Long-term persistence of a nonintegrated lentiviral vector in mouse hematopoietic stem cells.

OBJECTIVE: Lentiviral transduction is an established method for efficiently modifying the gene expression program of primary cells, but the ability of the introduced construct to persist as an episome has not been well studied. MATERIAL AND METHODS: Here we investigated this issue in lethally irradiated female mice injected with 300 or 3000 doubly sorted male lin(neg), Sca-1(high), c-kit(high), Thy-1.1(low) mouse bone marrow cells that had been exposed in vitro to self-inactivating lentivirus vector encoding a green fluorescence protein (GFP) cDNA. Seven to sixteen months later, bone marrow cells from primary mice were injected into secondary female recipients and another 8 months later into tertiary female recipients. Integration study was performed on individual spleen colonies by Southern blot analysis. Inverse polymerase chain reaction (PCR) and sequence of amplified vector-derived DNA was used to verify Southern blot results. RESULTS: Spleen colony-forming cell study revealed that a small fraction of the spleen colonies contained integrated provirus as shown by Southern blot analysis. Unexpectedly, many spleen colonies were found to contain a nonintegrated episomal form of the provirus, which was confirmed by an inverse PCR analysis. In some of the spleen colonies containing only the episomal form, GFP-expressing cells were also detected. Lentiviral sequences were present in hematopoietic tissues of primary mice but not in other tissues. CONCLUSIONS: These results demonstrate that lentiviral vectors produce episomal circles in hematopoietic stem cells that can be transferred through many cell generations and expressed in their progeny.

Developmental fate of hematopoietic stem cells: the study of individual hematopoietic clones at the level of antigen-responsive B lymphocytes.

We have shown previously that hematopoiesis in mice reconstituted with retrovirally marked hematopoietic stem cells (HSCs) is provided by multiple, mainly short-lived clones, as measured by retroviral insertion site analysis of individual spleen colony-forming unit (CFU-S)-derived colonies. However, the CFU-S is the relatively early progenitor and the contribution of each CFU-S in the steady-state hematopoiesis is uncertain. Here, we have studied the fate of individual mature B cells, as well as CFU-S, representing the progeny of retrovirally transduced marrow-repopulating cells (MRC). B-cells-generated hybridomas and CFU-S-derived colonies were used to determine the clonal composition of hematolymphopoiesis at the single-cell level. Bone marrow (BM) cells and splenocytes (approximately 1/3-1/2 of spleen at a time) from mice reconstituted with retrovirally marked syngeneic BM cells were repeatedly collected at 3, 10, and 16 months post-transplant. The percentage of retrovirally marked CFU-S and B-cell-produced hybridomas was about 50% at 3 months and decreased to 10-15% at 10 months after reconstitution in spite of stable degree of chimerism. The clonal origin of BM-derived CFU-S and spleen-derived B-cell hybridomas was detected by Southern blot analysis. Overall, DNA obtained from 159 retrovirally marked spleen colonies, 287 hybridomas and 43 BM samples were studied. Multiple simultaneously functioning clones of MRC-derived B cells were observed. The same individual clones among hybridomas and CFU-S were identified in three out of 11 mice. Thus, hematopoiesis is generated by multiple hematopoietic clones some of which can simultaneously contribute to both mature lymphoid cells and myeloid progenitors. These data establish that the stem cell compartment functions by continuously producing progeny, which fully but transiently repopulate all lineages.

Neoplastic transformation is not the cause of extremely long (more than 100 weeks) hematopoiesis maintenance of long-term bone marrow culture from TNF-deficient mice.

Total cell production and longevity of hematopoiesis in long-term bone marrow culture of tumor necrosis factor (TNF)-deficient mice (LTBM-TNFko) are increased. The rate of apoptosis is decreased during the first 40 weeks in culture, then the level of apoptosis reaches levels of wild-type cultures. Extended lifespan of primary cultures usually is the consequence of the neoplastic transformation. We set out to check this possibility in the LTBM-TNFko. Telomerase activity in suspension fraction (SF) of LTBM-TNFko increases with time and reaches maximum a year after culture initiation. Cytogenetic study reveals genome instability in SF and hyperploidy in the adhesion cell layer (ACL) of LTBM-TNFko. All of the above indicate the possibility of neoplastic transformation. However, histological study of cells and CFU-S-derived colonies of SF does not reveal a block of differentiation. Cells of SF are unable to grow without ACL. Although those cells could proliferate in the presence of exogenous growth factors, they are not able to be passaged. Attempts of passaging ACL cells failed as well. Neither healthy nor sublethally irradiated recipients injected intravenously or intraperitoneally with cells of SF develop tumors within 8 months of observation. In conclusion, abnormal dynamics of long-term bone marrow culture of TNF-deficient mice could not be explained by neoplastic transformation.