title: Induction of pluripotency in long-term cryopreserved human neonatal fibroblasts in feeder-free condition.
abstract: A novel approach for stem cell generation is the attempt to induce conversion of the adult somatic cells into pluripotent stem cells so called induced pluripotent stem cells (iPSCs) by introducing specific transcription factors. iPSCs have two essential cell characteristics, they are pluripotent and posses long term cell-renewal capacity. Additionally, iPSCs can be derived from patient-specific somatic cells, thus bypassing ethical and immunological issues. The aim of our study was to reprogram long-term cryopreserved human neonatal fibroblasts by new method using lipid nano-particle technology (Lipofectamine 3000 reagent transfection system) in combination with Epi 5 reprogramming vectors. Obtained iPSCs were characterized by several sophisticated methods of molecular biology and microscopy. Distinct colonies of iPSCs started to appear by day 20 after reprogramming. The presence of iPSCs colonies was proved by alkaline phosphatase (AP) live staining. After manual picking the colonies and their subsequent passaging, they did not lose ability to form embryoid bodies, they were positive for AP, Tra-1-60, and SSEA-5. Moreover, obtained iPSCs expressed pluripotency markers Oct4, Sox2 and Nanog, and the expression levels of chondrogenic, osteogenic and adipogenic markers were significantly higher in comparison to control (p < 0.05). In summary, we have demonstrated that long-term cryopreserved human neonatal fibroblasts can be reprogrammed into iPSCs and after further analysis concerns on their biological safety they may be used as patient-specific cells in regenerative medicine.
authors.affiliation: Faculty of Medicine, Institute of Histology and Embryology, Comenius University, Sasinkova 4, 811 08, Bratislava, Slovakia.
authors.forename: Maria
authors.initials: M
authors.lastname: Csobonyeiova
authors.affiliation: Faculty of Medicine, Institute of Medical Biology, Genetics and Clinical Genetics, Comenius University, Sasinkova 4, 811 08, Bratislava, Slovakia.
authors.forename: Lubica
authors.initials: L
authors.lastname: Krajciova
authors.affiliation: Faculty of Medicine, Institute of Medical Biology, Genetics and Clinical Genetics, Comenius University, Sasinkova 4, 811 08, Bratislava, Slovakia.
authors.forename: Andreas
authors.initials: A
authors.lastname: Nicodemou
authors.affiliation: Faculty of Medicine, Institute of Histology and Embryology, Comenius University, Sasinkova 4, 811 08, Bratislava, Slovakia.
authors.forename: Stefan
authors.initials: S
authors.lastname: Polak
authors.affiliation: Faculty of Medicine, Institute of Medical Biology, Genetics and Clinical Genetics, Comenius University, Sasinkova 4, 811 08, Bratislava, Slovakia. lubos.danisovic@fmed.uniba.sk.
authors.forename: Lubos
authors.identifier: http://orcid.org/0000-0002-5074-9621
authors.initials: L
authors.lastname: Danisovic
mesh_terms: D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D065150:Cellular Reprogramming; D015925:Cryopreservation; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D010957:Plasmids / Q000235:genetics
chemical_list: D008055:Lipids; C086724:Lipofectamine
keywords: Episomal vectors; Human neonatal fibroblasts; Multilineage differentiation; Reprogramming; iPSCs