title: Efficient oncoretroviral transduction of extended long-term culture-initiating cells and NOD/SCID repopulating cells: enhanced reconstitution with gene-marked cells through an ex vivo expansion approach.
abstract: Recent developments of surrogate assays for human hematopoietic stem cells (HSC) have facilitated efforts at improving HSC gene transfer efficiency. Through the use of xenograft transplantation models, such as nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, successful oncoretroviral gene transfer to transplantable hematopoietic cells has been achieved. However, because of the low frequency and/or homing efficiency of SCID repopulating cells (SRC) in bone marrow (BM), studies have primarily focused on cord blood (CB). The recently developed extended (> 60 days) long-term culture-initiating cell (ELTC-IC) assay detects an infrequent and highly quiescent candidate stem cell population in BM as well as CB of the CD34(+)CD38(-) phenotype. Although these characteristics suggest that ELTC-IC and SRC might be closely related, attempts to oncoretrovirally transduce ELTC-IC have been unsuccessful. Here, recently developed conditions (high concentrations of SCF + FL + Tpo in serum-free medium) supporting expansion of BM CD34(+)CD38(-) 12 week ELTC-IC promoted efficient oncoretroviral transduction of BM and CB ELTC-IC. Although SRC can be transduced with oncoretroviral vectors, this is frequently associated with loss of reconstituting activity, posing a problem for development of clinical HSC gene therapy. However, previous attempts at expanding transduced HSC posttransduction resulted in compromised rather than improved gene marking. Utilizing conditions promoting cell divisions and transduction of ELTC-IC we show that although 5 days of ex vivo culture is sufficient to obtain maximum gene transfer efficiency to SRC, extension of the expansion period to 12 days significantly enhances multilineage reconstitution activity of transduced SRC, supporting the feasibility of improving gene marking through ex vivo expansion.
authors.affiliation: Department of Stem Cell Biology, Institute of Laboratory Medicine, Klinikgatan 26, University Hospital of Lund, 221 84 Lund, Sweden.
authors.forename: Helga
authors.initials: H
authors.lastname: Björgvinsdóttir
authors.forename: David
authors.initials: D
authors.lastname: Bryder
authors.forename: Ewa
authors.initials: E
authors.lastname: Sitnicka
authors.forename: Veslemøy
authors.initials: V
authors.lastname: Ramsfjell
authors.forename: Ineke
authors.initials: I
authors.lastname: De Jong
authors.forename: Karin
authors.initials: K
authors.lastname: Olsson
authors.forename: Corinne
authors.initials: C
authors.lastname: Rusterholz
authors.forename: Stefan
authors.initials: S
authors.lastname: Karlsson
authors.forename: Sten Eirik W
authors.initials: SE
authors.lastname: Jacobsen
mesh_terms: D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D002455:Cell Division; D002460:Cell Line; D003470:Culture Media; D005312:Fetal Blood / Q000378:metabolism; D018014:Gene Transfer Techniques; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009244:NAD+ Nucleosidase; D012190:Retroviridae / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D014161:Transduction, Genetic
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D003470:Culture Media; D008562:Membrane Glycoproteins; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1