Transient Inhibition of Endogenous Transforming Growth Factor-β1 (Tgfβ1) in Hematopoietic Stem Cells Accelerates Engraftment and Enhances Multi-Lineage Repopulating Efficiency

HSC transplantation efficiency can be improved by modulating cell surface molecules such as CD26 and CXCR4.1,2 This is important in cases where donor cell numbers are limiting, as in cord blood stem cell transplantation in adults where high morbidity and slow engraftment occur3 due to limiting numbers of CD34+ cells and possibly more differentiate progeny.4 The approach involving ex vivo expansion of primitive HSC to increase the total transplantable cell number has not been clinically useful.5,6 Here, we took the approach of enhancing HSC engraftment without expansion by transiently inhibiting endogenous TGFβ1 expression in HSC using minimal ex vivo manipulation. We previously reported that TGFβ1 directly and reversibly inhibits the initial cell divisions of murine LTR-HSC as well hematopoietic progenitor cells in vitro.7,8 TGFβ1 has been shown to be a primary regulator of LTR-HSC quiescence (G0) in bone marrow niches.9 Adult mice with a conditional knock-out of the TGFβ1 receptor exhibited essentially normal in vivo hematopoiesis.10 However, HSC/progenitor cells released from G0 quiescence in vitro by TGFβ neutralizing antibodies resulted in improved retroviral gene transfer.11,12 Furthermore, inhibiting Smad signaling, the intracellular regulators of TGFβ1 signaling, promoted HSC self-renewal in vivo.13 Here we studied highly enriched murine LTRHSC from lineagenegative bone marrow cells followed by FACS sequential selection of low retention of the fluorescent, viable dyes Höechst 33342 (Hö) (binding A-T base pairs of DNA) and Rhodamine 123 (Rh) (binding predominately activated mitochondria membranes). Low retention of the dyes is due to both high efflux rates and low target binding. Our previous studies showed that selecting cells that are lin-, Hö low (~G0),14 c-kit+7,15 identifies both LTRHSC and short -term repopulating (STR-HSC). A further selection step based on Rh fluorescence resolves the LTR-HSC (low Rh) and STR-HSC (high Rh). LTR-HSC are predominantly quiescent9 compared to STR-HSC, and are unique in their long-term repopulating ability due a high probability of selfreplication while at the same time generating large numbers of STRHSC16 Untreated LTR-HSC have poor short-term repopulating ability early after transplant, however over time in vivo give rise to daughter cells that possess efficient short-term repopulating ability. We show that transient inhibition of endogenous TGFβ1 in LTR-HSC ex vivo