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    • To test the efficiency of gene knockdown the

    2018-11-06

    To test the efficiency of gene knockdown, the ES cell lines expressing the shPU.1 constructs were differentiated into hematopoietic cells. The transcription factor PU.1 is not expressed in ES cytotoxicity assay at day 0 of differentiation and becomes expressed by day 9 when hematopoietic progenitor cells are generated (Fig. 3A). At this time point, the expression of PU.1 in the two knockdown cell lines was >80% lower than expression in the control GFP-expressing ES cell line. The day 9 hematopoietic progenitor cells were then put in liquid culture for an additional four days in a cytokine cocktail favoring myeloid cell generation. At day 13 of differentiation, the expression of PU.1 in the two knockdown lines was >90% lower than expression in the control cell line (Fig. 3A). These data demonstrate that constitutively expressed AAVS1-targeted knockdown constructs against PU.1 can successfully decrease expression levels throughout differentiation. To determine the effect of decreased PU.1 levels on hematopoietic cell development, day 9 progenitor cells were put into liquid expansion cultures and analyzed for erythroid (CD235+CD41−) and myeloid (CD18+CD45+) lineages. Both knockdown cell lines expressed almost undetectable levels of CD18 and CD45 compared to ~50% co-expression of these markers on control GFP-expressing cells, indicative of a block in myelopoiesis (Fig. 3B). Furthermore, the absolute yield of myeloid cells in the two knockdown lines was much lower compared to the GFP control cell line (Fig. 3C). These findings were confirmed by gene expression analysis of the myeloid marker cFMS, which was also drastically reduced in the PU.1 knockdown cell lines (Fig. 3D). Analysis of the erythroid lineage demonstrated an ~4-fold expansion in the percentage of CD235+ cells (Fig. 3B) with a concomitant increase in absolute yield of cells, especially in the knockdown line set 2 (Fig. 3C). These findings were confirmed by an increase in gene expression of the erythrocyte transcription factor EKLF in the two knockdown cell lines (Fig. 3D). The pan-hematopoietic marker GATA1 was unaffected and there was a minor increase in the megakaryocyte marker PF4. These data demonstrate that AAVS1-targeted knockdown of PU.1 specifically decreases the myeloid lineage and skews the progenitor cells to an erythroid cell fate.
    Discussion The CD43 promoter fragment we describe here allows tissue specific, controlled expression of a transgene specifically in cells of the hematopoietic lineages. During the differentiation of the CD43-GFP hematopoietic progenitors into the erythroid lineage it was observed that these cells began to lose the expression of GFP over time in culture. Since endogenous CD43 also decreases, this observation is most likely due to increased sensitivity of the CD43 antibody over GFP and not due to silencing of transgene expression. It has been shown that mature erythrocytes lose expression of CD43, hence this loss of expression is anticipated (Remold-O\'Donnell et al., 1987). Importantly, myeloid cells, which maintain CD43 expression in the adult, do not lose GFP expression in the reporter lines while in culture (Fig. 2C). The CD43 reporter represents a valuable tool to easily follow hematopoietic specification from ESCs in live cultures with specificity that rivals CD41 or CD45 as all hematopoietic progenitors are CD43+ (Vodyanik et al., 2006). In mouse models, a homozygous loss of PU.1 is embryonic lethal (Scott et al., 1994) or if mice are born alive, they die shortly after birth (McKercher et al., 1996). Mice that are born are found to lack any mature B and T cells, macrophages and neutrophils but have erythrocytes and megakaryocytes (McKercher et al., 1996) indicating that PU.1 is required for myeloid cell lineage development. A heterozygous loss of PU.1 is non-lethal, however it leads to a loss in the formation of multipotent myeloid progenitors (Scott et al., 1994). We demonstrate that a >80% knockdown of PU.1 in human hematopoietic cells results in a loss of myeloid lineage cells, phenocopying what occurs with a loss of PU.1 in the mouse. The differentiation of ES cells into primitive hematopoietic cells can be considered to mimic human development, thus our data suggests that during early human hematopoiesis PU.1 is also required for myeloid lineage development.