title: Decitabine suspends human CD34+ cell differentiation and proliferation during lentiviral transduction.
abstract: Efficient ex vivo transduction of hematopoietic stem cells (HSCs) is encumbered by differentiation which reduces engraftment. We hypothesized that inhibiting DNA methyltransferase with decitabine would block differentiation of transduced CD34+ cells under cytokine stimulation and thus improve transduction efficiency for engrafting HSCs. Human CD34+ cells in cytokine-containing media were treated with or without decitabine for 24 or 48 hours, and then these cells were transduced with a GFP-expressing lentiviral vector. Utilizing decitabine pre-treatment for 48 hours, we observed an equivalent percentage of successfully transduced cells (GFP-positivity) and a higher percentage of cells that retained CD34 positivity, compared to no decitabine exposure. Cell proliferation was inhibited after decitabine exposure. Similar results were observed among CD34+ cells from six different donors. Repopulating activity was evaluated by transplantation into NOD/SCID/IL2Rγnull mice and demonstrated an equivalent percentage of GFP-positivity in human cells from decitabine-treated samples and a trend for higher human cell engraftment (measured 20-24 weeks after transplantation), compared to no decitabine exposure. In conclusion, ex vivo decitabine exposure inhibits both differentiation and proliferation in transduced human CD34+ cells and modestly increases the engraftment ability in xenograft mice, while the transduction efficiency is equivalent in decitabine exposure, suggesting improvement of lentiviral transduction for HSCs.
authors.affiliation: Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, Maryland, United States of America.
authors.forename: Naoya
authors.initials: N
authors.lastname: Uchida
authors.affiliation: Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, Maryland, United States of America.
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Hsieh
authors.affiliation: Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, Maryland, United States of America.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Platner
authors.affiliation: Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, Ohio, United States of America.
authors.forename: Yogen
authors.initials: Y
authors.lastname: Saunthararajah
authors.affiliation: Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, Maryland, United States of America.
authors.forename: John F
authors.initials: JF
authors.lastname: Tisdale
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000235:genetics / Q000276:immunology; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology; D000077209:Decitabine; D015870:Gene Expression; D005822:Genetic Vectors; D006085:Graft Survival; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D019650:Hematopoietic Stem Cell Mobilization; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D061251:Primary Cell Culture; D014161:Transduction, Genetic; D014183:Transplantation, Heterologous
chemical_list: D018952:Antigens, CD34; D000964:Antimetabolites, Antineoplastic; D016179:Granulocyte Colony-Stimulating Factor; D000077209:Decitabine; D004248:DNA (Cytosine-5-)-Methyltransferases; D001374:Azacitidine
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