Background:Currently,direct conversion from somatic cells to neurons requires virus-mediated delivery of at least one transcriptional factor or a combination of several small-molecule compounds.Delivery of transcripti...Background:Currently,direct conversion from somatic cells to neurons requires virus-mediated delivery of at least one transcriptional factor or a combination of several small-molecule compounds.Delivery of transcriptional factors may affect genome stability,while small-molecule compounds may require more evaluations when applied in vivo.Thus,a defined medium with only conventional growth factors or additives for cell culture is desirable for inducing neuronal trans-differentiation.Results:Here,we report that a defined medium(5C)consisting of basic fibroblast growth factor(bFGF),N2 supplement,leukemia inhibitory factor,vitamin C(Vc),andβ-mercaptoethanol(βMe)induces the direct conversion of somatic cells to cells with neuronal characteristics.Application of 5C medium converted mouse embryonic fibroblasts(MEFs)into TuJ+neuronal-like cells,which were capable of survival after being transplanted into the mouse brain.The same 5C medium could convert primary rat astrocytes into neuronal-like cells with mature electrophysiology characteristics in vitro and facilitated the recovery of brain injury,possibly by inducing similar conversions,when infused into the mouse brain in vivo.Crucially,5C medium could also induce neuronal characteristics in several human cell types.Conclusions:In summary,this 5C medium not only provides a means to derive cells with neuronal characteristics without viral transfection in vitro but might also be useful to produce neurons in vivo for neurodegenerative disease treatment.展开更多
Mesenchymal stem cells(MSCs)have been intensively investigated for their therapeutic potentials.In addition to their ability to differentiate into multiple cell types,they can orchestrate immune responses and modulate...Mesenchymal stem cells(MSCs)have been intensively investigated for their therapeutic potentials.In addition to their ability to differentiate into multiple cell types,they can orchestrate immune responses and modulate tissue microenvironments,resulting in tissue regeneration1,2.However,therapeutic inconsistency is one of the major obstacles to clinical application.This therapeutic inconsistency may result from the population heterogeneity,tissue origin,passage number or in vitro expansion system of MSCs.Human platelet lysate(PL)has been demonstrated to support human MSCs’expansion and used as the standard culture medium for MSCs’clinical applications3.However,batch variation in human PL preparations may affect the phenotype and function of MSCs.Indeed,it has been noted that human PL products from different commercial suppliers show different therapeutic efficacies in treating a mouse model of systemic lupus erythematosus(SLE)4.Furthermore,previous investigations have shown that PL upregulates the expression of the costimulatory factors CD40 and CD865,6,which may induce immunorecognition and rejection by the host immune system.展开更多
Background Keratinoyte serum-free medium （K-SFM） is a defined medium used to support the growth of primary keratinocytes and embryonic stem cell. The aim of this research was to optimize enrichment of breast cancer ...Background Keratinoyte serum-free medium （K-SFM） is a defined medium used to support the growth of primary keratinocytes and embryonic stem cell. The aim of this research was to optimize enrichment of breast cancer stem cells （CSCs） using K-SFM. Methods＇ A K-SFM was used to enrich CSCs from two breast cancer cell lines and a primary culture of breast cancer. RPMI-1640 supplemented with 10% fetal calf serum （FCS） was used as a control. CSCs were identified with flow cytometry using CD44＋/CD24-as molecular markers. The expression of a variety of CSC markers （Oct-4, ABCG2, Nanog, N-cadherin, and E-cadherin） was analyzed with real-time PCR. Results Much higher percentage of CSCs was achieved with K-SFM： 17.3% for MCF-7 cells, 17.4% for SKBR-3, and 20.0% for primary breast cancer culture. Less than 1% CSC was achieved using RPMI-1640 supplemented with 10% FCS. In comparison to the CSCs obtained with RPMI-1640, CSCs in the K-SFM expressed higher levels of Oct-4, ABCG2, Nanog and N-cadherin, and lower level of E-cadherin. Conclusion K-SFM is an optimal culture medium to maintain and to enrich breast CSCs.展开更多
Wholly defined ex vivo expansion conditions for biliary tree stem cell(BTSC)organoids were established,consisting of a defined proliferative medium(DPM)used in combination with soft hyaluronan hydrogels.The DPM consis...Wholly defined ex vivo expansion conditions for biliary tree stem cell(BTSC)organoids were established,consisting of a defined proliferative medium(DPM)used in combination with soft hyaluronan hydrogels.The DPM consisted of commercially available Kubota's Medium(KM),to which a set of small molecules,particular paracrine signals,and heparan sulfate(HS)were added.The small molecules used were DNA methyltransferase inhibitor(RG108),TGF-βType I receptor inhibitor(A83-01),adenylate cyclase activator(Forskolin),and L-type Ca2+channel agonist(Bay K8644).A key paracrine signal proved to be R-spondin 1(RSPO1),a secreted protein that activates Wnts.Soluble hyaluronans,0.05%sodium hyaluronate,were used with DPM to expand monolayer cultures.Expansion of organoids was achieved by using DPM in combination with embedding organoids in Matrigel that was replaced with a defined thiol-hyaluronan triggered with PEGDA to form a hydrogel with a rheology[G*]of less than 100 Pa.The combination is called the BTSC-Expansion-Glycogel-System(BEX-gel system)for expanding BTSCs as a monolayer or as organoids.The BTSC organoids were expanded more than 3000-fold ex vivo in the BEX-gel system within 70 days while maintaining phenotypic traits indicative of stem/progenitors.Stem-cell-patch grafting of expanded BTSC organoids was performed on the livers of Fah-/-mice with tyrosinemia and resulted in the rescue of the mice and restoration of their normal liver functions.The BEX-gel system for BTSC organoid expansion provides a strategy to generate sufficient numbers of organoids for the therapeutic treatments of liver diseases.展开更多
基金This work was supported by“Strategic Priority Research Program of the Chinese Academy of Sciences(XDA01020302)”the“National Natural Science Foundation of China(31422032,31421004)”+4 种基金the“Guangdong Natural Science Foundation(2014A030308002)”the“Guangdong Science and Technology Planning Project(2013B010404040),”the“Guangzhou Health Care Collaborative Innovation Program(201508020250)”We sincerely thank Dr.Chen Ling(mouse macrophages)Dr.Wang Lihui(HFFs)in GIBH,and Dr.Peng Xiang(BM-hMSCs)in Sun Yat-Sen University for providing cells。
文摘Background:Currently,direct conversion from somatic cells to neurons requires virus-mediated delivery of at least one transcriptional factor or a combination of several small-molecule compounds.Delivery of transcriptional factors may affect genome stability,while small-molecule compounds may require more evaluations when applied in vivo.Thus,a defined medium with only conventional growth factors or additives for cell culture is desirable for inducing neuronal trans-differentiation.Results:Here,we report that a defined medium(5C)consisting of basic fibroblast growth factor(bFGF),N2 supplement,leukemia inhibitory factor,vitamin C(Vc),andβ-mercaptoethanol(βMe)induces the direct conversion of somatic cells to cells with neuronal characteristics.Application of 5C medium converted mouse embryonic fibroblasts(MEFs)into TuJ+neuronal-like cells,which were capable of survival after being transplanted into the mouse brain.The same 5C medium could convert primary rat astrocytes into neuronal-like cells with mature electrophysiology characteristics in vitro and facilitated the recovery of brain injury,possibly by inducing similar conversions,when infused into the mouse brain in vivo.Crucially,5C medium could also induce neuronal characteristics in several human cell types.Conclusions:In summary,this 5C medium not only provides a means to derive cells with neuronal characteristics without viral transfection in vitro but might also be useful to produce neurons in vivo for neurodegenerative disease treatment.
基金supported by the Natural Science Foundation of Shenzhen(JCYJ20180305163407913 and KQJSCX20180328093434771)Guangdong Provincial Science and Technology Program(No.2019B030301009)the Medical Foundation of Guangdong(A2018308).
文摘Mesenchymal stem cells(MSCs)have been intensively investigated for their therapeutic potentials.In addition to their ability to differentiate into multiple cell types,they can orchestrate immune responses and modulate tissue microenvironments,resulting in tissue regeneration1,2.However,therapeutic inconsistency is one of the major obstacles to clinical application.This therapeutic inconsistency may result from the population heterogeneity,tissue origin,passage number or in vitro expansion system of MSCs.Human platelet lysate(PL)has been demonstrated to support human MSCs’expansion and used as the standard culture medium for MSCs’clinical applications3.However,batch variation in human PL preparations may affect the phenotype and function of MSCs.Indeed,it has been noted that human PL products from different commercial suppliers show different therapeutic efficacies in treating a mouse model of systemic lupus erythematosus(SLE)4.Furthermore,previous investigations have shown that PL upregulates the expression of the costimulatory factors CD40 and CD865,6,which may induce immunorecognition and rejection by the host immune system.
基金This work was supported by a grant from the National Natural Science Foundation of China （No. 81001102）.
文摘Background Keratinoyte serum-free medium （K-SFM） is a defined medium used to support the growth of primary keratinocytes and embryonic stem cell. The aim of this research was to optimize enrichment of breast cancer stem cells （CSCs） using K-SFM. Methods＇ A K-SFM was used to enrich CSCs from two breast cancer cell lines and a primary culture of breast cancer. RPMI-1640 supplemented with 10% fetal calf serum （FCS） was used as a control. CSCs were identified with flow cytometry using CD44＋/CD24-as molecular markers. The expression of a variety of CSC markers （Oct-4, ABCG2, Nanog, N-cadherin, and E-cadherin） was analyzed with real-time PCR. Results Much higher percentage of CSCs was achieved with K-SFM： 17.3% for MCF-7 cells, 17.4% for SKBR-3, and 20.0% for primary breast cancer culture. Less than 1% CSC was achieved using RPMI-1640 supplemented with 10% FCS. In comparison to the CSCs obtained with RPMI-1640, CSCs in the K-SFM expressed higher levels of Oct-4, ABCG2, Nanog and N-cadherin, and lower level of E-cadherin. Conclusion K-SFM is an optimal culture medium to maintain and to enrich breast CSCs.
基金Major Program of National Key Research and Development Project(2020YFA0112600,2019YFA0801502)National Natural Science Foundation of China(82173019,82270638,8220374,82300718)+1 种基金Project of Shanghai Science and Technology Commission(22ZR1451100,22Y11908500)Peak Disciplines(Type IV)of Institutions of Higher Learning in Shanghai,and Shanghai Engineering Research Center of Stem Cells Translational Medicine(20DZ2255100).
文摘Wholly defined ex vivo expansion conditions for biliary tree stem cell(BTSC)organoids were established,consisting of a defined proliferative medium(DPM)used in combination with soft hyaluronan hydrogels.The DPM consisted of commercially available Kubota's Medium(KM),to which a set of small molecules,particular paracrine signals,and heparan sulfate(HS)were added.The small molecules used were DNA methyltransferase inhibitor(RG108),TGF-βType I receptor inhibitor(A83-01),adenylate cyclase activator(Forskolin),and L-type Ca2+channel agonist(Bay K8644).A key paracrine signal proved to be R-spondin 1(RSPO1),a secreted protein that activates Wnts.Soluble hyaluronans,0.05%sodium hyaluronate,were used with DPM to expand monolayer cultures.Expansion of organoids was achieved by using DPM in combination with embedding organoids in Matrigel that was replaced with a defined thiol-hyaluronan triggered with PEGDA to form a hydrogel with a rheology[G*]of less than 100 Pa.The combination is called the BTSC-Expansion-Glycogel-System(BEX-gel system)for expanding BTSCs as a monolayer or as organoids.The BTSC organoids were expanded more than 3000-fold ex vivo in the BEX-gel system within 70 days while maintaining phenotypic traits indicative of stem/progenitors.Stem-cell-patch grafting of expanded BTSC organoids was performed on the livers of Fah-/-mice with tyrosinemia and resulted in the rescue of the mice and restoration of their normal liver functions.The BEX-gel system for BTSC organoid expansion provides a strategy to generate sufficient numbers of organoids for the therapeutic treatments of liver diseases.
