AIM： To investigate and compare the hepatogenic transdifferentiation of adipose tissue-derived stem cells （ADSC） and bone marrow-derived mesenchymal stem cells （BMSC） in vitro. Transdifferentiation of BMSC into h...AIM： To investigate and compare the hepatogenic transdifferentiation of adipose tissue-derived stem cells （ADSC） and bone marrow-derived mesenchymal stem cells （BMSC） in vitro. Transdifferentiation of BMSC into hepatic cells in vivo has been described. Adipose tissue represents an accessible source of ADSC, with similar characteristics to BMSC. METHODS： BMSCs were obtained from patients undergoing total hip arthroplasty and ADSC from human adipose tissue obtained from lipectomy. Cells were grown in medium containing 15% human serum. Cultures were serum deprived for 2 d before cultivating under similar pro-hepatogenic conditions to those of liver development using a 2-step protocol with sequential addition of growth factors, cytokines and hormones. Hepatic differentiation was RT-PCR-assessed and liver-marker genes were immunohistochemically analysed.RESULTS： BMSC and ADSC exhibited a fibroblastic morphology that changed to a polygonal shape when cells differentiated. Expression of stem cell marker Thyl decreased in differentiated ADSC and BMSC. However, the expression of the hepatic markers, albumin and CYPs increased to a similar extent in differentiated BMSC and ADSC. Hepatic gene activation could be attributed to increased liver-enriched transcription factors （C/EBPβ and HNF4α）, as demonstrated by adenoviral expression vectors.CONCLUSION： Mesenchymal stem cells can be induced to hepatogenic transdifferentiation in vitro. ADSCs have a similar hepatogenic differentiation potential to BMSC, but a longer culture period and higher proliferation capacity. Therefore, adipose tissue may be an ideal source of large amounts of autologous stem cells, and may become an alternative for hepatocyte regeneration, liver cell transplantation or preclinical drug testing.展开更多
Objective： To induce adipocyte differentiation in vitro by adipose-derived stromal cells （ASCs） harvested from transgenic mice with green fluorescent protein （GFP） and assess the possibility of constructing adipo...Objective： To induce adipocyte differentiation in vitro by adipose-derived stromal cells （ASCs） harvested from transgenic mice with green fluorescent protein （GFP） and assess the possibility of constructing adipose tissues via attachment of ASCs to type I collagen scaffolds. Methods： Inguinal fat pads from GFP transgenic mice were digested by enzymes for isolation of ASCs （primary culture ）. After expansion to three passages of ASCs, the cells were incubated in an adipogenic medium for two weeks, and the adipocyte differentiation by ASCs in vitro was assessed by morphological observation and Off Red O staining. Then they were attached to collagen scaffolds and co-cultured for 12 hours, followed by hypodermic implantation to the dorsal skin of nude mice for 2 months. The newly-formed tissues were detected by HE staining. Results： The cultured primary stem cells were fibroblast-like and showed active proliferation. After being incubated in an adipocyte differentiation medium, the lipid droplets in the cytoplasm accumulated gradually and finally developed into mature adipocytes, which showed positive in Oil Red O staining. A 0.5-cm^3 new tissue clot was found under the dorsal skin of the nude mice and it was confirmed as mature adipose tissues by fluorescent observation and HE staining. Conclusions： ASCs can successfully differentiate adipose tissues into mature adipocytes, which exhibit an adipocyte-like morphology and express as intracytoplasmic lipid droplets. It is an efficient model of adipose tissues engineered with ASCs and type I collagen scaffolds.展开更多
Objective： To testify the effect of the stem cells derived from the widely distributed fat tissue on repairing full-thickness hyaline cartilage defects. Methods： Adipose-derived stem cells （ADSCs） were derived fro...Objective： To testify the effect of the stem cells derived from the widely distributed fat tissue on repairing full-thickness hyaline cartilage defects. Methods： Adipose-derived stem cells （ADSCs） were derived from adipose tissue and cultured in vitro. Twenty- seven New Zealand white rabbits were divided into three groups randomly. The cultured ADSCs mixed with calcium alginate gel were used to fill the full-thickness hyaline cartilage defects created at the patellafemoral joint, and the defects repaired with gel or without treatment served as control groups. After 4, 8 and 12 weeks, the reconstructed tissue was evaluated macroscopically and microscopically. Histological analysis and qualitative scoring were also performed to detect the outcome.Results： Full thickness hyaline cartilage defects were repaired completely with ADSCs-derived tissue. The result was better in ADSCs group than the control ones. The microstructure of reconstructed tissue with ADSCs was similar to that of hyaline cartilage and contained more cells and regular matrix fibers, being better than other groups. Plenty of collagen fibers around cells could be seen under transmission electron microscopy. Statistical analysis revealed a significant difference in comparison with other groups at each time point （t=4.360, P〈0.01）. Conclusion： These results indicate that stem cells derived from mature adipose without induction possess the ability to repair cartilage defects展开更多
Objective： To differentiate rat adipose tissue-derived mesenchymal stem cells （ADSCs） into cells with a nucleus pulposus-like phenotype in vitro, so as to lay a foundation for the cell-based transplantation therapy...Objective： To differentiate rat adipose tissue-derived mesenchymal stem cells （ADSCs） into cells with a nucleus pulposus-like phenotype in vitro, so as to lay a foundation for the cell-based transplantation therapy of degenerated intervertebral discs. Methods： Rat ADSCs were isolated only from the subcutaneous inguinal region and purified by limited dilution. ADSCs of the third passages were analyzed by fluorescence activated cell sorter （FACS） to detect the cell surface markers （Sca-1, CD44, CD45, CDI lb）. To induce ADSCs to- wards a nucleus pulposus-like phenotype, ADSCs were immobilized in 3-dimensional alginate hydrogels and cultured in an inducing medium containing transforming growth factor-beta1 （TGF- β1） under hypoxia （2% O2）, while control groups under normoxia （21% O2） in alginate beads in medium with or without the presence of TGF-β 1. Semiquantitative reverse transcription polymerase chain reaction （RT-PCR） was carried out to evaluate phenotypic and biosynthetic activities in the process of differentiation. Meanwhile, Alcian blue staining were used to detect the formation of sulfated glycosaminoglycans （GAGs） in the differentiated cells. Results： The purified ADSCs were fibroblast-like and proliferated rapidly in vitro. The flow cytometry showed that ADSCs were positive for Sca-1 and CD44, negative for CD45 and CD11b. The results of RT-PCR manifested that the gene expressions of Sox-9, aggrecan and collagen Ⅱ, which were chondrocyte specific, were upregulated in medium containing TGF-β1 under hypoxia （2% O2）. Likewise, gene expression of HIF-1 a, which was characteristics of in- tervertebral discs, was also upregulated. Simultaneously, Alcian blue staining exhibited the formation of many GAGs. Conclusions： The approach in our experiment is a simple and effective way to acquire a large quantity of homogenous ADSCs. Rat ADSCs can be differentiated into nucleus pulposus-like cells. ADSCs may replace bone marrow mesenchymal stem cells as a new kind of seed cells in regeneration of degenerated intervertebral discs using cell transolantation therarw.展开更多
Objective： To explore the therapeutic ef- fect of osteogenically induced adipose-derived stem cells （ADSCs） on vascular deprivation-induced osteonecrosis of the femoral head （ONFH） in rabbit model. Methods： Vas...Objective： To explore the therapeutic ef- fect of osteogenically induced adipose-derived stem cells （ADSCs） on vascular deprivation-induced osteonecrosis of the femoral head （ONFH） in rabbit model. Methods： Vascular deprivation-induced ONFH was established by intramuscular injection of methylpre- dnisolone, and vascular occlusion of the capital femoral epiphysis by electrocoagulation in adult New Zealand white rabbits. Eight weeks afLer the establishment of vascular dep- rivation-induced ONFH, animals were randomly divided into three equal groups. In Group A （control）, no therapy was given. In Group B, core decompression was performed by drilling a hole （1.2 mm in diameter） from the outer cortex 2.5 cm distal to the proximal end of the greater trochanter. In Group C, 1 x 107 osteogenically induced ADSCs were resus- pended in 0.5 ml PBS, and then injected directly into the femoral head. Femoral head specimens were obtained at postoperative 8 weeks. The bone formation and three-di- mensional microstructure of the femoral head was evaluated by micro-computed tomography scans （u-CT）. Immunohistochemical analysis was performed to detect the expression ofosteocalcin. Angiogenesis and repair of the femoral head were observed histologically. Results： In trabecular bone at the proximal femur region, the trabecular volume was higher in Group C （130.70 mm3± 4.33 mm3） than that in Groups A （101.07 mm3±7.76 mm3） and B （107.89 mm3±8.6g mm3, P〈0.01）. Bone volume was significantly increased in Group C （40.09 mm3±6.35 mm3） than in Groups A （29.65 mm3±4.61 mm3） and B （31.80 mm3± 4.01 mm3, P〈0.01）. The trabecular number was higher in Groups C （1.58±0.25） than other two groups （1.15±0.18, 1.16± 0.21, P〈0.01）. Bone mineral density showed statistically significant difference between Groups C and A or B （375.38± 23.06） mg HA/ccm, vs （313.73 ± 19.30） mg HA/ccm and （316.09± 16.45） mg HA/ccm, P〈0.01）. Histological examination indicated that there was more new bone formation in Group C than in other groups. Conclusion： Treatment with autologous osteogenically induced ADSCs transplantation results in an enhanced osteogenesis and microstructure of the vascular deprivation-induced osteonecrosis in rabbits.展开更多
基金Supported by the ALIVE Foundation, the FIS from Instituto de Salud Carlos III, Spain, No. 03/0339, and the European Commission, No. LSHB-CT-2004-504761
文摘AIM： To investigate and compare the hepatogenic transdifferentiation of adipose tissue-derived stem cells （ADSC） and bone marrow-derived mesenchymal stem cells （BMSC） in vitro. Transdifferentiation of BMSC into hepatic cells in vivo has been described. Adipose tissue represents an accessible source of ADSC, with similar characteristics to BMSC. METHODS： BMSCs were obtained from patients undergoing total hip arthroplasty and ADSC from human adipose tissue obtained from lipectomy. Cells were grown in medium containing 15% human serum. Cultures were serum deprived for 2 d before cultivating under similar pro-hepatogenic conditions to those of liver development using a 2-step protocol with sequential addition of growth factors, cytokines and hormones. Hepatic differentiation was RT-PCR-assessed and liver-marker genes were immunohistochemically analysed.RESULTS： BMSC and ADSC exhibited a fibroblastic morphology that changed to a polygonal shape when cells differentiated. Expression of stem cell marker Thyl decreased in differentiated ADSC and BMSC. However, the expression of the hepatic markers, albumin and CYPs increased to a similar extent in differentiated BMSC and ADSC. Hepatic gene activation could be attributed to increased liver-enriched transcription factors （C/EBPβ and HNF4α）, as demonstrated by adenoviral expression vectors.CONCLUSION： Mesenchymal stem cells can be induced to hepatogenic transdifferentiation in vitro. ADSCs have a similar hepatogenic differentiation potential to BMSC, but a longer culture period and higher proliferation capacity. Therefore, adipose tissue may be an ideal source of large amounts of autologous stem cells, and may become an alternative for hepatocyte regeneration, liver cell transplantation or preclinical drug testing.
文摘Objective： To induce adipocyte differentiation in vitro by adipose-derived stromal cells （ASCs） harvested from transgenic mice with green fluorescent protein （GFP） and assess the possibility of constructing adipose tissues via attachment of ASCs to type I collagen scaffolds. Methods： Inguinal fat pads from GFP transgenic mice were digested by enzymes for isolation of ASCs （primary culture ）. After expansion to three passages of ASCs, the cells were incubated in an adipogenic medium for two weeks, and the adipocyte differentiation by ASCs in vitro was assessed by morphological observation and Off Red O staining. Then they were attached to collagen scaffolds and co-cultured for 12 hours, followed by hypodermic implantation to the dorsal skin of nude mice for 2 months. The newly-formed tissues were detected by HE staining. Results： The cultured primary stem cells were fibroblast-like and showed active proliferation. After being incubated in an adipocyte differentiation medium, the lipid droplets in the cytoplasm accumulated gradually and finally developed into mature adipocytes, which showed positive in Oil Red O staining. A 0.5-cm^3 new tissue clot was found under the dorsal skin of the nude mice and it was confirmed as mature adipose tissues by fluorescent observation and HE staining. Conclusions： ASCs can successfully differentiate adipose tissues into mature adipocytes, which exhibit an adipocyte-like morphology and express as intracytoplasmic lipid droplets. It is an efficient model of adipose tissues engineered with ASCs and type I collagen scaffolds.
基金This work was funded by the National Natural Science Foundation of China （No. 30801167）.
文摘Objective： To testify the effect of the stem cells derived from the widely distributed fat tissue on repairing full-thickness hyaline cartilage defects. Methods： Adipose-derived stem cells （ADSCs） were derived from adipose tissue and cultured in vitro. Twenty- seven New Zealand white rabbits were divided into three groups randomly. The cultured ADSCs mixed with calcium alginate gel were used to fill the full-thickness hyaline cartilage defects created at the patellafemoral joint, and the defects repaired with gel or without treatment served as control groups. After 4, 8 and 12 weeks, the reconstructed tissue was evaluated macroscopically and microscopically. Histological analysis and qualitative scoring were also performed to detect the outcome.Results： Full thickness hyaline cartilage defects were repaired completely with ADSCs-derived tissue. The result was better in ADSCs group than the control ones. The microstructure of reconstructed tissue with ADSCs was similar to that of hyaline cartilage and contained more cells and regular matrix fibers, being better than other groups. Plenty of collagen fibers around cells could be seen under transmission electron microscopy. Statistical analysis revealed a significant difference in comparison with other groups at each time point （t=4.360, P〈0.01）. Conclusion： These results indicate that stem cells derived from mature adipose without induction possess the ability to repair cartilage defects
文摘Objective： To differentiate rat adipose tissue-derived mesenchymal stem cells （ADSCs） into cells with a nucleus pulposus-like phenotype in vitro, so as to lay a foundation for the cell-based transplantation therapy of degenerated intervertebral discs. Methods： Rat ADSCs were isolated only from the subcutaneous inguinal region and purified by limited dilution. ADSCs of the third passages were analyzed by fluorescence activated cell sorter （FACS） to detect the cell surface markers （Sca-1, CD44, CD45, CDI lb）. To induce ADSCs to- wards a nucleus pulposus-like phenotype, ADSCs were immobilized in 3-dimensional alginate hydrogels and cultured in an inducing medium containing transforming growth factor-beta1 （TGF- β1） under hypoxia （2% O2）, while control groups under normoxia （21% O2） in alginate beads in medium with or without the presence of TGF-β 1. Semiquantitative reverse transcription polymerase chain reaction （RT-PCR） was carried out to evaluate phenotypic and biosynthetic activities in the process of differentiation. Meanwhile, Alcian blue staining were used to detect the formation of sulfated glycosaminoglycans （GAGs） in the differentiated cells. Results： The purified ADSCs were fibroblast-like and proliferated rapidly in vitro. The flow cytometry showed that ADSCs were positive for Sca-1 and CD44, negative for CD45 and CD11b. The results of RT-PCR manifested that the gene expressions of Sox-9, aggrecan and collagen Ⅱ, which were chondrocyte specific, were upregulated in medium containing TGF-β1 under hypoxia （2% O2）. Likewise, gene expression of HIF-1 a, which was characteristics of in- tervertebral discs, was also upregulated. Simultaneously, Alcian blue staining exhibited the formation of many GAGs. Conclusions： The approach in our experiment is a simple and effective way to acquire a large quantity of homogenous ADSCs. Rat ADSCs can be differentiated into nucleus pulposus-like cells. ADSCs may replace bone marrow mesenchymal stem cells as a new kind of seed cells in regeneration of degenerated intervertebral discs using cell transolantation therarw.
基金This work is supported by a grant from the National Natural Science Foundation of China （30960390） and Doctoral Innovation Fund of Xinjiang Medical University （DC2010-12）.
文摘Objective： To explore the therapeutic ef- fect of osteogenically induced adipose-derived stem cells （ADSCs） on vascular deprivation-induced osteonecrosis of the femoral head （ONFH） in rabbit model. Methods： Vascular deprivation-induced ONFH was established by intramuscular injection of methylpre- dnisolone, and vascular occlusion of the capital femoral epiphysis by electrocoagulation in adult New Zealand white rabbits. Eight weeks afLer the establishment of vascular dep- rivation-induced ONFH, animals were randomly divided into three equal groups. In Group A （control）, no therapy was given. In Group B, core decompression was performed by drilling a hole （1.2 mm in diameter） from the outer cortex 2.5 cm distal to the proximal end of the greater trochanter. In Group C, 1 x 107 osteogenically induced ADSCs were resus- pended in 0.5 ml PBS, and then injected directly into the femoral head. Femoral head specimens were obtained at postoperative 8 weeks. The bone formation and three-di- mensional microstructure of the femoral head was evaluated by micro-computed tomography scans （u-CT）. Immunohistochemical analysis was performed to detect the expression ofosteocalcin. Angiogenesis and repair of the femoral head were observed histologically. Results： In trabecular bone at the proximal femur region, the trabecular volume was higher in Group C （130.70 mm3± 4.33 mm3） than that in Groups A （101.07 mm3±7.76 mm3） and B （107.89 mm3±8.6g mm3, P〈0.01）. Bone volume was significantly increased in Group C （40.09 mm3±6.35 mm3） than in Groups A （29.65 mm3±4.61 mm3） and B （31.80 mm3± 4.01 mm3, P〈0.01）. The trabecular number was higher in Groups C （1.58±0.25） than other two groups （1.15±0.18, 1.16± 0.21, P〈0.01）. Bone mineral density showed statistically significant difference between Groups C and A or B （375.38± 23.06） mg HA/ccm, vs （313.73 ± 19.30） mg HA/ccm and （316.09± 16.45） mg HA/ccm, P〈0.01）. Histological examination indicated that there was more new bone formation in Group C than in other groups. Conclusion： Treatment with autologous osteogenically induced ADSCs transplantation results in an enhanced osteogenesis and microstructure of the vascular deprivation-induced osteonecrosis in rabbits.
