title: Apoptotic susceptibility to DNA damage of pluripotent stem cells facilitates pharmacologic purging of teratoma risk.
abstract: Pluripotent stem cells have been the focus of bioengineering efforts designed to generate regenerative products, yet harnessing therapeutic capacity while minimizing risk of dysregulated growth remains a challenge. The risk of residual undifferentiated stem cells within a differentiated progenitor population requires a targeted approach to eliminate contaminating cells prior to delivery. In this study we aimed to validate a toxicity strategy that could selectively purge pluripotent stem cells in response to DNA damage and avoid risk of uncontrolled cell growth upon transplantation. Compared with somatic cell types, embryonic stem cells and induced pluripotent stem cells displayed hypersensitivity to apoptotic induction by genotoxic agents. Notably, hypersensitivity in pluripotent stem cells was stage-specific and consistently lost upon in vitro differentiation, with the mean half-maximal inhibitory concentration increasing nearly 2 orders of magnitude with tissue specification. Quantitative polymerase chain reaction and Western blotting demonstrated that the innate response was mediated through upregulation of the BH3-only protein Puma in both natural and induced pluripotent stem cells. Pretreatment with genotoxic etoposide purged hypersensitive pluripotent stem cells to yield a progenitor population refractory to teratoma formation upon transplantation. Collectively, this study exploits a hypersensitive apoptotic response to DNA damage within pluripotent stem cells to decrease risk of dysregulated growth and augment the safety profile of transplant-ready, bioengineered progenitor cells.
authors.affiliation: Department of Medicine and Transplant Center, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: Alyson J
authors.initials: AJ
authors.lastname: Smith
authors.forename: Natalie G
authors.initials: NG
authors.lastname: Nelson
authors.forename: Saji
authors.initials: S
authors.lastname: Oommen
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Hartjes
authors.forename: Clifford D
authors.initials: CD
authors.lastname: Folmes
authors.forename: Andre
authors.initials: A
authors.lastname: Terzic
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Nelson
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002454:Cell Differentiation; D002478:Cells, Cultured; D004249:DNA Damage; D058732:Embryoid Bodies / Q000166:cytology / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005047:Etoposide / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D005434:Flow Cytometry; D042822:Genomic Instability; D020128:Inhibitory Concentration 50; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D009152:Mutagenicity Tests / Q000379:methods; D009928:Organ Specificity; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012307:Risk Factors; D013724:Teratoma / Q000473:pathology / Q000517:prevention & control*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D017304:Annexin A5; D051017:Apoptosis Regulatory Proteins; C488588:PUMA protein, mouse; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D005047:Etoposide