Loss of Krüppel-Like Factor 4 (KLF4) Leads to Increased Self-Renewal Under Stress Conditions and Improved Survival of Hematopoietic Stem Cells

Abstract 861

Hematopoiesis is a highly regulated process in which a small number of hematopoietic stem cells (HSC) generate all mature blood cells. In order to preserve homeostasis of the hematopoietic system throughout lifetime, this pool of HSC must be maintained by the processes of self-renewal and survival. Self-renewal requires a coordination of survival signals and control of proliferation uncoupled from differentiation. Even though extrinsic signals from the microenvironment and cell-intrinsic regulators are required for self-renewal of HSCs, the intricate transcriptional machinery that selectively regulates HSC self-renewal and survival is still poorly understood. Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor that regulates proliferation, differentiation, apoptosis, and self-renewal. The role of KLF4 in reprogramming adult somatic cells into pluripotent stem cells along with OCT3/4, c-Myc and SOX2 suggests that KLF4 is required for preservation of an undifferentiated state. To investigate the function of KLF4 in HSC maintenance, we used conditional Klf4 knockout mice (Klf4^fl/flVav-iCre⁺) to specifically delete KLF4 gene in hematopoietic cells. We first analyzed the frequency of HSC and progenitor cells in the bone marrow (BM) of Klf4^fl/flVav-iCre^– (control) and Klf4^fl/flVav-iCre⁺ (knockout) by flow cytometry. We found that KLF4 deficiency leads to a 2.4-fold increase in the number of long-term HSC (Lin^–Sca-1⁺c-Kit⁺ CD150⁺ CD48^–) and a 2.2-fold increase in short-term HSC compartements (Lin^–Sca-1⁺c-Kit⁺ CD150⁺ CD48⁺) whereas no significant changes were found in myeloid and lymphoid progenitor cells. Consistent with this phenotypic analysis, KLF4 expression in HSC is higher than hematopoietic progenitor cells and mature lineages (n=3; P<0.05). Even though ablation of Klf4 gene does not affect multi-lineage potential of HSC upon transplantation, its deletion leads to a reduction of monocytes and T cell expansion. To assess the effect of Klf4 ablation in self-renewal, we performed serial competitive repopulation assays using a 1:1 mixture of BM cells from control (Klf4^fl/flVav-iCre^–; CD45.2⁺) or knockout (Klf4^fl/flVav-iCre⁺; CD45.2⁺) with B6.SJL (CD45.1⁺) mice. In primary transplants, the contribution of knockout BM cells in peripheral blood was similar to controls. Interestingly, loss of KLF4 led to enhanced contribution to peripheral blood in secondary transplants (4.5-fold; P<0.005) and tertiary transplants (2.6-fold; P<0.005). Consistent with this result, we found a significant increased number of colony forming units only in the third replating on methylcellulose (P<0.0005). To further characterize the role of KLF4 in HSC proliferation, we determined expression of Ki-67 and DNA content in nuclei of LSK CD150⁺ cells. The fraction of G0 cells defined as Ki-67^– within 2n DNA in Klf4-knockout LSK CD150⁺ cells was similar to control (control 74.3 ± 0.7% vs 73.2 ± 2.3%). However, Annexin V staining revealed a 2.4-fold increased survival of LSK CD150⁺ cells in Klf4-knockout mice compared to control mice but not in myeloid progenitor cells (Lin^–c-Kit⁺Sca-1^–) suggesting that KLF4 selectively regulates the survival of HSC. These studies indicate that KLF4 controls steady state HSC survival and self-renewal under stress conditions.