AIM: To development of an improved p38 MAPK inhibitor-based serum-free medium for embryoid body cardiomyocyte differentiation of human pluripotent stem cells. METHODS: Human embryonic stem cells (hESC) differentiated ...AIM: To development of an improved p38 MAPK inhibitor-based serum-free medium for embryoid body cardiomyocyte differentiation of human pluripotent stem cells. METHODS: Human embryonic stem cells (hESC) differentiated to cardiomyocytes (CM) using a p38 MAPK inhibitor (SB203580) based serum-free medium (SB media). Nutrient supplements known to increase cell viability were added to SB medium. The ability of these supplements to improve cardiomyogenesis was evaluated by measurements of cell viability, total cell count, and the expression of cardiac markers via flow cytometry. An improved medium containing Soy hydrolysate (HySoy) and bovine serum albumin (BSA) (SupSB media) was developed and tested on 2 additional cell lines (H1 and Siu-hiPSC). Characterization of the cardiomyocytes was done by immunohistochemistry, electrophysiology and quantitative real-time reverse transcriptionpolymerase chain reaction. RESULTS: hESC cell line, HES-3, differentiating in SB medium for 16 d resulted in a cardiomyocyte yield of 0.07 ± 0.03 CM/hESC. A new medium (SupSB media) was developed with the addition of HySoy and BSA to SB medium. This medium resulted in 2.6 fold increase in cardiomyocyte yield (0.21 ± 0.08 CM/hESC). The robustness of SupSB medium was further demonstrated using two additional pluripotent cell lines (H1, hESC and Siu1, hiPSC), showing a 15 and 9 fold increase in cardiomyocyte yield respectively. The age (passage number) of the pluripotent cells did not affect the cardiomyocyte yields. Embryoid body (EB) cardiomyocytes formed in SupSB medium expressed canonical cardiac markers (sarcomeric α-actinin, myosin heavy chain and troponin-T) and demonstrated all three major phenotypes: nodal-, atrial- and ventricular-like. Electrophysiological characteristics (maximum diastolic potentials and action potential durations) of cardiomyocytes derived from SB and SupSB media were similar. CONCLUSION: The nutrient supplementation (HySoy and BSA) leads to increase in cell viability, cell yield and cardiac marker expression during cardiomyocyte differentiation, translating to an overall increase in cardiomyocyte yield.展开更多
Several approaches have been used to encourage the differentiation of cardiomyocytes from human embryonic stem cells.However,the differentiation efficiency is low,and appropriate culture protocols are needed to produc...Several approaches have been used to encourage the differentiation of cardiomyocytes from human embryonic stem cells.However,the differentiation efficiency is low,and appropriate culture protocols are needed to produce adequate numbers of cardiomyocytes for therapeutic cell transplantation.This study investigated the effects of serum on cardiomyocyte differentiation in suspension culture medium during embryoid body(EB) formation by human embryonic stem cells.The addition of ascorbic acid,dimethylsulfoxide and 5-aza-2'-deoxycytidine during days 5-7 at the EB-forming stage resulted in an increase in the numbers of rhythmically contracting clusters of derived cardiomyocytes.Treatment with 0.1 mmol L-1 ascorbic acid alone,or more notably in combination with 10 μmol L-1 5-aza-2'-deoxycytidine,induced the formation of beating cells within EBs.Most of the beating clusters had spontaneous contraction rates similar to those found in human adults,and their contractile ac-tivity lasted for up to 194 days.展开更多
Previous research has shown that mouse embryonic stem (ES) cells can be induced to form neural cells in adherent monocultures.In this study,pluripotent stem (iPS) C5 cells derived from meningeal membranes were convert...Previous research has shown that mouse embryonic stem (ES) cells can be induced to form neural cells in adherent monocultures.In this study,pluripotent stem (iPS) C5 cells derived from meningeal membranes were converted successfully into neural-like cells using the same protocol generally used for ES cells.Meningeal-iPS C5 cells were induced to express neural markers Sox1,Sox3,Pax6,Nestin and Tuj1 and to reduce the expression of ES markers Oct4 and Nanog during neural differentiation,and can be differentiated into Pax6 and Nestin positive neural progenitors,and further into neuronal,astrocytic,and oligodendrocytic cells.In vitro differentiation of iPS cells into patient-specific neural cells could serve as a model to study mechanisms of genetic diseases and develop promising candidates for therapeutic applications in dysfunctional or aging neural tissues.Meningeal cells express a high level of the embryonic master regulator Sox2,allowing them to be reprogrammed into iPS cells more easily than other somatic cells.展开更多
基金Supported by Science Technology and Research (A*STAR)Hong Hong Kong Research Grant Council Collaborative Research Fund (HKU8/CRF/09)+1 种基金Theme-based Research Scheme (T12-705/11)Tse HT and Oh SKW contributed to financial support
文摘AIM: To development of an improved p38 MAPK inhibitor-based serum-free medium for embryoid body cardiomyocyte differentiation of human pluripotent stem cells. METHODS: Human embryonic stem cells (hESC) differentiated to cardiomyocytes (CM) using a p38 MAPK inhibitor (SB203580) based serum-free medium (SB media). Nutrient supplements known to increase cell viability were added to SB medium. The ability of these supplements to improve cardiomyogenesis was evaluated by measurements of cell viability, total cell count, and the expression of cardiac markers via flow cytometry. An improved medium containing Soy hydrolysate (HySoy) and bovine serum albumin (BSA) (SupSB media) was developed and tested on 2 additional cell lines (H1 and Siu-hiPSC). Characterization of the cardiomyocytes was done by immunohistochemistry, electrophysiology and quantitative real-time reverse transcriptionpolymerase chain reaction. RESULTS: hESC cell line, HES-3, differentiating in SB medium for 16 d resulted in a cardiomyocyte yield of 0.07 ± 0.03 CM/hESC. A new medium (SupSB media) was developed with the addition of HySoy and BSA to SB medium. This medium resulted in 2.6 fold increase in cardiomyocyte yield (0.21 ± 0.08 CM/hESC). The robustness of SupSB medium was further demonstrated using two additional pluripotent cell lines (H1, hESC and Siu1, hiPSC), showing a 15 and 9 fold increase in cardiomyocyte yield respectively. The age (passage number) of the pluripotent cells did not affect the cardiomyocyte yields. Embryoid body (EB) cardiomyocytes formed in SupSB medium expressed canonical cardiac markers (sarcomeric α-actinin, myosin heavy chain and troponin-T) and demonstrated all three major phenotypes: nodal-, atrial- and ventricular-like. Electrophysiological characteristics (maximum diastolic potentials and action potential durations) of cardiomyocytes derived from SB and SupSB media were similar. CONCLUSION: The nutrient supplementation (HySoy and BSA) leads to increase in cell viability, cell yield and cardiac marker expression during cardiomyocyte differentiation, translating to an overall increase in cardiomyocyte yield.
文摘Several approaches have been used to encourage the differentiation of cardiomyocytes from human embryonic stem cells.However,the differentiation efficiency is low,and appropriate culture protocols are needed to produce adequate numbers of cardiomyocytes for therapeutic cell transplantation.This study investigated the effects of serum on cardiomyocyte differentiation in suspension culture medium during embryoid body(EB) formation by human embryonic stem cells.The addition of ascorbic acid,dimethylsulfoxide and 5-aza-2'-deoxycytidine during days 5-7 at the EB-forming stage resulted in an increase in the numbers of rhythmically contracting clusters of derived cardiomyocytes.Treatment with 0.1 mmol L-1 ascorbic acid alone,or more notably in combination with 10 μmol L-1 5-aza-2'-deoxycytidine,induced the formation of beating cells within EBs.Most of the beating clusters had spontaneous contraction rates similar to those found in human adults,and their contractile ac-tivity lasted for up to 194 days.
基金supported by the National Basic Research Program of China (2007CB947804)the Joint Funds of the National Natural Science Foundation of China-Guangdong Province (U0972001/L02)+1 种基金the National Natural Science Foundation of China (30700213/C090204)the Natural Science Foundation of Guangdong Province (07007215)
文摘Previous research has shown that mouse embryonic stem (ES) cells can be induced to form neural cells in adherent monocultures.In this study,pluripotent stem (iPS) C5 cells derived from meningeal membranes were converted successfully into neural-like cells using the same protocol generally used for ES cells.Meningeal-iPS C5 cells were induced to express neural markers Sox1,Sox3,Pax6,Nestin and Tuj1 and to reduce the expression of ES markers Oct4 and Nanog during neural differentiation,and can be differentiated into Pax6 and Nestin positive neural progenitors,and further into neuronal,astrocytic,and oligodendrocytic cells.In vitro differentiation of iPS cells into patient-specific neural cells could serve as a model to study mechanisms of genetic diseases and develop promising candidates for therapeutic applications in dysfunctional or aging neural tissues.Meningeal cells express a high level of the embryonic master regulator Sox2,allowing them to be reprogrammed into iPS cells more easily than other somatic cells.
