Journal: STAR Protocols

Article Title: Generation of hematopoietic lineage cell-specific chimeric mice using retrovirus-transduced fetal liver cells

doi: 10.1016/j.xpro.2022.101526

Figure Lengend Snippet:

Article Snippet: Mouse SCF, research grade , Miltenyi Biotec , 130-101-741.

Techniques: Virus, Recombinant, Marker, Modification, Saline, Transfection, Western Blot, Plasmid Preparation, Purification, Gel Extraction, Retroviral, Sequencing, Software, Flow Cytometry

Journal: PLoS ONE

Article Title: Membrane-Bound Steel Factor Maintains a High Local Concentration for Mouse Primordial Germ Cell Motility, and Defines the Region of Their Migration

doi: 10.1371/journal.pone.0025984

Figure Lengend Snippet: (A) There was no significant change in PGC numbers at E7.5 in Steel d/d embryos compared to their littermates. “n” indicates the number of embryos used for quantitation. (B) PGC number after 24 hours in vitro culture in medium with or without soluble recombinant Steel factor on different feeder layer cells. Y axis represents the ratio of PGC number 24 hours after plating versus 3 hours after plating. ΔMEF: primary MEF from Steel-null embryos. M220: stromal cell line express only membrane-bound Steel factor. ** = p<0.01. (C) PGC number reduced significantly in E8.0 Steel d/d embryos compared to their littermates. “n” indicates the number of embryos used for quantitation. * = p<0.05.

Article Snippet: For the Steel factor addition assay, 200 ng/ml soluble mouse recombinant Steel factor (R&D Systems) was added into the embryo culture medium immediately before time-lapse analysis was started.

Techniques: Quantitation Assay, In Vitro, Recombinant, Membrane

Journal: PLoS ONE

Article Title: Membrane-Bound Steel Factor Maintains a High Local Concentration for Mouse Primordial Germ Cell Motility, and Defines the Region of Their Migration

doi: 10.1371/journal.pone.0025984

Figure Lengend Snippet: (Column A, B) Frames at t = 0 and t = 6 hours respectively from movies of E7.5 Steel d/d embryos with or without addition of 200 ng/ml soluble recombinant Steel factor. (Column C) Tracks were made from PGCs in the allantois (white boxes) that remained in the plane of the confocal image throughout the movies. The white line indicates the boundary between the extraembryonic tissues (EEM), and the posterior end of the embryo (PEM). Scale bars in (A–C): 100 µm. (D) The maximum velocity, average velocity, and displacement of PGCs in E7.5 Steel d/d embryos were significantly increased by adding of 200 ng/ml soluble recombinant Steel factor into culture medium for 6 hours. “n” indicates the number of PGCs used for quantitation. Units on the “Y” axis vary based upon parameter, and are indicated below the bar charts. ** = p<0.01. (E) The maximum velocity, average velocity, and displacement of PGCs after 24 hours in vitro culture with increasing concentration of soluble recombinant Steel factor on Steel-null MEFs (ΔMEF). Units on the “Y” axis vary based upon parameter, and are indicated below the bar charts. * = p<0.05.

Article Snippet: For the Steel factor addition assay, 200 ng/ml soluble mouse recombinant Steel factor (R&D Systems) was added into the embryo culture medium immediately before time-lapse analysis was started.

Techniques: Recombinant, Quantitation Assay, In Vitro, Concentration Assay

Journal: PLoS ONE

Article Title: Membrane-Bound Steel Factor Maintains a High Local Concentration for Mouse Primordial Germ Cell Motility, and Defines the Region of Their Migration

doi: 10.1371/journal.pone.0025984

Figure Lengend Snippet: Directions of individual PGC migration in Steel d/d embryos (A) or wild type embryos (B) with or without addition of 200 ng/ml soluble recombinant Steel factor. The boundary between the extraembryonic tissues (EEM), and the posterior end of the embryo (PEM), is marked by a line. Column I, II, and III are representative images from 3 different embryos of the same genotype labeled on the left. (C) The maximum velocity, average velocity, and displacement of PGCs in E7.5 wild type embryos with or without addition of 200 ng/ml soluble recombinant Steel factor into culture medium for 6 hours. “n” indicates the number of PGCs used for quantitation. Units on the “Y” axis vary based upon parameter, and are indicated below the bar charts. ** = p<0.01.

Article Snippet: For the Steel factor addition assay, 200 ng/ml soluble mouse recombinant Steel factor (R&D Systems) was added into the embryo culture medium immediately before time-lapse analysis was started.

Techniques: Migration, Recombinant, Labeling, Quantitation Assay

Journal: PLoS ONE

Article Title: Membrane-Bound Steel Factor Maintains a High Local Concentration for Mouse Primordial Germ Cell Motility, and Defines the Region of Their Migration

doi: 10.1371/journal.pone.0025984

Figure Lengend Snippet: (Column A-C) Frames at t = 0, t = 6 and t = 12 hours respectively from movies of E11.0 wild type embryo slices with or without addition of 200 ng/ml soluble recombinant Steel factor. (D) The relative expression level of membrane-bound and soluble Steel factor mRNA in E10.5 genital ridges, E11.5 genital ridges and E11.5 midline mysenchyme as determined by real-time RT-PCR. *p<0.05, ** p <0.01. (E and F) E-cadherin expression in PGCs without (E) or with (F) addition of Steel factor. Upper panels show E-cadherin (red) staining. The lower panels show the merged images with PGC marker Stella-GFP (green). No difference in expression of E-cadherin was observed between groups.

Article Snippet: For the Steel factor addition assay, 200 ng/ml soluble mouse recombinant Steel factor (R&D Systems) was added into the embryo culture medium immediately before time-lapse analysis was started.

Techniques: Recombinant, Expressing, Membrane, Quantitative RT-PCR, Staining, Marker

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Targeting of Deciduous Tooth Pulp Stem Cell-Derived Extracellular Vesicles on Telomerase-Mediated Stem Cell Niche and Immune Regulation in Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2001312

Figure Lengend Snippet: FIGURE 1. Systemic transplantation of SHED rescues hematopoietic niche-forming and immunomodulatory functions of BMMSC in recipient MRL/lpr mice. (AC) Representative images of in vivo hematopoietic niche formation were detected in s.c. implants of BMMSCs with an HA/TCP (HA) carrier into NOD-SCID mice. The images represent one of three independent experiments with five mice per group. H&E staining. Dot-line circled area: de novo BM- like niche components. B6-BMMSC, lpr-BMMSC, and SHED-BMMSC are BMMSCs isolated from C57BL/6 (B6) mice, nontransplanted MRL/lpr (lpr) mice, and SHED-transplanted MRL/lpr (SHED-lpr) mice, respectively. Scale bars, 100 mm (A). The graph shows the percentages of BM-like niche area in implants (B). The representative histograms show the expression of Sca-1, c-Kit, and CD45 in implants by FCM analysis. The images represent one of three independent experiments with five mice per group. The graphs show the percentages of Sca-1-, c-Kit-, and CD45-positive cells in implants by FCM analysis (C). (D) The graph shows the levels of SCF in CM of BMMSCs by ELISA. (E) The graph shows the number of hematopoietic CFC of B6 mouse-derived BM cells (B6-BMC) cocultured with BMMSCs. (F and G) BMMSCs were cocultured with preactivated B6 mouse-derived T cells (B6-T). The presentative dot-blots show the expression of CD41IL-171IFN-g and CD41CD251Foxp31 cells by FCM analysis. The images represent one of three independent ex- periments with five mice per group. The graphs show the positive percentages of CD41IL-171IFN-g and CD41CD251Foxp31 cells by FCM analysis. (F). The presentative dot-blots show the expression of CD41Annexin-V(AV)17AAD1 cells by FCM analysis. The images represent one of three independent experiments with five mice per group. The graphs show the positive percentages of CD41Annexin-V(AV)17AAD1 cells by FCM analysis (G). (H) The graph shows the survival ratio of mice using the KaplanMeier curve. n = 7 mice per group. (BG) The data are representative of three independent experi- ments (mean ± SEM) with five mice per group in each experiment. *p < 0.05, **p < 0.01, ***p < 0.005. B, de novo bone matrix; B6-BMMSCT, B6-Tadopted mice transplanted with B6-BMMSC; B6-TAdopt, B6-Tadopted mice; Cont, nonadoption control mice; lpr-BMMSCT, B6-Tadopted mice transplanted with lpr-BMMSC; ns, not significant; R-PE, R-phycoerythrin; SHED-BMMSCT, B6-Tadopted mice transplanted with SHED-BMMSC.

Article Snippet: The level of stem cell factor (SCF) was measured in the CM of mBMMSCs using a mouse SCF Quantikine ELISA kit (R&D Systems)

Techniques: Transplantation Assay, In Vivo, Staining, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Targeting of Deciduous Tooth Pulp Stem Cell-Derived Extracellular Vesicles on Telomerase-Mediated Stem Cell Niche and Immune Regulation in Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2001312

Figure Lengend Snippet: FIGURE 2. Tert-mediated telomerase activity regulates hematopoietic niche formation and immunomodulatory function in BMMSCs of SHED-trans- planted MRL/lpr mice. (A) The graph shows the telomerase activity in BMMSCs by RQ-TRAP. HEK 293T cells (293T), n = 5; heat-inactivated HEK 293T cells (293T H.I.), n = 5. (B) The graph shows the ratio of Tert expression in BMMSCs detected by RT-qPCR. The results are shown as a ratio to the expres- sion in B6-BMMSC (B6-BMMSC = 1). (C) Representative images of in vivo hematopoietic niche formation were detected in s.c. implants of BMMSCs into NOD-SCID mice. H&E staining. The images represent one of three independent experiments with five mice per group. Dot-line circled area: de novo BM- like niche components. Scale bars, 50 mm. (D) The graph shows the percentages of BM-like niche area in implants. (E) The graph shows the percentages of Sca-11, c-Kit1, and CD451 cells in implants by FCM analysis. (F) The graph shows the levels of SCF in CM of BMMSCs by ELISA. (G) The graph shows the number of hematopoietic CFC cells in B6-BMC cocultured with BMMSCs. (H) The graph shows the positive percentages of CD41IL-171IFN-g and CD41CD251Foxp31 cells in B6-T cocultured with BMMSCs by FCM analysis. (I) The graph shows the positive percentages of CD41AV17AAD1 cells in B6-T cocultured with BMMSCs by FCM analysis. (J) The graph shows the survival ratio of mice using the KaplanMeier curve. n = 7 mice per group. siRNAContT-SHED-BMMSCT and siRNATert-SHED-BMMSCT are B6-Tadopted mice transplanted with siRNACont-pretreated SHED-BMMSCs and siRNATert-pretreated SHED-BMMSCs. (A, B, DI) The data are representative of three independent experiments (mean ± SEM) with five mice per group in each experiment. *p < 0.05, **p < 0.01, ***p < 0.005. ns, not significant; SQ, threshold cycle.

Article Snippet: The level of stem cell factor (SCF) was measured in the CM of mBMMSCs using a mouse SCF Quantikine ELISA kit (R&D Systems)

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, In Vivo, Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Targeting of Deciduous Tooth Pulp Stem Cell-Derived Extracellular Vesicles on Telomerase-Mediated Stem Cell Niche and Immune Regulation in Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2001312

Figure Lengend Snippet: FIGURE 3. Effects of transplantation of RAB27A mRNAknocked-down SHED on SLE-like phenotypes in MRL/lpr mice. (A) The graph shows the ratio of RAB27A expression in SHED detected by RT-qPCR. The results are shown as a ratio to the expression in siRNACont-pretreated SHED (siRNACont- SHED = 1). siRNARAB27A, pretreatment with siRNA for RAB27. (BE) The therapeutic efficacy was assessed 4 wk after the transplantation of SHED into MRL/lpr mice. The graphs show the serum levels of ANA, anti-dsDNA IgG, and anti-dsDNA IgM by ELISA (B). The graphs show the levels of urine pro- tein and serum creatinine by colorimetry (C). The graphs show the positive percentages of CD41IL-171IFN-g (Th17) and CD41CD251Foxp31 (Treg) cells in PBMCs by FCM analysis (D). The graph shows the ratio of Treg to Th17 (Treg/Th17) (E). (AE) The data are representative of three independent ex- periments (mean ± SEM) with five mice per group in each experiment. *p < 0.05, ***p < 0.005. lpr, MRL/lpr mice; ns, not significant; SHEDT, systemic transplantation of SHED.

Article Snippet: The level of stem cell factor (SCF) was measured in the CM of mBMMSCs using a mouse SCF Quantikine ELISA kit (R&D Systems)

Techniques: Transplantation Assay, Expressing, Quantitative RT-PCR, Drug discovery, Enzyme-linked Immunosorbent Assay, Colorimetric Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Targeting of Deciduous Tooth Pulp Stem Cell-Derived Extracellular Vesicles on Telomerase-Mediated Stem Cell Niche and Immune Regulation in Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2001312

Figure Lengend Snippet: FIGURE 4. Systemic administration of SHED-EVs improves SLE-like phenotypes in MRL/lpr mice. (A) Representative fluorescent images of lpr- BMMSCs were shown 3 d after the loading of CSFE-labeled SHED-EVs pretreated with MOCK (MOCK-EV) and RNase (5 U/ml; RNase-EV) for 3 h. The images represent one of three independent experiments with five mice per group. Nuclei were stained with DAPI. Scale bars, 100 mm. (B) The graph shows the ratio of Tert expression in BMMSCs by RT-qPCR. The results are shown as a ratio to the expression in B6-BMMSCs (B6-BMMSC = 1). (C) The graph shows the telomerase activity in BMMSCs by RQ-TRAP assay. (DH) Therapeutic efficacy of MOCKSHED-EVs and RNaseSHED-EVs was analyzed 4 wk after systemic administration into MRL/lpr mice. The graphs show the serum levels of ANA, anti-dsDNA IgG, and anti-dsDNA IgM by ELISA (D). Representative images of the kidney were detected by Masson trichrome staining. The images represent one of three independent experiments with five mice per group. Scale bars, 100 mm (E). The graphs show the levels of urine protein and serum creatinine by colorimetry (F). The graphs show the positive percen- tages of CD41IL-171IFN-g and CD41CD251Foxp31 PBMCs by FCM analysis (G). The graphs show the ratio of Treg/Th17 by FCM analysis (H). (BD and FH) The data are representative of three independent experiments (mean ± SEM) with five mice per group in each experiment. *p < 0.05, **p < 0.01, ***p < 0.005. B6, control C57B/6 mice; G, glomerulus; lpr, MRL/lpr mice; MOCK-EVA, MRL/lpr mice administrated with MOCKSHED-EV; RNase- EVA, MRL/lpr mice administrated with RNaseSHED-EV; ns, not significant.

Article Snippet: The level of stem cell factor (SCF) was measured in the CM of mBMMSCs using a mouse SCF Quantikine ELISA kit (R&D Systems)

Techniques: Labeling, Staining, Expressing, Quantitative RT-PCR, Activity Assay, TRAP Assay, Drug discovery, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Targeting of Deciduous Tooth Pulp Stem Cell-Derived Extracellular Vesicles on Telomerase-Mediated Stem Cell Niche and Immune Regulation in Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2001312

Figure Lengend Snippet: FIGURE 5. Systemic SHED-EV administration rescued the hematopoietic niche-forming function of recipient MRL/lpr mouse-derived BMMSCs. (A) Representative fluorescent micrographs were detected in the BM of MRL/lpr mice 3 d after the infusion of SHED-EV (100 mg/mouse) labeled with CFSE (CFSE-EV). The images represent one of three independent experiments with five mice per group. Nuclei were stained with DAPI. Scale bars, 100 mm. (BH) The graph shows the ratio of Tert expression in BMMSCs by RT-qPCR. The data are shown as a ratio to the expression in B6-BMMSC (B6- BMMSC = 1) (B). The graph shows the telomerase activity in BMMSCs by RQ-TRAP (C). Representative images were detected in s.c. implants of BMMSCs. H&E staining. The images represent one of three independent experiments with five mice per group. Scale bars, 100 mm (D). The graph shows the percentages of BM-like niche area in implants (E). The graph shows the percentages of Sca-11, c-Kit1, and CD451 cells in implants by FCM analysis (F). The graph shows the levels of SCF in CM of BMMSCs by ELISA (G). The graph shows the number of hematopoietic CFC cells in B6-BMC cocultured with BMMSCs (H). (BH) The data are representative of three independent experiments (mean ± SEM) with five mice per group in each experiment. *p < 0.05, ***p < 0.005. MOCK-EV-BMMSC, BMMSCs isolated from MOCK-EVinfused MRL/lpr mice; ns, not significant; RNase-EV-BMMSC, BMMSCs isolated from RNase-EVinfused MRL/lpr mice.

Article Snippet: The level of stem cell factor (SCF) was measured in the CM of mBMMSCs using a mouse SCF Quantikine ELISA kit (R&D Systems)

Techniques: Derivative Assay, Labeling, Staining, Expressing, Quantitative RT-PCR, Activity Assay, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Targeting of Deciduous Tooth Pulp Stem Cell-Derived Extracellular Vesicles on Telomerase-Mediated Stem Cell Niche and Immune Regulation in Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2001312

Figure Lengend Snippet: FIGURE 6. Systemic SHED-EV administration rescued the immunomodulatory function of the recipient MRL/lpr mouse-derived BMMSCs. (A) The graphs show the positive percentages of CD41IL-171IFN-g and CD41CD251Foxp31 cells in B6-T cocultured with MOCK-EV-BMMSC and RNase-EV- BMMSC by FCM analysis. (B) The graphs show the positive percentages of CD41AV17AAD1 cells in B6-T cocultured with MOCK-EV-BMMSC and RNase-EV-BMMSC by FCM analysis. Graph bars represent mean ± SEM. (C) The graph shows the survival ratio of mice using the KaplanMeier curve. n = 7 mice per group. MOCK-EV-BMMSCT and RNase-EV-BMMSCT, B6-Tadopted mice transplanted with MOCK-EV-BMMSC and RNase-EV- BMMSC. (DG) Therapeutic efficacy was analyzed 4 wk after the transplantation of MOCK-EV-BMMSC and RNase-EV-BMMSC into MRL/lpr mice. The graphs show the serum levels of ANA, anti-dsDNA IgG, and anti-dsDNA IgM by ELISA (D). The graphs show the levels of urine protein and serum creati- nine by colorimetry (E). The graphs show the percentages of CD41IL-171IFN-g and CD41CD251Foxp31 PBMCs by FCM analysis (F). The graphs show the ratio of Treg/Th17 by FCM analysis (G). MOCK-EV-BMMSCT and RNase-EV-BMMSCT, MRL/lpr mice transplanted with MOCK-EV-BMMSC and RNase-EV-BMMSC. (AG) The data are representative of three independent experiments (mean ± SEM) with five mice per group in each experiment. *p < 0.05, ***p < 0.005. B6, control C57B/6 mice; lpr, MRL/lpr mice; ns, not significant.

Article Snippet: The level of stem cell factor (SCF) was measured in the CM of mBMMSCs using a mouse SCF Quantikine ELISA kit (R&D Systems)

Techniques: Derivative Assay, Drug discovery, Transplantation Assay, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, Control

Journal: Cell death & disease

Article Title: SCF and IL-33 regulate mouse mast cell phenotypic and functional plasticity supporting a pro-inflammatory microenvironment.

doi: 10.1038/s41419-023-06139-7

Figure Lengend Snippet: Fig. 7 SCF controls connective tissue-like MC accumulation in tumor lesions. A Schematic representation of anti-SCF neutralizing Ab administration in vivo. AOM/DSS-treated mice were i.p. injected three times with anti-SCF or control Ab (100 μg/mouse) starting from the fourth DSS cycle. Treated and control mice were sacrificed at 13 weeks from AOM administration. B Colon paraffin-embedded sections from AOM/DSS mice treated with Ctrl-Ig or anti-SCF neutralizing antibodies as described in (A) were stained with anti-MCP4 Ab followed by Alexa Fluor 488 secondary Abs (green). Nuclei were counterstained with DAPI (blue) and images were acquired with a Zeiss LSM980 confocal microscopy using a 20× objective. The frequencies of MCs positive for mMCP4 protease were analyzed in 20 fields randomly acquired from tumor lesions and shown as mean ± SD cells/field. Paired Student’s t test: *p < 0.05. Number of adenomas and colon lengths are shown in (C). Paired Student’s t test: *p < 0.05. Graphs are representative of two independent experiments with a total of 5 mice/group.

Article Snippet: For blocking experiments, mice were i.p. injected with anti-SCF neutralizing antibody (AB-455-NA R&D Systems) or normal goat control IgG (AB-108-C R&D Systems) (100μg/mouse) three times every 10 days starting one day before the fourth DSS cycle (see Fig. 7A).

Techniques: In Vivo, Injection, Control, Staining, Confocal Microscopy