Cord blood is a recent advance in marrow replacement therapy, allowing much less frequent and severe graft-vs-host disease from a reliable, easily procured source. Unfortunately, delayed production of white cells and platelets, especially in larger patients, is a life-threatening and expensive problem. Ways of expanding stem and progenitor cells have been reported, and even used in human trials. To date, no improvement in speed of recovery has been reported. This may be due to inadequate numbers of early stem cells, or to lack of more committed progenitors. The proposed study will alter the earliest hematopoietic cells by inserting genes to be controlled by exogenous small molecules. Genes that promote stem cell self-renewal and, separately, inhibit programmed cell death (apoptosis), will be used. With this new control system, the cells will respond without altering the extant cellular machinery. The control molecules will either not be naturally occurring or will be found in very low amounts in animals. Once the genes are inserted, the cells will be signaled to multiply without differentiation (self-renew) and without apoptosis, causing birth of many more early blood-forming cells. Then a portion of the cells will be expanded with growth factors that will cause differentiation, by turning off the self-renewal gene and continuing to block apoptosis. For in vivo use, apoptosis will be allowed to start again under normal cell control before the cells are infused, to prevent possible uncontrolled growth. The early cells will provide long-term engraftment and the differentiated cells will provide rapid recovery. Control of the two inserted genes will be possible after infusion, should it be needed. The effect of the control molecules on blood cell formation kinetics once the cells are in place in the animals will also be studied. At each stage of the project, expanded cells will be evaluated for surface markers and gene expression using flow cytometry, in situ hybridization, and PCR, as well as in vitro colony forming and long term culture initiating cell systems, and in vivo in appropriately selected mice. This project will introduce a control system that will be silent in the absence of control molecules, and will allow exploration of the use of large numbers of blood-forming cells both in the lab and, possibly, clinically. In addition to marrow replacement, the cells may be usable for the production of other cells and tissues, especially given recent discoveries regarding stem cell plasticity.
| Zhong, Yiming; Sullenbarger, Brent; Lasky, Larry C (2010) Effect of increased HoxB4 on human megakaryocytic development. Biochem Biophys Res Commun 398:377-82 |
| Sullenbarger, Brent; Bahng, Joong Hwan; Gruner, Ryan et al. (2009) Prolonged continuous in vitro human platelet production using three-dimensional scaffolds. Exp Hematol 37:101-10 |
| Vanderbyl, Sandra L; Sullenbarger, Brent; White, Nicole et al. (2005) Transgene expression after stable transfer of a mammalian artificial chromosome into human hematopoietic cells. Exp Hematol 33:1470-6 |
