Investigation into an engraftment defect induced by culturing primitive hematopoietic cells with cytokines.
BACKGROUND
Strategies for transplanting primitive hematopoietic progenitor (PHP) cells are under development that require in vitro manipulation of cells for several hours to several days prior to transplantation. This applies to gene-therapy protocols involving transduction with adenoviral or lentiviral vectors (typically 1 day of ex vivo culture) or retroviral vectors (up to 3 days of culture).


METHODS
Human mobilized peripheral blood (MPB) CD34(+) cells were cultured with the cytokines thrombopoietin mimetic peptide (mTPO), flt3 ligand (FL), and c-kit ligand (KL). Equal numbers of CD34(+) cells, either uncultured or cultured for various time periods up to 5 days, were tested for engraftment in sublethally irradiated 8-10 week-old NOD/SCID mice. Cells were also compared for expression and function of several key surface molecules.


RESULTS
At a limiting dose of 1 million cells, mice receiving uncultured cells had a mean of 20% CD45(+) (human) cells in their BM 6 weeks post-transplantation, versus 3% for mice receiving 3-5 day cultured cells. Analysis of 10 surface molecules, CD11a, CD18, CD29, CD49d, CD49e, CXCR-4, CD62L, CD31, CD43, and CD44 over a 5-day culture period showed that their expression levels were either maintained or up-regulated on CD34(+) cells and the primitive Thy-1(+) subset. Similar percentages of uncultured and 3-day cultured MPB CD34(+) cells bound to plates coated with vascular cell adhesion molecule-1 (VCAM-1) under both static and physiological flow conditions, and chemotaxis of cultured cells towards stromal-derived factor-1 (SDF-1) was not impaired, suggesting that VLA-4 and CXCR-4 were functional on cultured cells. CD34(+) Thy-1(+) MPB cells cultured with cytokines expressed increasing levels of Fas receptor beginning at 20 h in culture, with peak expression levels after 3 days (mean Day 0 expression, 39%; mean Day 3 expression, 86%), without increased apoptosis. Including inhibitors of caspases in the media of cells cultured for 24-48 h significantly improved their engraftment in a SCID-hu bone-engraftment model.


CONCLUSIONS
Increased susceptibility to apoptosis upon in vivo injection may contribute to impaired engraftment of in vitro manipulated cells. Inhibitors of apoptosis may increase their engrafting capacity in clinical settings.
Young
J C
JC
SyStemix Inc., Palo Alto, CA 94070 (a subsidiary of Novartis Pharmaceuticals), USA.
Lin
K
K
Travis
M
M
Hansteen
G
G
Abitorabi
A
A
Sirenko
O
O
Murray
L
L
Hill
B
B
D000818:Animals; D018952:Antigens, CD34 / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology; D002448:Cell Adhesion / Q000187:drug effects / Q000276:immunology; D015815:Cell Adhesion Molecules / Q000276:immunology; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology* / Q000627:therapeutic use; D006085:Graft Survival / Q000187:drug effects* / Q000276:immunology; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology* / Q000627:therapeutic use; D019650:Hematopoietic Stem Cell Mobilization / Q000379:methods*; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006801:Humans; D008214:Lymphocytes / Q000187:drug effects / Q000276:immunology; D008565:Membrane Proteins / Q000494:pharmacology / Q000627:therapeutic use; D051379:Mice; D016688:Mice, Inbred NOD; D019089:Stem Cell Factor / Q000494:pharmacology / Q000627:therapeutic use; D013926:Thrombopoietin / Q000494:pharmacology / Q000627:therapeutic use; D015854:Up-Regulation / Q000187:drug effects / Q000276:immunology; D019014:fas Receptor / Q000276:immunology
D018952:Antigens, CD34; D000954:Antigens, Surface; D015815:Cell Adhesion Molecules; D016207:Cytokines; D016298:Hematopoietic Cell Growth Factors; D008565:Membrane Proteins; D019089:Stem Cell Factor; D019014:fas Receptor; C084881:flt3 ligand protein; D013926:Thrombopoietin