Date of Award
Open Access Thesis
Medical Doctor (MD)
Laura E. Niklason
Tissue-engineered vascular grafts (TEVGs) have the potential to provide life-saving arterial replacements to patients requiring vascular bypass, hemodialysis access, and pediatric coronary surgery. Recent years have seen impressive strides towards widespread clinical use, but significant work still remains in optimizing cell source and graft growth, including the suppression of unwanted calcification during culture.
In this study, I examine oxygen concentration and two small-molecule bone morphogenic protein (BMP) inhibitors, DMH-1 and LDN193189, as potential avenues of increased control over mesenchymal stem cell (MSC) differentiation of into vascular smooth muscle cells (SMCs). Applying BMP-inhibitor concentrations from 0.01 to 10 µM at oxygen tensions of 2 & 20% over two weeks of growth, I use reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) to evaluate resultant expression levels of smooth muscle (SM22-α), bone (OCN), and cartilage (Col2a) marker genes. Via multiple linear regression, I demonstrate that low oxygen growth causes a statistically significant SM22-α downregulation (∆∆Cq = 1.77 ± 0.22, mean ± standard error, p <0.0%) coupled with increased Col2a (∆∆Cq = -2.85 ± 0.73, p =0.1%) and type-I collagen expression (∆∆Cq = -1.75 ± 0.65, p = 1.2%), suggesting that physiological oxygen tensions increase the incidence of chondrogenic differentiation. In contrast, LDN193189 increases SM22-α expression (∆∆Cq = -0.78 ± 0.27 per µM, p =0.8%) and reduces Col2a expression (∆∆Cq = -2.17 ± 0.89, p =2.2%), seeming to usefully suppress chondrogenesis.
Additionally, I evaluate the effects of pulsatile vessel growth conditions on an attractive new cell source: MSCs derived from induced pluripotent stem cells (iPSCs). Using PicoGreen and modified Bradford assays, I demonstrate that pulsatile growth conditions significantly increase dry weights of double-stranded DNA (dsDNA) from 0.0780 ± 0.002% to 0.1414 ± 0.008% (mean ± standard error, p = 0.015%) and collagen from 25 ± 2% to 46 ± 2% (p = 2.3%), approaching a sample of native aorta at 55% collagen.
Overall, results suggest normoxic growth conditions remain superior for SMC differentiation, but that the BMP-inhibitor LDN193189 may have a future role in suppressing cartilage production during TEVG growth. Furthermore, iPSC-derived MSCs demonstrate similar responses to traditional bone marrow MSCs, and may well represent an attractive cell source in future TEVG production.
Siewert, Joshua Morgan Arthur, "Differentiation Control In Tissue-Engineered Vascular Grafts With Oxygen & Small Molecule Inhibitors" (2017). Yale Medicine Thesis Digital Library. 2171.