A rapid, simple,reliable method is described for assaying androgen receptor (AR) in dispersed, whole,cultured human genital skin fibroblasts (GSF) with a synthetic androgen, H-methyltrienolone (3H- R1881). Receptors f...A rapid, simple,reliable method is described for assaying androgen receptor (AR) in dispersed, whole,cultured human genital skin fibroblasts (GSF) with a synthetic androgen, H-methyltrienolone (3H- R1881). Receptors for androgen in GSF exhibit high affinity (Kd=3.01 0.1 nmol/L), low binding capacity and androgen sped/icity.The content of AR in cultured GSF from 40 normal men varying in age from 1.5-60 years was also investigated by this assay.Scatchard analysis and single plot revealed the presence of 4500-8500 binding sites per cell, mean number of AR in GSF of these men is 628811082 binding sites/cell.NO signijicant difference was observed in the content of AR in different age groups. This result showed that the content of AR in these cells did not change with age.展开更多
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
文摘A rapid, simple,reliable method is described for assaying androgen receptor (AR) in dispersed, whole,cultured human genital skin fibroblasts (GSF) with a synthetic androgen, H-methyltrienolone (3H- R1881). Receptors for androgen in GSF exhibit high affinity (Kd=3.01 0.1 nmol/L), low binding capacity and androgen sped/icity.The content of AR in cultured GSF from 40 normal men varying in age from 1.5-60 years was also investigated by this assay.Scatchard analysis and single plot revealed the presence of 4500-8500 binding sites per cell, mean number of AR in GSF of these men is 628811082 binding sites/cell.NO signijicant difference was observed in the content of AR in different age groups. This result showed that the content of AR in these cells did not change with age.
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.
