Sheena Abraham

Characterization of human fibroblast-derived extracellular matrix components for human pluripotent stem cell propagation.

Recent studies from our laboratory have shown that acellular substrates generated from human fibroblasts successfully maintained human pluripotent stem cells (hPSCs) in their undifferentiated state for extended periods. Aiming at better characterization, we conducted proteomic analyses to identify the extracellular matrix (ECM) proteins in mouse embryonic- and two human fibroblast-derived acellular substrates. Our studies identified heparan sulfate proteoglycan (HSPG) as a core component of these substrates and immunocytochemical analyses confirmed the presence of HSPG as well as other ECM proteins identified through proteomic analyses. In our attempt to develop surfaces that mimic fibroblast-deposited ECM and their self-renewal capabilities, substrates comprising HSPG and other core ECM proteins were formulated and assessed for the function of hPSC self-renewal. WA09 and BG01v hPSCs maintained on these substrates exhibit multiple characteristics of pluripotency, including (i) tight colony formation with typical stem cell morphology; (ii) positive expression of alkaline phosphatase, (iii) positive expression of SSEA3, SSEA4 and Oct4 based on immunocytochemical analyses; (iv) POU5F1, NANOG and SOX2 mRNA expression; and (v) in vitro differentiation and expression of germ-layer-specific markers. Our studies also reveal that although HSPG by itself-does not support hPSC self-renewal, a substrate that combines HSPG and fibronectin is sufficient for undifferentiated propagation of hPSCs. These studies form the basis for identification of appropriate ECM components in a substrate that synergistically promotes activation of adhesion and signaling pathways responsible for hPSC self-renewal.

High Antimicrobial Effectiveness with Low Hemolytic and Cytotoxic Activity for PEG/Quaternary Copolyoxetanes

The alkyl chain length of quaternary ammonium/PEG copolyoxetanes has been varied to discern effects on solution antimicrobial efficacy, hemolytic activity and cytotoxicity. Monomers 3-((4-bromobutoxy)methyl)-3-methyloxetane (BBOx) and 3-((2-(2-methoxyethoxy)ethoxy)methyl)-3-methyloxetane (ME2Ox) were used to prepare precursor P[(BBOx)(ME2Ox)-50:50–4 kDa] copolyoxetane via cationic ring opening polymerization. The 1:1 copolymer composition and Mn (4 kDa) were confirmed by 1H NMR spectroscopy. After C–Br substitution by a series of tertiary amines, ionic liquid Cx-50 copolyoxetanes were obtained, where 50 is the mole percent of quaternary repeat units and “x” is quaternary alkyl chain length (2, 6, 8, 10, 12, 14, or 16 carbons). Modulated differential scanning calorimetry (MDSC) studies showed Tgs between −40 and −60 °C and melting endotherms for C14–50 and C16–50. Minimum inhibitory concentrations (MIC) were determined for Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. A systematic dependence of MIC on alkyl chain length was found. The most effective antimicrobials were in the C6–50 to C12–50 range. C8–50 had better overall performance with MICs of 4 μg/mL, E. coli; 2 μg/mL, S. aureus; and 24 μg/mL, P. aeruginosa. At 5 × MIC, C8–50 effected >99% kill in 1 h against S. aureus, E. coli, and P. aeruginosa challenges of 108 cfu/mL; log reductions (1 h) were 7, 3, and 5, respectively. To provide additional insight into polycation interactions with bacterial membranes, a geometric model based on the dimensions of E. coli is described that provides an estimate of the maximum number of polycations that can chemisorb. Chain dimensions were estimated for polycation C8–50 with a molecular weight of 5 kDa. Considering the approximations for polycation chemisorption (PCC), it is surprising that a calculation based on geometric considerations gives a C8–50 concentration within a factor of 2 of the MIC, 4.0 (±1.2) μg/mL for E. coli. Cx-50 copolyoxetane cytotoxicity was low for human red blood cells, human dermal fibroblasts (HDF), and human foreskin fibroblasts (HFF). Selectivities for bacterial kill over cell lysis were among the highest ever reported for polycations indicating good prospects for biocompatibility.

Role of bioinspired polymers in determination of pluripotent stem cell fate

Human pluripotent stem cells, including embryonic and induced pluripotent stem cells, hold enormous potential for the treatment of many diseases, owing to their ability to generate cell types useful for therapeutic applications. Currently, many stem cell culture propagation and differentiation systems incorporate animal-derived components for promoting self-renewal and differentiation. However, use of these components is labor intensive, carries the risk of xenogeneic contamination and yields compromised experimental results that are difficult to duplicate. From a biomaterials perspective, the generation of an animal- and cell-free biomimetic microenvironment that provides the appropriate physical and chemical cues for stem cell self-renewal or differentiation into specialized cell types would be ideal. This review presents the use of natural and synthetic polymers that support propagation and differentiation of stem cells, in an attempt to obtain a clear understanding of the factors responsible for the determination of stem cell fate.

Stable propagation of human embryonic and induced pluripotent stem cells on decellularized human substrates

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have gained enormous interest as potential sources for regenerative biomedical therapies and model systems for studying early development. Traditionally, mouse embryonic fibroblasts have been used as a supportive feeder layer for the sustained propagation of hPSCs. However, the use of nonhuman‐derived feeders presents concerns about the possibility of xenogenic contamination, labor intensiveness, and variability in experimental results in hPSC cultures. Toward addressing some of these concerns, we report the propagation of three different hPSCs on feeder‐free extracellular matrix (ECM)‐based substrates derived from human fibroblasts. hPSCs propagated in this setting were indistinguishable by multiple criteria, including colony morphology, expression of pluripotency protein markers, trilineage in vitro differentiation, and gene expression patterns, from hPSCs cultured directly on a fibroblast feeder layer. Further, hPSCs maintained a normal karyotype when analyzed after 15 passages in this setting. Development of this ECM‐based culture system is a significant advance in hPSC propagation methods as it could serve as a critical component in the development of humanized propagation systems for the production of stable hPSCs and its derivatives for research and therapeutic applications.

Propagation of human embryonic and induced pluripotent stem cells in an indirect co-culture system

We have developed and validated a microporous poly(ethylene terephthalate) membrane-based indirect co-culture system for human pluripotent stem cell (hPSC) propagation, which allows real-time conditioning of the culture medium with human fibroblasts while maintaining the complete separation of the two cell types. The propagation and pluripotent characteristics of a human embryonic stem cell (hESC) line and a human induced pluripotent stem cell (hiPSC) line were studied in prolonged culture in this system. We report that hPSCs cultured on membranes by indirect co-culture with fibroblasts were indistinguishable by multiple criteria from hPSCs cultured directly on a fibroblast feeder layer. Thus this co-culture system is a significant advance in hPSC culture methods, providing a facile stem cell expansion system with continuous medium conditioning while preventing mixing of hPSCs and feeder cells. This membrane culture method will enable testing of novel feeder cells and differentiation studies using co-culture with other cell types, and will simplify stepwise changes in culture conditions for staged differentiation protocols.

Molecularly engineered hydrogels for implant biocompatibility

The biocompatibility of biosmart polymer membranes synthesized from cross-linkable (2-hydroxyethyl methacrylate) (HEMA) and tetraethylene glycol diacrylate and containing different mole-percent polyethylene glycol methacrylate (PEGMA) and methacryloyloxyethyl phosphorylcholine (MPC), a phosphorylcholine-containing co-monomer, was investigated. The cytotoxicity (cell viability and proliferation) and the adhesion of extra cellular matrix proteins to these hydrogel surfaces were separately tested. Cell proliferation assays were conducted by cultivating human skeletal muscle fibroblasts onto the surfaces of these polymeric membranes prepared by in-situ polymerization in chemically derivatized 8-well cell-culture plates. The compositions containing MPC and PEGMA concentrations greater than 1.0 and 0.05 mole% respectively demonstrated good protein adhesion and cell viability (>90%) of human muscle fibroblast cells. Morphological deviances and partial colonization of the hydrogel surface has been noticed and suggests good compatibility of hydrogels for cellular viability but restricted proliferation. It is well known that the adsorption of proteins onto biomaterial surfaces modulates the cellular interaction with these surfaces. The extent of adsorption of fluorescein labeled proteins (laminin, collagen, and fibronectin) onto these polymer membrane surfaces was evaluated by measuring the resultant fluorescence intensity using a confocal fluorescence scanner.

A BIAXIAL SHAPE MEMORY ALLOY ACTUATED CELL/TISSUE STRETCHING SYSTEM

In this article, we describe the design of a shape memory alloy-based system to stretch cells cultured on top of a flexible membrane in multi-directions (longitudinal and transverse). Mechanical cues (such as strain and force) can affect the state and behavior of cells, such as, morphology, the differentiation process, and apoptosis. Therefore, a thorough understanding of the effects of mechanical perturbations on cells/tissues will have a deep impact in the biological sciences. The proposed design allows application of anisotropic (multi-axial) strain with high-precision. Certain cells, for example endothelial cells that line the inside of blood vessels, experience multi-axial (circumferential and longitudinal) stresses and strains. A cell stretching device that enables controlled application of biaxial strain will allow for systematic and accurate studies of the effects of externally applied mechanical perturbation throughout the cell, tissue, or organ. A preliminary design is proposed that exploits the strain recovery property of the shape memory alloy (SMA) actuators. We describe the design of the mechanical system and show experimental results to demonstrate stretching of a thin PDMS membrane in the longitudinal and transverse directions. To account for the inherent nonlinearity of the SMA, a feedback controller is implemented to achieve high-precision control of the stretching process. Additionally, the design can be integrated with an atomic force microscope (AFM) for high spatial and temporal resolution studies.Copyright

Molecularly Engineered p(HEMA)-based Hydrogels Possessing Poly(Ethylene Glycol) and Phosphorylcholine for Implant Biocompatibility

Hydrogels based on 2-hydroxyethyl methacrylate (HEMA) crosslinked with tetraethylene glycol (TEGDA) and molecularly engineered using two methacrylate-based monomers, poly (ethylene glycol) (200) monomethacrylate (PEGMA) and 2-methacryloyloxyethyl phosphorylcholine (MPC) in the range of 0.0 - 0.5 mol % and 0-10 mol % respectively were investigated. Hydration studies demonstrated up to a 93.8% increase in the hydration with an increase in the MPC content. Data obtained from the measurement of the fluorescence intensity of FITC-dye tagged protein adsorbed onto various hydrogel substrates when exposed to solutions of different protein concentration solutions at 25degC were modeled to the Langmuir isotherm. Variables Kd and Qm confirmed the reduction in the adsorption of protein onto hydrogels with the increase in the MPC concentration and with extensive hydration of the hydrogels, 5 days. Cell viability studies using human aortic muscle endothelial cells exhibited greater than 80% viability with all the hydrogel formulations studied. Cell retention in the hydrogel matrix was investigated by staining cells that remained in the hydrogel matrix following trypsinization, with a fluorescent dye DAPI. It was observed, using fluorescence microscopy, that the higher the MPC content in the hydrogel the greater the cell retention capacity of the hydrogel