Inhibition of endogenous transforming growth factor beta-1 in long term repopulating hematopoietic stem cells by morpholinos prior to transplant accelerates engraftment while reducing the number of stem cells required for long-term repopulation

Transforming growth factor-beta 1 (TGF-beta) has pleiotrophic effects on stem and progenitor cells. We previously showed that TGF-beta directly inhibits the initial cell divisions of murine long term repopulating-hematopoietic stem cells (LTR-HSC) in vitro (Sitnicka et al. BLOOD 88:82,1996). We recently reported that a cell surface blockade of endogenous, autocrine TGF-beta results in the survival of single or multiple LTR-HSC cultured in the absence of growth factors (Ruscetti et al. BLOOD 94, 466a,1999). These studies have been extended to include the kinetics of hematopoietic reconstitution of LTR-HSC after a blockade of endogenous TGF-beta. We found that even a short (20 minute) exposure of purified LTR-HSC to a neutralizing anti-TGF-beta antibody dramatically reduced the time required for engraftment of LTR-HSC. Subsequent experiments have shown that MAS also induce LTR-HSC survival, reduce the number of LTR-HSC required for hematopoietic repopulation, and decrease the time to engraftment. Briefly, LTR-HSC were purified from B6SJL mice(CD45.1+), treated ex vivo with anti-TGF-beta MAS (or scrambled MAS) or neutralizing anti-TGF-beta monoclonal antibodies (or isotype antibodies) then transplanted into 950 rad irradiated congenic C57B16 (CD45.2). As expected, transplantation of 100 untreated LTR-HSC or control treated LTR-HSC (scramble MAS or isotype antibodies) required competitor or support marrow to prevent hematopoietic death and generated only low donor cell chimeras (10-30%) between 1.5 to 6 months. In contrast, 100 LTR-HSC exposed to anti-TGF-beta MAS for 16 hours engrafted rapidly to reach donor cell chimerism of 50-60% by 3 Weeks and 70-85% by 1.5 months. The early engraftment was predominantly donor neutrophils followed by B-cells and then T-cells by 1.5 months. This rapid engraftment of LTR-HSC did not impair their LTR ability as measured by sustained high % donor chimeras. Finally, we found that a blockade of endogenous TGF-beta in LTR-HSC dramatically reduced the number required to rescue a lethally irradiated congenic recipient While more than 1000 control LTR-HSC per mouse were required for hematopoietic rescue, as few as 50-100 anti-TGF-beta MAS treated cells would rescue a recipient. Translation of this murine ex vivo method to human cord blood or adult stem cell transplants could reduce the time to engraftment plus lower the number of stem cells required for a durable graft.