Enhanced effect of vascular endothelial growth factor, thrombopoietin peptide agonist, SCF, and Flt3-L on LTC-IC and reporter gene transduction from umbilical cord blood CD34+ cells.
BACKGROUND
Hemangioblastic precursors have been identified that give rise to both endothelial cells and HPCs, suggesting that common growth factor requirements may exist.


METHODS
The effect of vascular endothelial growth factor (VEGF) in combination with thrombopoietin peptide agonist (TPOA), Flt-3 L (F), and SCF (S) on long-term culture-initiating cell (LTC-IC), CFU, differentiation, and transduction of cord blood (CB) CD34+ were evaluated up to 4 weeks in culture.


RESULTS
At Week 4, in cultures containing T/F/S and VEGF, the LTC-IC increased 1000-fold (from 37 +/- 8 to 37,012 +/- 14,329) with a frequency of 3.4 in 10,000 cells. In the T/F/S cultures without VEGF, the LTC-IC increased 675-fold (to 25,086 +/- 12,102) with a frequency of one LTC-IC in 10,000 cells. The addition of VEGF significantly increased (p < 0.05) the LTC-IC per 10,000 CB CD34+ cells. Transduction with reporter gene enhanced green fluorescent protein (EGFP), resulted in an increase in EGFP+ CFU at Week 1 and EGFP + LTC-IC at Weeks 1 and 4. The cells maintained their multilineage expression when cultured in conditions for erythroid, myeloid, or megakaryocytic differentiation. Peak percentage EGFP coexpression of GlyA and CD11b was 51 +/- 6 percent and 63 +/- 15 percent, respectively, at Week 2, while CD41a was 34 +/- 17 percent at Week 4.


CONCLUSIONS
T/F/S and VEGF have an enhanced effect on total LTC-IC output and frequency but do not appear to significantly alter the differentiation or transducibility characteristics of CB HPCs in vitro.
Institute for Transfusion Medicine, Stem Cell Services, Glenview, Illinois, USA. smiths2@baxter.com
Stephen L
SL
Smith
Joseph
J
Kiss
Christopher
C
Siatskas
Jeffrey A
JA
Medin
Richard L
RL
Moldwin
D018952:Antigens, CD34 / Q000032:analysis; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage / Q000187:drug effects; D002478:Cells, Cultured; D004357:Drug Synergism; D005312:Fetal Blood / Q000166:cytology*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000166:cytology / Q000187:drug effects; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008533:Megakaryocytes / Q000166:cytology; D008565:Membrane Proteins / Q000494:pharmacology*; D010455:Peptides / Q000494:pharmacology; D012190:Retroviridae / Q000235:genetics; D019089:Stem Cell Factor / Q000494:pharmacology*; D013926:Thrombopoietin / Q000819:agonists*; D014161:Transduction, Genetic; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
D018952:Antigens, CD34; D008164:Luminescent Proteins; D008565:Membrane Proteins; D010455:Peptides; D019089:Stem Cell Factor; D042461:Vascular Endothelial Growth Factor A; C084881:flt3 ligand protein; D049452:Green Fluorescent Proteins; D013926:Thrombopoietin