Permanently Hypoxic Cell Culture Yields Rat Bone Marrow Mesenchymal Cells with Higher Therapeutic Potential in the Treatment of Chronic Myocardial Infarction

Abstract

Background: The mismatch between traditional in vitro cell culture conditions and targeted chronic hypoxic myocardial tissue could potentially hamper the therapeutic effects of implanted bone marrow mesenchymal stem cells (BMSCs). This study sought to address (i) the extent of change to BMSC biological characteristics in different in vitro culture conditions and (ii) the effectiveness of permanent hypoxic culture for cell therapy in treating chronic myocardial infarction (MI) in rats. Methods: rat BMSCs were harvested and cultured in normoxic (21% O₂, n=27) or hypoxic conditions (5% O₂, n=27) until Passage 4 (P4). Cell growth tests, flow cytometry, and Bio-Plex assays were conducted to explore variations in the cell proliferation, phenotype, and cytokine expression, respectively. In the in vivo set-up, P3-BMSCs cultured in normoxia (n=6) or hypoxia (n=6) were intramyocardially injected into rat hearts that had previously experienced 1-month-old MI. The impact of cell therapy on cardiac segmental viability and hemodynamic performance was assessed 1 month later by 2-Deoxy-2[¹⁸F]fluoro-D-glucose (¹⁸F-FDG) positron emission tomography (PET) imaging and pressure-volume catheter, respectively. Additional histomorphological examinations were conducted to evaluate inflammation, fibrosis, and neovascularization. Results: Hypoxic preconditioning significantly enhanced rat BMSC clonogenic potential and proliferation without altering the multipotency. Different profiles of inflammatory, fibrotic, and angiogenic cytokine secretion were also documented, with a marked correlation observed between in vitro and in vivo proangiogenic cytokine expression and tissue neovessels. Hypoxic-preconditioned cells presented a beneficial effect on the myocardial viability of infarct segments and intrinsic contractility. Conclusion: Hypoxic-preconditioned BMSCs were able to benefit myocardial perfusion and contractility, probably by modulating the inflammation and promoting angiogenesis.

Repetitive Transient Ischemia-Induced Cardiac Angiogenesis is Mediated by Camkii Activation

“…Stimulation of coronary collateral formation provides a broad prospect for the treatment of CAD patients [39][40][41]. Various therapeutic angiogenesis attempts focused on VEGF and FGF to induce coronary collateral growth have been reported in recent decades [10,42,43], but none of them really works in the patients.…”

Preconditioned and Genetically Modified Stem Cells for Myocardial Infarction Treatment

“…Long-term hypoxic preconditioning enhanced the cardioprotective properties of bone marrow stem cells (BMSCs) and downregulated the expression of pro-inflammatory and pro-fibrotic cytokines, whereas the expression of anti-inflammatory and angiogenic cytokines was upregulated in MI tissue. Compared to normoxic conditions, hypoxic preconditioned BMSCs showed better clonogenic potential and cell proliferation [ 51 ]. In addition, the studies demonstrated the increased viability of BMSCs at the site of infarction, enhanced heart function, and an increase in VEGF and transforming growth factor-β (TGF-β) expression.…”

“…In addition, the studies demonstrated the increased viability of BMSCs at the site of infarction, enhanced heart function, and an increase in VEGF and transforming growth factor-β (TGF-β) expression. The latter proteins were associated with collagen deposition in fibroblasts via PI3K/Akt and TGF-β/mothers against decapentaplegic homolog 2 (SMAD2) pathways, and VEGF also promoted vascularization at the MI site [ 51 ]. Although preconditioning for 24 h significantly enhanced paracrine secretion and the survival of MSCs in post myocardial tissue through the regulation of autophagy, 48 h preconditioning diminished the expression of VEGF, basic fibroblast growth factor (bFGF or FGF2), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1) and enhanced the level of apoptosis and autophagy in tissue [ 52 ].…”

Hypoxic Preconditioning as a Strategy to Maintain the Regenerative Potential of Mesenchymal Stem Cells

“…Significantly higher number of cells as well as increased viability of ADSC occurs in hypoxic conditions [93]. Rat bone marrow MSC cultured at 5% oxygen levels exhibited increased number of colonies and shorter population doubling time as compared to normoxic cultured cells [94]. Asadpoor Dezaki et al showed increased expansion, population doublings, viability and colony forming unit fibroblasts in a group cultured in 2.5% oxygen tension than normoxia group [95].…”