Purpose: Stem cell therapy for angiogenesis is limited by insufficient expression of angiogenic factors and poor cell viability after transplantation. Viral gene delivery raises safety concerns, while non-viral methods often suffer from low transfection efficiency and toxicity. A safe, efficient, biodegradable polymeric nanoparticle system is needed to transiently engineer human stem cells to overexpress VEGF for therapeutic angiogenesis.
Hypothesis: If human mesenchymal stem cells (hMSCs) and human embryonic stem cell–derived cells (hESdCs) are transfected with hVEGF plasmid DNA using biodegradable poly(β-amino ester) (PBAE) nanoparticles, then they will produce markedly enhanced VEGF, survive and engraft better after transplantation, and promote greater angiogenesis, limb salvage, and tissue repair than untransfected cells or cells transfected with Lipofectamine 2000.
Aims: Synthesize and use end-modified C32 PBAE polymers (C32-103, C32-117, C32-122) for VEGF gene delivery to hMSCs and hESdCs. - Assess in vitro VEGF production, cell viability, and paracrine effects. - Evaluate angiogenesis in a subcutaneous scaffold implantation model in mice. - Evaluate therapeutic efficacy in a mouse ischemic hindlimb model: angiogenesis, limb salvage, muscle degeneration, fibrosis, cell survival, and engraftment.
Delivery system:
Component: Polymer; Details: Biodegradable poly(β-amino esters) (PBAEs); C32 base polymer end-modified with amines 103, 117, or 122
Component: Payload; Details: Plasmid DNA encoding human VEGF (hVEGF); controls: EGFP or luciferase plasmid
Component: Nanoparticle formation; Details: Polymer–DNA electrostatic self-assembly
Component: Target cells; Details: Human bone marrow–derived mesenchymal stem cells (hMSCs); human embryonic stem cell–derived cells (hESdCs)
Component: Targeting ligand; Details: None
Component: Delivery route; Details: Ex vivo transfection, then cell transplantation; in vivo: subcutaneous scaffold implantation or intramuscular injection into ischemic hindlimb
Component: Scaffold; Details: PLGA/PLLA scaffolds seeded with transfected stem cells for s.c. implantation
Component: Key feature; Details: Transient, non-viral, biodegradable gene delivery to stem cells for paracrine VEGF secretion
Approach: In vitro: hMSCs and hESdCs transfected with PBAE/VEGF or controls; VEGF ELISA and viability. - Subcutaneous model: Athymic mice implanted with stem cell–seeded scaffolds. hMSC groups: C32-103/VEGF, C32-117/VEGF, C32-122/VEGF; controls: C32-103/Luc, Lipo/VEGF, acellular scaffold. hESdC groups: C32-117/VEGF, C32-117/Luc, acellular scaffold (n = 3). Harvested at 2 or 3 weeks. - Ischemic hindlimb model: Mice with induced hindlimb ischemia. Five groups (n = 8): PBS, no transfection, hMSC-C32-122/EGFP, hMSC-Lipo/VEGF, hMSC-C32-122/VEGF. 1 × 10⁶ cells injected intramuscularly. Analysis at 4 weeks. - Disease context: Therapeutic angiogenesis, ischemic disease, tissue regeneration.
Key methods: VEGF production: ELISA (in vitro supernatant and in vivo muscle). - Cell viability: Viability assessment after transfection. - Angiogenesis: Immunohistochemistry for MECA (mouse endothelial cell antigen), smooth muscle α-actin (SMA), von Willebrand factor (vWF); vessel density quantification. - Cell engraftment/survival: RT-PCR for human chromosome 17α satellite; immunofluorescence for human nuclear antigen (HNA). - Homing factors: SDF-1α and CXCR4 expression. - Tissue repair: H&E, Masson’s trichrome, TTC staining; limb salvage scoring. - Statistics: ANOVA with Bonferroni test; P < 0.05 considered significant.
Key results: In vitro VEGF: PBAE-transfected hMSCs/hESdCs secreted ~1–3-fold more VEGF than untransfected controls and ~1–2-fold more than Lipofectamine 2000 at day 4 (P < 0.05); viability 80–90%. - Subcutaneous model: Scaffolds with VEGF-expressing stem cells led to 2- to 4-fold higher vessel densities at 2–3 weeks vs controls (C32-103/Luc, Lipo/VEGF, acellular scaffold). - Ischemic hindlimb: C32-122/VEGF hMSCs produced 6-fold higher VEGF than untransfected cells at day 2; 3-fold higher MECA-positive microvessels vs PBS and 50% higher than Lipo/VEGF. Limb salvage increased from 12.5% to 50%; limb loss decreased from 60% to 20%. Reduced muscle degeneration and fibrosis; enhanced engraftment and CXCR4 expression. - hESdCs: Also showed enhanced vessel density in s.c. model.
Interpretation: The authors claim that human stem cells transiently engineered with biodegradable PBAE nanoparticles to express VEGF can promote therapeutic angiogenesis, improve limb salvage, and reduce tissue degeneration/fibrosis in ischemic disease models. This combined polymer–stem cell approach may provide a safer, non-viral alternative for vascularizing tissue constructs and treating ischemic diseases, with potential utility in bone, muscle, and complex tissue regeneration.
Limitations: Transient expression: VEGF production lasted ~2 weeks, lower and shorter than adenoviral vectors (which can last ~30 days and produce higher VEGF levels). - In vivo models only: Mouse subcutaneous and hindlimb ischemia models; no large-animal or human validation. - Ex vivo manipulation: Requires cell isolation, transfection, and transplantation; not an injectable in vivo gene delivery system. - No targeting ligand: Delivery relies on ex vivo transfection; no active targeting after transplantation. - Limited sample sizes: s.c. model n = 3; hindlimb model n = 8 per group. - Single time point for hindlimb analysis: 4 weeks; longer-term outcomes and safety not assessed. - No direct comparison with viral vectors in same study; comparison to adenovirus drawn from literature. - No long-term safety/tumorigenicity data. - No dose–response optimization for VEGF plasmid or polymer reported.
