Forward RNAi screens in primary human hematopoietic stem/progenitor cells

Abstract

The mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral short hairpin RNA (shRNA) library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation, we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex, pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion, respectively, thereby demonstrating the potential of this forward screening approach in primary human stem cell populations.

Figure 1

RNAi screen in human CB CD34 cells. (A) Overall design of the screening strategy. Large numbers of primary CB CD34⁺ cells are infected with the lentiviral short hairpin RNA (shRNA) library and subsequently passaged in long-term cultures (10 weeks) followed by colony-forming cell (CFC) assays to positively select for clones that have acquired enhanced self-renewal/proliferation ability. Potential hits are identified by sequence analysis of proviral inserts from the selected cells. (B) CFC levels after 10 weeks culture. Twelve pools of library-transduced cells were independently assayed. Control pools were transduced with shRNA against GFP in 2 pools, and 1 pool was left untransduced (mock). Pool 7 showed high levels of BFU-E growth indicated by *. (C) Distribution of proviral shRNAs among the screening pools that showed increased CFC levels. The graph shows relative abundance of shRNAs in each screening pool as an overlay on the colony numbers shown in panel B.

Figure 2

Validation of candidate shRNAs. Cells were transduced with individual shRNA vectors and cultured for several weeks. Total colony-forming cell numbers (A) as well as the frequency of CFU-E/BFU-E colonies (B) were measured weekly. Results from 2 experiments are shown.

Figure 3

Target validation for EXT1. (A) shRNA vectors targeting different regions of the EXT1 transcript were tested for their ability to induce an effect on erythroid progenitor activity as measured by frequency of erythroid progenitors 1 week after transduction Results are given as mean values (± SEM); *P < .05 (Student t test). (B) The same constructs were tested for their ability to knock down the EXT1 transcript as measured by quantitative (q)–PCR. Results from 3 independent experiments are shown. To assess differences in relative knockdown efficiency, a paired t test was used to test significance when comparing shEXT1 and shEXT1-3 with any of the other shRNAs. P values are indicated for the comparisons with shEXT1-4 and shEXT1-5. For comparisons with the other shRNAs P values were below .02.

Figure 4

Markedly increased numbers of LTC-ICs induced by shSTK38. The frequency of LTC-ICs 1 week after transduction is shown for 2 independent experiments. The error bars give the 95% confidence interval from the limiting-dilution LTC-IC calculations.

Figure 5

Expansion of NOD/SCID repopulating cells by shSTK38. (A) Transduced CD34⁺ cells were transplanted into NOD/SCID mice, either 1 day after transduction or after 11 and 26 days of ex vivo culture. The transplanted cell doses were expansion equivalents of the indicated numbers of input day 0 CD34⁺ cells before transduction. The level of human engraftment in bone marrow 7 weeks after transplantation is shown. (B) Day 26 shSTK38 expanded cells show lymphoid and myeloid engraftment in NOD/SCID mice. FACS plots from a representative mouse. (C) Day 26 shSTK38 expanded cells engraft in secondary recipient NOD/SCID mice. Half a femur equivalent of bone marrow from primary recipient mice was transplanted to a total of 10 secondary recipients. Engraftment levels were analyzed in bone marrow after 7 weeks. FACS plots showing engrafted human cells in 1 of 2 recipients with a clear contribution of human cells.