recombinant mouse pdgfa  (Novus Biologicals)

Journal: bioRxiv

Article Title: Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells

doi: 10.1101/2020.12.20.423651

Figure Lengend Snippet: Data for 46C cells (which have Sox1 promoter driving GFP expression) differentiating towards NE lineage in N2B27 (without any inducers such as RA) that were previously self-renewing in serum+LIF (see STAR Methods). Same RNA-Seq dataset as in . To identify secreted factors other than FGF4 that might contribute to determining a population survival, we performed RNA-Seq to detect expression of any secreted factors that are known to control cell proliferation and/or death. We performed RNA-Seq on four populations: (1) pluripotent population prior to differentiation; (2) low-density (862 cells/cm 2 ) population; (3) high-density (5172 cells/cm 2 ) population; and (4) medium-density (1931 cells/cm 2 ) population that is near the threshold density. For the three differentiating populations, we collected their cells on the first and second day after triggering differentiation. Expression levels (FPKMs) of secreted factors that are known to control proliferation and/or apoptosis in ES cells and that fall within the range of molecular weights that the membranefilter experiments identified (50 – 300 kDa with +/-50% error) . Shown are the following genes: Ctgf, Scf, Ppia, Clu, Vegfa, Vegfb, Cyr61, Fgf5, Pdgfa, Fgf4 and Hspa8 . Below each gene name is the molecule’s weight (kDa) according to two online resources: Uniprot and ExPASy. n = 3 for all plots; Error bars are s.e.m.

Article Snippet: After 2 days of culturing in N2B27, we added 500 nM of Retinoic Acid, and one or combinations of the following recombinant proteins to the medium: 200 ng/mL of recombinant mouse FGF4 (R&D Systems, #7486-F4), 200 ng/mL of recombinant human FGF5 (R&D Systems, #237-F5), 100 ng/mL of recombinant mouse PDGFA (Novus, NBP1-43148), 100 ng/mL of recombinant mouse VEGFB 186 (Novus, #767-VE), 100 ng/mL of recombinant mouse VEGFA (Novus, #493-MV), 500 ng/mL of recombinant human CYR61/CCN1 (Novus, #4055-CR), 500 ng/mL of recombinant human CTGF/CCN2 (Novus, #9190-CC), 200 ng/mL of recombinant mouse CLU (Novus, #2747-HS), 500 ng/mL of recombinant human HSPA8/HSC70 (Novus, #NBP1-30278), 1000 ng/mL of recombinant human Cyclophilin A (PPIA) (Novus, #NBC1-18425), or 2000 ng/mL of mouse recombinant SCF (STEMCELL, #78064).

Techniques: Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells

doi: 10.1101/2020.12.20.423651

Figure Lengend Snippet: The RNA-Seq revealed that 11 secreted factors that are known to control cell proliferation/and or death were highly expressed in differentiating, high-density populations. We thus reasoned that one or combinations of these factors may be the secreted molecule(s) that determine the survival-versus-extinction fate of a population. Data in (A) and (B) for 46C cells (which have Sox1 promoter driving GFP expression) differentiating towards NE lineage in N2B27+RA that were previously self-renewing in serum+LIF (see STAR Methods). (A) We tested these molecules by adding them one-by-one into the medium of a low-density population (862 cells/cm 2 ) that would ordinarily become extinct. We added the following molecules individually, each at a saturating concentration (also see STAR Methods): version of recombinant mouse FGF4 used in (200 ng/mL), recombinant human FGF5 (200 ng/mL), recombinant mouse PDGFA (100 ng/mL), recombinant mouse VEGFB 186 (100 ng/mL), recombinant mouse VEGFA (100 ng/mL), recombinant human CYR61 (500 ng/mL), recombinant human CTGF (500 ng/mL), recombinant mouse CLU (200 ng/mL), recombinant human HSPA8 (500 ng/mL), recombinant human CYPA (1000 ng/mL), and recombinant mouse SCF (2000 ng/mL). After 6 days in a medium containing one of these molecules, we measured the fold-change in density (black bars) and differentiation efficiency (green bars) of the low-density population. n = 3; error bars are s.e.m. These results show that only the recombinant mouse FGF4 causes the fold-change in population density to be higher than one. All the other factors resulted in the low-density population either approaching extinction (fold change much less than 1) or becoming extinct (indicated with an asterisk). The black dashed line marks the maximum fold-change in population density achieved when the low-density population grows in the medium of a high-density population. The green dashed line marks the maximum differentiation efficiency achieved when the low-density population grows in the medium of a high-density population. The box beneath the plot shows which signaling factors were mixed together and then given to the low-density population in (B). (B) Results obtained by giving combinations of the 11 factors together to the low-density population, with the ingredients of the mixture indicated in the box below (A). Giving all 11 factors together at once yielded the highest growth (~4-fold increase in population density; black bar), which was virtually identical to the growth obtained with a high-density population’s (5172 cells/cm 2 ) medium (black dashed line). But, with the 11 molecules added together at once, the differentiation efficiency (green) remained rather low at ~20% compared to the ~40% (green dashed line) that we get from incubating the low-density population in the medium of a high-density population. As we progressively reduced the number of signaling factors in the mixture from 11 to 2, we observed only a modest decrease in population growth, down to about ~2 fold. Importantly, recombinant FGF4 was included in all these mixtures.

Article Snippet: After 2 days of culturing in N2B27, we added 500 nM of Retinoic Acid, and one or combinations of the following recombinant proteins to the medium: 200 ng/mL of recombinant mouse FGF4 (R&D Systems, #7486-F4), 200 ng/mL of recombinant human FGF5 (R&D Systems, #237-F5), 100 ng/mL of recombinant mouse PDGFA (Novus, NBP1-43148), 100 ng/mL of recombinant mouse VEGFB 186 (Novus, #767-VE), 100 ng/mL of recombinant mouse VEGFA (Novus, #493-MV), 500 ng/mL of recombinant human CYR61/CCN1 (Novus, #4055-CR), 500 ng/mL of recombinant human CTGF/CCN2 (Novus, #9190-CC), 200 ng/mL of recombinant mouse CLU (Novus, #2747-HS), 500 ng/mL of recombinant human HSPA8/HSC70 (Novus, #NBP1-30278), 1000 ng/mL of recombinant human Cyclophilin A (PPIA) (Novus, #NBC1-18425), or 2000 ng/mL of mouse recombinant SCF (STEMCELL, #78064).

Techniques: RNA Sequencing Assay, Expressing, Concentration Assay, Recombinant