Project VIII

Agneta Simionescu

Stem Cell Differentiation and Cardiovascular Tissue Engineering in Diabetes

Target Investigator
Dr. Agneta Simionescu
Assistant Professor of Bioengineering
Clemson University

Tissue engineered constructs based on scaffolds and autologous progenitor cells are currently being developed1, but very little information exists regarding the fate of tissue engineered devices in the compromised patient, and more specifically in diabetic environments. Diabetes is a major risk factor for cardiovascular diseases and diabetics have a significantly greater frequency of cardiovascular disorders. As a consequence, diabetics are more prone to undergo surgery for repair or replacement of tissues such as blood vessels and heart valves. Diabetes is characterized by elevated levels of blood glucose, which interacts irreversibly with proteins, lipids and nucleic acids via oxidation and crosslinking processes, resulting in formation of advanced glycosylation end products (AGEs). Glycoxidation induces severe cell and matrix alterations that result in endothelial dysfunction, accelerated atherosclerosis, activation of inflammation, fibrosis, impaired healing and ectopic calcifications all of which are not conducive to the desired integration and remodeling of tissue engineered constructs.

Two specific aims will be pursued:

  1. To Assess Human Stem Cell Differentiation in Diabetes
    Rationale and Hypothesis: Since high glucose levels are expected to chemically alter most cell components, we hypothesize that diabetes might also alter differentiation of human stem cells into target cardiovascular cells by slowing down the process or reducing the efficacy of differentiation.
    Approach: Human adipose derived stem cells (hADSCs) will be isolated from healthy individuals and diabetic patients. Control non-diabetic stem cells (ND-hADSCs) and diabetic stem cells (D-hADSCs) will be propagated, maintained and tested in normal (100mg glucose/dl) and diabetic media (250mg glucose/dl) respectively. Cells will be seeded onto and into stabilized vascular grafts (lumen, media, adventitia) and separately to stabilized aortic valve scaffolds (valve surfaces and interstitium) and mounted in bioreactors for physiologic mechanical conditioning for up to 4 weeks in media enriched with growth factors for differentiation. Matching cell-seeded scaffolds will be cultured in static conditions as controls. Cells will be analyzed for extent of differentiation using specific markers for endothelial, smooth muscle, valvular interstitial cells and fibroblasts by gene and protein expression.
    Innovative features: We will report for the first time on fundamental cell biology aspects of diabetic stem cells and on their ability to serve as tissue engineering building blocks.
  2. To Evaluate the Need for Stem Cell Pre-differentiation in Diabetes
    Rationale and Hypothesis: Seeding scaffolds with freshly prepared autologous ADSCs before implantation is an attractive option for intra-operative procedures, as long as we can prove that stem cells would differentiate into desired cells as a response to mechanical stimuli. Such differentiation might be impaired in diabetes, requiring in vitro pre-differentiation before scaffold seeding and implantation.
    Approach: Control non-diabetic human adipose derived stem cells stem cells (ND-hADSCs) and diabetic stem cells (D-hADSCs) will be pre-differentiated in vitro into target cardiovascular cells by incubating them with specific growth factors for up to 4 weeks. Cells will be then seeded onto the appropriate histo-anatomical areas (e.g. endothelial cells onto luminal surfaces, smooth muscle cells into medial layers, fibroblasts onto adventitia, etc.) and constructs mounted in bioreactors for physiologic mechanical conditioning for up to 4 weeks. Cells will be analyzed for preservation of differentiation markers using specific labels for endothelial, smooth muscle, valvular interstitial cells and fibroblasts by gene and protein expression and results compared to the data obtained from Aim 1, where stem cells were seeded onto scaffolds without pre-differentiation.
    Innovative features: This experiment will determine whether diabetic stem cells require “conditioning” before implantation as autologous-cell seeded scaffolds for cardiovascular tissue engineering.

My long-term goal is to develop constructs adapted to withstand high glycoxidation stress for translational and clinical applications. My working hypothesis is that both scaffolds and cells are susceptible to diabetes-induced complications and that stabilization of scaffolds would improve the outcome of tissue engineering in diabetes. To test this hypothesis we propose to test constructs and autologous stem cells in vitro and in vivo diabetic models and compare their properties to non-diabetic control environments. In preliminary studies I have shown that experimental diabetes in rats significantly affects stem cells and matrix-derived scaffolds and that treatment of scaffolds with an extracellular matrix-binding polyphenolic antioxidant stabilizes the scaffolds and renders them “diabetes-resistant” (Patent pending) and calcification resistant, without impeding on stem cell seeding, infiltration and matrix remodeling after subdermal implantation in diabetic rats. For the upcoming RO1 submission I would propose pre-clinical validation of tissue engineered, autologous stem cell seeded vascular grafts and heart valves in large animal models of diabetes. As a bridge to the RO1 grant, I am proposing to evaluate the effect of diabetes on human stem cell differentiation and to optimize an autologous cell sourcing protocol that would be applicable for large animal studies.

Please click here for more information about Dr. Simionescu's laboratory.