title: Restoration of Physiologically Responsive Low-Density Lipoprotein Receptor-Mediated Endocytosis in Genetically Deficient Induced Pluripotent Stem Cells.
abstract: Acquiring sufficient amounts of high-quality cells remains an impediment to cell-based therapies. Induced pluripotent stem cells (iPSC) may be an unparalleled source, but autologous iPSC likely retain deficiencies requiring correction. We present a strategy for restoring physiological function in genetically deficient iPSC utilizing the low-density lipoprotein receptor (LDLR) deficiency Familial Hypercholesterolemia (FH) as our model. FH fibroblasts were reprogrammed into iPSC using synthetic modified mRNA. FH-iPSC exhibited pluripotency and differentiated toward a hepatic lineage. To restore LDLR endocytosis, FH-iPSC were transfected with a 31 kb plasmid (pEHZ-LDLR-LDLR) containing a wild-type LDLR (FH-iPSC-LDLR) controlled by 10 kb of upstream genomic DNA as well as Epstein-Barr sequences (EBNA1 and oriP) for episomal retention and replication. After six months of selective culture, pEHZ-LDLR-LDLR was recovered from FH-iPSC-LDLR and transfected into Ldlr-deficient CHO-a7 cells, which then exhibited feedback-controlled LDLR-mediated endocytosis. To quantify endocytosis, FH-iPSC ± LDLR were differentiated into mesenchymal cells (MC), pretreated with excess free sterols, Lovastatin, or ethanol (control), and exposed to DiI-LDL. FH-MC-LDLR demonstrated a physiological response, with virtually no DiI-LDL internalization with excess sterols and an ~2-fold increase in DiI-LDL internalization by Lovastatin compared to FH-MC. These findings demonstrate the feasibility of functionalizing genetically deficient iPSC using episomal plasmids to deliver physiologically responsive transgenes.
authors: Cardiovascular Innovation Institute, University of Louisville School of Medicine and Jewish Hospital, Louisville, Kentucky 40202, USA.|Department of Physiology, University of Louisville School of Medicine, Louisville, Kentucky 40202, USA.
authors: Venkat M
authors: VM
authors: Ramakrishnan
authors: Regenerative Bioscience Center, University of Georgia, Athens, Georgia 30602, USA.|Department of Animal and Dairy Sciences, University of Georgia, Athens, GA 30206, USA.
authors: Jeong-Yeh
authors: JY
authors: Yang
authors: Cardiovascular Innovation Institute, University of Louisville School of Medicine and Jewish Hospital, Louisville, Kentucky 40202, USA.
authors: Kevin T
authors: KT
authors: Tien
authors: Cardiovascular Innovation Institute, University of Louisville School of Medicine and Jewish Hospital, Louisville, Kentucky 40202, USA.
authors: Thomas R
authors: TR
authors: McKinley
authors: Cardiovascular Innovation Institute, University of Louisville School of Medicine and Jewish Hospital, Louisville, Kentucky 40202, USA.|Georgetown College, Georgetown, KY 40324, USA.
authors: Braden R
authors: BR
authors: Bocard
authors: Cardiovascular Innovation Institute, University of Louisville School of Medicine and Jewish Hospital, Louisville, Kentucky 40202, USA.|Department of Surgery, University of Louisville School of Medicine, Louisville, KY 40202, USA.
authors: John G
authors: JG
authors: Maijub
authors: Cardiovascular Innovation Institute, University of Louisville School of Medicine and Jewish Hospital, Louisville, Kentucky 40202, USA.
authors: Patrick O
authors: PO
authors: Burchell
authors: Cardiovascular Innovation Institute, University of Louisville School of Medicine and Jewish Hospital, Louisville, Kentucky 40202, USA.|Department of Physiology, University of Louisville School of Medicine, Louisville, Kentucky 40202, USA.
authors: Stuart K
authors: SK
authors: Williams
authors: Cardiovascular Innovation Institute, University of Louisville School of Medicine and Jewish Hospital, Louisville, Kentucky 40202, USA.|Department of Surgery, University of Louisville School of Medicine, Louisville, KY 40202, USA.
authors: Marvin E
authors: ME
authors: Morris
authors: Cardiovascular Innovation Institute, University of Louisville School of Medicine and Jewish Hospital, Louisville, Kentucky 40202, USA.|Department of Physiology, University of Louisville School of Medicine, Louisville, Kentucky 40202, USA.
authors: James B
authors: JB
authors: Hoying
authors: Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford OX1 3QX, UK.
authors: Richard
authors: R
authors: Wade-Martins
authors: Regenerative Bioscience Center, University of Georgia, Athens, Georgia 30602, USA.|Department of Animal and Dairy Sciences, University of Georgia, Athens, GA 30206, USA.
authors: Franklin D
authors: FD
authors: West
authors: Cardiovascular Innovation Institute, University of Louisville School of Medicine and Jewish Hospital, Louisville, Kentucky 40202, USA.|Department of Physiology, University of Louisville School of Medicine, Louisville, Kentucky 40202, USA.
authors: Nolan L
authors: NL
authors: Boyd
mesh_terms: D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D004705:Endocytosis / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000235:genetics*; D057026:Induced Pluripotent Stem Cells / Q000473:pathology* / Q000502:physiology*; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics*; D011973:Receptors, LDL / Q000235:genetics*; D020127:Recovery of Function
chemical_list: D011973:Receptors, LDL