Nfi Genes Are Novel Regulators Of Murine Hematopoietic Stem- and Progenitor Cell Survival

Hematopoietic stem cells (HSCs) are responsible for life-long maintenance of hematopoiesis.

HSC transplantation represents one of the most heavily exploited cell based therapies, routinely used to treat a myriad of life threating disorders, such as leukemia and bone marrow failure. Identifying the molecular pathways that regulate HSC engraftment is crucial to further improving outcomes in patients that rely on HSC transplantation as a curative therapy.

By examining the global gene expression profiles of highly purified HSC (Lineage-Sca-1+c-Kit+CD150+CD48-), we recently identified the following members of the Nfi gene family of transcription factors as highly expressed by HSC (McKinney-Freeman et al., Cell Stem Cell, 2012): Nfix, Nfia, and Nfic. These data suggest that Nfi genes may play a novel role in regulating HSC function. To test this hypothesis, HSCs were enriched from adult bone marrow (Lineage-, c-kit+, Sca-1+ (LSK) cells) and then transduced, individually, with lentiviruses carrying shRNAs targeting each Nfi gene. Twenty-four hours post-transduction, cells were injected into lethally irradiated mice along with untransduced bone marrow LSK competitor cells congenic at the CD45 allele. The peripheral blood of recipient mice was then analyzed periodically over 16 weeks for engraftment of the Nfi-depleted cells. Although shRNA mediated knockdown of Nfi gene expression had no effect on the in vitro cell growth or viability of LSK cells, Nfi-depleted HSCs displayed a significant loss of short- and long-term in vivo hematopoietic repopulating activity. This was true for Nfia-, Nfic-, and Nfix-deficient HSC. While Nfia and Nfic are only expressed by bone marrow HSC, Nfix is highly expressed by both bone marrow and fetal liver HSC. When Nfix was depleted by shRNAs from LSK cells purified from E14.5 fetal liver, a similar loss in competitive repopulating potential was seen. Lineage analysis of peripheral blood of recipients showed no significant differences in the distribution of the major blood lineages derived from LSK cells transduced with Nfi-specific shRNAs compared to controls. When the bone marrow of recipients transplanted with Nfix- depleted cells was examined 4 and 16 weeks post-transplant, a general loss of all hematopoietic stem- and progenitor compartments examined was seen relative to control. Thus, the observed decrease in repopulating activity occurs at the level of HSCs and multipotent progenitors. To confirm an essential role for an Nfi gene family member in the regulation of HSC engraftment post-transplant, LSK cells were purified from Nfix fl/fl mice, transduced with lentiviral Cre recombinase and subsequently introduced into lethally irradiated recipients alongside congenic competitor cells. Like LSK transduced with Nfix-specific shRNAs, Nfix^-/- LSK cells failed to repopulate the peripheral blood of recipient mice as efficiently as control and similar trends were detected in all stem- and progenitor cell populations examined.

Time-course experiments immediately following transplantation revealed that Nfix-depleted LSK cells establish themselves in the marrow of recipient mice as efficiently as control at 5 days post-transplant, but thereafter exhausted rapidly. Examination 10 days post-transplant revealed a 5-fold increase in apoptosis specifically in the LSK compartment, but not in its differentiated progeny, in recipients transplanted with Nfix-depleted LSK cells compared to control. The increase in apoptosis was not associated with any apparent change in the cell cycle status of the LSK cells. These data suggest that Nfi genes are necessary for the survival of HSC post-transplantation.

In an effort to identify the molecular pathways regulated by Nfi genes in HSC, we acquired the global gene expression profiles of Nfix-depleted HSC. In agreement with our observation that Nfix-deficient HSC displays elevated levels of apoptosis following transplantation in vivo, we observed a significant decrease in multiple genes known to be important for HSC survival, such as Erg, Mecom and Mpl, in Nfix-depleted HSC.

In summary, we have for the first time established a role for the Nfi gene family in HSC biology, as evident by a decrease in bone marrow repopulating activity in Nfi-depleted HSCs. By dissecting the precise role of Nfi genes in HSC biology, we will glean insights that could improve our understanding of graft failure in clinical bone marrow transplantations.