Journal: Cell stem cell

Article Title: Jak1 Integrates Cytokine Sensing to Regulate Hematopoietic Stem Cell Function and Stress Hematopoiesis

doi: 10.1016/j.stem.2017.08.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PE anti-mouse CD150 (SLAM) , eBiosciences , 12-1502-82.

Techniques: Recombinant, Microscopy, Software

Journal: bioRxiv

Article Title: A murine model with JAK2V617F expression in both hematopoietic cells and vascular endothelial cells recapitulates the key features of human myeloproliferative neoplasm

doi: 10.1101/2021.08.24.457585

Figure Lengend Snippet: Decreased marrow hematopoiesis in the Tie2FF1 mice during aging. ( A ) Colony formation assays in marrow cells isolated from young (n=4 mice in each group) and old (n=6-7 mice in each group) Tie2-cre control and Tie2FF1 mice. ( B ) Representative flow cytometry plots showing gating strategy (left) of marrow Lin - cKit + Sca1 + CD150 + CD48 - HSCs frequency (right) in young (n=7 mice in each group) and old (n=5-6 mice in each group) Tie2-cre control and Tie2FF1 mice. ( C-D ) Cell proliferation of marrow Lin-HSPCs isolated from young (C) and old (D) Tie2-cre control and Tie2FF1 mice. Cells were cultured on SFEM medium containing recombinant mouse SCF (100ng/mL), recombinant mouse IL3 (6ng/mL), and recombinant human IL6 (10ng/mL). Data are from one of two independent experiments (with triplicates in each experiment) that gave similar results. * P <0.05

Article Snippet: For analysis of HSC (Lin - cKit + Sca1 + CD150 + CD48 - ) proliferation, Lin - cells were first enriched using the Lineage Cell Depletion Kit (Miltenyi Biotec) before staining with fluorescent antibodies specific for cell surface HSC markers, followed by fixation and permeabilization using the Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA), DNase digestion (Sigma, St. Louis, MO), and anti-BrdU antibody (Biolegend, San Diego, CA) staining to analyze BrdU incorporation .

Techniques: Isolation, Control, Flow Cytometry, Cell Culture, Recombinant

Journal: bioRxiv

Article Title: A murine model with JAK2V617F expression in both hematopoietic cells and vascular endothelial cells recapitulates the key features of human myeloproliferative neoplasm

doi: 10.1101/2021.08.24.457585

Figure Lengend Snippet: Expanded splenic extramedullary hematopoiesis in the Tie2FF1 mice. ( A ) Colony formation assays in spleen cells isolated from young (n=3 mice in each group) and old (n=5-6 mice in each group) Tie2-cre control and Tie2FF1 mice. ( B ) Spleen Lin - cKit + Sca1 + CD150 + CD48 - HSCs frequency in young (n=3 mice in each group) and old (n=5 mice in each group) Tie2-cre control and Tie2FF1 mice. ( C-D ) Cell proliferation of spleen Lin-HSPCs isolated from young (C) and old (D) Tie2-cre control and Tie2FF1 mice. Cells were cultured in SFEM medium containing recombinant mouse SCF (100ng/mL), recombinant mouse IL3 (6ng/mL), and recombinant human IL6 (10ng/mL). Data are from one of two independent experiments (with triplicates in each experiment) that gave similar results. * P <0.05

Article Snippet: For analysis of HSC (Lin - cKit + Sca1 + CD150 + CD48 - ) proliferation, Lin - cells were first enriched using the Lineage Cell Depletion Kit (Miltenyi Biotec) before staining with fluorescent antibodies specific for cell surface HSC markers, followed by fixation and permeabilization using the Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA), DNase digestion (Sigma, St. Louis, MO), and anti-BrdU antibody (Biolegend, San Diego, CA) staining to analyze BrdU incorporation .

Techniques: Isolation, Control, Cell Culture, Recombinant

Journal: Nature Communications

Article Title: Outside-in integrin signalling regulates haematopoietic stem cell function via Periostin-Itgav axis

doi: 10.1038/ncomms13500

Figure Lengend Snippet: ( a ) Flowcytometry analysis of the BM cells derived from 16-week-old FVB/NJ (WT; upper panel) and Postn −/− (KO; lower panel) mice ( N =12). ( b ) Frequency of SLAM KLS cells per million BM cells derived from 16-week-old FVB/NJ (WT) and Postn −/− (KO) mice ( N =12, t test: * P <0.008). ( c , d ) BrdU incorporation assays to examine the proliferation status of KLS cells (ST-HSCs; c ) and SLAM KLS cells (LT-HSCs; d ) in WT and Postn −/− mice. BrdU staining in addition to HSC markers in BM cells following 3 ( c ) or 7 ( d ) days of BrdU infusion ( n =3, N =9, t test: * P <0.02). ( e ) Schematic representation of the competitive repopulation assays. 50,000 total BM cells derived from WT/ Postn −/− mice (CD45.2) were transplanted into sub-lethally irradiated Rag2 −/− γC −/− mice. PB chimerism was followed for 12 weeks, after which secondary transplantations were performed. ( f , g ) Donor-derived PB chimerism in primary ( f ) and secondary ( g ) recipients transplanted with total BM cells from 8-week-old WT/ Postn −/− mice ( n =3, N =18, t test: * P <0.03). ( h – m ) Blood obtained from 16-week-old wild-type (WT) and Postn −/− mice was assessed for WBC count ( h ), RBC count ( i ), haematocrit value ( j ), haemoglobin level ( k ), lymphocyte ( l ) and granulocytes ( m ) numbers ( N =12, t test: *** P <0.001, ** P <0.01, * P <0.05). ( n , o ) Donor-derived PB chimerism in primary ( n ) and secondary ( o ) recipients transplanted with sorted primitive HSCs (CD150 + CD48 − KLS cells) total BM cells from 16-week-old WT/ Postn −/− mice ( n =3, N =18, t test: * P =0.02). ( p ) Frequency of primitive HSCs in the donor-derived fraction of BM cells from secondary recipients ( n =3, N =6, t test: * P =0.007). ( q ) Proportion of donor-derived primitive HSCs in secondary recipients in G0 stage of cell cycle ( n =3, N =6, t test: ** P =0.001). ( n =independent experiments, N =number of mice. Error bars indicate mean±s.e.m.).

Article Snippet: Flow cytometric analysis for primitive HSCs and haematopoietic progenitors was performed using anti-mouse CD48 APC and CD150 PECy7 (0.25 μg ml −1 ; ebiosciences) along with KLS cells staining (as for sorting).

Techniques: Derivative Assay, BrdU Incorporation Assay, BrdU Staining, Irradiation

Journal: Nature Communications

Article Title: Outside-in integrin signalling regulates haematopoietic stem cell function via Periostin-Itgav axis

doi: 10.1038/ncomms13500

Figure Lengend Snippet: ( a – c ) Following sub-lethal irradiation PB counts were measured weekly for 7 weeks. Numbers of WBCs ( a ), granulocytes ( b ), and haematocrit values ( c ) were compared between FVB/NJ (WT) and Postn −/− (KO) mice ( n =3, N =18, t test: ** P <0.01, * P <0.05). ( d ) Following sub-lethal irradiation the cell cycle status of BM derived KLS cells was analysed. Two weeks after irradiation, BM cells were isolated and flowcytometry analysis was performed to assess proliferation of lin − c-kit + Sca-1 + (KLS) cells with Hoechst labelling ( n =3, N =9). ( e , f ) Flowcytometry based analysis performed during 8 weeks following sub-lethal irradiation to compare the number of SLAM KLS ( e ) and various lineage committed ( f ) cells in the BM of FVB/NJ (WT) and Postn −/− (KO) mice ( n =3, N =9, t test * P <0.05). ( n =independent experiments, N =number of mice. Error bars indicate mean ±s.e.m.).

Article Snippet: Flow cytometric analysis for primitive HSCs and haematopoietic progenitors was performed using anti-mouse CD48 APC and CD150 PECy7 (0.25 μg ml −1 ; ebiosciences) along with KLS cells staining (as for sorting).

Techniques: Irradiation, Derivative Assay, Isolation

Journal: Nature Communications

Article Title: Outside-in integrin signalling regulates haematopoietic stem cell function via Periostin-Itgav axis

doi: 10.1038/ncomms13500

Figure Lengend Snippet: ( a – c ) BM derived KLS cells were cultured for 5 days in serum-free medium with SCF and TPO in the absence (ST) or presence (STP) of Postn. ( a ) Cultured BM KLS cells harvested after 5 days were labelled with the PKH-26 dye and plated on ST2 cell feeders. The relative proportion of cells that adhered after 3 h was plotted ( n =4, t test: NS P >0.05). ( b ) Migration of cultured BM KLS cell progeny following 5 days of culture, and labelled with the PKH-26 dye, towards SDF-1α was assessed using a trans-well system. The relative proportion of cells that migrated after 3 h was plotted ( n =4, t test: NS P >0.05). ( c ) Cultured BM KLS cells harvested after 5 days were tested for their in vivo homing capacity, by infusing into lethally irradiated animals. The percentage of transplanted CFCs that homed into the BM within 16 h was plotted ( n =4, N =12, t test: NS P >0.05). ( d ) Expression of Itgav (CD51) and Itgab3 (CD61) on SLAM KLS cells assessed by flowcytometry ( n =6). ( e ) KLS cells incubated with SCF+TPO without (ST) or with (STP) Postn for 3 h were allowed to adhere on cyclo-RGDfK coated plates. The percentage of cells that adhered to the plates after 3 h was plotted for each condition ( n =4, t test: * P =0.018). ( f , g ) BM derived KLS cells were cultured in serum-free medium containing SCF and TPO for 2 days without (ST) or with (STP) Postn alone, or in combination with neutralizing antibodies against Itgav (STP+αItgav) or Itgb3 (STP+αItgb3) for 2–5 days. ( n =4, t test: ** P <0.01; scale bar, 50 μm). ( f ) Bright field image showing cell expansion after 2 days. The proliferating cells appear as a cluster of cells in the middle of the round-bottom 96-well plates. ( g ) After 5 days of culture, cells were harvested and fold expansion was compared. ( n =independent experiments, N =number of mice. Error bars indicate mean ±s.e.m.).

Article Snippet: Flow cytometric analysis for primitive HSCs and haematopoietic progenitors was performed using anti-mouse CD48 APC and CD150 PECy7 (0.25 μg ml −1 ; ebiosciences) along with KLS cells staining (as for sorting).

Techniques: Derivative Assay, Cell Culture, Migration, In Vivo, Irradiation, Expressing, Incubation

Journal: Nature Communications

Article Title: Outside-in integrin signalling regulates haematopoietic stem cell function via Periostin-Itgav axis

doi: 10.1038/ncomms13500

Figure Lengend Snippet: ( a – d ) Counts for various blood cell types in Vav-iCre + ; Itgav fl/fl (KO), Vav-iCre + ; Itgav fl/+ (HT) and Vav-iCre + ; Itgav +/+ (WT) mice. WBC ( a ), monocyte ( b ), granulocyte ( c ) lymphocyte ( d ) counts in WT, HT and KO mice ( N =10, t test: ** P <0.01, * P <0.05). ( e – g ) Phenotypic analysis of BM cells by flowcytometry ( e ), to compare the numbers of KLS ( f ) and SLAM KLS ( g ) cells ( N =12, t test: ** P <0.01, * P <0.05). ( h ) Schematic representation of the competitive repopulation assay. 200 KLS cells or 10,000 WBMCs from Vav;Itgav −/− mice (CD45.2) along with 100,000 or 90,000 whole BM competitor cells (CD45.1), respectively, were transplanted into sub-lethally irradiated CD45.1 WT CD45.1 mice. PB chimerism was followed for 12 weeks, after which secondary transplantations were performed. ( i , j ) PB chimerism in primary and secondary recipients transplanted with 10,000 total BM cells from Vav-iCre + ; Itgav fl/fl (KO) and Vav-iCre + ; Itgav +/+ (WT) mice, together with 90,000 competitor cells ( n =3, N =15, t test: ** P <0.01, * P <0.05). ( k , l ) PB chimerism in primary and secondary recipients transplanted with 200 KLS cells from the BM of Vav-iCre + ; Itgav fl/fl (KO) and Vav-iCre + ; Itgav +/+ (WT) mice, together with 100,000 competitor cells ( n =3, N =12, t test: ** P <0.01, * P <0.05). ( n =independent experiments, N =number of mice. Error bars indicate mean ±s.e.m.).

Article Snippet: Flow cytometric analysis for primitive HSCs and haematopoietic progenitors was performed using anti-mouse CD48 APC and CD150 PECy7 (0.25 μg ml −1 ; ebiosciences) along with KLS cells staining (as for sorting).

Techniques: Irradiation

Journal: Nature Communications

Article Title: Outside-in integrin signalling regulates haematopoietic stem cell function via Periostin-Itgav axis

doi: 10.1038/ncomms13500

Figure Lengend Snippet: ( a ). Representative primitive HSCs (SLAM KLS cells) isolated by FACS and stained with anti-γH2AX antibodies (pseudo-color red) and Hoechst 33342 (pseudo-color green). White arrows indicate foci. ( n =4). ( b ). Representative example of primitive HSCs (SLAM KLS cells) isolated by FACS and stained with anti-RPA antibodies (pseudo-color red) and Hoechst 33342 (pseudo-color green). White arrows indicate foci. ( n =4). ( c ). Percentage of HSCs with γH2AX-marks from young Postn −/− mice (right), young WT (left), and old WT (middle) mice. ( n =4, t test: * P <0.05). ( d ). Average number of γH2AX-positive foci in primitive HSCs from young Postn −/− mice (right), young WT (left) and old WT (middle) mice. ( n =4, t test: * P <0.05). ( n =independent experiments, Error bars indicate mean ±s.e.m.).

Article Snippet: Flow cytometric analysis for primitive HSCs and haematopoietic progenitors was performed using anti-mouse CD48 APC and CD150 PECy7 (0.25 μg ml −1 ; ebiosciences) along with KLS cells staining (as for sorting).

Techniques: Isolation, Staining