Effects of a GSK-3 inhibitor on retroviral-mediated gene transfer to human CD34+ hematopoietic progenitor cells.

摘要

Hematopoietic stem cells (HSC) are rare cells of the hematopoietic system with the ability to self-renew and differentiate into all mature blood lineages. Although HSC are an extremely attractive target for HSC gene therapy and despite successful use of HSCs in the clinic and in clinical gene therapy trials, limitations exist. One limitation is the transduction protocol used requiring a combination of cytokines to promote HSC into cell cycle, since gamma-retroviral vectors can only transduce dividing cells. The Wnt/beta-catenin pathway has been shown to be involved in stem cell fate determination and self-renewal. In these studies we activate the Wnt/beta-catenin pathway using a GSK3 inhibitor, BIO, and test its effects on gamma-retroviral transduction, homing, engraftment and hematopoiesis potential of human umbilical cord blood (UCB) CD34+ cells. We initially explored different methods to activate the Wnt/beta-catenin pathway in CD34+ cells. We designed and tested siRNA's to Axin and used GSK-inhibitors in order to disrupt the destruction complex thereby stabilizing beta-catenin. GSK3 inhibitor BIO showed the most promise. We found BIO treatment led to increased accumulation of beta-catenin in UCB CD34+ cells. We observed increased beta-catenin activity following BIO treatment, analyzed by TOPflash reporter assay and upregulation of Ccnd1 expression compared to the untreated control. We determined that BIO initially increased UCB CD34+ cells progression through cell cycle but then imposed an arrest in cell division. BIO decreased cell apoptosis and did not impair UCB CD34+ cells homing, engraftment and hematopoiesis potential. BIO treatment of CD34+ cells allowed an increased extent of gene transfer using a gamma-retroviral vector. Future studies on the effects of the BIO induced cell arrest will allow for a better understanding of the effects that GSK inhibition has on HSC function. BIO mediated manipulation of the Wnt pathway shows promise as a useful method to support gamma-retroviral vector mediated gene transfer to HSC for gene therapy.