title: Generation of human β-thalassemia induced pluripotent cell lines by reprogramming of bone marrow-derived mesenchymal stromal cells using modified mRNA.
abstract: Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with β-thalassemia (β-thal) with the aim to generate trangene-free β-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4, Klf4, Sox2, cMyc, and Lin28 resulted in formation of five iPSC colonies, from which three were picked up and expanded in β-thal-iPSC lines. After 10 serial passages in vitro, β-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs, whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%, but with a decreased hematopoietic colony-forming capability. In conclusion, we report herein the generation of transgene-free β-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover, it was demonstrated that the mRNA-based reprogramming method, used mainly in fibroblasts, is also suitable for reprogramming of human BM-MSCs.
authors.affiliation: 1 Stem Cell Transplant Unit, Aghia Sophia Children's Hospital , Athens, 11527, Greece .
authors.forename: Ioanna
authors.initials: I
authors.lastname: Varela
authors.forename: Angeliki
authors.initials: A
authors.lastname: Karagiannidou
authors.forename: Vasilis
authors.initials: V
authors.lastname: Oikonomakis
authors.forename: Maria
authors.initials: M
authors.lastname: Tzetis
authors.forename: Marianna
authors.initials: M
authors.lastname: Tzanoudaki
authors.forename: Elena-Konstantina
authors.initials: EK
authors.lastname: Siapati
authors.forename: George
authors.initials: G
authors.lastname: Vassilopoulos
authors.forename: Stelios
authors.initials: S
authors.lastname: Graphakos
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Kanavakis
authors.forename: Evgenios
authors.initials: E
authors.lastname: Goussetis
mesh_terms: D002454:Cell Differentiation; D002460:Cell Line; D065150:Cellular Reprogramming; D055028:Comparative Genomic Hybridization; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D000090062:Kruppel-Like Factor 4; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D012333:RNA, Messenger / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D017086:beta-Thalassemia
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D012333:RNA, Messenger; D014157:Transcription Factors