Effects of Megakaryocyte Growth and Development Factor on Survival and Retroviral Transduction of T Lymphoid Progenitor Cells

Abstract

Murine retroviral vectors have the potential to mediate stable gene transfer into hematopoietic progenitor cells. A known drawback to the use of these vectors is that transduction can only take place in cells actively progressing through the cell cycle. Thrombopoietin, the c-mpl ligand, is known to support division of hematopoietic precursors of primitive origin. Polyethylene glycol (PEG)-conjugated recombinant human megakaryocyte growth and development factor (MGDF) is a polypeptide related to thrombopoietin that stimulates megakaryocyte production. To investigate whether MGDF would also induce stem cell division and support retroviral transduction of CD34⁺ cells, we compared the effects of MGDF, stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, alone or in combination, using amphotropic and vesicular stomatitis virus (VSV-G) pseudotyped murine retroviral vectors. Similar transduction efficiency was observed when CD34⁺ cells were transduced in the presence of SCF and MGDF as compared to SCF, IL-3, and IL-6. Using the SCID-hu mouse model of thymopoiesis, we investigated whether CD34⁺ cells transduced in the presence of these cytokines could reconstitute irradiated thymic implants, and whether vector sequences were present in mature thymocytes. At early timepoints, no significant differences were observed on engraftment of donor progenitors incubated with each cytokine combination. However, a significant difference in the percentage of donor derived CD4⁺/CD8⁺ immature thymocytes was observed 9 weeks after implantation of CD34⁺ cells exposed to the combination of SCF and MGDF as compared to SCF, IL-3, and IL-6 (p = 0.04), indicating that MGDF/SCF better supported the survival of thymocyte precursor cells. Approximately 4% of thymocytes in both cytokine groups harbored vector sequences. These studies provide evidence that MGDF and SCF in combination can mediate transduction of hematopoietic progenitors capable of contributing to long-term thymopoiesis. These results may have important applications for the implementation of gene therapy strategies in disorders affecting the T lymphoid system.

Overview summary

Cell cycle induction is a necessary step for transduction of hematopoietic precursors. Cytokine combinations are used during transduction to induce cell cycle progression. Using in vitro cultures and the SCID-hu mouse model of human thymopoiesis, we present evidence that megakaryocyte growth and development factor (MGDF) can substitute for interleukin-3 (IL-3) and IL-6, in combination with stem cell factor (SCF), to allow transduction of CD34⁺ progenitor cells. SCID-hu thymic implants reconstituted with exogenous CD34⁺ cells, incubated in the presence of MGDF and SCF, display longer-lasting engraftment of donor-derived human thymocytes and prolonged persistence of double-positive CD4/CD8 immature precursor T lymphocytes, as compared to exogenous CD34⁺ cells cultured with SCF, IL-3, and IL-6. These findings demonstrate that, in addition to its regulatory role in megakaryopoiesis, MGDF can support survival of immature progenitors. These results have importance for gene therapeutic approaches involving T lymphoid disorders, such as acquired immunodeficiency syndrome (AIDS).