Epidermal Growth Factor Rejuvenates Aging Hematopoietic Stem Cells

Aging hematopoietic stem cells display distinct abnormalities such as myeloid skewing, decreased repopulating capacity, and leukemia predisposition. Radiation exposure potentiates aging of the hematopoietic system, as evidenced by premature and persistent senescence of HSCs in mice exposed to moderate dose irradiation (Blood, 2014. 123(20): p. 3105-15). Our laboratory demonstrated that EGF promotes hematopoietic stem cell (HSC) regeneration after radiation injury (Nat Med, 2013. 19(3): p. 295-304). Based on these results, we hypothesized that EGF and EGFR signaling may have rejuvenating effects on aging HSCs.

For comparisons of young and aged mice, we utilized 2-4 month old mice and >18-24 month old female C57BL/6 mice, respectively. We discovered that aged C57BL/6 mice have decreased levels of EGF in the peripheral blood compared to young mice (p= 0.02) and decreased expression of EGFR on bone marrow (BM) ckit+sca-1+lin- (KSL) stem/progenitor cells (p= 0.03). BM KSL cells from aged mice displayed increased DNA damage in culture with thrombopoietin, SCF and Flt-3 ligand (TSF) compared to young KSL cells (p< 0.0001). Aged BM KSL cells treated with 100 ng/ml EGF had decreased DNA damage compared to aged BM KSL cells treated with TSF only (p< 0.01) Treatment with EGF for 7 days increased the colony forming capacity of aged BM KSL cells (p< 0.01). Importantly, competitive repopulation assays revealed that 7 day in vitro treatment of aged BM KSL cells with EGF significantly increased primary donor cell engraftment (p< 0.01) at 16 weeks compared to aged BM KSL cells cultured with TSF alone.

Based on these in vitro observations, we next tested whether systemic administration of a recombinant, pegylated EGF (pEGF), 20 mcg three times weekly for 4 weeks could alter the hematopoietic characteristics of aged C57BL/6 mice. pEGF treatment decreased BM myeloid skewing (p< 0.01) and increased CD3 T cell content (p< 0.01) in aged mice compared to saline-injected controls. Furthermore, competitive repopulation assays revealed that pEGF treatment of aged mice caused a significant increase in functional HSCs capable of competitive engraftment in recipient congenic mice. Total primary donor cell (p< 0.01), myeloid cell (p< 0.01), T cell (p< 0.01) and B cell (p= 0.01) engraftment were significantly increased in recipient mice transplanted with BM cells from aged, pEGF-treated donors compared to recipients of aged, saline-treated donors at 20 weeks. Additionally, secondary transplantation also resulted in increased donor engraftment (p< 0.01) and increased donor myeloid engraftment (p< 0.05) at 12 weeks. These results suggested that systemic administration of pEGF rejuvenates the repopulating ability of aged HSCs.

We next sought to determine if deficiency in EGFR signaling could accelerate hematopoietic aging in mice. For this purpose, we utilized a doxycycline-inducible, hematopoietic cell specific EGFR dominant negative mutant model (SCL-tTA;EGFR-DN mice). Interestingly, adult (13 month old) SCL-tTA;EGFR-DN mice displayed increased myeloid skewing in the peripheral blood (p< 0.05) and BM cells from SCL-tTA;EGFR-DN mice generated significantly decreased colony forming cells, compared to age-matched, EGFR-expressing mice (p< 0.01). Mechanistically, BM KSL cells from 13 month old SCL-tTA;EGFR-DN mice demonstrated increased senescence (p< 0.01) and increased expression of p16 (p< 0.05) compared to young mice. In contrast, BM KSL cells from aged C57BL/6 mice that were treated with 100 ng/ml EGF (plus TSF) for 7 days displayed decreased senescence and decreased p16 expression compared to BM KSL cells from aged mice treated with TSF alone. Taken together, these studies suggest that EGF/EGFR signaling declines with age and that reactivation of EGFR signaling via EGF treatment can ameliorate clinically relevant features of hematopoietic aging, including HSC self-renewal capacity.