Long-term maintenance of chicken primordial germ cells (PGCs) in vitro has tremendous potential for transgenic chicken production. Feeder cells are essential for the establishment and culture of chicken PGCs in vitro....Long-term maintenance of chicken primordial germ cells (PGCs) in vitro has tremendous potential for transgenic chicken production. Feeder cells are essential for the establishment and culture of chicken PGCs in vitro. Buffalo rat liver (BRL) cells are the most commonly used feeder cells for PGCs culture;however, this feeder layers from other animal species usually cause immunogenic contaminations, compromising the potential of PGCs in applications. Therefore, we tested chicken source mensenchymal stem cell (MSCs) derived from bone marrow as feeder cells to further improve PGC culture conditions. MSCs derived from chicken bone marrow have a powerful capacity to proliferate and secrete cytokines. We found chicken primordial germ cells derived from circulating blood (cPGCs) and gonads (gPGCs) can be maintained and proliferated with MSCs feeder layer cells. PGCs co-cultured on MSCs feeder retained their pluripotency, expressed PGCs specific genes and stemness markers, and maintained undifferentiated state. Our study indicated that the xeno-free MSCs-feeders culture system is a good candidate for growth and expansion of PGCs as the stepping stone for transgenic chicken research.展开更多
Background Transplantation of mensenchymal stem cells （MSCs） has been proposed as a promising way for tissue engineering. However, the application of MSCs for transplantation will undergo apoptosis due to the extrem...Background Transplantation of mensenchymal stem cells （MSCs） has been proposed as a promising way for tissue engineering. However, the application of MSCs for transplantation will undergo apoptosis due to the extremely harsh microenvironment such as excessive inflammation. Apigenin （API） has been reported to protect cells against inflammatory damage and cell death by exhibiting anti-inflammatory and anti-oxidative capacity. Here we investigated the modulatory effects of API in lipopolysaccharide （LPS）-mediated inflammation and apoptosis of MSCs, and further defined the underlying mechanism. Methods Effects of different concentrations of API （0, 5, 10, 20, 40 and 80 IJmol/L） for 24 hours, and LPS （0, 0.5 and 5.0 pg/ml） for 6 hours and 24 hours on MSCs viability were assayed by MTT. Based on this, MSCs were pretreated with different concentrations of API （0-40μmol/L） at the indicated times （6, 12 and 24 hours） followed by exposure to 5μg/ml LPS for 24 hours. MTT, phase-contrast microscopy, annexinV/propidium iodide （PI） double stain flow cytometry （FCM） and Hoechst staining were applied to explore the effects of API on MSCs induced by 5 μg/ml LPS for 24 hours. In addition, reverse-transcription polymerase chain reaction （RT-PCR） was applied to detect the mRNA expression of pro-inflammatory factors including cyclooxygenase-2 （COX-2）, inducible nitric oxide synthase （iNOS）, nuclear factor-kappa B （NF-KB）, pro-apoptotic gene caspase-3, Bad, and anti-apoptotic gene Bcl-2. Moreover, AutoDock software was used to imitate the docking score of API and vitamin D receptor （VDR）. In parallel, Western blotting and RT-PCR were used to investigate protein and mRNA expression of VDR. Results MSCs stimulated with LPS 5 IJg/ml for 24 hours was used as a model of apoptosis induced by over inflammatory stimulus. API （0-40μmol/L） had non-toxic effect on MSCs; however, it could decrease mRNA expression of COX-2, iNOS and NF-KB at different time points in MSCs induced by LPS, except for API at the concentration of 5 μmol/L Results from phase-contrast microscopy, MTT, Hoechst staining and AnnexinV/PI double stain FCM demonstrated that with the increasing concentrations of API and extension of administrating time, significant morphological changes of MSCs occurred, viability of cells was strongly inhibited, and meanwhile, apoptosis of LPS-administrated MSCs was exacerbated, compared with LPS individual group. In addition, API promoted caspase-3, Bad mRNA expression and inhibited Bcl-2 mRNA expression in a time-dePendent and concentration-dependent manner. Further study found that pro-apoptosis effect of API was related to suppress VDR expression. Conclusions API could inhibit the expression of inducible inflammatory factors, therefore exert the strong anti-inflammatory function. However, API could not protect MSC apoptosis induced by LPS but amplified the apoptosis, The apoptosis is related to Bad/Bcl-2 increasing and caspase-3 activation, which is mediated through suppressing VDR expression.展开更多
文摘Long-term maintenance of chicken primordial germ cells (PGCs) in vitro has tremendous potential for transgenic chicken production. Feeder cells are essential for the establishment and culture of chicken PGCs in vitro. Buffalo rat liver (BRL) cells are the most commonly used feeder cells for PGCs culture;however, this feeder layers from other animal species usually cause immunogenic contaminations, compromising the potential of PGCs in applications. Therefore, we tested chicken source mensenchymal stem cell (MSCs) derived from bone marrow as feeder cells to further improve PGC culture conditions. MSCs derived from chicken bone marrow have a powerful capacity to proliferate and secrete cytokines. We found chicken primordial germ cells derived from circulating blood (cPGCs) and gonads (gPGCs) can be maintained and proliferated with MSCs feeder layer cells. PGCs co-cultured on MSCs feeder retained their pluripotency, expressed PGCs specific genes and stemness markers, and maintained undifferentiated state. Our study indicated that the xeno-free MSCs-feeders culture system is a good candidate for growth and expansion of PGCs as the stepping stone for transgenic chicken research.
文摘Background Transplantation of mensenchymal stem cells （MSCs） has been proposed as a promising way for tissue engineering. However, the application of MSCs for transplantation will undergo apoptosis due to the extremely harsh microenvironment such as excessive inflammation. Apigenin （API） has been reported to protect cells against inflammatory damage and cell death by exhibiting anti-inflammatory and anti-oxidative capacity. Here we investigated the modulatory effects of API in lipopolysaccharide （LPS）-mediated inflammation and apoptosis of MSCs, and further defined the underlying mechanism. Methods Effects of different concentrations of API （0, 5, 10, 20, 40 and 80 IJmol/L） for 24 hours, and LPS （0, 0.5 and 5.0 pg/ml） for 6 hours and 24 hours on MSCs viability were assayed by MTT. Based on this, MSCs were pretreated with different concentrations of API （0-40μmol/L） at the indicated times （6, 12 and 24 hours） followed by exposure to 5μg/ml LPS for 24 hours. MTT, phase-contrast microscopy, annexinV/propidium iodide （PI） double stain flow cytometry （FCM） and Hoechst staining were applied to explore the effects of API on MSCs induced by 5 μg/ml LPS for 24 hours. In addition, reverse-transcription polymerase chain reaction （RT-PCR） was applied to detect the mRNA expression of pro-inflammatory factors including cyclooxygenase-2 （COX-2）, inducible nitric oxide synthase （iNOS）, nuclear factor-kappa B （NF-KB）, pro-apoptotic gene caspase-3, Bad, and anti-apoptotic gene Bcl-2. Moreover, AutoDock software was used to imitate the docking score of API and vitamin D receptor （VDR）. In parallel, Western blotting and RT-PCR were used to investigate protein and mRNA expression of VDR. Results MSCs stimulated with LPS 5 IJg/ml for 24 hours was used as a model of apoptosis induced by over inflammatory stimulus. API （0-40μmol/L） had non-toxic effect on MSCs; however, it could decrease mRNA expression of COX-2, iNOS and NF-KB at different time points in MSCs induced by LPS, except for API at the concentration of 5 μmol/L Results from phase-contrast microscopy, MTT, Hoechst staining and AnnexinV/PI double stain FCM demonstrated that with the increasing concentrations of API and extension of administrating time, significant morphological changes of MSCs occurred, viability of cells was strongly inhibited, and meanwhile, apoptosis of LPS-administrated MSCs was exacerbated, compared with LPS individual group. In addition, API promoted caspase-3, Bad mRNA expression and inhibited Bcl-2 mRNA expression in a time-dePendent and concentration-dependent manner. Further study found that pro-apoptosis effect of API was related to suppress VDR expression. Conclusions API could inhibit the expression of inducible inflammatory factors, therefore exert the strong anti-inflammatory function. However, API could not protect MSC apoptosis induced by LPS but amplified the apoptosis, The apoptosis is related to Bad/Bcl-2 increasing and caspase-3 activation, which is mediated through suppressing VDR expression.
