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


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2015-10

AUTHORS

Venkat M. Ramakrishnan, Jeong-Yeh Yang, Kevin T. Tien, Thomas R. McKinley, Braden R. Bocard, John G. Maijub, Patrick O. Burchell, Stuart K. Williams, Marvin E. Morris, James B. Hoying, Richard Wade-Martins, Franklin D. West, Nolan L. Boyd

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. More... »

PAGES

13231

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/srep13231

DOI

http://dx.doi.org/10.1038/srep13231

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1023370425

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/26307169


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