Human pluripotent stem cells (hPSC) differentiated to retinal pigment epithelial cells (RPE) provide a promising tool for cell replacement therapies of retinal degenerative diseases. The in vitro differentiation of hP...Human pluripotent stem cells (hPSC) differentiated to retinal pigment epithelial cells (RPE) provide a promising tool for cell replacement therapies of retinal degenerative diseases. The in vitro differentiation of hPSC-RPE is still poorly understood and current differentiation protocols rely on spontaneous differentiation on fibroblast feeder cells or as floating cell aggregates in suspension. The fibroblast feeder cells may have an inductive effect on the hPSC-RPE differentiation, providing variable signals mimicking the extraocular mesenchyme that directs the differentiation in vivo. The effect of the commonly used fibroblast feeder cells on the hPSCRPE differentiation was studied by comparing suspension differentiation in standard RPEbasic (no bFGF) medium to RPEbasic medium conditioned with mouse embryonic (mEF-CM) and human foreskin (hFF-CM) fibroblast feeder cells. The fibroblast secreted factors were found to enhance early hPSC-RPE differentiation. The onset of pigmentation was faster in the conditioned media (CM) compared to RPEbasic for both human embryonic (hESC) and induced pluripotent (iPSC) stem cells, with the first pigments appearing around two weeks of differentiation. After four weeks of differentiation, CM conditions consistently contained higher number of pigmented cell aggregates. The ratio of PAX6 and MITF positive cells was quantified to be clearly higher in the CM conditions, with mEFCM containing most positive cells. The mEF cells were found to secrete low levels of activin A growth factor that is known to regulate eye field differentiation. As RPEbasic was supplemented with corresponding, low level (10 ng/ml) of recombinant human activin A, a clear increase in the hPSC-RPE differentiation was achieved. Thus, inductive effect provided by feeder cells was at least partially driven by activin A and could be substituted with a low level of recombinant growth factor in contrasts to previously reported much higher concentrations.展开更多
Objective:To compare the expression levels of pluripotent genes among incomplete reprogrammed colonies and induced pluripotent stem cells(iPSCs),to explore the relationship between the expression of pluripotent genes ...Objective:To compare the expression levels of pluripotent genes among incomplete reprogrammed colonies and induced pluripotent stem cells(iPSCs),to explore the relationship between the expression of pluripotent genes and incomplete reprogramming.Methods:four genes(Oct4,Sox2,Klf4,C-Myc) were introduced into human foreskin fibroblasts(HFFs) by retroviruses.The HFFs were induced to reprogramming.Different forms of colonies were picked up,analyzed,and compared with iPSCs from different aspects,including the morphology of clones,alkaline phosphatase(AP) staining,immuno-fluorescence,and Q-PCR.Results:In the reprogramming process,different colonies were emerged,some of them exhibited typical human embryonic stem cell morphology(eg.,compact colonies,high nucleus-to-cytoplasm ratios,and prominent nucleoli).However,these colonies couldn't maintain these characters after passage.There was an intermediate state,named partially reprogramming.Through analysis and identification,AP staining results were weakly positive,compared with iPSC colonies.The immuno-fluorescence staining demonstrated these colonies just expressed pluripotent protein Oct4.Q-PCR indicated that the expression of exogenous transcription factors was inappropriate,either at a high level or at a low level.Most of the endogenous pluripotency genes were expressed at a low level.Conclusions:It may be one of the causes of incomplete reprogramming that the exogenous pluripotent gene is low-expressed or over-expressed,and successful reprogramming may depend on a specific stoichiometric balance of Oct4,Sox2,Klf4 and c-Myc.展开更多
文摘Human pluripotent stem cells (hPSC) differentiated to retinal pigment epithelial cells (RPE) provide a promising tool for cell replacement therapies of retinal degenerative diseases. The in vitro differentiation of hPSC-RPE is still poorly understood and current differentiation protocols rely on spontaneous differentiation on fibroblast feeder cells or as floating cell aggregates in suspension. The fibroblast feeder cells may have an inductive effect on the hPSC-RPE differentiation, providing variable signals mimicking the extraocular mesenchyme that directs the differentiation in vivo. The effect of the commonly used fibroblast feeder cells on the hPSCRPE differentiation was studied by comparing suspension differentiation in standard RPEbasic (no bFGF) medium to RPEbasic medium conditioned with mouse embryonic (mEF-CM) and human foreskin (hFF-CM) fibroblast feeder cells. The fibroblast secreted factors were found to enhance early hPSC-RPE differentiation. The onset of pigmentation was faster in the conditioned media (CM) compared to RPEbasic for both human embryonic (hESC) and induced pluripotent (iPSC) stem cells, with the first pigments appearing around two weeks of differentiation. After four weeks of differentiation, CM conditions consistently contained higher number of pigmented cell aggregates. The ratio of PAX6 and MITF positive cells was quantified to be clearly higher in the CM conditions, with mEFCM containing most positive cells. The mEF cells were found to secrete low levels of activin A growth factor that is known to regulate eye field differentiation. As RPEbasic was supplemented with corresponding, low level (10 ng/ml) of recombinant human activin A, a clear increase in the hPSC-RPE differentiation was achieved. Thus, inductive effect provided by feeder cells was at least partially driven by activin A and could be substituted with a low level of recombinant growth factor in contrasts to previously reported much higher concentrations.
基金The 973 program of Ministry of Science and Technology of China(No.2012CB966502)The Key International Cooperation Program of Science and Technology Department of Hainan Province(No.GJXM201106,KJHZ2014-11)MOST of China(No.2014DFA30180)
文摘Objective:To compare the expression levels of pluripotent genes among incomplete reprogrammed colonies and induced pluripotent stem cells(iPSCs),to explore the relationship between the expression of pluripotent genes and incomplete reprogramming.Methods:four genes(Oct4,Sox2,Klf4,C-Myc) were introduced into human foreskin fibroblasts(HFFs) by retroviruses.The HFFs were induced to reprogramming.Different forms of colonies were picked up,analyzed,and compared with iPSCs from different aspects,including the morphology of clones,alkaline phosphatase(AP) staining,immuno-fluorescence,and Q-PCR.Results:In the reprogramming process,different colonies were emerged,some of them exhibited typical human embryonic stem cell morphology(eg.,compact colonies,high nucleus-to-cytoplasm ratios,and prominent nucleoli).However,these colonies couldn't maintain these characters after passage.There was an intermediate state,named partially reprogramming.Through analysis and identification,AP staining results were weakly positive,compared with iPSC colonies.The immuno-fluorescence staining demonstrated these colonies just expressed pluripotent protein Oct4.Q-PCR indicated that the expression of exogenous transcription factors was inappropriate,either at a high level or at a low level.Most of the endogenous pluripotency genes were expressed at a low level.Conclusions:It may be one of the causes of incomplete reprogramming that the exogenous pluripotent gene is low-expressed or over-expressed,and successful reprogramming may depend on a specific stoichiometric balance of Oct4,Sox2,Klf4 and c-Myc.
