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

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

doi:10.1038/srep13231

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.

Highlights

Challenged by their genetic homology with patient DNA, as they may retain functional deficiencies
Karyotyping (Supplemental Figure S3) and DNA fingerprinting (a) revealed no reprogramming-attributed chromosomal abnormalities and (b) demonstrated homology between the Familial Hypercholesterolemia (FH)-Induced pluripotent stem cells (iPSC) and their source fibroblasts. These results demonstrate that the GM01355 FH fibroblasts were successfully reprogrammed to into pluripotent FH-iPSC
The fact that our efficiency was considerably lower than published figures could potentially be attributed to the low-density lipoprotein cholesterol (LDL) receptor (LDLR) deficiency of our source FH fibroblasts, other reports describing the generation of FH-iPSC using viral vectors do not report reprogramming efficiencies[12,29,30]

Summary

Introduction

Challenged by their genetic homology with patient DNA, as they may retain functional deficiencies. We restored FH-iPSC LDLR-mediated endocytosis using a novel 31 kb plasmid containing (a) wild-type LDLR physiologically controlled by 10 kb upstream genomic regulatory control sequence and (b) the minimum number of required Epstein-Barr Virus (EBV) replication and retention sequences (Epstein-Barr Nuclear Association 1 (EBNA1) and origin of plasmid replication (oriP))[8]. This large plasmid was transfected into iPSC via electroporation and retained as a stable episome. The technologies and methods discussed represent a unique approach for functionalizing genetically deficient iPSC using episomal plasmids that contain genomic transgene control sequences

Methods

Results

Conclusion