Low level of gene transfer to and engraftment of murine bone marrow cells from long-term bone marrow cultures.
OBJECTIVE
We wanted to determine whether the long-term bone marrow culture (LTBMC) transduction system would lead to efficient gene transfer and engraftment of murine repopulating hematopoietic stem cells (HSC), particularly in nonablated recipients.


METHODS
Congenic mouse strains expressing Ly 5.1 or Ly 5.2 and the GP+E86 cell line producing the MGirL22Y vector carrying the gene for enhanced GFP were used. Murine LTBMCs were established and demi-depopulated on days 7 and 14 with addition of vector supernatant on days 8 and 15.


RESULTS
Cell recovery on day 21 was 21.3%+/-3.8% of input cells and CFU-C recovery was 9.7+/-3.4% as compared with CFU-C of input cells. In vitro transduction efficiency determined by CFU-C expressing GFP was 22.2%+/-1.6%. In irradiated (950 cGy) mice transplanted with 2x10(6) LTBMC cells, 94% of nucleated cells in the blood at week 16 were of donor origin. However, GFP was only detected at low level in a few animals at week 4 and not later. Analysis of bone marrow from these mice at week 20 did not show any GFP expression and semiquantitative PCR revealed a transgene level of <1%. When 3.5-20.8x10(6) LTBMC cells (corresponding to 20-100x10(6) fresh cells) were transplanted to nonablated recipients, no engraftment or GFP expression were detected. Competitive repopulation experiments showed that the long-term repopulation ability (LTRA) of the LTMC cells was only 7% of fresh cells.


CONCLUSIONS
These results indicate that LTBMC transduction of murine cells leads to low-level transduction of progenitors, no gene transfer to repopulating stem cells, and reduction in LTRA in ablated and nonablated recipients.
Relander
T
T
Department of Molecular Medicine and Gene Therapy, Wallenberg Neuroscience Center, University of Lund, Lund, Sweden. thomas.relander@molmed.lu.se
Fahlman
C
C
Karlsson
S
S
Richter
J
J
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects; D016026:Bone Marrow Transplantation / Q000379:methods*; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D018920:Coculture Techniques; D003470:Culture Media; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D006085:Graft Survival / Q000235:genetics*; D006494:Heparin Antagonists / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D051379:Mice; D020297:Mice, Congenic; D008810:Mice, Inbred C57BL; D011479:Protamines / Q000494:pharmacology; D019089:Stem Cell Factor / Q000494:pharmacology; D015996:Survival Rate; D013995:Time; D014162:Transfection; D019172:Transplantation Conditioning
D003470:Culture Media; D006494:Heparin Antagonists; D015850:Interleukin-6; D011479:Protamines; D019089:Stem Cell Factor