Restoration of Physiologically Responsive Low-Density Lipoprotein Receptor-Mediated Endocytosis in Genetically Deficient Induced Pluripotent Stem Cells

• Published in: Scientific reports 2015-08, Vol.5 (1), p.13231-13231, Article 13231
• Main Authors: Ramakrishnan, Venkat M., Yang, Jeong-Yeh, Tien, Kevin T., McKinley, Thomas R., Bocard, Braden R., Maijub, John G., Burchell, Patrick O., Williams, Stuart K., Morris, Marvin E., Hoying, James B., Wade-Martins, Richard, West, Franklin D., Boyd, Nolan L.
• Format: Article
• Language: English
• Subjects: 692/308/2171; 692/699/75/2099; Autografts; Cell culture; Cell Differentiation - genetics; Cells, Cultured; Deoxyribonucleic acid; DNA; Endocytosis; Endocytosis - genetics; Ethanol; Feedback; Fibroblasts; Genetic Enhancement - methods; Humanities and Social Sciences; Humans; Hypercholesterolemia; Hyperlipoproteinemia Type II - genetics; Induced Pluripotent Stem Cells - pathology; Induced Pluripotent Stem Cells - physiology; Internalization; Lovastatin; Low density lipoprotein; Low density lipoprotein receptors; Mesenchyme; mRNA; multidisciplinary; Nucleotide sequence; Physiology; Plasmids; Plasmids - administration & dosage; Plasmids - genetics; Pluripotency; Receptor density; Receptors, LDL - genetics; Recovery of Function; Science; Stem cells; Sterols; Transgenes
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep13231

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.