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.

The success of vascular intervention including angioplasty, stenting, and arterial bypass remains limited by negative remodeling resulted in lumen restenosis. This study was to characterize the global transcription profile reflecting concurrent events along arterial remodeling and neointima formation in a rat carotid artery balloon-injury model. Expression profiling of injured and control common carotid arteries on days 4, 7, 14 post-injury that mark the major pathohistological progression stages of neointimal formation were recorded on high-density oligonucleotide arrays. A subset of genes from microarray-based data was further studied using quantitative real time RT-PCR and in situ hybridization with sequential arterial samples from days 1 to 28 post-injury. The gene-encoded proteins were validated with Western blot. Besides temporal induction of a large cluster of genes over-represented by cell proliferation and macromolecule metabolism gene ontology categories, a fast-evolving inflammation could be demonstrated by the induction of Tgfb and other anti-inflammatory genes (e.g., C1qtnf3 (C1q and tumor necrosis factor related protein 3 (predicted))) and a shift from type 1 to 2 helper T cell response. The most significant signature of the induced neointimal profile is enrichment of genes functionally related to angiogenesis and extracellular matrix (ECM) remodeling (e.g., Spp1 (secreted phosphoprotein 1), CD44 (CD44 antigen), and Cxcl12 (chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)). Some of the genes represent stress-responsive mesenchymal stromal cell cytokines. This study highlighted mesenchymal stromal cell cytokines-driven inflammatory extracellular matrix remodeling, as target processes for potential clinical therapeutic intervention.

Vasoactivity, an important aspect of tissue healing, is often compromised in disease and tissue injury. Dysfunction in the smaller vasoactive arteries is most impactful, given the role of these vessels in controlling downstream tissue perfusion. The adipose stromal vascular fraction (SVF) is a mix of homeostatic cells shown to promote tissue healing. Our objective was to test the hypothesis that autologous SVF cells therapeutically modulate peripheral artery vasoactivity in syngeneic mouse models of small artery function. Analysis of vasoactivity of saphenous arteries isolated from normal mice 1 week after intravenous injection of freshly isolated SVF cells revealed that pressure-dependent artery vasomotor tone was decreased by the SVF cell isolate, but not one depleted of CD11b(+) cells. Scavenging hydrogen peroxide in the vessel wall abrogated the artery relaxation promoted by the SVF cell isolate. Consistent with a CD11b(+) cell being the relevant cell type, SVF-derived F4/80-positive macrophages were present within the adventitia of the artery wall coincident with vasorelaxation. In a model of artery inflammation mimicking a common disease condition inducing vasoactive dysfunction, the SVF cells potentiated relaxation of saphenous arteries without structurally remodeling the artery via a CD11b(+) cell-dependent manner. Our findings demonstrate that freshly isolated, adipose SVF cells promote vasomotor relaxation in vasoactive arteries via a hydrogen peroxide-dependent mechanism that required CD11b(+) cells (most likely macrophages). Given the significant impact of small artery dysfunction in disease, we predict that the intravenous delivery of this therapeutic cell preparation would significantly improve tissue perfusion, particularly in diseases with diffuse vascular involvement.