The HGFIN Gene Mediates Cell Cycle Quiescence of CD34+/CD38-: Implications for Hematopoietic Stem Cell Expansion and Gene Therapy.

Hematopoietic stem cells (HSC) are difficult to expand in vitro since expansion media mostly include exogenous factor, e.g., cytokines. Growth factors cause lineage commitment with the HSC exhibiting short-term immune reconstitution. The recently cloned HGFIN (or nmb) gene has been detected in CD34⁺/CD38⁻ and differentiated immune cells. HGFIN is a single transmembrane protein with multiple consensus regions for p53. In malignant cells, HGFIN mediate tumor progression and in normal cells, HGFIN maintain cell cycle quiescence. We hypothesize that HGFIN negatively regulates proliferation of HSC by prolonging the G₀ phase. Short-term knockdown of HGFIN in HSC could cause the cells to exit G₀/G₁ phase so as to accommodate the insertion of a replacement gene, without changing the pluripotent property of HSC. RT-PCR detected HGFIN mRNA in CD34⁺/CD38⁻ cells, but not in CD34⁺/CD38⁺ cells from bone marrow (BM) aspirates. Treatment of CD34⁺/CD38⁻ cells with double stranded HGFIN siRNA oligos for 2 h led to their exit from G₁ to G₂/S (flow cytometry). During this time, adenovirus-LacZ (ß-galactosidase assay) was incorporated in >80% CD34⁺/CD38⁻ cells, compared to 10–20% exposed to mutant siRNA, or untreated. After 24 h exposure to siRNA oligos, the CD34⁺/CD38⁻ cells reverted to G_0/1 phase. The latter cells (with adenovirus-LacZ) were studied in 12-wk long-term culture initiating assay. The results (ß-gal positive progenies) from the LTC-IC assay indicate that despite siRNA treatment, the CD34⁺/CD38⁻ cells retained multipotential properties. Future investigations with specific HGFIN antibody will map pathways among HGFIN and other molecules that regulate cell cycle progression in CD34⁺/CD38⁻ cells. In summary, the results show that HGFIN regulate cell cycle quiescence of CD34⁺/CD38⁻ cells. Transient knock down of HGFIN in these cells allow adenovirus to be inserted without loss of multipotential properties. Understanding the biology of HSC at the molecular level will be relevant to HSC expansion and restoration of immune competence via gene therapy.