Abstract
Several studies have demonstrated that homeobox (Hox) genes are involved in the regulation of hematopoietic stem cells (HSC), and overexpression with retroviral vectors containing HOXB4 generate increased numbers of repopulating stem cells in vitro, but may also perturb differentiation of hematopoietic cells when the concentration of HOXB4 is very high. HOXA10 is expressed in primitive hematopoietic cells and myeloid progenitors. To study the effect of this gene we generated an inducible system based on a tetracycline transactivator, controlling the expression of HOXA10, aiming to study how different concentrations of HOXA10 affect the fate of hematopoietic progenitors and stem cells. We mated our tetO-HOXA10 mouse with the Rosa26rtTA strain, allowing activation of HOXA10 in all hematopoietic tissues after administration of doxycycline. Mice were born at normal ratios with no hematopoietic pathology. Inducible bone marrow was harvest and cultured for 12 days in 6 different concentrations of doxycyclin, revealing an increased proliferation at low concentrations, but a decline in proliferation capacity with higher concentrations. To verify that hematopoietic progenitors were affected, a CFU-GM colony assay was performed on cells cultured for 12 days, showing a two fold increase in the number of CFU-GM formed from the highly proliferating cells compared to wt and uninduced HOXA10 cells (p = 0.01). To study the effect of HOXA10 in more primitive cells, sorted inducible HOXA10 lin−, Sca1+, c-kit+ (LSK) cells were cultured for 13 days in different concentrations of doxycyclin. Lower concentrations of doxycyclin resulted in increased proliferation, while increasing concentrations resulted in decreased proliferation. Furthermore, using Q-RT-PCR, we found that the expression of HOXA10 was directly proportional to the concentration of doxycycline and no leakiness was detected in the uninduced LSK cells. The cultured cells were transplanted in a competitive setting into lethally irradiated mice to evaluate the repopulating ability of the expanded cells. Three weeks post BMT (short-term repopulation), intermediate levels of HOXA10 (0.08–0.2 mg/ml doxycyclin) resulted in a three-fold increase in repopulating capacity of the HOXA10 LSK cells whereas uninduced and higher levels of HOXA10 resulted in decreased reconstitution compared with fresh LSK cells (fresh LSK = 100%, intermediate: 313±182%, high: 45±35%, uninduced 35±33%, n=7 p< 0.01). However, sixteen weeks after transplantation we found that cells cultured for 13 days at intermediate levels of HOXA10 (0.08–0.2 mg/ml doxycyclin) preserved the stem cell reconstitution capacity compared to fresh LSK cells (fresh LSK = 100%, 0.2 mg/ml 153±82% n=7). Furthermore, uninduced LSK cells and higher levels of HOXA10 resulted in a 3 fold lower long-term reconstitution compared to Fresh LSK cells (0 mg/ml 34±32 %, high HOXA10 9±8% significant to both fresh cells and cells cultured in 0.2 mg/ml, p<0.003, n=7). These findings show that intermediate expression of HOXA10 can increase the short-term HSCs repopulating potential and can maintain the long-term repopulating stem cells for up to 13 days of in vitro culture. These results suggest that HOXA10 plays an important role in the regulation of HSCs and indicate that the effect of HOXA10 on stem cell fate decisions is dependent on the level of HOXA10 expression.
NHR-49 Acts in Distinct Tissues to Promote Longevity versus Innate Immunity
