PTEN is a non-redundant phosphatase that functions as a negative regulator of the PI3K-AKT pathway, which has important roles in cell proliferation, survival and differentiation (5
). PTEN deficiency leads to accumulation of PIP3
which in turn activates several signaling molecules. Among them, Akt has been best characterized and its overexpression results in enhanced self-renewal of HSPC (9
). A number of substrates have been identified for Akt including the proapoptotic factors BAD, caspase 3, and 9 (10
). Phosphorylation of these molecules leads to changes in their subcellular localization, activities, or half lives, which in turn controls cell metabolism, cell death, cell cycle progression, and cell differentiation. Permanent inactivation of PTEN showed short-term expansion of HSPCs, but long-term decline as determined by their impaired ability to sustain hematopoietic reconstitution. Mice with PTEN-deficient BM had abnormal lineage fate determination and eventually developed myeloproliferative disorders (3
In the present study, we sought to determine whether siRNA-based transient silencing of PTEN in human CD34+ cells could promote HSPC proliferative potential in vitro
and improve human cell engraftment in NSG mice. siRNA are relatively unstable in blood and serum, as they are degraded by endo- and exonucleases, so their action is transient (11
). They have been directly delivered into mammalian cells via nucleofection or, alternatively, using engineered viral vectors. Viral strategies are time consuming, require special safety precautions and, unless viral vectors are modified to eliminate their innate ability to integrate in the genome, their silencing effects are permanent. In this study, we directly transferred siRNA via nucleofection to achieve the desired transient silencing of PTEN. The reported (12
) and our observed cell survival rates after nucleofection were approximately 50%. Less toxic methodologies for introducing the siRNA molecules into the target human HPSCs must be developed, for instance non-integrating lentiviral vectors, before this approach could be applied clinically.
Consistent with findings in the mouse, suppression of PTEN in human CD34+ cells led to their proliferation in vitro
and progression from quiescence into active cycle. We hypothesized that the increased proportion of cycling CD34+ cells by transient PTEN suppression may enhance their susceptibility to retroviral transduction and may improve results in gene therapy protocols. Transduction efficiencies in the bulk CD34+ cells transfected with PTEN1 and PTEN2 siRNAs were significantly higher compared with CD34+ cells transfected with a control siRNA. Others have reported that reducing the levels of both TGF-β with antibodies and p27kip-1 with antisense oligonucleotides were required to stimulate cell cycle entry and increase gene transduction efficiencies in human hematopoietic cells (13
). In contrast, the siRNA approach used in this study provides a relatively simple method for ex vivo
manipulation and gene transduction of human CD34+ cells. Transplantation studies in NSG mice will be required to determine if improved transduction of cells with in vivo
repopulating potential is achieved.
In mice, permanent deletion of PTEN increased HSPC proliferation and led to their depletion by inhibition of self-renewal (3
). PTEN was also found to have a role in controlling hematopoietic lineage fate, as evidenced by an increased representation of myeloid and T-lymphoid lineages, and the decline in B-lineage numbers in PTEN mutants (3
). We investigated the impact of transient PTEN silencing on the proliferative and engraftment potential of human CD34+ cells after transplantation into NSG mice. Mice were transplanted with PTEN or control siRNA treated CD34+ cells immediately after nucleofection. An increase in the number of human CD45+ cells was found in mice transplanted with CD34+
cells treated with PTEN1 siRNA compared with mice transplanted with control siRNA treated CD34+
cells. Both control and PTEN silenced groups showed similar multilineage differentiation, with equivalent percentages of myeloid and lymphoid human progeny cells. In addition, while PTEN mutant mice developed myeloproliferative diseases within days and transplantable leukemias (4
), transient PTEN depletion in human CD34+ cells did not lead to leukemogenesis, although follow-up was short, and the xenograft model may not be adequate for testing the leukemic potential of perturbations in human hematopoietic cells.
Overall, our data suggest that the transient suppression of PTEN in CD34+ cells results in increased proliferation of HSPCs in vitro and increased self-renewal of cells capable of in vivo reconstitution in NSG mice, without an appreciable change in their capacity for multipotential differentiation. Optimization of technologies for transfer of siRNA in primary CD34+ cells will be required to allow improved cell survival and permit clinical applications in transplant-related therapies, including gene therapy protocols and adult cord blood stem cell transplantation where the number of HSPCs is limited.