CDMC Research Interests
The current research in the CDMC lab includes the following:
Endothelial Promoters to Direct the Differentiation of Embryonic Stem Cells for Vascular Tissue Engineering
In tissue engineering of small diameter vascular grafts, an ample source of endothelial cells is desirable in order to improve the graft's patency. Pluripotent embryonic stem cells (ESCs) have been recognized to be a suitable source due to their rapid expansion rate and easy availability. However, in order for ESCs to be usable, an effective means of selecting pure endothelial populations from diverse phenotypes of differentiating cells is required. FACS has been used widely to enrich endothelial populations from differentiating cells, but it is costly and not 100% pure. In this study, we examined a new method of selecting endothelial cells using genetically transformed mouse ESCs with a GFP/Puror fusion protein under the control of different endothelial promoters (Flk1, PECAM, tie1, and VE-Cadherin). Upon differentiation in the presence of VEGF, cells stably expressing these constructs were treated with puromycin to select the puromycin resisistant cells. The use of multiple promoters enabled us to select cells at different stages in endothelial differentiation; for Flk1 at day 5 (D5), PECAM at D6, tie1 at D7, and VE-Cadherin at D8. After 7 days of additional selection in puromycin, the surviving cells were characterized for endothelial phenotype by Dil-Ac-LDL uptake and immunostaining of PECAM and vWF. Although these cells expressed a very low level of GFP/Puror fusion protein reflected by their low GFP fluorescence, the expression level was enough to provide puromycin resistance, and therefore a highly pure population of endothelial cells was achieved.
Mechanical Cues and Gene Regulation to Direct Differentiation of Embryonic Stem Cells to Cardiomyocytes
The prevalence of myocardial infarction (MI) in 2005 reached 8.1 million Americans. MI causes ischemia, which can lead to permanent injury to heart tissue, since cardiomyocytes do not regenerate following cell death. Treatments can reduce MI risk or mechanically restore blood flow in ischemia; however, currently no viable clinical options exist to replace lost or damaged tissue. Embryonic stem cells have potential in cellular replacement therapy, but therapeutic applications are currently limited due to teratoma formation. For injected or scaffold-seeded mature cardiomyocytes, extracellular matrix must be given time to grow, which may lead to undesired remodeling if proper mechanical stimulation is lacking.
We propose to construct a wholly cellular cardiac patch intended to replace myocardium damaged by MI. We will create a device that can mechanically condition in vitro extracellular matrix growth from stem-cell derived cardiomyocytes, and then allow for non-damaging detachment of cells and matrix for therapeutic use. Using silicone and thermally responsive polymer poly(N-isopropylacrylamide) (PIPAAm), we will make a elastic substrate that can also change surface properties with temperature change. Silicone's elastic properties can be used for controlled mechanical stimulation of differentiated cardiomyocytes to produce tissue that more closely mimics native myocardium, making it more readily viable for transplantation. PIPAAm's thermally responsive properties can cause cell attachment at body temperature, and spontaneous detachment at room temperature, allowing for cell removal from a tissue culture surface without damaging enzymatic treatments. We will then test for functional attachment and detachment of cells from PIPAAm-coated silicone surfaces using a genetically modified mouse embryonic stem cell line that features a selection gene under control of the cardiac-specific alpha myosin heavy chain promoter. Differentiated cardiomyocytes will then be subjected to mechanical conditioning using the FlexCell system and evaluated for gene expression levels of cardiac-specific proteins compared to nonconditioned cells using PCR and immunohistochemistry. With this proposed technology, we can create the first step to harvesting a cardiac patch that is ready to implant for replacement of myocardium damaged by MI. In future years, we plan to evaluate the functional characteristics of these patches in vivo with the help of our collaborators.
Reversible Attachment of Molecules to Surfaces for Drug Delivery and Tissue Engineering
Conjugation of biomolecules to polymeric surfaces has been used to control biocompatibility, cell attachment, and proliferation. For example, poly(tetrafluoroethylene) (PTFE) scaffolds containing immobilized VEGF on their surfaces have been demonstrated to promote endothelial cell migration (angiogenesis) in vitro. However, surfaces containing only immobilized VEGF lack the cell adhesion signal that is required at the onset of the healing process. Thus, it is desirable to design a scaffold that is capable of expressing various bioactive factors (e.g. RGD, VEGF, etc.) at different times. Beta-cyclodextrin (CD) has been widely used to form selective and reversible, high affinity inclusion complexes with hydrophobic molecules such as adamantane (Ad). In this study, we examine the potential of using inclusion complexes formed between CD and Ad derivatives to selectively and reversibly bind bioactive molecules to biomaterial surfaces.
We have demonstrated a new method for reversibly attaching molecules to biomaterial surfaces using a CD-Ad inclusion complex. Initial studies demonstrated reversible attachment of fluorescent molecules. Cell culture studies demonstrated that this observation could be translated to incorporation of biomolecules influencing cell attachment. Future research will examine using more complex bioactive molecules (e.g. EGF, bFGF, VEGF, etc.), with which we can examine how spatial and temporal arrangements influence cell proliferation, migration and differentiation.
Self-assembled Hydrogels for Protein Release in HIV Vaccine Adjuvants
The goal is to develop a safe, degradable hydrogel for protein delivery of Interlukin-7 (IL-7) protein, an adjuvant being examined clinically to boost the immune system of HIV patients. In the past two decades, the field of drug delivery has produced many different polymers for controlled release applications. However, these systems have been mainly developed to deliver pharmaceuticals (small molecular weight drug molecules) in a controlled manner; very few systems have been developed for proteins release. Of those studied for delivering proteins, few have the desired biodegradable and biocompatibility properties while maintaining the tunability for accommodating release rate or of different sized proteins. With the completion of the Human Genome Project, delivery of many new therapeutic proteins has become increasingly important. One such protein, IL7, has potential as an HIV therapy because it enhances T cell survival and plays a role in the conversion of effector cells into long lived memory T cells, both of which are impaired in HIV disease. Mouse studies examining IL-7 therapy relied on repeated administration to demonstrate effectiveness of the IL-7 as an adjuvant. The persistent delivery of this cytokine in a hydrogel provides a means to reduce the administration of IL-7 to a single injection while providing the same therapeutic benefit. The objective is to examine a novel degradable hydrogel platform which can be used for IL-7 release with tunable release rate. The first aim is to synthesize the polymers, characterize them chemically, and test for controlled release in vitro. The second aim is to examine IL-7 release from the polymeric device in conjunction with an animal model developed by collaborators at the Center for AIDS Research. The hydrogel platform will examine reversible crosslinking of poly(ethylene glycol), PEG, a polymer which has long been used in drug delivery due to its high biocompatibility. Using a disulfide crosslinker, this resultant category of polymers, called thoimers, are able to crosslink in biological environments, avoiding the use of harsh organic solvents or highly reactive crosslinking molecules, and therefore have shown great potential in protein delivery. The PEG platform can be explored by using biocompatible components 2,6 dithiopurine(DP) and functionalized polyethylene PEG. Using this system, a ladder-type network polymer can be synthesized, where each "rung" can be made using different molecular weights. The method again takes advantage of the thiomer based chemistry, which again can polymerize in situ without toxic by products, and its natural degradation by the ubiquitous endosomal enzyme glutathione S-transferase.
Once the polymers are synthesized, characterized and tested, degradable formulations will be provided to our research collaborators to test in an IL-7 adjuvant small animal model.
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