recombinant murine stem cell factor (mscf  (PeproTech)

Journal: iScience

Article Title: CD244 expression represents functional decline of murine hematopoietic stem cells after in vitro culture

doi: 10.1016/j.isci.2021.103603

Figure Lengend Snippet: Transplantation of HSCs cultured with various bile acids (A) The gating strategy for isolating CD150 + CD48 − KSL cells from murine bone marrow. (B) Experimental design of the competitive reconstitution assay. Fifty (50) CD150 + CD48 − KSL cells were isolated from BM of Ly5.1 mice (donor, two mice per experiment were used) and cultured in Stemspan SFEM medium supplemented with 100 ng/mL mSCF and 100 ng/mL hTPO with or without 100 μM of different types of bile acids (BA) for 7 or 14 days. Cells were then transplanted into lethally irradiated Ly5.2 mice (recipient, 5–18 mice) along with 2 × 10 5 total BM cells derived from Ly5.1/5.2 (F1) mice (competitor). Donor contribution (chimerism) was monitored by analyzing peripheral blood (PB) every month. (C) Peripheral blood analysis of the transplanted mice. Chimerism in PB at 16 weeks of transplanted mice are shown. Mean ± SD from three independent experiments (n = 5–18) are displayed. Significance was calculated using one-way ANOVA within the same culture days. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (D–F) Cell surface expression of CD48 on KSL fraction of HSCs cultured with or without BA. Representative FACS plots (C) and summary of the flow cytometry analyses (D and E) are shown. Mean ± SD (n = 3) are displayed.

Article Snippet: Recombinant Murine stem cell factor (mSCF) , PEPROTECH , Cat# 250-03-10 μg.

Techniques: Transplantation Assay, Cell Culture, Reconstitution Assay, Isolation, Irradiation, Derivative Assay, Expressing, Flow Cytometry

Journal: iScience

Article Title: CD244 expression represents functional decline of murine hematopoietic stem cells after in vitro culture

doi: 10.1016/j.isci.2021.103603

Figure Lengend Snippet: Gene expression changes in CD48 − KSL cells upon in vitro culture and aging (A) Experimental design of the gene expression analysis. Five hundred CD48 − KSL cells were sorted from BM of young (10 weeks old, YF) mice. Cells were also cultured in Stemspan SFEM medium supplemented with 100 ng/mL mSCF and 100 ng/mL hTPO for 14 days, and CD48 − KSL cells were re-sorted. (B) Volcano plot showing differentially expressed genes between YF and YC. Significant difference was defined as p < 0.001 and log 2 fold change < −2 (blue) or >2 (red). (C) Heatmaps showing a list of differentially expressed genes. Significant difference was defined as p < 0.0001 and log 2 fold change < −3 or >3. Blue arrowheads indicate CD markers. (D) Gene set enrichment analysis (GSEA) of the microarray data comparing YF and YC. NES, normalized enrichment score; NOM p-val, nominal p value; FDR q-val, false discovery rate q-value. See also .

Article Snippet: Recombinant Murine stem cell factor (mSCF) , PEPROTECH , Cat# 250-03-10 μg.

Techniques: Gene Expression, In Vitro, Cell Culture, Microarray

Journal: iScience

Article Title: CD244 expression represents functional decline of murine hematopoietic stem cells after in vitro culture

doi: 10.1016/j.isci.2021.103603

Figure Lengend Snippet: CD244 expression divides CD48 − KSL cells into functionally distinct subpopulations after in vitro culture (A) Experimental design of the in vitro culture experiment. One hundred CD48 − KSL cells were sorted from BM of young mice and cultured in Stemspan SFEM medium supplemented with 100 ng/mL mSCF and 100 ng/mL hTPO for 7 days. (B) Expression patterns of CD244 and CD48 on the cell surface of fresh and 14 days cultured KSL cells. Representative FACS plots on KSL population are shown. (C and D) qRT-PCR analysis for HSC-related genes and mast cell-related genes in CD244 + CD48 − KSL cells compared with the CD244 + CD48 − KSL counterpart. Representative FACS plot and gating of CD244 + CD48 − KSL cells (green) and CD244 − CD48 − KSL cells (red) on 7 days cultured KSL cells are shown in (C). Relative expression levels of genes in CD244 + CD48 − KSL cells to CD244 − CD48 − KSL are shown in (D). (E) Competitive reconstitution assay. After 7 days’ culture, two subpopulations were sorted and 1,000 of CD244 - or 1,500 of CD244 + CD48 − KSL cells were separately transplanted into lethally irradiated recipient mice (seven mice) with 2 × 10 5 total BM cells (competitor). Chimerism was monitored by analyzing PB every month. Significance was calculated using Student's t test at each time point. Mean ± SD from two independent experiments (n = 7) are displayed. ∗∗∗∗ p < 0.0001. (F) Lineage balance of donor-derived cells in the PB of recipient mice. Mean ± SD from two independent experiments (n = 6) are displayed. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. Each color represents different lineages. (G) Analysis of BM from engrafted mice after 16 weeks. Chimerism in each cell fraction is shown. Significance was calculated using Student's t test. Mean ± SD from two independent experiments (n = 6) are displayed. ∗∗ p < 0.01. (H and I) Limiting dilution assay for CD244 − CD48 − KSL cells. CD244 − CD48 − KSL cells were re-sorted after 7 days’ in vitro culture and 200, 40, or 10 cells were transplanted to recipient mice in a competitive manner. Chimerism above 1% was judged as successful engraftment. The frequency of functional HSC was calculated using ELDA ( http://bioinf.wehi.edu.au/software/elda/ ). Chimerism of individual mice is shown in (I).

Article Snippet: Recombinant Murine stem cell factor (mSCF) , PEPROTECH , Cat# 250-03-10 μg.

Techniques: Expressing, In Vitro, Cell Culture, Quantitative RT-PCR, Reconstitution Assay, Irradiation, Derivative Assay, Limiting Dilution Assay, Functional Assay, Software

Journal: iScience

Article Title: CD244 expression represents functional decline of murine hematopoietic stem cells after in vitro culture

doi: 10.1016/j.isci.2021.103603

Figure Lengend Snippet: ER stress induction and cytokine signals affect CD244 expression (A) Expression patterns of CD244 and CD48 on KSL cells after in vitro culture in Stemspan SFEM medium supplemented with 100 ng/mL mSCF and 100 ng/mL hTPO for 7 days, and with or without 0.5 μg/mL Tunicamycin or 100 μM TUDCA. Representative FACS plots are shown. (B) Summary of the flow cytometry analyses. Frequencies of KSL cells, CD48 - fraction in KSL population, and CD244 − CD48 - fraction in KSL population are shown. Mean ± SD from two independent experiments (n = 5–11) are displayed. Significance was calculated using one-way ANOVA within each population. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. (C) A heatmap showing frequencies of CD244 − CD48 − KSL cells after 7 days’ culture with various SCF/TPO concentrations. Mean values of Z score (n = 3) in each fraction are displayed.

Article Snippet: Recombinant Murine stem cell factor (mSCF) , PEPROTECH , Cat# 250-03-10 μg.

Techniques: Expressing, In Vitro, Flow Cytometry

Journal: iScience

Article Title: CD244 expression represents functional decline of murine hematopoietic stem cells after in vitro culture

doi: 10.1016/j.isci.2021.103603

Figure Lengend Snippet: Gene expression profiling of CD244 - and CD244 + KSL cells (A) Experimental design of the gene expression analysis. Five hundred CD244 - 48 − KSL cells were sorted from BM of 10-week-old mice (N) and were also cultured in Stemspan SFEM medium supplemented with 100 ng/mL mSCF and 100 ng/mL hTPO for 7 days, and CD244 − KSL (CN) and CD244 + KSL (CP) cells were re-sorted. (B) Volcano plot showing differentially expressed genes between CP versus N (left), CN versus N (center), and CP versus CN (right). Significant difference was defined as p < 0.01 and log 2 fold change < −2 (blue) or >2 (red). (C) K-mean clustering of genes showing different expression patterns between the three cohorts and heatmaps of selected genes. Significant difference was defined as p < 0.001 and log 2 fold change < −2 or >2. (D) Overlap of hallmarks and GO terms between the three groups based on GSEA. See also .

Article Snippet: Recombinant Murine stem cell factor (mSCF) , PEPROTECH , Cat# 250-03-10 μg.

Techniques: Gene Expression, Cell Culture, Expressing

Journal: iScience

Article Title: CD244 expression represents functional decline of murine hematopoietic stem cells after in vitro culture

doi: 10.1016/j.isci.2021.103603

Figure Lengend Snippet: CD244 expression distinguishes functionally distinct subpopulations after in vitro culture in PVA medium (A) Experimental design of the in vitro culture experiment. One hundred CD48 − KSL cells were sorted from BM of young mice and cultured in PVA containing medium ( Wilkinson et al., 2019 ) supplemented with 10 ng/mL mSCF and 100 ng/mL hTPO for 7 days. (B) Expression patterns of CD244 and CD48 on the cell surface of KSL cells after in vitro culture in PVA medium. A representative FACS plot on KSL population is shown. (C) Competitive reconstitution assay. After 7 days’ culture, three subpopulations were sorted and 500 of CD244 − CD48 − KSL cells, CD244 − CD48 + KSL cells, or CD244 + CD48 + KSL cells were separately transplanted into lethally irradiated recipient mice with 2 × 10 5 total BM cells. Chimerism was monitored by analyzing PB every month. Significance was calculated using one-way ANOVA at each time point. Mean ± SD from two independent experiments (n = 12) are displayed. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 (D) Lineage balance of donor-derived cells in the PB of recipient mice. Significance was calculated using one-way ANOVA at each time point. Mean ± SD from two independent experiments (n = 12) are displayed. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. Each color represents different lineages. (E) Analysis of BM from engrafted mice after 16 weeks. Chimerism in each cell fraction is shown. Significance was calculated using one-way ANOVA within each population. Mean ± SD from two independent experiments (n = 5) are displayed. ∗∗∗∗ p < 0.0001.

Article Snippet: Recombinant Murine stem cell factor (mSCF) , PEPROTECH , Cat# 250-03-10 μg.

Techniques: Expressing, In Vitro, Cell Culture, Reconstitution Assay, Irradiation, Derivative Assay

Journal: iScience

Article Title: CD244 expression represents functional decline of murine hematopoietic stem cells after in vitro culture

doi: 10.1016/j.isci.2021.103603

Figure Lengend Snippet:

Article Snippet: Recombinant Murine stem cell factor (mSCF) , PEPROTECH , Cat# 250-03-10 μg.

Techniques: Recombinant, Staining, Microarray, Gene Expression, Real-time Polymerase Chain Reaction, Software