The backbone vector without a gene insert was rescued as a negative control vector.
Flow cytometric analysis of UCB BM cells transduced with a negative control vector revealed a robust engraftment of human nucleated haematopoietic cell (CD45+) in transplanted recipient mice at 16 weeks after transplantation (Fig. 5b).
(a) Bar chart showing relative mRNA levels of NAP1L3 determined by qPCR of enriched CD34+ UCBs, transduced with shRNA vectors targeting NAP1L3 (NAP1L3 shRNA) or negative control vector (SC shRNA).
Subsequently, downregulation of Nap1l3 caused a marked reduction (approximately three-fold) in the total number of colony-forming units (CFUs) in the HSPCs compared to the control cells transduced with a negative control vector (Fig. 1d).
To investigate if the downregulation of Nap1l3 affects the reconstitution of haematopoietic cells in vivo, we transplanted cKit+ donor HSPCs transduced with either Nap1l3 shRNA or a negative control vector into lethally irradiated recipient mice, as in Fig. 2b e.
Wanting to further investigate the role of NAP1L3 in proliferation and differentiation of haematopoietic cells in human cells, we transduced CD34+ enriched UCB HSPCs with one of two shRNAs against human NAP1L3 or a negative control vector.
(e) Percentage of Lin+ UCBs transduced with NAP1L3 shRNA (NAP1L3 shRNA), or a negative control vector expressing scrambled shRNA (SC shRNA), determined by flow cytometry analysis according to (b), 48 hours post transduction and sorting of the cells.
When analysing the UCB HSCs transduced with NAP1L3 shRNAs, we observed a significant reduction of these primitive cells in the NSG-SGM3 recipient mice, compared to the control cells transduced with a negative control vector (Fig. 5c).
Flow cytometric analysis of bone marrow cells from the recipient mice five weeks after transplantation revealed that downregulation of Nap1l3 caused a distinct reduction of donor LSK cells (median percent of 0.4%), compared to transplanted LSK HSCs transduced with the negative control vector expressing scrambled shRNA (median percent of 2.6%) (Fig. 2c,d).
Thus, to investigate early cellular responses of primitive UCBs to downregulation of NAP1L3, we transduced sorted human (Lin−CD34+CD38−) UCB enriched HSCs (hereinafter referred to as UCB HSCs) with either two independent shRNAs against NAP1L3 or a negative control vector.
Remarkably, downregulation of Nap1l3 in LSK HSPCs led to a significant reduction of mixed myelo-erythroid CFUs (CFU-GEM) (ten-fold reduction) and a significant reduction in the number of granulocyte/macrophage CFUs (CFU-GM) (50% reduction), compared to cells transduced with a negative control vector (Fig. 1e).
