title: Improved generation of patient-specific induced pluripotent stem cells using a chemically-defined and matrigel-based approach.
abstract: Reprogramming of somatic cells into patient-specific pluripotent analogues of human embryonic stem cells (ESCs) emerges as a prospective therapeutic angle in molecular medicine and a tool for basic stem cell biology. However, the combination of relative inefficiency and high variability of non-defined culture conditions precluded the use of this technique in a clinical setting and impeded comparability between laboratories. To overcome these obstacles, we sequentially devised a reprogramming protocol using one lentiviral-based polycistronic reprogramming construct, optimized for high co-expression of OCT4, SOX2, KLF4 and MYC in conjunction with small molecule inhibitors of non-permissive signaling cascades, such as transforming growth factor β (SB431542), MEK/ERK (PD0325901) and Rho-kinase signaling (Thiazovivin), in a defined extracellular environment. Based on human fetal liver fibroblasts we could efficiently derive induced pluripotent stem cells (iPSCs) within 14 days. We attained efficiencies of up to 10.97±1.71% resulting in 79.5- fold increase compared to non-defined reprogramming using four singular vectors. We show that the overall increase of efficiency and temporal kinetics is a combinatorial effect of improved lentiviral vector design, signaling inhibition and definition of extracellular matrix (Matrigel®) and culture medium (mTESR®1). Using this protocol, we could derive iPSCs from patient fibroblasts, which were impermissive to classical reprogramming efforts, and from a patient suffering from familial platelet disorder. Thus, our defined protocol for highly efficient reprogramming to generate patient-specific iPSCs, reflects a big step towards therapeutic and broad scientific application of iPSCs, even in previously unfeasible settings.
authors: Department of Pediatric Hematology & Oncology, Hannover Medical School, D-30625 Hannover, Germany.
authors: B
authors: B
authors: Groß
authors: M
authors: M
authors: Sgodda
authors: M
authors: M
authors: Rasche
authors: A
authors: A
authors: Schambach
authors: G
authors: G
authors: Göhring
authors: B
authors: B
authors: Schlegelberger
authors: B
authors: B
authors: Greber
authors: T
authors: T
authors: Linden
authors: D
authors: D
authors: Reinhardt
authors: T
authors: T
authors: Cantz
authors: J-H
authors: JH
authors: Klusmann
mesh_terms: D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D001791:Blood Platelet Disorders / Q000235:genetics / Q000473:pathology; D049109:Cell Proliferation; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D003094:Collagen / Q000737:chemistry*; D050676:Core Binding Factor Alpha 2 Subunit / Q000235:genetics; D003470:Culture Media / Q000737:chemistry; D004149:Dioxoles / Q000494:pharmacology; D004159:Diphenylamine / Q000031:analogs & derivatives / Q000494:pharmacology; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000502:physiology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000187:drug effects / Q000502:physiology*; D007231:Infant, Newborn; D000090062:Kruppel-Like Factor 4; D007797:Laminin / Q000737:chemistry*; D051379:Mice; D011509:Proteoglycans / Q000737:chemistry*; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics
chemical_list: C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; D050676:Core Binding Factor Alpha 2 Subunit; D003470:Culture Media; D004149:Dioxoles; D004338:Drug Combinations; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D007797:Laminin; D011509:Proteoglycans; C493728:RUNX1 protein, human; D018125:Receptors, Transforming Growth Factor beta; D014157:Transcription Factors; C059485:matrigel; C506614:mirdametinib; D003094:Collagen; D004159:Diphenylamine