title: Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status.
abstract: BACKGROUND
  The reprogramming of a patient's somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known.
  
  
  METHODS
  In this study, we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry, karyotyping and teratoma formation, and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions.
  
  
  RESULTS
  We found that a STEMCCA-derived iPSC line that contains a single integration, found to be located in an intronic location in an actively transcribed gene, PRPF39, displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs, differentiated them into multiple clinically relevant cell types (including oligodendrocytes, hepatocytes, and cardiomyocytes), and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status.
  
  
  CONCLUSIONS
  For the first time, these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and, in light of the limited availability of current good manufacturing practice cellular manufacturing facilities, highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics.
authors.forename: Jason P
authors.initials: JP
authors.lastname: Awe
authors.forename: Patrick C
authors.initials: PC
authors.lastname: Lee
authors.forename: Cyril
authors.initials: C
authors.lastname: Ramathal
authors.forename: Agustin
authors.initials: A
authors.lastname: Vega-Crespo
authors.forename: Jens
authors.initials: J
authors.lastname: Durruthy-Durruthy
authors.forename: Aaron
authors.initials: A
authors.lastname: Cooper
authors.forename: Saravanan
authors.initials: S
authors.lastname: Karumbayaram
authors.forename: William E
authors.initials: WE
authors.lastname: Lowry
authors.forename: Amander T
authors.initials: AT
authors.lastname: Clark
authors.forename: Jerome A
authors.initials: JA
authors.lastname: Zack
authors.forename: Vittorio
authors.initials: V
authors.lastname: Sebastiano
authors.forename: Donald B
authors.initials: DB
authors.lastname: Kohn
authors.forename: April D
authors.initials: AD
authors.lastname: Pyle
authors.forename: Martin G
authors.initials: MG
authors.lastname: Martin
authors.forename: Gerald S
authors.initials: GS
authors.lastname: Lipshutz
authors.forename: Patricia E
authors.initials: PE
authors.lastname: Phelps
authors.forename: Renee A Reijo
authors.initials: RA
authors.lastname: Pera
authors.forename: James A
authors.initials: JA
authors.lastname: Byrne
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D065150:Cellular Reprogramming; D015870:Gene Expression; D023281:Genomics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D051379:Mice; D019076:Transgenes
references.citation: Nat Rev Genet. 2011 Apr;12(4):231-42
references.pmid: 21339765
references.citation: Neurobiol Dis. 2012 Apr;46(1):41-51
references.pmid: 22405424
references.citation: Cell. 2002 Aug 23;110(4):521-9
references.pmid: 12202041
references.citation: Glia. 2005 Feb;49(3):385-96
references.pmid: 15538751
references.citation: Cell Res. 2012 Jan;22(1):219-36
references.pmid: 22143566
references.citation: Cell Stem Cell. 2011 Jun 3;8(6):633-8
references.pmid: 21620789
references.citation: Stem Cells. 2010 Oct;28(10):1728-40
references.pmid: 20715179
references.citation: J Cell Physiol. 2013 Feb;228(2):267-75
references.pmid: 22767332
references.citation: Nat Protoc. 2010 Aug;5(8):1379-95
references.pmid: 20671722
references.citation: Eur Heart J. 2013 Jun;34(21):1575-86
references.pmid: 22621821
references.citation: Blood. 2003 Feb 15;101(4):1284-9
references.pmid: 12393514
references.citation: EMBO Rep. 2005 May;6(5):458-63
references.pmid: 15832171
references.citation: Nature. 2007 Jul 19;448(7151):313-7
references.pmid: 17554338
references.citation: Genome Biol. 2009;10(3):R25
references.pmid: 19261174
references.citation: Stem Cell Res. 2012 Jan;8(1):134-40
references.pmid: 22099027
references.citation: Hum Mol Genet. 2008 Apr 15;17(R1):R37-41
references.pmid: 18632695
references.citation: Science. 2009 May 8;324(5928):797-801
references.pmid: 19325077
references.citation: Cell. 2009 Mar 6;136(5):964-77
references.pmid: 19269371
references.citation: Cell Res. 2009 Nov;19(11):1233-42
references.pmid: 19736565
references.citation: Cell Stem Cell. 2009 Jun 5;4(6):472-6
references.pmid: 19481515
references.citation: Eur Heart J. 2013 Sep;34(33):2618-29
references.pmid: 22798560
references.citation: PLoS One. 2009;4(1):e4241
references.pmid: 19156207
references.citation: Hum Gene Ther. 2010 Aug;21(8):943-56
references.pmid: 20210626
references.citation: Biores Open Access. 2012 Jan;1(1):25-33
references.pmid: 23514702
references.citation: Cell Res. 2009 Mar;19(3):296-306
references.pmid: 19238173
references.citation: Stem Cells Transl Med. 2012 Jan;1(1):36-43
references.pmid: 23197638
references.citation: Stem Cells. 2010 Sep;28(9):1541-9
references.pmid: 20665739
references.citation: Cell Stem Cell. 2010 Nov 5;7(5):618-30
references.pmid: 20888316
references.citation: J Clin Invest. 1990 Nov;86(5):1752-6
references.pmid: 2243144
references.citation: Proc Natl Acad Sci U S A. 2006 May 2;103(18):6907-12
references.pmid: 16632596
references.citation: Cell. 2007 Nov 30;131(5):861-72
references.pmid: 18035408
references.citation: Exp Neurol. 2009 Mar;216(1):95-104
references.pmid: 19100263
references.citation: Nat Biotechnol. 2006 Jun;24(6):687-96
references.pmid: 16732270
references.citation: Stem Cells. 2010 Jan;28(1):64-74
references.pmid: 19904830
references.citation: J Allergy Clin Immunol. 2011 Jun;127(6):1400-7.e4
references.pmid: 21185069
references.citation: Hum Mol Genet. 2012 Oct 1;21(19):4348-55
references.pmid: 22773733
references.citation: Cell Res. 2012 Jan;22(1):178-93
references.pmid: 21844894
references.citation: PLoS One. 2009 Sep 23;4(9):e7118
references.pmid: 19774082
references.citation: Cell Stem Cell. 2011 Apr 8;8(4):376-88
references.pmid: 21474102
references.citation: Stem Cell Rev Rep. 2010 Sep;6(3):367-80
references.pmid: 20336395
references.citation: Nat Biotechnol. 2011 Jan;29(1):73-8
references.pmid: 21151124
references.citation: Nat Methods. 2010 Mar;7(3):197-9
references.pmid: 20139967
references.citation: Science. 2003 Oct 17;302(5644):415-9
references.pmid: 14564000
references.citation: Mol Cell Neurosci. 2007 Mar;34(3):310-23
references.pmid: 17196394
references.citation: Stem Cell Res Ther. 2010 Aug 10;1(3):26
references.pmid: 20699015
references.citation: Stem Cells. 2009 Mar;27(3):543-9
references.pmid: 19096035
references.citation: Proc Jpn Acad Ser B Phys Biol Sci. 2009;85(8):348-62
references.pmid: 19838014
references.citation: Nat Protoc. 2009;4(11):1614-22
references.pmid: 19834476
references.citation: Science. 2007 Dec 21;318(5858):1917-20
references.pmid: 18029452
references.citation: Cell Stem Cell. 2010 Jul 2;7(1):15-9
references.pmid: 20621044
references.citation: Nat Med. 2005 Feb;11(2):228-32
references.pmid: 15685172