The high proliferative potential-quiescent (HPP-Q) cell assay allows an optimized evaluation of gene transfer efficiency into primitive hematopoietic stem/progenitor cells.
Various protocols have been described to optimize gene transfer into hematopoietic cells. However, most of these methods do not specify whether they are associated with an improved transduction of the more primitive stem/progenitor cells, the best candidates for long-term engraftment. The majority of these primitive cells remains in quiescence because of the negative control of TGF-beta1, effective on these cells at low concentrations (10 pg/ml). In this study, CD34- cells were activated by a 10 h pretreatment with anti-TGF-beta1 followed by four successive retroviral supernatant incubations of 6 h each. After 12 h (two incubations), a significant increase in TGF-beta1 mRNA in CD34+ cells was observed. We wondered whether neo-synthesized autocrine TGF-beta1 could induce reversion to quiescence of the more primitive CD34+ cells transduced after one cell cycle. This would prevent their subsequent detection in a classic clonal assay. Using the HPP-Q assay comparing a rapid mixed colony assay with or without anti-TGF-beta1, we indeed observed, that in clonal growth conditions the more primitive transduced cells were activated and detectable only with anti-TGF-beta1. Therefore, this assay represents not only a rapid means to detect quiescent multipotent stem/progenitor cells but also a necessary step for the detection of the more primitive transduced cells which have returned to quiescence after retroviral induction of TGF-beta1 secretion.
Ducos
K
K
Laboratoire de Biologie des Cellules Souches Somatiques Humaines, Centre National de la Recherche Scientifique, UPR 1983, Villejuif, France.
Hatzfeld
A
A
Héron
A
A
Fortunel
N
N
Kisselev
S
S
Héron
C
C
Monier
M N
MN
Hatzfeld
J
J
D015703:Antigens, CD / Q000378:metabolism; D051927:CD24 Antigen; D018929:Cell Culture Techniques; D002455:Cell Division; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006412:Hematopoietic Stem Cells; D006801:Humans; D008562:Membrane Glycoproteins; D012333:RNA, Messenger / Q000235:genetics; D014161:Transduction, Genetic; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
D015703:Antigens, CD; D051927:CD24 Antigen; C497889:CD24 protein, human; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta