Lakeshia J. Taite

The expansion of human ES and iPS cells on porous membranes and proliferating human adipose-derived feeder cells

For clinical application of human embryonic stem cells (hESCs), it is critical to develop hESC culture techniques that completely exclude the use of animal feeder cells, mitotic inhibition, and enzyme treatments used in conventional hESC culture systems. Toward this goal, we attempted to maintain hESCs and induced pluripotent stem (iPS) cells on porous membranes (PMs) with proliferative human adipose-derived stromal cells (ASCs) seeded on the bottom surface of inverted PMs. This culture condition will ensure that the two cell types are separate from each other, yet retain the ability to interact through the pores of the membrane. We found that hESCs and iPS cells can be maintained stably and mechanically transferred without the need for enzyme treatment. In addition, the pluripotency of hESCs and iPS cells was stably maintained, as evidenced by immunostaining of Oct4, SSEA3/4 and TRA-1-60 as well as RT-PCR analyses of Nanog, Oct4 and Sox2 expression. Furthermore, hESCs cultured on PMs showed a normal karyotype and in vivo teratoma formation containing all three germ layers.

Poly(ethylene glycol)-lysine dendrimers for targeted delivery of nitric oxide

We have synthesized dendrimers of the amino-acid lysine bound to a central poly(ethylene glycol) (PEG) core, and then formed multiple diazeniumdiolate nitric oxide (NO) donors on the lysine residues. NO release from these materials occurred for up to 60 days under physiological conditions. These materials display the ability to regulate vascular cell proliferation and inhibit platelet adhesion to thrombogenic surfaces. When modified with a targeting ligand specific for inflamed endothelium (Sialyl Lewis X), we were able to demonstrate binding of fluorescently-labeled dendrimers to endothelial cells activated by interleukin 1β (IL-1β).

Bioactive Hydrogel Substrates: Probing Leukocyte Receptor–Ligand Interactions in Parallel Plate Flow Chamber Studies

The binding of activated integrins on the surface of leukocytes facilitates the adhesion of leukocytes to vascular endothelium during inflammation. Interactions between selectins and their ligands mediate rolling, and are believed to play an important role in leukocyte adhesion, though the minimal recognition motif required for physiologic interactions is not known. We have developed a novel system using poly(ethylene glycol) (PEG) hydrogels modified with either integrin-binding peptide sequences or the selectin ligand sialyl Lewis X (SLeX) within a parallel plate flow chamber to examine the dynamics of leukocyte adhesion to specific ligands. The adhesive peptide sequences arginine–glycine–aspartic acid–serine (RGDS) and leucine–aspartic acid–valine (LDV) as well as sialyl Lewis X were bound to the surface of photopolymerized PEG diacrylate hydrogels. Leukocytes perfused over these gels in a parallel plate flow chamber at physiological shear rates demonstrate both rolling and firm adhesion, depending on the identity and concentration of ligand bound to the hydrogel substrate. This new system provides a unique polymer-based model for the study of interactions between leukocytes and endothelium as well as a platform to develop improved scaffolds for cardiovascular tissue engineering.

Self-assembly of elastin-based peptides into the ECM: the importance of integrins and the elastin binding protein in elastic fiber assembly.

The formation of a suitable extracellular matrix (ECM) that promotes cell adhesion, organization, and proliferation is essential within biomaterial scaffolds for tissue engineering applications. In this work, short elastin mimetic peptide sequences, EM-19 and EM-23, were engineered to mimic the active motifs of human elastin in hopes that they can stimulate ECM development in synthetic polymer scaffolds. Each peptide was incubated with human aortic smooth muscle cells (SMCs) and elastin and desmosine production were quantified after 48 h. EM-19 inhibited elastin production through competitive binding phenomena with the elastin binding protein (EBP), whereas EM-23, which contains an RGDS domain, induces recovery of elastin production at higher concentrations, alluding to a higher binding affinity for the integrins than for the EBP and the involvement of integrins in elastin production. Colocalization of each peptide with the elastin matrix was confirmed using immunofluorescent techniques. Our data suggest that with appropriate cell-binding motifs, we can simulate the cross-linking of tropoelastin into the developing elastin matrix using short peptide sequences. The potential for increased cell adhesion and the incorporation of elastin chains into tissue engineering scaffolds make these peptides attractive bioactive moieties that can easily be incorporated into synthetic biomaterials to induce ECM formation.

Targeted diazeniumdiolates: Localized nitric oxide release from glioma-specific peptides and proteins

Nitric oxide (NO) is a small yet important biological messenger, which at sufficient concentrations has been shown to induce apoptosis as well as increase radiosensitization in tumor cells. However, the short half-life of NO gas itself has limited its utility as a therapeutic agent. The objective of this study was the development of targeted NO donors and we illustrate their utility as a potential therapeutic for treatment of glioblastoma multiforme, the most common and aggressive malignant primary brain tumor in adults. We have synthesized two diazeniumdiolate NO donors by reacting NO gas with glioma-specific targeting sequences, VTWTPQAWFQWVGGGSKKKKK (VTW) and chlorotoxin (CTX), and achieved repeatable NO release from both donors. FITC-labeled biomolecules, when incubated with glioma and control cells preferentially bound to the glioma cells and showed only minimal binding to the control cells. Additionally, tumor cell viability was significantly decreased when cells were incubated with the NO donors whereas control cell viability was not affected.

Targeted nitric oxide delivery preferentially induces glioma cell chemosensitivity via altered p53 and O6‐Methylguanine‐DNA Methyltransferase activity

Glioblastoma multiforme is the most common malignant central nervous system tumor, and also among the most difficult to treat due to a lack of response to chemotherapeutics. New methods of countering the mechanisms that confer chemoresistance to malignant gliomas could lead to significant advances in the quest to identify novel drug combinations or targeted drug delivery systems for cancer therapy. In this study, we investigate the use of a targeted nitric oxide (NO) donor as a pretreatment to sensitize glioma cells to chemotherapy. The protein chlorotoxin (CTX) has been shown to preferentially target glioma cells, and we have developed CTX–NO, a glioma‐specific, NO‐donating CTX derivative. Pretreatment of cells with CTX–NO followed by 48‐h exposure to either carmustine (BCNU) or temozolomide (TMZ), both common chemotherapeutics used in glioma treatment, resulted in increased efficacy of both therapeutics. After CTX–NO exposure, both T98G and U‐87MG human malignant glioma cells show increased sensitivity to BCNU and TMZ. Further investigation revealed that the consequences of this combination therapy was a reduction in active levels of the cytoprotective enzyme MGMT and altered p53 activity, both of which are essential in DNA repair and tumor cell resistance to chemotherapy. The combination of CTX–NO and chemotherapeutics also led to decreased cell invasion. These studies indicate that this targeted NO donor could be an invaluable tool in the development of novel approaches to treat cancer. Biotechnol. Bioeng. 2013; 110: 1211–1220.

Synergistic activity of αvβ3 integrins and the elastin binding protein enhance cell-matrix interactions on bioactive hydrogel surfaces.

Engineering materials suitable for vascular prostheses has been a significant challenge, especially in promoting extracellular matrix (ECM) development within synthetic materials. Herein we have utilized two different elastin mimetic peptide sequences, EM-19 and EM-23, to provide a template for ECM deposition when covalently incorporated into scaffold materials. Both peptides contain the hexapeptide sequence VGVAPG, which interacts with the cell surface receptor known as the elastin binding protein (EBP). Additionally, EM-23 contains an RGDS sequence intended for the peptides interaction with the α(v)β(3) integrin. We first confirm that the presence of both peptides approximates the synergistic mechanism for elastic fiber assembly in vivo, a process that utilizes both the EBP and α(v)β(3). Peptides were then grafted onto the surface of a poly(ethylene glycol) diacrylate (PEG-DA) hydrogel and their efficacy as templates for promoting cell adhesion, spreading, and elastin deposition was evaluated. Although both peptides were able to encourage smooth muscle cell (SMC) adhesion and elastin deposition over PEG-DA and PEG-RGDS controls, PEG-grafted EM-23 was proven to be the more promising motif for inclusion in synthetic substrates to be used in the engineering of vascular tissues, enhancing cell adhesion 60-fold and elastin content 2-fold compared with PEG-RGDS.