53. Inducable FGFr-1 stimulates self renewal of hematopoietic cells
Authors: Michael A. Weinreich, C. Anthony Blau
Journal: Molecular Therapy (2004)

Fibroblast growth factor receptor-1 (FGFr-1) signaling has been implicated in self renewal and proliferation of hematopoietic stem cells. Our group is interested in exploiting the proliferative signals in hematopoietic cells to selectively expand transduced hematopoietic cells after transplantation. We have studied the effect of chemical induced dimerization (CID)/AP20187-inducible FGFr-1 on primary mouse bone marrow cells. For these studies we used a bicistronic vector encoding green fluorescence protein (GFP) as a marker and a fusion protein made up of the intracellular portion of FGFr-1 and the CID binding domain. To test the construct in vitro we plated transduced mouse bone marrow cells in 10% serum with or without AP20187 (100 nanomolar). While the cells without CID died within two weeks, in the culture with CID the transduced, GFP marked cells proliferated. These hematopoietic cells have proliferated in vitro for over 30 weeks. This makes FGFr-1 only the second CID dependent receptor, other than mpl, that is able to support long-term self renewal of primary hematopoietic cells. To test their differentiation potential we transplanted these cells (after 28 days of culture, and having expanded from 106 to 1.2×1012 cells) IV into lethally irradiated recipients (1050 rads) with 105 fresh bone marrow cells. We were able to detect mature, GFP + red cells and lymphocytes, B and T cells in the recipients. The production of donor derived mature cells was transient. In another experiment with cells that had been cultured for 78 days and had expanded from 106 to 4.4 × 1020 we again observed the transient production of GFP positive red blood cells. This indicates that these cells do not self-renew in vivo but did have both lymphoid and myeloid potential. We performed bone marrow transplants of freshly transduced cells and have preliminary data from the in vivo CID induced activation of FGFr-1. Two transplanted mice were treated with two weeks of daily AP20187 IP injections. GFP marked red cells increased in one mouse from 16% before CID treatment to 49 % at the peak of response three months after treatment began and in the other mouse from 10 % to 50%. A response was also seen in the platelets of both mice where the percentage of GFP positive cells at two weeks from the beginning of treatment had increased from 23 % to 47 % and from 19 % to 46 % in the respective mice. In one of the mice there was an additional response in the lymphocytes. 20 % of the T cells were GFP + at baseline and after CID treatment more than 40 % of the T cells were GFP +. Even more striking was in the B cell compartment where CID treatment drove the percentage of GFP + B cells from 25 percent to over 80 percent. From this data it seems clear that CID induced activation of FGFr-1 has a distinct in vivo effect in comparison to mpl. We currently are in the process of treating more mice to investigate this differential in vivo response to CID induced activation of FGFr-1. The CID controlled activation of FGFr-1 in hematopoietic cells may have utility for control of genetically modified cells after transplantation.