Journal: Circulation research

Article Title: Differentiated Smooth Muscle Cells Generate a Subpopulation of Resident Vascular Progenitor Cells in the Adventitia Regulated by KLF4

doi: 10.1161/CIRCRESAHA.116.309322

Figure Lengend Snippet: Two-month old Myh11-CreERT-βGal or -YFP mice received I mg IP tamoxifen injections once a day for five consecutive days. Aortae, carotid arteries plus aortic arch, and femoral arteries were harvested 8-weeks post-tamoxifen. (A). Aortas were whole mount stained for X-Gal; histological section from a representative aorta showing βGalactosidase activity (blue reaction color). Arrows = X-Gal-positive SMC-derived adventitial cell. Arrowheads = external elastic lamina. Dashed line represents media-adventitia boundary. (B–E). Carotid or femoral arteries were immunofluorescently stained for αSMA (red) and YFP (green; B), Sca1 (red) and YFP (green; C&D), or CD34 (red) and YFP (green; E). Representative carotid arteries are shown in (B,C,&E); representative femoral artery shown in (D). Arrow = SMC-derived adventitial cells. A = adventitia; M = media. Dashed lines represent media-adventitia boundary. (F&G). Higher magnification images of inset boxes in (B&C). Scale bars=50μm. A minimum of 8 mice were analyzed.

Article Snippet: For flow sorting, single cell suspensions were incubated with a rat anti-mouse monoclonal APC-Sca1 antibody (eBiosciences); live cells were sorted on a MoFlo high-speed cell sorter based on Sca1-APC and endogenous YFP expression.

Techniques: Staining, Activity Assay, Derivative Assay

Journal: Circulation research

Article Title: Differentiated Smooth Muscle Cells Generate a Subpopulation of Resident Vascular Progenitor Cells in the Adventitia Regulated by KLF4

doi: 10.1161/CIRCRESAHA.116.309322

Figure Lengend Snippet: (A). Carotid arteries (CA) plus aortic (Ao) arch, descending aorta, and femoral arteries were harvested from two-month old SM22α-Cre-YFP mice. Arteries from 5–7 mice were pooled, digested into single cell suspensions, labeled with an APC-conjugated anti-Sca1 antibody, and flow sorted based on endogenous YFP and Sca1 expression. The percent±SE SMC-derived YFP(+)Sca1(+) (quadrant B; Supplemental Figure 5) of total Sca1(+) cells (quadrants A + B; Supplemental Figure 5) from 7 independent sorts is shown in the table. (B). Representative immunofluorescent stains of carotid artery sections from two-month old SM22α-Cre-YFP mice for αSMA (red) and YFP (green; top panels), Sca1 (red) and YFP (green; middle panels), or CD34 (red) and YFP (green; bottom panels). Arrows = YFP(+) adventitial cells. Dashed lines represent media-adventitia boundary. A minimum of 8 mice were analyzed. Scale bars=50μm. (C). Higher magnification image of inset box in (B, middle panel). (D). Representative immunofluorescent stain of a carotid artery section from a five day-old SM22α-Cre-YFP mouse for YFP (green), Sca1 (red), and αSMA (white). Arrows = SMC-derived AdvSca1+ cells expressing residual levels of αSMA; Arrowheads = SMC-derived adventitial cells expressing residual levels of αSMA and low/absent levels of Sca1. Dashed line represent media-adventitia boundary. Scale bars=50μm.

Article Snippet: For flow sorting, single cell suspensions were incubated with a rat anti-mouse monoclonal APC-Sca1 antibody (eBiosciences); live cells were sorted on a MoFlo high-speed cell sorter based on Sca1-APC and endogenous YFP expression.

Techniques: Labeling, Expressing, Derivative Assay, Staining

Journal: Circulation research

Article Title: Differentiated Smooth Muscle Cells Generate a Subpopulation of Resident Vascular Progenitor Cells in the Adventitia Regulated by KLF4

doi: 10.1161/CIRCRESAHA.116.309322

Figure Lengend Snippet: Total RNA was isolated from cell populations from pooled, digested arteries from SM22α-Cre-YFP mice and analyzed by qPCR for the indicated mRNAs. Shown are fold changes in mRNA copy number±SE from an N=3 independent experiments using arteries from 10 pooled mice per experiment; *P<0.05. β-actin was used for normalization. (A). SMC markers. To compare among individual experiments, data was normalized to YFP(+) SMCs. (B). Progenitor cell markers expressed by both Sca1+ populations. To compare among individual experiments, data was normalized to AdvSca1-MA cells. (C). mRNAs selectively expressed in Sca1(+)YFP(−) cells. To compare among individual experiments, data was normalized to AdvSca1-MA cells. Shown for all panels are data obtained from cell populations isolated from pooled carotid arteries plus aortic arch. ND = not detectable.

Article Snippet: For flow sorting, single cell suspensions were incubated with a rat anti-mouse monoclonal APC-Sca1 antibody (eBiosciences); live cells were sorted on a MoFlo high-speed cell sorter based on Sca1-APC and endogenous YFP expression.

Techniques: Isolation

Journal: Circulation research

Article Title: Differentiated Smooth Muscle Cells Generate a Subpopulation of Resident Vascular Progenitor Cells in the Adventitia Regulated by KLF4

doi: 10.1161/CIRCRESAHA.116.309322

Figure Lengend Snippet: Single cell suspensions were obtained from digested arteries from SM22α-Cre-YFP mice. (A). Representative flow cytometry plots showing expression of CD45 and Ly6C in YFP(−) and YFP(+) cells. (B). Representative flow cytometry plots showing expression of Sca1 in CD45(−) and CD45(+) cells from YFP(−) and YFP(+) cell populations. (C). Representative flow cytometry plots showing expression of Ly6C in Sca1(+)CD45(−) and Sca1(+)CD45(+) cells from YFP(−) and YFP(+) cell populations. (D). Representative flow cytometry plots showing expression of Ly6C and CD115 in YFP(−) and YFP(+) cells. (E). Representative flow cytometry plots for expression of CD140b and CD31 in Sca1(+)CD45(−)Ly6C(−) and Sca1(+)CD45(−)Ly6C(+) cells from YFP(−) and YFP(+) cell populations. (F). Pie charts illustrating the various subpopulations of Sca1(+) progenitor cells within Sca1(+)YFP(−) and Sca1(+)YFP(+) groups. Shown for all are data obtained from cell populations isolated from carotid arteries plus aortic arch; N=4 independent analyses using pooled arteries from 5 mice.

Article Snippet: For flow sorting, single cell suspensions were incubated with a rat anti-mouse monoclonal APC-Sca1 antibody (eBiosciences); live cells were sorted on a MoFlo high-speed cell sorter based on Sca1-APC and endogenous YFP expression.

Techniques: Flow Cytometry, Expressing, Isolation

Journal: Circulation research

Article Title: Differentiated Smooth Muscle Cells Generate a Subpopulation of Resident Vascular Progenitor Cells in the Adventitia Regulated by KLF4

doi: 10.1161/CIRCRESAHA.116.309322

Figure Lengend Snippet: Two-month old SM22α-Cre-YFP mice were subjected to carotid artery ligation injury and injured left and uninjured right arteries were harvested 3-days post injury for immunofluorescence analysis. (A). Representative stains of injured vessels for αSMA (red) and YFP (green) from 2 independent mice showing expansion of SMC-derived adventitial cells. (B). Representative stain of an injured vessel for Sca1 (red) and YFP (green). (C). Representative stain of an injured vessel for CD45 (red) and YFP (green). Arrows = YFP(+)CD45(+) adventitial cells. M = arterial media; A = arterial adventitia; arrowheads = internal (A) and external elastic laminae. (D). Higher magnification images of arrows in panel (C). For A–C, dashed lines represent media-adventitia boundary. (E&F). Total numbers of adventitial Sca1(−)YFP(+), Sca1(+)YFP(+), or Sca1(+)YFP(−) cells (panel E) or CD45(−)YFP(+), CD45(+)YFP(+), or CD45(+)YFP(−) cells (panel F) in uninjured compared to injured arteries were quantitated as described in Material and Methods. Total numbers±SE are recorded in the graphs. N=3 independent mice; *P<0.05. Scale bars=50μm.

Article Snippet: For flow sorting, single cell suspensions were incubated with a rat anti-mouse monoclonal APC-Sca1 antibody (eBiosciences); live cells were sorted on a MoFlo high-speed cell sorter based on Sca1-APC and endogenous YFP expression.

Techniques: Ligation, Immunofluorescence, Derivative Assay, Staining

Journal: Circulation research

Article Title: Differentiated Smooth Muscle Cells Generate a Subpopulation of Resident Vascular Progenitor Cells in the Adventitia Regulated by KLF4

doi: 10.1161/CIRCRESAHA.116.309322

Figure Lengend Snippet: Aorta and carotid arteries plus aortic arch were harvested from 8–10-day old SM22α-CreKI-YFP WT (A) or SM22α-CreKI-YFP Klf4 KO (B) mice. Representative immunofluorescent stains of aortic sections for Sca1 (red) and YFP (green); aortae from two independent WT and KO mice shown; N=6 per genotype total. Scale bars=50μm. Dashed lines represent media-adventitia boundary. (C&D). FACS analysis was used to quantitate SMC-derived AdvSca1-SM (C) or non-SMC-derived AdvSca1-MA (D) cells in WT versus Klf4 KO mice. Each symbol represents data from an individual mouse. N=7 WT and N=5 KO mice.

Article Snippet: For flow sorting, single cell suspensions were incubated with a rat anti-mouse monoclonal APC-Sca1 antibody (eBiosciences); live cells were sorted on a MoFlo high-speed cell sorter based on Sca1-APC and endogenous YFP expression.

Techniques: Derivative Assay

Journal: Circulation research

Article Title: Differentiated Smooth Muscle Cells Generate a Subpopulation of Resident Vascular Progenitor Cells in the Adventitia Regulated by KLF4

doi: 10.1161/CIRCRESAHA.116.309322

Figure Lengend Snippet: (A&B). Isolated AdvSca1 cells from WT mice were cultured for 24 h, transfected with siRNAs targeting GFP (control) or KLF4, and evaluated 72 hrs after siRNA transfection. (A) Representative images from treated cultures with immunostaining for αSMA (green), Sca1 (red), and DAPI (blue). Sca1-positive and αSMA-positive cells from each treatment group were counted and normalized to total cell numbers. N=3 independent experiments; *P<0.05. (C&D). Isolated AdvSca1 cells from WT mice were transduced with an empty adenovirus (control) or a Klf4-expressing adenovirus then cultured for 7 days. (C) Representative images from treated cultures with immunostaining for αSMA (green), Sca1 (red), and DAPI (blue). (D) Sca1-positive and αSMA-positive were counted and normalized to total cell numbers. N=3 independent experiments; *P<0.05.

Article Snippet: For flow sorting, single cell suspensions were incubated with a rat anti-mouse monoclonal APC-Sca1 antibody (eBiosciences); live cells were sorted on a MoFlo high-speed cell sorter based on Sca1-APC and endogenous YFP expression.

Techniques: Isolation, Cell Culture, Transfection, Immunostaining, Transduction, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Cardiac Bmi1 + cells contribute to myocardial renewal in the murine adult heart

doi: 10.1186/s13287-015-0196-9

Figure Lengend Snippet: Characterization and evolution of B-CPC throughout Bmi1 -YFP mouse lifespan. a Generation of Bmi1 CreER/+ ; Rosa26 YFP/+ ( Bmi1 -YFP) mice. b Detection of the YFP + fraction (2.7 ± 0.2%, n = 21) of freshly isolated mononuclear non-cardiomyocyte heart cells from Bmi1 -YFP mice, analyzed five days post TM induction (5d-postTM); inset , Bmi1 -YFP NI (non-induced) and TM-induced Rosa26 YFP/+ negative controls. Data shown as mean ± SEM. PE, phycoerythrin. c Immunofluorescence analysis of BMI1 and SCA-1 in freshly isolated YFP + cells. Bars, 50 μm. d The B-CPC population is a subset of the SCA-1 + population (5.4 ± 0.4 %, n = 18). The plots show from ( left to right and top up to bottom ) the YFP + fraction of Bmi1 -YFP hearts 5d-postTM, the negative control from SCA-1 staining in the non-CM fraction, staining for SCA-1, and the fraction of the YFP + SCA-1 + population. Data represented as mean ± SEM. PE, phycoerythrin, SSC, side scatter. e RT-qPCR of freshly sorted Bmi1 + (YFP + ) and SCA-1 + YFP − cells ( n = three replicates; two to three mice per replicate). Data shown as mean ± SD. P values were calculated by paired Student’s t-test. * P < 0.05, ** P < 0.01. f Analysis of the YFP + compartment in Bmi1 -YFP mice at one year post-TM induction (1y-postTM) ( left ) (5.8 ± 0.74 %) and analysis of B-CPC (YFP + ) in one-year-old mice 5d-postTM ( right ) (2.2 ± 0.22 %). PE, phycoerythrin, SSC, side scatter. g YFP + cell number at 5d-postTM in young mice (two-month-old; n = 15), in mice 1y-postTM induction at six to eight weeks of age ( n = 4), and in one-year-old mice at 5d-postTM ( n = 7). Data shown as mean ± SEM. P values were calculated by unpaired Student’s t-test with Welch’s correction, compared to 5d-postTM young mice. * P < 0.05, *** P < 0.0001

Article Snippet: For flow cytometry analysis, cardiac cells from hearts of TM-induced Bmi1 -YFP mice were incubated with the following primary and secondary antibodies as indicated: APC (allophycocyanin)-conjugated rat anti-c-KIT, APC-rat anti-SCA-1/Ly6a, biotin-rat anti-SCA-1/Ly6a, biotin-rat anti-CD45, biotin-rat anti-CD31 (all at 1:100; all from BD Pharmingen, Madrid, Spain), and streptavidin-Alexa Fluor 405 conjugate (1:500; Invitrogen).

Techniques: Isolation, Immunofluorescence, Negative Control, Staining, Quantitative RT-PCR

Journal: Stem Cell Research & Therapy

Article Title: Cardiac Bmi1 + cells contribute to myocardial renewal in the murine adult heart

doi: 10.1186/s13287-015-0196-9

Figure Lengend Snippet: Evaluation of extracardiac Bmi1 + cells for de novo YFP + CM generation. a Scheme for BM (RFP + ) transplant into a lethally irradiated, TM-induced Bmi1 -YFP mouse. b - e FACS analysis of the non-CM fraction of Bmi1 -YFP mice after BM transplant ( n = 3). b No YFP + or RFP + heart cells were detected in non-induced Bmi1 -YFP mice. c FACS analysis of induced chimeric Bmi1 -YFP hearts two months post-BM transplant confirmed the presence of YFP + (3.53 ± 0.87 %) and RFP + cells (45.5 ± 15 %). d Negative control for SCA-1 and CD45 staining of the non-CM fraction of Bmi1 -YFP hearts ( left ) and percentage of SCA-1 + CD45 − cells after the staining (20 ± 3.74 %; right ). e Percentage of SCA-1 + CD45 − in RFP + cells in the non-CM fraction of chimeric Bmi1 -YFP hearts two months post-transplant (0.26 ± 0.06 %). ( f - h ) FACS and ICC of the CM compartment of chimeric Bmi1 -YFP mice two months post-transplant ( n = 3). f No RFP or YFP expression was detected in Bmi1 -YFP CM analyzed by FACS 5d-postTM ( left ). RFP + CM from Act -RFP mice ( right ). g No RFP + CM were detected in chimeric Bmi1 -YFP mice two months post-transplant. h ICC of chimeric Bmi1 -YFP CM two months post-transplant. Bars, 100 μm. Data shown as mean ± SD. YFP yellow fluorescent protein, CM cardiomyocytes, FACS fluorescence-activated cell sorting

Article Snippet: For flow cytometry analysis, cardiac cells from hearts of TM-induced Bmi1 -YFP mice were incubated with the following primary and secondary antibodies as indicated: APC (allophycocyanin)-conjugated rat anti-c-KIT, APC-rat anti-SCA-1/Ly6a, biotin-rat anti-SCA-1/Ly6a, biotin-rat anti-CD45, biotin-rat anti-CD31 (all at 1:100; all from BD Pharmingen, Madrid, Spain), and streptavidin-Alexa Fluor 405 conjugate (1:500; Invitrogen).

Techniques: Irradiation, Negative Control, Staining, Expressing, Fluorescence, FACS

Journal: Cell stem cell

Article Title: Organoids model transcriptional hallmarks of oncogenic KRAS activation in lung epithelial progenitor cells

doi: 10.1016/j.stem.2020.07.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rat monoclonal anti-Ly-6A/E (Sca1) APC/Cy7 [D7] , Thermo Fisher Scientific , RRID:AB_1727552; Cat#560654.

Techniques: Virus, Plasmid Preparation, Recombinant, Multiplex Assay, Lysis, Reverse Transcription, Mass Spectrometry, Gene Expression, Software, Saline, Gentle, Modification