No.V106B-024Yen Tjing Ling Medical Foundation,No.CI-106-20+1 种基金Cheng Hsin General Hospital,No.CY10716Taiwan Ministry of Science and Technology,No.MOST 105-2314-B-010-010-MY3 and No.MOST 106-2314-B-010-009
文摘BACKGROUND Despite the availability of current therapies,including oral antidiabetic drugs and insulin,for controlling the symptoms caused by high blood glucose,it is difficult to cure diabetes mellitus,especially type 1 diabetes mellitus.AIM Cell therapies using mesenchymal stem cells(MSCs)may be a promising option.However,the therapeutic mechanisms by which MSCs exert their effects,such as whether they can differentiate into insulin-producing cells (IPCs) beforetransplantation, are uncertain.METHODSIn this study, we used three types of differentiation media over 10 d to generateIPCs from human Wharton’s jelly MSCs (hWJ-MSCs). We further transplantedthe undifferentiated hWJ-MSCs and differentiated IPCs derived from them intothe portal vein of rats with streptozotocin-induced diabetes, and recorded thephysiological and pathological changes.RESULTSUsing fluorescent staining and C-peptide enzyme-linked immunoassay, we wereable to successfully induce the differentiation of hWJ-MSCs into IPCs.Transplantation of both IPCs derived from hWJ-MSCs and undifferentiated hWJMSCshad the therapeutic effect of ameliorating blood glucose levels andimproving intraperitoneal glucose tolerance tests. The transplanted IPCs homedto the pancreas and functionally survived for at least 8 wk after transplantation,whereas the undifferentiated hWJ-MSCs were able to improve the insulitis andameliorate the serum inflammatory cytokine in streptozotocin-induced diabeticrats.CONCLUSIONDifferentiated IPCs can significantly improve blood glucose levels in diabetic ratsdue to the continuous secretion of insulin by transplanted cells that survive in theislets of diabetic rats. Transplantation of undifferentiated hWJ-MSCs cansignificantly improve insulitis and re-balance the inflammatory condition indiabetic rats with only a slight improvement in blood glucose levels.
