The effect of pegylated antisense acetylcholinesterase on hematopoiesis.
To determine whether the efficacy of entry and action of antisense oligonucleotides (AS-ODN) on hematopoietic stem cells in vitro could be improved by the addition of polyethylene glycol (PEG), a molecule of PEG was bound to AS- or sense-acetylcholinesterase (AS-ACHE or S-ACHE). The introduction of 0.1-0.5 microM PEG-AS-ACHE or 0.5 microM AS-ACHE into methylcellulose bone marrow (BM) cultures produced a doubling in number of colony-forming unit-granulocyte-erythrocyte-macrophage-megakaryocyte (CFU-GEMM) and a 5-fold increase in cell number of the PEG-ODN. Further increase in concentration of the PEG-ODN reduced colony numbers. PEG-AS-ACHE induced higher colony numbers and greatly increased megakaryocyte (MK) formation when compared with PEG and AS-ACHE added separately to the culture. In addition, differentials of the CFU-GEMMs indicated there was a direct relationship between MK number and PEG-AS-ACHE concentration. Under these culture conditions, 5 microM PEG alone gave control values of CFU-GEMM. On addition of FITC-PEG-AS-ACHE to the cell cultures, using confocal microscopy, the nuclei of both early and mature MKs were labeled specifically, whereas all other cellular nuclei were negative to the stain. The use of PEG-AS-ODN, affording specific delivery of AS-ODN to target cells, increased cell proliferation, and enhanced ODN uptake, may be of potential importance in stem cell expansion for BM transplantation and gene therapy.
Department of Bone Marrow Transplantation, Hadassah University Hospital, Jerusalem, Israel 91120. patinkin@md.huji.ac.il
Deborah
D
Patinkin
Adel
A
Hidmi
Lola
L
Weiss
Shimon
S
Slavin
Joshua
J
Katzhendler
D000110:Acetylcholinesterase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001854:Bone Marrow Cells; D002217:Carbachol / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D006410:Hematopoiesis / Q000187:drug effects*; D051379:Mice; D008809:Mice, Inbred C3H; D008916:Miotics / Q000494:pharmacology; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D011092:Polyethylene Glycols / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D008916:Miotics; D016376:Oligonucleotides, Antisense; D011092:Polyethylene Glycols; D002217:Carbachol; D000110:Acetylcholinesterase