In vitro conversion of adult murine endothelial cells to hematopoietic stem cells.
In this method article, we are describing a multi-phasic approach for converting adult murine endothelial cells \(ECs) to bona fide haematopoietic stem cells \(HSCs) \(rEC-HSCs) via transient expression of four transcription factors FosB, Gfi1, Runx1, and Spi1 \(FGRS) and stimulation with angiocrine factors supplied by the vascular niche. Adult mouse ECs were isolated from Runx1-IRES-GFP; Rosa26-rtTa and maintain in culture with EC growth factor and tgfb inhibition. Induction phase \(day 0-8) of conversion is initiated by expressing FGRS in mature ECs resulting in endogenous Runx1 expression. During specification phase \(day 8-20), endogenous Runx1+ FGRS-transduced ECs commit to a haematopoietic fate and no longer require FGRS expression. The vascular-niche drives robust self-renewal and expansion of rEC-HSCs \(day 20-28). Upon conversion, rEC-HSCs are endowed with a transcriptomic signature and long-term self-renewal capacity similar to adult HSCs, and are competent for clonal engraftment and multi-lineage reconstituting potential, including antigen-dependent adaptive immune function. Our work provides a tractable strategy to interrogate the generation of engraftable hematopoietic cell, and advances the mechanistic understanding of hematopoietic development and HSC self-renewal.
Figure 1
Figure 2
Figure 3
Posted 22 May, 2017
In vitro conversion of adult murine endothelial cells to hematopoietic stem cells.
Posted 22 May, 2017
In this method article, we are describing a multi-phasic approach for converting adult murine endothelial cells \(ECs) to bona fide haematopoietic stem cells \(HSCs) \(rEC-HSCs) via transient expression of four transcription factors FosB, Gfi1, Runx1, and Spi1 \(FGRS) and stimulation with angiocrine factors supplied by the vascular niche. Adult mouse ECs were isolated from Runx1-IRES-GFP; Rosa26-rtTa and maintain in culture with EC growth factor and tgfb inhibition. Induction phase \(day 0-8) of conversion is initiated by expressing FGRS in mature ECs resulting in endogenous Runx1 expression. During specification phase \(day 8-20), endogenous Runx1+ FGRS-transduced ECs commit to a haematopoietic fate and no longer require FGRS expression. The vascular-niche drives robust self-renewal and expansion of rEC-HSCs \(day 20-28). Upon conversion, rEC-HSCs are endowed with a transcriptomic signature and long-term self-renewal capacity similar to adult HSCs, and are competent for clonal engraftment and multi-lineage reconstituting potential, including antigen-dependent adaptive immune function. Our work provides a tractable strategy to interrogate the generation of engraftable hematopoietic cell, and advances the mechanistic understanding of hematopoietic development and HSC self-renewal.
Figure 1
Figure 2
Figure 3
© Research Square 2021