Figure 2
Figure 2. The low megakaryocyte and erythroid potentials of LMPPs are highly enriched in LMPPs coexpressing cell-surface Mpl. (A) In vitro megakaryocyte (Mk) potential of BM LSKFlt3−, LSKFlt3hiMplhi (Flt3hiMplhi), and LSKFlt3hiMpl− (Flt3hiMpl−) cells, as described in “Methods” after 10 days of culture. Mean plus or minus SEM values from 7 experiments. Cell morphology pictures from typical cultures of LSKFlt3hiMplhi cells and LSKFlt3hiMpl− cells, respectively. (B) Mk potential of LSKFlt3hiMplhi and LSKFlt3hiMpl− BM cells, after 4 and 6 days of culture. Mean plus or minus SEM values from 2 experiments. (C) In vitro erythroid potential of BM LSKFlt3−, LSKFlt3hiMplhi, and LSKFlt3hiMpl− cells, as established by DAF staining of methylcellulose cultures after 12 days of culture, as described in “Methods.” Mean plus or minus SEM values from 4 experiments. (D) Total cloning frequencies (left) and erythroid potential (right) of LSKFlt3hiMplhi and LSKFlt3hiMpl− cells evaluated after 4, 6, 8, and 10 days of methylcellulose culture. Mean plus or minus SEM values from 3 experiments. (E) Left panel shows results from clonal assays of single-cell deposited LSKFlt3hiMplhi and LSKFlt3hiMpl− cells cultured in cytokines promoting GM development (“Methods”). Open bars show cloning frequencies as established after 10 days of culture, and black bars show frequency of high proliferative clones (covering > 50% of the well). Mean plus or minus SEM values from 3 experiments. Middle panel shows relative distribution between clones with monocyte (M) or combined granulocyte-monocyte (GM) contents, derived from single LSKFlt3hiMplhi and LSKFlt3hiMpl− cells as established by morphologic evaluation of MGG-stained cytospin preparations (right panels). Mean plus or minus SEM values from 2 experiments. (F) T-cell potential of single-cell deposited LSKFlt3hi (Flt3hi) and LSKFlt3hiMpl− BM cells grown for 3 to 4 weeks on OP9-DL1, as evaluated by FACS, and defined as NK1.1−Thy1.2hiCD25hi and/or NK1.1−CD4+CD8+ and negative for the viability dye propidium iodide (PI) as previously described.10,15 Mean plus or minus SEM values from 2 experiments (n = 24 per group and experiment). Right panel shows representative FACS profiles of analyzed clone derived from a single cell. The T-cell identity of NK1.1−Thy1.2hiCD25hi clones was as previously shown15 and also confirmed by nested PCR analysis demonstrating expression of CD3 antigen, epsilon polypeptide (Cd3e), and pre–T-cell antigen receptor alpha (Ptcra) (data not shown).

The low megakaryocyte and erythroid potentials of LMPPs are highly enriched in LMPPs coexpressing cell-surface Mpl. (A) In vitro megakaryocyte (Mk) potential of BM LSKFlt3−, LSKFlt3hiMplhi (Flt3hiMplhi), and LSKFlt3hiMpl− (Flt3hiMpl−) cells, as described in “Methods” after 10 days of culture. Mean plus or minus SEM values from 7 experiments. Cell morphology pictures from typical cultures of LSKFlt3hiMplhi cells and LSKFlt3hiMpl− cells, respectively. (B) Mk potential of LSKFlt3hiMplhi and LSKFlt3hiMpl− BM cells, after 4 and 6 days of culture. Mean plus or minus SEM values from 2 experiments. (C) In vitro erythroid potential of BM LSKFlt3−, LSKFlt3hiMplhi, and LSKFlt3hiMpl− cells, as established by DAF staining of methylcellulose cultures after 12 days of culture, as described in “Methods.” Mean plus or minus SEM values from 4 experiments. (D) Total cloning frequencies (left) and erythroid potential (right) of LSKFlt3hiMplhi and LSKFlt3hiMpl− cells evaluated after 4, 6, 8, and 10 days of methylcellulose culture. Mean plus or minus SEM values from 3 experiments. (E) Left panel shows results from clonal assays of single-cell deposited LSKFlt3hiMplhi and LSKFlt3hiMpl− cells cultured in cytokines promoting GM development (“Methods”). Open bars show cloning frequencies as established after 10 days of culture, and black bars show frequency of high proliferative clones (covering > 50% of the well). Mean plus or minus SEM values from 3 experiments. Middle panel shows relative distribution between clones with monocyte (M) or combined granulocyte-monocyte (GM) contents, derived from single LSKFlt3hiMplhi and LSKFlt3hiMpl− cells as established by morphologic evaluation of MGG-stained cytospin preparations (right panels). Mean plus or minus SEM values from 2 experiments. (F) T-cell potential of single-cell deposited LSKFlt3hi (Flt3hi) and LSKFlt3hiMpl− BM cells grown for 3 to 4 weeks on OP9-DL1, as evaluated by FACS, and defined as NK1.1−Thy1.2hiCD25hi and/or NK1.1−CD4+CD8+ and negative for the viability dye propidium iodide (PI) as previously described.10,15  Mean plus or minus SEM values from 2 experiments (n = 24 per group and experiment). Right panel shows representative FACS profiles of analyzed clone derived from a single cell. The T-cell identity of NK1.1−Thy1.2hiCD25hi clones was as previously shown15  and also confirmed by nested PCR analysis demonstrating expression of CD3 antigen, epsilon polypeptide (Cd3e), and pre–T-cell antigen receptor alpha (Ptcra) (data not shown).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal