| Effect of Csf1 knockdown on 5G3 hematopoietic support capacity. (A) The sorting strategy to isolate MPP and MDP is shown. Lin -bone marrow cells were prepared by antibody depletion using MACS ® column technology and anti-biotin microbeads (Miltenyi Biotec). For sorting progenitor subsets, cells were stained with lineage antibodies as well as fluorochrome-conjugated antibodies specific for progenitors: Sca-1, cKit, Flt3, CD150 and CD115. Cells were gated initially as Lin -PI -(live) cells. "Fluorescence minus one" controls were used to set gates to identify specific antibody binding. MPP were gated as cKit + Sca-1 + Flt3 + CD150 -cells, and MDP were further gated as cKit + Sca-1 -Flt3 + CD115 -cells. (B) Co-cultures were established by overlay of equal numbers (1 × 10 3 cells/flask) of sorted MDP and MPP above control 5G3 stroma, Csf1 knockdown stroma (CSF1(5) KD), and Igf2 knockdown stroma (IGF2 (2) KD). Non-adherent cells were collected weekly and stained with fluorochrome-conjugated antibodies specific for CD11c, CD11b, MHC-II and F4/80 to identify subsets of CD11b -CD11c -progenitor cells, CD11b + CD11c -myeloid cells, CD11b + CD11c + MHC-II + cDC-like cells and CD11b + CD11c + MHC-II -L-DC. Data are presented as cumulative cell production over time. Cultures showing significant (p 0.0417) increasing subset production over 4 time points are shown by *. (C) A model for the role of Csf1 in hematopoiesis is shown. Red bars show blocking effects of Csf1 knockdown on one of two subsets of MPP.

Contexts in source publication

Context 1

... similar finding was reported previously ( Petvises and O'Neill, 2014a). MPP co-cultured over control stroma also gave significant production of myeloid progenitors in this experiment, which was not lost through either Csf1 or Igf2 knockdown ( Figure 4B). Csf1 knockdown stroma however supported significant production of L-DC. ...

Context 2

... knockdown stroma however supported significant production of L-DC. The best explanation for this result is that L-DC progenitors are a subset of MPP and are not the cells dependent on CSF1 ( Figure 4C), so that when this gene is knocked down, there is a rebound effect with increased significant production of L-DC ( Figure 4B). In contrast, MDP co-cultures gave significant production of only the L-DC subset which was not inhibited by knockdown of either Csf1 or Igf2 in 5G3 stroma ( Figure 4B). ...

Context 3

... knockdown stroma however supported significant production of L-DC. The best explanation for this result is that L-DC progenitors are a subset of MPP and are not the cells dependent on CSF1 ( Figure 4C), so that when this gene is knocked down, there is a rebound effect with increased significant production of L-DC ( Figure 4B). In contrast, MDP co-cultures gave significant production of only the L-DC subset which was not inhibited by knockdown of either Csf1 or Igf2 in 5G3 stroma ( Figure 4B). ...

Context 4

... best explanation for this result is that L-DC progenitors are a subset of MPP and are not the cells dependent on CSF1 ( Figure 4C), so that when this gene is knocked down, there is a rebound effect with increased significant production of L-DC ( Figure 4B). In contrast, MDP co-cultures gave significant production of only the L-DC subset which was not inhibited by knockdown of either Csf1 or Igf2 in 5G3 stroma ( Figure 4B). The production of L-DC, suggests the presence of an L-DC progenitor within the sorted MDP population (Lin -Sca1 + ckit - Flt3 + CD115). ...