msc marker validation kit  (R&D Systems)

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: Isolation and characterization of MSCs from human amniotic membrane. (A) Harvesting of human amniotic membrane, a thin translucent layer attached to the chorionic membrane. (B) Phase contrast images taken 24 h after isolation. Culture cells display a fibroblast-like morphology and spindle shape similar to that displayed by human MSCs. Scale bar, 50 μm. (C) Fluorescence-activated cell sorting analysis. hAmMSCs were separated using antibodies for positive marker genes (CD90, CD73, and CD105) and negative marker genes (CD45, CD34, CD11b, CD79A, and HLA-DR). Almost all cells were CD90-positive, 80% were CD73-positive, and only 4% were CD105-positive. (D) The expression of several representative genes involved in gonadal development in hAmMSCs. SRY expression was only observed in hAmMSCs derived from male babies. Data are presented as the mean ± SD from three independent experiments. N.D., not detectable. MSCs, mesenchymal stem cells; hAmMSCs, human amniotic membrane-derived mesenchymal stem cells; SSC, side-scattered; FSC, forward scattered; SRY, sex-determining region Y.

Article Snippet: The cell suspensions were sorted using the Human Mesenchymal Stem Cell Validation Flow Kit (R&D Systems, Abingdon, UK) and stained with the following antibodies: APC-conjugated CD90 antibody, FITC-conjugated CD73 antibody, PerCP-conjugated CD105 antibody, and phycoerythrin-conjugated negative markers (CD45, CD34, CD11b, CD79A, and HLA-DR).

Techniques: Isolation, Membrane, Fluorescence, FACS, Marker, Expressing, Derivative Assay

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: Differentiation capacity of hAmMSCs. (A) Adipogenic differentiation was evidenced by Oil Red O staining. Lipid vacuoles were formed in the hAmMSCs. (B) Osteogenic differentiation was evidenced by Alizarin Red S staining. Mineralized matrix was formed in the hAmMSCs. (C) The hAmMSCs cultured with chondrogenic differentiation medium formed pellets. Note that the cells did not form pellets in the control medium (RPMI 1640 plus 10% Fetal Calf Serum). Chondrogenic differentiation was evidenced by Alcian blue staining. Scale bar, 100 µm. hAmMSCs, human amniotic membrane-derived mesenchymal stem cells.

Article Snippet: The cell suspensions were sorted using the Human Mesenchymal Stem Cell Validation Flow Kit (R&D Systems, Abingdon, UK) and stained with the following antibodies: APC-conjugated CD90 antibody, FITC-conjugated CD73 antibody, PerCP-conjugated CD105 antibody, and phycoerythrin-conjugated negative markers (CD45, CD34, CD11b, CD79A, and HLA-DR).

Techniques: Staining, Cell Culture, Control, Membrane, Derivative Assay

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 cell suspensions were sorted using the Human Mesenchymal Stem Cell Validation Flow Kit (R&D Systems, Abingdon, UK) and stained with the following antibodies: APC-conjugated CD90 antibody, FITC-conjugated CD73 antibody, PerCP-conjugated CD105 antibody, and phycoerythrin-conjugated negative markers (CD45, CD34, CD11b, CD79A, and HLA-DR).

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: 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 cell suspensions were sorted using the Human Mesenchymal Stem Cell Validation Flow Kit (R&D Systems, Abingdon, UK) and stained with the following antibodies: APC-conjugated CD90 antibody, FITC-conjugated CD73 antibody, PerCP-conjugated CD105 antibody, and phycoerythrin-conjugated negative markers (CD45, CD34, CD11b, CD79A, and HLA-DR).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Membrane, Derivative Assay, Wilms Tumor Assay, 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: Effects of SF-1 and WT1 on adrenal steroidogenic cell lineage. (A) SF-1 significantly increased the mRNA expression of NRDB1 ( DAX-1 ) but not of OSR2 , ACTHR , and AR , in hAmMSCs. (B) SF-1, WT1+KTS, and WT1−KTS did not change the mRNA levels of CYP21A2 , CYP11B1 , and CYP11B2 . (C) Co-expression of SF-1 and WT1−KTS did not alter the mRNA levels of adrenal steroidogenic enzymes in hAmMSCs. Data are presented as the mean ± SD from three independent experiments. *, p < 0.05. hAmMSCs, human amniotic membrane-derived mesenchymal stem cells; WT1, Wilms’ tumor 1; SF-1, steroidogenic factor 1; SD, standard deviation; KTS, lysine, threonine, and serine.

Article Snippet: The cell suspensions were sorted using the Human Mesenchymal Stem Cell Validation Flow Kit (R&D Systems, Abingdon, UK) and stained with the following antibodies: APC-conjugated CD90 antibody, FITC-conjugated CD73 antibody, PerCP-conjugated CD105 antibody, and phycoerythrin-conjugated negative markers (CD45, CD34, CD11b, CD79A, and HLA-DR).

Techniques: Expressing, Membrane, Derivative Assay, Wilms Tumor Assay, 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: Effects of SF-1 and WT1 on androgen-producing cell lineage. (A) SF-1 did not change the mRNA levels of NR2F2 , LIFR , PDGFRα , and LHCGR . (B) SF-1 significantly increased the mRNA expression of CYP17A1 in hAmMSCs. In contrast, WT1+KTS and WT1−KTS did not alter the mRNA expression of androgen-producing enzymes. (C) SF-1 enhanced DHEA and androstenedione production, but not testosterone production, by hAmMSCs. (D) No difference was observed in the mRNA levels of androgen-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; N.D., not detectable. hAmMSCs, human amniotic membrane-derived mesenchymal stem cells; WT1, Wilms’ tumor 1; SF-1, steroidogenic factor 1; DHEA, dehydroepiandrosterone; SD, standard deviation; KTS, lysine, threonine, and serine.

Article Snippet: The cell suspensions were sorted using the Human Mesenchymal Stem Cell Validation Flow Kit (R&D Systems, Abingdon, UK) and stained with the following antibodies: APC-conjugated CD90 antibody, FITC-conjugated CD73 antibody, PerCP-conjugated CD105 antibody, and phycoerythrin-conjugated negative markers (CD45, CD34, CD11b, CD79A, and HLA-DR).

Techniques: Expressing, Membrane, Derivative Assay, Wilms Tumor Assay, 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: Effects of SF-1 and WT1 on estrogen-producing cell lineage. (A) WT1−KTS significantly increased the mRNA expression of ALDH1A2 , AMHR2, LHX9, GPX3 , and NR5A2 in hAmMSCs. (B) WT1−KTS, but not WT1+KTS or SF-1, significantly increased the mRNA expression of CYP19A1 in hAmMSCs. SF-1 increased the mRNA level of HSD17B1 . (C) Immunocytochemical analysis confirmed that WT1−KTS increased aromatase expression in hAmMSCs. (D) WT1−KTS and SF-1 did not increase estrogen production by hAmMSCs even in the presence of testosterone. (E) Co-expression of SF-1 and WT1−KTS also did not alter the mRNA levels of estrogen-producing enzymes in hAmMSCs. Data are presented as the mean ± SD from three independent experiments. *, p < 0.05; **, p < 0.01; ****, p < 0.0001; N.S., not significant. hAmMSCs, human amniotic membrane-derived mesenchymal stem cells; WT1, Wilms’ tumor 1; SF-1, steroidogenic factor 1; KTS, lysine, threonine, and serine.

Article Snippet: The cell suspensions were sorted using the Human Mesenchymal Stem Cell Validation Flow Kit (R&D Systems, Abingdon, UK) and stained with the following antibodies: APC-conjugated CD90 antibody, FITC-conjugated CD73 antibody, PerCP-conjugated CD105 antibody, and phycoerythrin-conjugated negative markers (CD45, CD34, CD11b, CD79A, and HLA-DR).

Techniques: Expressing, Membrane, Derivative Assay, Wilms Tumor Assay

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: Activation of CYP19A1 PII promoter by WT1–KTS. (A) Tissue-specific promoters of human CYP19A1. The mRNA expression of I.1 (placenta), I.f (brain), and PII (ovary) transcripts was observed in hAmMSCs. Transient expression of WT1−KTS significantly increased the mRNA level of the ovary-specific PII promoter in hAmMSCs. (B) Ovary-specific PII promoter of human CYP19A1. Luciferase assay revealed that WT1−KTS activated the CYP19A1 PII promoter. Data are presented as the mean ± SD from three independent experiments. *, p <0.05. hAmMSCs, human amniotic membrane-derived mesenchymal stem cells; WT1, Wilms’ tumor 1; KTS, lysine, threonine, and serine.

Article Snippet: The cell suspensions were sorted using the Human Mesenchymal Stem Cell Validation Flow Kit (R&D Systems, Abingdon, UK) and stained with the following antibodies: APC-conjugated CD90 antibody, FITC-conjugated CD73 antibody, PerCP-conjugated CD105 antibody, and phycoerythrin-conjugated negative markers (CD45, CD34, CD11b, CD79A, and HLA-DR).

Techniques: Activation Assay, Expressing, Luciferase, Membrane, Derivative Assay, Wilms Tumor Assay

Journal: International Journal of Bioprinting

Article Title: Graphene Oxide-Embedded Extracellular Matrix-Derived Hydrogel as a Multiresponsive Platform for 3D Bioprinting Applications

doi: 10.18063/ijb.v7i3.353

Figure Lengend Snippet: SISMA-graphene oxide (GO) bioprinting. Adequate filament formation during extrusion aided by a 3D bioprinter ( A ) and shape fidelity upon deposition ( B ) was observed for SISMA-GO composite hydrogels, highlighting their suitability for bioprinting applications. ( C ) Flow sweep experiments of SISMA-GO and SISMA hydrogels depict an appropriate shear-thinning behavior, with no observable difference in the presence of GO. ( D ) Cell viability of human adipose tissue-derived mesenchymal stem cells (hAD-MSC) 2 h, 3 days, and 7 days after bioprinting indicates high biocompatibility with SISMA-GO hydrogels ( n = 4). ( E ) Confocal microscopy images of hAD-MSCs embedded in bioprinted SISMA and SISMA-GO constructs with stained nuclei in blue and stained F-actin filaments in green. Elongated morphologies are observed after 6 days of incubation, indicating the presence of cell-matrix interactions that mediate cell adhesion and spreading. ( F ) hAD-MSC proliferation in SISMA and SISMA-GO constructs, as measured by resazurin reduction as a consequence of metabolic activity.

Article Snippet: Their stem cell status was then characterized through flow cytometry (BD FACSCantoTM Flow Cytometer, BD Biosciences, San Jose, CA, USA) with the aid of a human mesenchymal stem cell validation kit (Human Mesenchymal Stem Cell Multi-Color Flow Kit, R&D Systems, Minneapolis, MN, USA) and data acquisition and analysis was done with the BD FACSDivaTM software version 6.1.3.

Techniques: Shear, Derivative Assay, Confocal Microscopy, Construct, Staining, Incubation, Activity Assay