Maude L. Cuchiara

Integrating valve-inspired design features into poly(ethylene glycol) hydrogel scaffolds for heart valve tissue engineering.

The development of advanced scaffolds that recapitulate the anisotropic mechanical behavior and biological functions of the extracellular matrix in leaflets would be transformative for heart valve tissue engineering. In this study, anisotropic mechanical properties were established in poly(ethylene glycol) (PEG) hydrogels by crosslinking stripes of 3.4 kDa PEG diacrylate (PEGDA) within 20 kDa PEGDA base hydrogels using a photolithographic patterning method. Varying the stripe width and spacing resulted in a tensile elastic modulus parallel to the stripes that was 4.1-6.8 times greater than that in the perpendicular direction, comparable to the degree of anisotropy between the circumferential and radial orientations in native valve leaflets. Biomimetic PEG-peptide hydrogels were prepared by tethering the cell-adhesive peptide RGDS and incorporating the collagenase-degradable peptide PQ (GGGPQG↓IWGQGK) into the polymer network. The specific amounts of RGDS and PEG-PQ within the resulting hydrogels influenced the elongation, de novo extracellular matrix deposition and hydrogel degradation behavior of encapsulated valvular interstitial cells (VICs). In addition, the morphology and activation of VICs grown atop PEG hydrogels could be modulated by controlling the concentration or micro-patterning profile of PEG-RGDS. These results are promising for the fabrication of PEG-based hydrogels using anatomically and biologically inspired scaffold design features for heart valve tissue engineering.

Fabrication and Mechanical Evaluation of Anatomically-Inspired Quasilaminate Hydrogel Structures with Layer-Specific Formulations

A major tissue engineering challenge is the creation of multilaminate scaffolds with layer-specific mechanical properties representative of native tissues, such as heart valve leaflets, blood vessels, and cartilage. For this purpose, poly(ethylene glycol) diacrylate (PEGDA) hydrogels are attractive materials due to their tunable mechanical and biological properties. This study explored the fabrication of trilayer hydrogel quasilaminates. A novel sandwich method was devised to create quasilaminates with layers of varying stiffnesses. The trilayer structure was comprised of two “stiff” outer layers and one “soft” inner layer. Tensile testing of bilayer quasilaminates demonstrated that these scaffolds do not fail at the interface. Flexural testing showed that the bending modulus of acellular quasilaminates fell between the bending moduli of the “stiff” and “soft” hydrogel layers. The bending modulus and swelling of trilayer scaffolds with the same formulations were not significantly different than single layer gels of the same formulation. The encapsulation of cells and the addition of phenol red within the hydrogel layers decreased bending modulus of the trilayer scaffolds. The data presented demonstrates that this fabrication method can make quasilaminates with robust interfaces, integrating layers of different mechanical properties and biofunctionalization, and thus forming the foundation for a multilaminate scaffold that more accurately represents native tissue.

Covalent immobilization of stem cell factor and stromal derived factor 1α for in vitro culture of hematopoietic progenitor cells

Hematopoietic stem cells (HSCs) are currently utilized in the treatment of blood diseases, but widespread application of HSC therapeutics has been hindered by the limited availability of HSCs. With a better understanding of the HSC microenvironment and the ability to precisely recapitulate its components, we may be able to gain control of HSC behavior. In this work we developed a novel, biomimetic PEG hydrogel material as a substrate for this purpose and tested its potential with an anchorage-independent hematopoietic cell line, 32D clone 3 cells. We immobilized a fibronectin-derived adhesive peptide sequence, RGDS; a cytokine critical in HSC self-renewal, stem cell factor (SCF); and a chemokine important in HSC homing and lodging, stromal derived factor 1α (SDF1α), onto the surfaces of poly(ethylene glycol) (PEG) hydrogels. To evaluate the systems capabilities, we observed the effects of the biomolecules on 32D cell adhesion and morphology. We demonstrated that the incorporation of RGDS onto the surfaces promotes 32D cell adhesion in a dose-dependent fashion. We also observed an additive response in adhesion on surfaces with RGDS in combination with either SCF or SDF1α. In addition, the average cell area increased and circularity decreased on gel surfaces containing immobilized SCF or SDF1α, indicating enhanced cell spreading. By recapitulating aspects of the HSC microenvironment using a PEG hydrogel scaffold, we have shown the ability to control the adhesion and spreading of the 32D cells and demonstrated the potential of the system for the culture of primary hematopoietic cell populations.

Bioactive poly(ethylene glycol) hydrogels to recapitulate the HSC niche and facilitate HSC expansion in culture.

Hematopoietic stem cells (HSCs) have been used therapeutically for decades, yet their widespread clinical use is hampered by the inability to expand HSCs successfully in vitro. In culture, HSCs rapidly differentiate and lose their ability to self‐renew. We hypothesize that by mimicking aspects of the bone marrow microenvironment in vitro we can better control the expansion and differentiation of these cells. In this work, derivatives of poly(ethylene glycol) diacrylate hydrogels were used as a culture substrate for hematopoietic stem and progenitor cell (HSPC) populations. Key HSC cytokines, stem cell factor (SCF) and interferon‐γ (IFNγ), as well as the cell adhesion ligands RGDS and connecting segment 1 were covalently immobilized onto the surface of the hydrogels. With the use of SCF and IFNγ, we observed significant expansion of HSPCs, ∼97 and ∼104 fold respectively, while maintaining c‐kit+lin− and c‐kit+Sca1+lin− (KSL) populations and the ability to form multilineage colonies after 14 days. HSPCs were also encapsulated within degradable poly(ethylene glycol) hydrogels for three‐dimensional culture. After expansion in hydrogels, ∼60% of cells were c‐kit+, demonstrating no loss in the proportion of these cells over the 14 day culture period, and ∼50% of colonies formed were multilineage, indicating that the cells retained their differentiation potential. The ability to tailor and use this system to support HSC growth could have implications on the future use of HSCs and other blood cell types in a clinical setting. Biotechnol. Bioeng. 2016;113: 870–881.

Gene patents, patenting life and the impact of court rulings on US stem cell patents and research

In June 2013, the US Supreme Court ruled that naturally occurring genes were unpatentable in the case Association for Molecular Pathology v. Myriad Genetics. Up until this decision, Myriad Genetics was the only company in the USA that could legally conduct diagnostic testing for BRCA1 and 2, genes that are linked to familial breast and ovarian cancer. The court case and rulings garnered discussion in public about patenting biological materials. This paper will describe the progression of the Myriad Genetics case, similar US rulings and biological intellectual property policies. In addition, it will discuss the impact of the case on biological patents - specifically those for human embryonic stem cells.

Regulating the therapeutic translation of regenerative medicine

Regenerative medicine and stem cell research are exciting new fields. But as the fields progress toward clinical therapies, controversies emerge. Hype surrounding stem cell research has caused an increase in their use in interventions that are not clinically proven. Furthermore, the regulatory agencies have a lot of difficulty dealing with cell therapies, which are distinctly different from drugs and medical devices they more commonly approve. To move the field forward, advocates, regulators and scientists need to come together to find new options for stem cell research oversight that protects both the patients and the research field.

Micropatterning of Poly(ethylene glycol) Diacrylate Hydrogels

This protocol describes the techniques to synthesize and fabricate micropatterned poly(ethylene glycol) diacrylate-based hydrogels that can be used as substrates in cellular studies and tissue engineering scaffolds. These materials provide an essentially bioinert background material due to the very low protein adsorption characteristics of poly(ethylene glycol), but the materials can be modified with covalently grafted peptides, proteins, or other biomolecules of interest to impart specific biofunctionality to the material. Further, it is possible to use micropatterning technologies to control the localization of such covalent grafting of biomolecules to the hydrogel materials, thus spatially controlling the cell-material interactions. This protocol presents a relatively simple approach for mask-based photolithographic patterning, generally best suited for patterning the surface of hydrogel materials for 2D cell studies. A more sophisticated technique, two-photon laser scanning lithography, is also presented. This technique allows free-form, 3D micropatterning in hydrogels.

Defining “Research” in the US and EU: Contrast of Sherley v. Sebelius and Brüstle v. Greenpeace Rulings

In 2011, courts in both the United States and European Union handed down decisions related to human embryonic stem cell (hESC) research. In both cases, the definition of research was challenged – but the two courts reached different opinions. In the US case, Sherley v. Sebelius, research was defined as a specific project. The US District Court of Appeals did not link research utilizing existing hESC lines to the act of destroying a human embryo in order to create the line, which is not eligible for federal funding. In contrast, the Court of Justice of the European Union in the Brüstle v. Greenpeace case determined inventions related to hESCs were unpatentable since they resulted from research that involved the destruction of human embryos. In this article, we will compare and contrast these two court cases, the politics related to the rulings, and their impacts. We find that these cases significantly impacted current research and have the potential to negatively impact future stem cell research and development. However, the long-term effects of the cases remain to be seen, and there is a chance that these cases could actually strengthen this area of science. Ultimately, we feel that stem cell polices must be straightforward and supported by the public to prevent courts and judges from making decisions on science, which are disruptive to the progression of research.