In Vitro Expansion of Haematopoietic Stem Cells without Permanent Genetic Modification

Abstract

Limited availability of adult stem cells is a major draw back to their use both in regenerative medicine and in the treatment of malignant disease. A rapidly growing body of evidence has shown that enforced expression of small panel of genes, including Oct-3/4, KLF4, Sox2 and c-Myc, can induce the reprogramming of previously differentiated cells, to generate pluripotent stem (iPS) cells. However, the generation of iPS by genetic modification is highly desirable. Using GFP and Apoptin as model proteins, we have recently described a strategy for the generation of cell lines that secrete proteins that carry a modified HIV-TAT based secretable protein transduction domain. The presence of secreted Oct-3/4 and KLF4 in the culture medium of the producer 293T cell, we have not been able to demonstrate the uptake of this protrein by target cells. The biollogical consequences, and the reversibility of the induced effects of Oct-3/4 protein transduction into JURKAT and FDCP-1 cells are now under investigation. With a similar objective for the transient, non-genetic modification of target cells with induced pluripotency factors, we have also started the generation of non-integrating lentivirus vectors for the transient expression of the induced pluripotency factors. In preliminary studies we have already shown the efficient expression of Oct-3/4, KLF4 and Sox-2 in CD34+ primary human cells infected with integrating lentivirus vectors encoding each of these factors. The combined enforced expression of all three factors appear to generate a surface adherent, embryonic stem cell-like pheotype.

Date of Award	2011
Original language	English
Awarding Institution	King's College London
Supervisor	Farzin Farzaneh (Supervisor) & Joop Gaken (Supervisor)