title: Optimized surface markers for the prospective isolation of high-quality hiPSCs using flow cytometry selection.
abstract: hiPSC derivation and selection remains inefficient; with selection of high quality clones dependent on extensive characterization which is not amenable to high-throughput (HTP) approaches. We recently described the use of a cocktail of small molecules to enhance hiPSC survival and stability in single cell culture and the use of flow cytometry cell sorting in the HTP-derivation of hiPSCs. Here we report an enhanced protocol for the isolation of bona fide hiPSCs in FACS-based selection using an optimized combination of cell surface markers including CD30. Depletion of CD30(+) cells from reprogramming cultures almost completely abolished the NANOG and OCT4 positive sub-population, suggesting it is a pivotal marker of pluripotent cells. Combining CD30 to SSEA4 and TRA-1-81 in FACS greatly enhanced specificity and efficiency of hiPSC selection and derivation. The current method allows for the efficient and automated, prospective isolation of high-quality hiPSC from the reprogramming cell milieu.
authors: Fate Therapeutics Inc., 3535 General Atomics Court Suite 200, San Diego, CA 92121, USA.
authors: Ramzey
authors: R
authors: Abujarour
authors: Bahram
authors: B
authors: Valamehr
authors: Megan
authors: M
authors: Robinson
authors: Betsy
authors: B
authors: Rezner
authors: Florin
authors: F
authors: Vranceanu
authors: Peter
authors: P
authors: Flynn
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002469:Cell Separation; D065150:Cellular Reprogramming; D005434:Flow Cytometry; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D007621:Karyotyping; D017730:Ki-1 Antigen / Q000235:genetics / Q000378:metabolism; D051379:Mice; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D055746:Stage-Specific Embryonic Antigens / Q000378:metabolism; D013724:Teratoma / Q000473:pathology
chemical_list: D000954:Antigens, Surface; D018398:Homeodomain Proteins; D017730:Ki-1 Antigen; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D055746:Stage-Specific Embryonic Antigens; C519803:TRA-1-81 antigen, human; C066584:stage-specific embryonic antigen-4
references: Nat Biotechnol. 2012 May 27;30(6):531-42
references: 22634564
references: J Biol Chem. 1997 Jun 20;272(25):15708-14
references: 9188463
references: Dev Dyn. 2004 Feb;229(2):259-74
references: 14745951
references: Molecules. 2012 May 25;17(6):6196-236
references: 22634835
references: J Am Soc Nephrol. 2009 Aug;20(8):1669-76
references: 19578008
references: Proc Natl Acad Sci U S A. 2008 Feb 26;105(8):2883-8
references: 18287077
references: Cell Stem Cell. 2009 Dec 4;5(6):584-95
references: 19951687
references: Nat Biotechnol. 2009 Nov;27(11):1033-7
references: 19826408
references: Leuk Lymphoma. 2011 Sep;52(9):1641-54
references: 21619423
references: PLoS One. 2011 Mar 02;6(3):e17540
references: 21407814
references: Hum Reprod. 2009 Oct;24(10):2477-89
references: 19584135
references: Cell. 2007 Nov 30;131(5):861-72
references: 18035408
references: Exp Hematol. 2012 Aug;40(8):601-11
references: 22510344
references: Stem Cells. 2007 Mar;25(3):723-30
references: 17124010
references: J Cell Biochem. 2012 Oct;113(10):3061-8
references: 22573568
references: Nat Genet. 2009 Jul;41(7):843-8
references: 19483683
references: J Neurosci Res. 2007 Feb 1;85(2):250-9
references: 17131412
references: J Neurosci Res. 2006 Aug 15;84(3):471-82
references: 16721767
references: J Neurocytol. 1994 Sep;23(9):577-89
references: 7529299
references: Sci Rep. 2012;2:213
references: 22355727
references: Nature. 1999 Mar 25;398(6725):341-4
references: 10192335
references: Nat Rev Drug Discov. 2011 Nov 11;10(12):915-29
references: 22076509
references: Regen Med. 2011 Sep;6(5):623-34
references: 21916597
references: Nat Biotechnol. 2007 Jul;25(7):803-16
references: 17572666
references: Cell Res. 2011 Nov;21(11):1551-63
references: 21894191
references: J Anat. 2002 Mar;200(Pt 3):249-58
references: 12033729
references: Science. 2007 Dec 21;318(5858):1917-20
references: 18029452
references: Nat Biotechnol. 2011 Aug 14;29(9):829-34
references: 21841799