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.

Development of a vascularized, induced pluripotent stem cell-derived liver-tissue mimic for therapeutic applications.

“…Each experimental condition was quantified for length density (i.e., a ratio of the total EC length to the 3D scan volume; Figure 20B) as well as the Adipose SVF is a heterogeneous cell population that contains EC, perivascular cells, macrophage, fibroblasts and stem cells 9,369,400 . This endogenous heterogeneity is likely what imbues the cellular system with the specific capacity to form functional microvasculatures in vivo 1,9,370 . In prior efforts, we leveraged this functionality to support the engraftment of hepatic parenchymal cells 1,9 .…”

“…This endogenous heterogeneity is likely what imbues the cellular system with the specific capacity to form functional microvasculatures in vivo 1,9,370 . In prior efforts, we leveraged this functionality to support the engraftment of hepatic parenchymal cells 1,9 . Koh and colleagues demonstrated that the formation of vascular networks by mouse SVF (mSVF) depended on the presence of EC and is VEGF-dependent 299 .…”

“…Our data supports that Wnt5a plays a specific role in hSVF microvascular self-assembly in vitro. Therefore, we wanted to determine its role in the self-assembly that others and we have reported in vivo 1,9,299 . We initially exposed hSVF cultures to varying concentrations (0-20 µg/ml) of Wnt5a neutralizing antibody (referred to as anti-Wnt5a) 377,378,399 to determine an appropriate concentration for use (Figure 19).…”

“…For analysis of protein expression, wells were prepared for fluorescence microscopy as previously described 1 . Cells were permeabilized in 0.5% Triton X-100 [377][378][379] Media changes occurred every other day.…”

“…RT-PCR, and gel electrophoresis were all conducted as previously described 1 . RT-PCR reactions (primers can be found in Table 2) utilized the following settings: 25 cycles, with denaturation at 95˚C, annealing at 59˚C, and extension at 70˚C for 30 seconds.…”