title: mRNA transfection-based, feeder-free, induced pluripotent stem cells derived from adipose tissue of a 50-year-old patient.
abstract: Induced pluripotent stem cells (iPSC) have successfully been derived from somatic fibroblasts through transfection of synthetic modified mRNA encoding transcription factors. This technique obviates the use of recombinant DNA and viral vectors in cellular reprogramming. The present study derived iPSC from adipose-derived mesenchymal stem cells (of a 50-year-old female patient) by utilizing a similar technique, but with defined culture medium without feeder cells, during both reprogramming and propagation. Clonal selection was performed to yield 12 putative iPSC lines from individual colonies of nascent reprogrammed cells, starting from 150,000 cells. However, only seven lines maintained their undifferentiated state after 10 continuous serial passages. These seven lines were then subjected to a rigorous battery of analyses to confirm their identity as iPSC. These tests included immunostaining, flow cytometry, qRT-PCR, in vitro differentiation assay, and teratoma formation assay within SCID mice. Positive results were consistently observed in all analyses, thus verifying the cells as fully reprogrammed iPSC. While all 7 iPSC lines displayed normal karyogram up to passage 13, chromosomal anomalies occurred in 4 of 7 lines with extended in vitro culture beyond 24 serial passages. Only three lines retained normal karyotype of 46,XX. The remaining four lines displayed mosaicism of normal and abnormal karyotypes. Hence, this study successfully derived iPSC from abundant and easily accessible adipose tissues of a middle-aged patient; utilizing a mRNA-based integration-free technique under feeder-free conditions. This is a step forward in translating iPSC into personalized regenerative medicine within the clinic.
authors.affiliation: Department of Biosystems Science and Engineering-D-BSSE, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland.
authors.forename: Boon Chin
authors.initials: BC
authors.lastname: Heng
authors.forename: Karl
authors.initials: K
authors.lastname: Heinimann
authors.forename: Peter
authors.initials: P
authors.lastname: Miny
authors.forename: Giandomenica
authors.initials: G
authors.lastname: Iezzi
authors.forename: Katharina
authors.initials: K
authors.lastname: Glatz
authors.forename: Arnaud
authors.initials: A
authors.lastname: Scherberich
authors.forename: Henryk
authors.initials: H
authors.lastname: Zulewski
authors.forename: Martin
authors.initials: M
authors.lastname: Fussenegger
mesh_terms: D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D005260:Female; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008875:Middle Aged; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D012333:RNA, Messenger