AIM: To compare the phenotypic and neural differentiation potential of human bone marrow derived multipotent adult progenitor cells (MAPC) and mesenchymal stem cells (MSC). METHODS: Cultures of MAPC and MSC were estab...AIM: To compare the phenotypic and neural differentiation potential of human bone marrow derived multipotent adult progenitor cells (MAPC) and mesenchymal stem cells (MSC). METHODS: Cultures of MAPC and MSC were established in parallel from same samples of human bone marrow (n = 5). Both stem cell types were evaluated for expression of pluripotency markers including Oct-4 and Nanog by immunocytochemistry and reversetranscription polymerase chain reaction (RT-PCR) and expression of standard mesenchymal markers including CD14, CD34, CD44, CD45, CD73, CD90, CD105 andhuman leukocyte antigen (HLA)-ABC by flow cytometry. After treatment with neural induction medium both MAPC and MSC were evaluated for expression of neural proteins [neuronal filament-200 (NF-200) and glial fibrillar acidic protein (GFAP)] by immunocytochemistry and Western blotting and neural genes [NF-200, GFAP, Tau, microtubule-associated protein (MAP)-1B, MAP-2, neuron-specific enolase (NSE) and oligodendrocyte-1 (Olig-1)] by quantitative real-time-PCR. RESULTS: MAPC had small trigonal shaped while MSC had elongated spindle-shaped morphology. The MAPC expressed Oct-4 and Nanog both at gene and protein levels, whereas MSC were negative for these pluripotent markers. MAPC were negative for HLA-ABC while MSC had high expression of HLA-ABC. In addition, MAPC as compared to MSC had significantly lower expression of CD44 (36.56% ± 1.92% vs 98.23% ± 0.51%), CD73 (15.11% ± 2.24% vs 98.53% ± 2.22%) and CD105 (13.81% ± 3.82%vs 95.12% ± 5.65%) (P < 0.001, for all) MAPC cultures compared to MSC cultures treated with neural induction medium had significantly higher fold change expression of NF-200 (0.64), GFAP (0.52), Tau (0.59), MAP-2 (0.72), Olig-1 (0.18) and NSE (0.29) proteins (P < 0.01 for Olig-1 and P < 0.001 for rest) as well as higher fold change expression of genes of NF-200 (1.34),GFAP (1.12),Tau (1.08),MAP-1B (0.92), MAP-2 (1.14) andNSE (0.4) (P < 0.001 for all). CONCLUSION: MAPC can be differentially characterized from MSC as Oct-4 and Nanog positive stem cells with no expression of HLA-ABC and low expression of mesenchymal markers CD44, CD73 and CD105 and when compared to MSC they possess greater predilection for differentiation into neuro-ectodermal lineage.展开更多
AIM: To investigate whether fetal kidney stem cells(f KSC) ameliorate cisplatin induced acute renal failure(ARF) in rats and promote renal angiogenesis.METHODS: The f KSC were isolated from rat fetuses of gestation da...AIM: To investigate whether fetal kidney stem cells(f KSC) ameliorate cisplatin induced acute renal failure(ARF) in rats and promote renal angiogenesis.METHODS: The f KSC were isolated from rat fetuses of gestation day 16 and expanded in vitro up to 3rd passage. They were characterized for the expression of mesenchymal and renal progenitor markers by flow cytometry and immunocytochemistry, respectively. The in vitro differentiation of f KSC towards epithelial lineage was evaluated by the treatment with specific induction medium and their angiogenic potential by matrigel induced tube formation assay. To study the effect of f KSC in ARF, f KSC labeled with PKH26 were infused in rats with cisplatin induced ARF and, the blood and renal tissues of the rats were collected at different time points. Blood biochemical parameters were studied to evaluate renal function. Renal tissues were evaluated for renal architecture, renal cell proliferation and angiogenesis by immunohistochemistry, renal cell apoptosis by terminal deoxynucleotidyl transferase nickend labeling assay and early expression of angiogenic molecules viz. vascular endothelial growth factor(VEGF), hypoxia-inducible factor(HIF)-1α and endothelial nitric oxide synthase(eN OS) by western blot.RESULTS: The fK SC expressed mesenchymal markers viz. CD29, CD44, CD73, CD90 and CD105 as well asrenal progenitor markers viz. Wt1, Pax2 and Six2. They exhibited a potential to form CD31 and Von Willebrand factor expressing capillary-like structures and could be differentiated into cytokeratin(CK)18 and CK19 positive epithelial cells. Administration of fK SC in rats with ARF as compared to administration of saline alone, resulted in a significant improvement in renal function and histology on day 3(2.33 ± 0.33 vs 3.50 ± 0.34, P < 0.05) and on day 7(0.83 ± 0.16 vs 2.00 ± 0.25, P < 0.05). The infused PKH26 labeled fK SC were observed to engraft in damaged renal tubules and showed increased proliferation and reduced apoptosis(P < 0.05) of renal cells. The kidneys of fK SC as compared to saline treated rats had a higher capillary density on day 3 [13.30 ± 1.54 vs 7.10 ± 1.29, capillaries/high-power fields(HPF), P < 0.05], and on day 7(21.10 ± 1.46 vs 15.00 ± 1.30, capillaries/HPF, P < 0.05). In addition, kidneys of fK SC treated rats had an upregulation of angiogenic proteins hypoxia-inducible factor-1α, VEGF and eN OS on day 3(P < 0.05).CONCLUSION: Our study shows that fK SC ameliorate cisplatin induced ARF in rats and promote renal angiogenesis, which may be an important therapeutic mechanism of these stem cells in the disease.展开更多
AIM:To study the expression of embryonal markers by fetal cardiac mesenchymal stem cells(fC-MSC)and their differentiation into cells of all the germ layers. METHODS:Ten independent cultures of rat fCMSC were set up fr...AIM:To study the expression of embryonal markers by fetal cardiac mesenchymal stem cells(fC-MSC)and their differentiation into cells of all the germ layers. METHODS:Ten independent cultures of rat fCMSC were set up from cells derived from individual or pooled fetal hearts and studies given below were carried out at passages 3,6,15 and 21.The phenotypic markers CD29,CD31,CD34,CD45,CD73,CD90, CD105,CD166 and HLA-DR were analyzed by flow cytometry.The expression of embryonal markers Oct-4, Nanog,Sox-2,SSEA-1,SSEA-3,SSEA-4,TRA-1-60 and TRA 1-81 were studied by immunocytochemistry.The fC-MSC treated with specific induction medium were evaluated for their differentiation into(1)adipocytes and osteocytes(mesodermal cells)by Oil Red O and Alizarin Red staining,respectively,as well as by expression of lipoprotein lipase,PPARγ2 genes in adipocytes and osteopontin and RUNX2 genes in osteocytes by reverse-transcription polymerase chain reaction(RT- PCR);(2)neuronal(ectodermal)cells by expression of neuronal Filament-160 and Glial Fibrillar Acidic Protein by RT-PCR and immunocytochemistry;and(3)hepa- tocytic(endodermal)cells by expression of albumin by RT-PCR and immunocytochemistry,glycogen deposits by Periodic Acid Schiff staining and excretion of urea into the culture supernatant. RESULTS:The fC-MSC expressed CD29,CD73,CD90, CD105,CD166 but lacked expression of CD31,CD34, CD45 and HLA-DR.They expressed embryonal markers,viz.Oct-4,Nanog,Sox-2,SSEA-1,SSEA-3,SSEA-4, TRA-1-81 but not TRA-1-60.On treatment with specific induction media,they differentiated into adipocytes and osteocytes,neuronal cells and hepatocytic cells. CONCLUSION:Our results together suggest that fCMSC are primitive stem cell types with a high degree of plasticity and,in addition to their suitability for cardiovascular regenerative therapy,they may have a wide spectrum of therapeutic applications in regenerative medicine.展开更多
基金Supported by The Grant-in-Aid entitled"Stem cells for regenerative medicine:Isolation of Multipotent adult Progenitor Cells from Human Bone Marrow and their Clonal Expansion and Differentiation into Cardiomyocytes,Hepatocytes and Beta-islets"No.BT/PR6303/MED/14/776/2005,sanctioned by Department of Biotechnology,Government of India
文摘AIM: To compare the phenotypic and neural differentiation potential of human bone marrow derived multipotent adult progenitor cells (MAPC) and mesenchymal stem cells (MSC). METHODS: Cultures of MAPC and MSC were established in parallel from same samples of human bone marrow (n = 5). Both stem cell types were evaluated for expression of pluripotency markers including Oct-4 and Nanog by immunocytochemistry and reversetranscription polymerase chain reaction (RT-PCR) and expression of standard mesenchymal markers including CD14, CD34, CD44, CD45, CD73, CD90, CD105 andhuman leukocyte antigen (HLA)-ABC by flow cytometry. After treatment with neural induction medium both MAPC and MSC were evaluated for expression of neural proteins [neuronal filament-200 (NF-200) and glial fibrillar acidic protein (GFAP)] by immunocytochemistry and Western blotting and neural genes [NF-200, GFAP, Tau, microtubule-associated protein (MAP)-1B, MAP-2, neuron-specific enolase (NSE) and oligodendrocyte-1 (Olig-1)] by quantitative real-time-PCR. RESULTS: MAPC had small trigonal shaped while MSC had elongated spindle-shaped morphology. The MAPC expressed Oct-4 and Nanog both at gene and protein levels, whereas MSC were negative for these pluripotent markers. MAPC were negative for HLA-ABC while MSC had high expression of HLA-ABC. In addition, MAPC as compared to MSC had significantly lower expression of CD44 (36.56% ± 1.92% vs 98.23% ± 0.51%), CD73 (15.11% ± 2.24% vs 98.53% ± 2.22%) and CD105 (13.81% ± 3.82%vs 95.12% ± 5.65%) (P < 0.001, for all) MAPC cultures compared to MSC cultures treated with neural induction medium had significantly higher fold change expression of NF-200 (0.64), GFAP (0.52), Tau (0.59), MAP-2 (0.72), Olig-1 (0.18) and NSE (0.29) proteins (P < 0.01 for Olig-1 and P < 0.001 for rest) as well as higher fold change expression of genes of NF-200 (1.34),GFAP (1.12),Tau (1.08),MAP-1B (0.92), MAP-2 (1.14) andNSE (0.4) (P < 0.001 for all). CONCLUSION: MAPC can be differentially characterized from MSC as Oct-4 and Nanog positive stem cells with no expression of HLA-ABC and low expression of mesenchymal markers CD44, CD73 and CD105 and when compared to MSC they possess greater predilection for differentiation into neuro-ectodermal lineage.
基金Supported by An extramural grant from the Department of Biotechnology,Government of India(BT/PR6519/MED/14/826/2005)sanctioned(to Nityanand S)
文摘AIM: To investigate whether fetal kidney stem cells(f KSC) ameliorate cisplatin induced acute renal failure(ARF) in rats and promote renal angiogenesis.METHODS: The f KSC were isolated from rat fetuses of gestation day 16 and expanded in vitro up to 3rd passage. They were characterized for the expression of mesenchymal and renal progenitor markers by flow cytometry and immunocytochemistry, respectively. The in vitro differentiation of f KSC towards epithelial lineage was evaluated by the treatment with specific induction medium and their angiogenic potential by matrigel induced tube formation assay. To study the effect of f KSC in ARF, f KSC labeled with PKH26 were infused in rats with cisplatin induced ARF and, the blood and renal tissues of the rats were collected at different time points. Blood biochemical parameters were studied to evaluate renal function. Renal tissues were evaluated for renal architecture, renal cell proliferation and angiogenesis by immunohistochemistry, renal cell apoptosis by terminal deoxynucleotidyl transferase nickend labeling assay and early expression of angiogenic molecules viz. vascular endothelial growth factor(VEGF), hypoxia-inducible factor(HIF)-1α and endothelial nitric oxide synthase(eN OS) by western blot.RESULTS: The fK SC expressed mesenchymal markers viz. CD29, CD44, CD73, CD90 and CD105 as well asrenal progenitor markers viz. Wt1, Pax2 and Six2. They exhibited a potential to form CD31 and Von Willebrand factor expressing capillary-like structures and could be differentiated into cytokeratin(CK)18 and CK19 positive epithelial cells. Administration of fK SC in rats with ARF as compared to administration of saline alone, resulted in a significant improvement in renal function and histology on day 3(2.33 ± 0.33 vs 3.50 ± 0.34, P < 0.05) and on day 7(0.83 ± 0.16 vs 2.00 ± 0.25, P < 0.05). The infused PKH26 labeled fK SC were observed to engraft in damaged renal tubules and showed increased proliferation and reduced apoptosis(P < 0.05) of renal cells. The kidneys of fK SC as compared to saline treated rats had a higher capillary density on day 3 [13.30 ± 1.54 vs 7.10 ± 1.29, capillaries/high-power fields(HPF), P < 0.05], and on day 7(21.10 ± 1.46 vs 15.00 ± 1.30, capillaries/HPF, P < 0.05). In addition, kidneys of fK SC treated rats had an upregulation of angiogenic proteins hypoxia-inducible factor-1α, VEGF and eN OS on day 3(P < 0.05).CONCLUSION: Our study shows that fK SC ameliorate cisplatin induced ARF in rats and promote renal angiogenesis, which may be an important therapeutic mechanism of these stem cells in the disease.
基金Supported by Department of Biotechnology,Government of India,BT/PR6519/MED/14/826/2005,to Dr.Nityanand S
文摘AIM:To study the expression of embryonal markers by fetal cardiac mesenchymal stem cells(fC-MSC)and their differentiation into cells of all the germ layers. METHODS:Ten independent cultures of rat fCMSC were set up from cells derived from individual or pooled fetal hearts and studies given below were carried out at passages 3,6,15 and 21.The phenotypic markers CD29,CD31,CD34,CD45,CD73,CD90, CD105,CD166 and HLA-DR were analyzed by flow cytometry.The expression of embryonal markers Oct-4, Nanog,Sox-2,SSEA-1,SSEA-3,SSEA-4,TRA-1-60 and TRA 1-81 were studied by immunocytochemistry.The fC-MSC treated with specific induction medium were evaluated for their differentiation into(1)adipocytes and osteocytes(mesodermal cells)by Oil Red O and Alizarin Red staining,respectively,as well as by expression of lipoprotein lipase,PPARγ2 genes in adipocytes and osteopontin and RUNX2 genes in osteocytes by reverse-transcription polymerase chain reaction(RT- PCR);(2)neuronal(ectodermal)cells by expression of neuronal Filament-160 and Glial Fibrillar Acidic Protein by RT-PCR and immunocytochemistry;and(3)hepa- tocytic(endodermal)cells by expression of albumin by RT-PCR and immunocytochemistry,glycogen deposits by Periodic Acid Schiff staining and excretion of urea into the culture supernatant. RESULTS:The fC-MSC expressed CD29,CD73,CD90, CD105,CD166 but lacked expression of CD31,CD34, CD45 and HLA-DR.They expressed embryonal markers,viz.Oct-4,Nanog,Sox-2,SSEA-1,SSEA-3,SSEA-4, TRA-1-81 but not TRA-1-60.On treatment with specific induction media,they differentiated into adipocytes and osteocytes,neuronal cells and hepatocytic cells. CONCLUSION:Our results together suggest that fCMSC are primitive stem cell types with a high degree of plasticity and,in addition to their suitability for cardiovascular regenerative therapy,they may have a wide spectrum of therapeutic applications in regenerative medicine.
