Functional screen identifies novel regulators of hematopoietic stem cell in vivo repopulation

Hematopoietic stem cell (HSC) transplantation is used to treat hematologic disease. Improving HSC engraftment could ameliorate transplant morbidity. Thus, understanding mechanisms regulating HSC engraftment is key. We used our recently published gene expression profiles of HSC during ontogeny and other public databases to prioritize 60 genes as putative regulators of adult HSC function. 55/60 gene candidates were enriched in Lineage-Sca-1+c-Kit+ (LSK) cells relative to downstream populations, suggesting a role at this stage. To assess a role for each gene of interest (GOI) in HSC engraftment, we designed and validated &ge 2 independent shRNAs yielding > 70% knockdown in LSK cells (48/55 GOI). LSK cells were lentivirally transduced overnight with control or individual gene-specific shRNAs and then transplanted into ablated recipients with mock-transduced LSK competitor cells congenic at the CD45 allele. Transplant occurred within 24-hours of LSK cell isolation, thus avoiding extensive ex vivo culture and allowing us to detect genes critical for efficient HSC engraftment. Peripheral blood chimerism was analyzed for > 16 weeks post-transplant. We identified 17 genes as necessary for optimal HSC engraftment (i.e. knockdown induced a significant loss of repopulation) and, remarkably, two genes whose loss enhanced HSC repopulation. shRNAs targeting each &ldquo Hit were interrogated ex vivo for non-specific effects on LSK cell viability and expansion. A 2° screen was performed to validate the results of the primary screen. Thus far, 10/13 primary &ldquo Hits have been confirmed to perturb HSC repopulating potential. The transcription factor, FoxA3, is one hit identified here as necessary for HSC repopulation. We found that that FoxA3-/- bone marrow displays a significant loss of repopulating potential > 16 weeks post-transplant, confirming the results of our screen. As FoxA3-/- long-term HSC also display reduced colony forming potential in vitro and fail to contribute to downstream progenitor compartments in transplant recipients, we propose that FoxA3 is a novel regulator of HSC differentiation post-transplant. Hematopoietic stem cell (HSC) transplantation is used to treat hematologic disease. Improving HSC engraftment could ameliorate transplant morbidity. Thus, understanding mechanisms regulating HSC engraftment is key. We used our recently published gene expression profiles of HSC during ontogeny and other public databases to prioritize 60 genes as putative regulators of adult HSC function. 55/60 gene candidates were enriched in Lineage-Sca-1+c-Kit+ (LSK) cells relative to downstream populations, suggesting a role at this stage. To assess a role for each gene of interest (GOI) in HSC engraftment, we designed and validated &ge 2 independent shRNAs yielding > 70% knockdown in LSK cells (48/55 GOI). LSK cells were lentivirally transduced overnight with control or individual gene-specific shRNAs and then transplanted into ablated recipients with mock-transduced LSK competitor cells congenic at the CD45 allele. Transplant occurred within 24-hours of LSK cell isolation, thus avoiding extensive ex vivo culture and allowing us to detect genes critical for efficient HSC engraftment. Peripheral blood chimerism was analyzed for > 16 weeks post-transplant. We identified 17 genes as necessary for optimal HSC engraftment (i.e. knockdown induced a significant loss of repopulation) and, remarkably, two genes whose loss enhanced HSC repopulation. shRNAs targeting each &ldquo Hit were interrogated ex vivo for non-specific effects on LSK cell viability and expansion. A 2° screen was performed to validate the results of the primary screen. Thus far, 10/13 primary &ldquo Hits have been confirmed to perturb HSC repopulating potential. The transcription factor, FoxA3, is one hit identified here as necessary for HSC repopulation. We found that that FoxA3-/- bone marrow displays a significant loss of repopulating potential > 16 weeks post-transplant, confirming the results of our screen. As FoxA3-/- long-term HSC also display reduced colony forming potential in vitro and fail to contribute to downstream progenitor compartments in transplant recipients, we propose that FoxA3 is a novel regulator of HSC differentiation post-transplant.