An Improved Mouse Sca-1+ Cell-Based Bone Marrow Transplantation Model for Use in Gene- and Cell-Based Therapeutic Studies

Abstract

This study sought to develop a murine bone marrow transplantation strategy that would yield consistently high levels of long-term engraftment without significant morbidity and mortality. Hematopoietic stem cell (HSC)-enriched Sca-1+ cells were used for transplantation because of their propensity of homing to bone marrow. Green fluorescent protein (GFP)-expressing transgenic mice were used as donors. Murine Sca-1+ cells were enriched 13-fold from whole bone marrow with immunomagnetic column chromatography. Retroorbital injections yielded highly reproducible and higher levels of engraftment compared with tail vein injections. The combination of W⁴¹/W⁴¹ recipient mice and sublethal irradiation preconditioning produced long-term engraftment with minimal morbidity and mortality. A 24-hour delay between the sublethal irradiation and transplantation did not affect the efficiency and level of engraftment, but provided flexibility with respect to the timing of transplantation. Based on these findings, a mouse Sca-1+ cell-based strategy, involving the retroorbital injection of Sca-1+ cells into sublethally irradiated, myelosuppressed W⁴¹/W⁴¹ recipient mice within 24 h after irradiation, was developed. Transplantation of lentiviral vector-transduced wild-type Sca-1+ cells expressing GFP by this strategy led to consistently high levels of long-term engraftment. In summary, this murine Sca-1+ cell-based strategy could be used in studies of HSC-based gene or cell therapies.

Do Pluripotent Stem Cells Exist in Adult Mice as Very Small Embryonic Stem Cells?

“…The experimental design, summarized in Figure 4D, was similar to that described in a previous report (Hall et al., 2007). Three months after the mice underwent transplantation of GFP-expressing HSCs, 98% of their peripheral blood cells (not shown) and 96.8% of their BM granulocytes were GFP + (Figure S4A).…”

Chronic occupational exposure to arsenic induces carcinogenic gene signaling networks and neoplastic transformation in human lung epithelial cells

“…BEAS-2B and B-As cells were injected intravenously (iv) via the retroorbital plexus (Hall et al, 2007). Five and nine animals were used for BEAS-2B and B-As injections, respectively.…”

Significant differences in genotoxicity induced by retrovirus integration in human T cells and induced pluripotent stem cells

“…To construct pRV/C-GFP, a 2-kb CMV-GFP cassette was isolated from pHIV7/C-GFP with BamH1 and BglII digestion and cloned into the unique BamH1 site in pCLSA (Hall et al, 2007). …”

“…Cells grown in a 100-mm culture dish were cotransfected with 10 μg of the vector, 10 μg of pCMV-GP and 2 μg of pCMV-G (Hall et al, 2007). Vectors were harvested 40 h later and tittered in HT1080 cells using fluorescence-activated cell sorting (FACS) to detect GFP-positive cells.…”