ELMO1 downmodulation in human CB CD34+ cells does not alter growth, colony formation or differentiation, but significantly decreases stem cell frequency.

<p>(A) K562 cells were transduced with control scrambled shRNA vector (shSCR) or with ELMO1-targeting shRNA vectors (shELMO1), sorted and used for RNA extraction. Quantitative PCR was performed to measure ELMO1 expression in transduced cells. ELMO1 mRNA levels were normalized against RPL27 mRNA expression. Alternatively, cells were used for Western blot analysis to determine ELMO1 protein levels. (B) FACS plots showing transduction efficiencies of cord blood (CB) CD34⁺ stem/progenitor cells transduced with shSCR or shELMO1. (C) 3×10⁵ transduced and sorted cells per group were plated in liquid culture and followed for thirty days. Cumulative cell count is showed representative of 3 independent experiments. (D) 10⁵ transduced and sorted cells per group were plated on MS5 stromal cells and kept in the co-culture for 5 weeks; cultures were demi-depopulated weekly for analysis. Weekly cumulative cell growth is shown for a representative experiment of 3 independent experiments and the average of those 3 experiments is shown in (E). (F) Suspension cells from MS5 co-cultures as described in panel D were analyzed for progenitor frequency by CFC assay. 10⁴ cells from each co-culture were plated in a CFC assay in methylcellulose in duplicate, and colonies were evaluated 2 weeks after plating. CFC cells were then harvested and 10⁵ cells were re-plated to form secondary CFCs. CFU-GM and BFU-E numbers are shown from a representative of 3 independent experiments; error bars indicate standard deviation. (G) LTC-IC frequencies were determined in bulk on MS5 stromal cells. After 5 weeks of co-culture methylcellulose was added to and colonies were scored two weeks later. CFU -GM, colony-forming unit-granulocyte-macrophage; BFU-E, burst forming unit-erythroid. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.