Journal: Frontiers in Endocrinology

Article Title: Steroidogenic differentiation of human amniotic membrane-derived mesenchymal stem cells into a progesterone-/androgen-producing cell lineage by SF-1 and an estrogen-producing cell lineage by WT1−KTS

doi: 10.3389/fendo.2024.1410433

Figure Lengend Snippet: Lentivirus-mediated gene transfection into hAmMSCs. (A) The hAmMSCS 48 h after GFP transfection. Almost all hAmMSCs expressed GFP. (B) SF1 expression in hAmMSCs was confirmed via western blot analysis. (C) WT1+KTS and WT1−KTS mRNA expression in hAmMSCs was confirmed via quantitative PCR using isoform-specific primers. (D) Expression of WT1 in hAmMSCs was confirmed via western blot analysis. (E) WT1+KTS and WT1−KTS were mainly expressed in the cytoplasm and partially in the nucleus. (F) Neither the SF-1- nor WT1-transfected hAmMSCs showed any morphological changes compared with those transfected with GFP. Data are presented as the mean ± SD from three independent experiments. **, p <0.01, ***, p <0.001. hAmMSCs, human amniotic membrane-derived mesenchymal stem cells; GFP, green fluorescent protein; WT1, Wilms’ tumor 1; SF-1, steroidogenic factor 1; RT-PCR, reverse transcription polymerase chain reaction; SD, standard deviation.

Article Snippet: The transfected hAmMSCs were washed with PBS, and total RNA was extracted using SuperPrepII Cell Lysis & RT Kit for qPCR (Toyobo), followed by reverse transcription polymerase chain reaction (RT-PCR) using SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA) on the StepOnePlusTM RT-PCR system (Thermo Fisher Scientific).

Techniques: Transfection, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Membrane, Derivative Assay, Wilms Tumor Assay, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Polymerase Chain Reaction, Standard Deviation

Journal: Frontiers in Endocrinology

Article Title: Steroidogenic differentiation of human amniotic membrane-derived mesenchymal stem cells into a progesterone-/androgen-producing cell lineage by SF-1 and an estrogen-producing cell lineage by WT1−KTS

doi: 10.3389/fendo.2024.1410433

Figure Lengend Snippet: Lentivirus-mediated gene transfection into human fibroblasts. (A) SF-1, WT1+KTS, and WT1−KTS mRNA expression in human fibroblasts was confirmed via quantitative PCR. (B) SF-1 and WT1 expression in human fibroblasts was confirmed via western blot analysis. (C) Real-time RT-PCR analysis revealed that neither SF-1 nor WT1 transient expression affected the gene expression of steroidogenic factors in human fibroblasts. (D) SF-1 did not enhance pregnenolone and progesterone production by human fibroblasts. Data are presented as the mean ± SD from three independent experiments. *, p<0.05, **, p<0.01.

Article Snippet: The transfected hAmMSCs were washed with PBS, and total RNA was extracted using SuperPrepII Cell Lysis & RT Kit for qPCR (Toyobo), followed by reverse transcription polymerase chain reaction (RT-PCR) using SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA) on the StepOnePlusTM RT-PCR system (Thermo Fisher Scientific).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Gene Expression

Journal: Frontiers in Endocrinology

Article Title: Steroidogenic differentiation of human amniotic membrane-derived mesenchymal stem cells into a progesterone-/androgen-producing cell lineage by SF-1 and an estrogen-producing cell lineage by WT1−KTS

doi: 10.3389/fendo.2024.1410433

Figure Lengend Snippet: Effects of SF-1 and WT1 on progesterone-producing cell lineage. GFP, SF-1, WT1+KTS, and WT1−KTS were transiently expressed in hAmMSCs via lentivirus-mediated gene transfection and analyzed for progesterone-producing capacity. (A) The expression of steroidogenic genes involved in progesterone synthesis in hAmMSCs was measured via quantitative PCR. SF-1 considerably increased the expression of StAR , CYP11A1 , and HSD3B2 in hAmMSCs. In contrast, WT1+KTS and WT1−KTS did not alter the mRNA expression of progesterone-producing factors. (B) SF-1 significantly enhanced pregnenolone and progesterone production by the hAmMSCs. (C) No difference was observed in the mRNA levels of progesterone-producing factors when SF-1 and WT1−KTS were co-expressed compared with those when SF-1 was expressed alone. Data are presented as the mean ± SD from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001. hAmMSCs, human amniotic membrane-derived mesenchymal stem cells; GFP, green fluorescent protein; WT1, Wilms’ tumor 1; SF-1, steroidogenic factor 1; PCR, polymerase chain reaction; SD, standard deviation; KTS, lysine, threonine, and serine.

Article Snippet: The transfected hAmMSCs were washed with PBS, and total RNA was extracted using SuperPrepII Cell Lysis & RT Kit for qPCR (Toyobo), followed by reverse transcription polymerase chain reaction (RT-PCR) using SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA) on the StepOnePlusTM RT-PCR system (Thermo Fisher Scientific).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Membrane, Derivative Assay, Wilms Tumor Assay, Polymerase Chain Reaction, Standard Deviation