Said R. Bogatyrev

Combinatorial development of biomaterials for clonal growth of human pluripotent stem cells

Both human embryonic stem (hES) cells and induced pluripotent stem (hiPS) cells can self-renew indefinitely in culture, however current methods to clonally grow them are inefficient and poorly-defined for genetic manipulation and therapeutic purposes. Here we develop the first chemically-defined, xeno-free, feeder-free synthetic substrates to support robust self-renewal of fully-dissociated hES and hiPS cells. Materials properties including wettability, surface topography, surface chemistry and indentation elastic modulus of all polymeric substrates were quantified using high-throughput methods to develop structure/function relationships between materials properties and biological performance. These analyses show that optimal hES cell substrates are generated from monomers with high acrylate content, have a moderate wettability, and employ integrin αvβ3 and αvβ5 engagement with adsorbed vitronectin to promote colony formation. The structure/function methodology employed herein provides a general framework for the combinatorial development of synthetic substrates for stem cell culture.

Genetic engineering of human stem cells for enhanced angiogenesis using biodegradable polymeric nanoparticles

Stem cells hold great potential as cell-based therapies to promote vascularization and tissue regeneration. However, the use of stem cells alone to promote angiogenesis remains limited because of insufficient expression of angiogenic factors and low cell viability after transplantation. Here, we have developed vascular endothelial growth factor (VEGF) high-expressing, transiently modified stem cells for the purposes of promoting angiogenesis. Nonviral, biodegradable polymeric nanoparticles were developed to deliver hVEGF gene to human mesenchymal stem cells (hMSCs) and human embryonic stem cell-derived cells (hESdCs). Treated stem cells demonstrated markedly enhanced hVEGF production, cell viability, and engraftment into target tissues. S.c. implantation of scaffolds seeded with VEGF-expressing stem cells (hMSCs and hESdCs) led to 2- to 4-fold-higher vessel densities 2 weeks after implantation, compared with control cells or cells transfected with VEGF by using Lipofectamine 2000, a leading commercial reagent. Four weeks after intramuscular injection into mouse ischemic hindlimbs, genetically modified hMSCs substantially enhanced angiogenesis and limb salvage while reducing muscle degeneration and tissue fibrosis. These results indicate that stem cells engineered with biodegradable polymer nanoparticles may be therapeutic tools for vascularizing tissue constructs and treating ischemic disease.

Nanoparticulate cellular patches for cell-mediated tumoritropic delivery.

The targeted delivery of therapeutics to tumors remains an important challenge in cancer nanomedicine. Attaching nanoparticles to cells that have tumoritropic migratory properties is a promising modality to address this challenge. Here we describe a technique to create nanoparticulate cellular patches that remain attached to the membrane of cells for up to 2 days. NeutrAvidin-coated nanoparticles were anchored on cells possessing biotinylated plasma membrane. Human bone marrow derived mesenchymal stem cells with nanoparticulate patches retained their inherent tumoritropic properties as shown using a tumor model in a 3D extracellular matrix. Additionally, human umbilical vein endothelial cells with nanoparticulate patches were able to retain their functional properties and form multicellular structures as rapidly as unmodified endothelial cells. These results provide a novel strategy to actively deliver nanostructures and therapeutics to tumors utilizing stem cells as carriers and also suggest that nanoparticulate cellular patches may have applications in tissue regeneration.

Polymer surface functionalities that control human embryoid body cell adhesion revealed by high throughput surface characterization of combinatorial material microarrays

High throughput materials discovery using combinatorial polymer microarrays to screen for new biomaterials with new and improved function is established as a powerful strategy. Here we combine this screening approach with high throughput surface characterization (HT-SC) to identify surface structure-function relationships. We explore how this combination can help to identify surface chemical moieties that control protein adsorption and subsequent cellular response. The adhesion of human embryoid body (hEB) cells to a large number (496) of different acrylate polymers synthesized in a microarray format is screened using a high throughput procedure. To determine the role of the polymer surface properties on hEB cell adhesion, detailed HT-SC of these acrylate polymers is carried out using time of flight secondary ion mass spectrometry (ToF SIMS), X-ray photoelectron spectroscopy (XPS), pico litre drop sessile water contact angle (WCA) measurement and atomic force microscopy (AFM). A structure-function relationship is identified between the ToF SIMS analysis of the surface chemistry after a fibronectin (Fn) pre-conditioning step and the cell adhesion to each spot using the multivariate analysis technique partial least squares (PLS) regression. Secondary ions indicative of the adsorbed Fn correlate with increased cell adhesion whereas glycol and other functionalities from the polymers are identified that reduce cell adhesion. Furthermore, a strong relationship between the ToF SIMS spectra of bare polymers and the cell adhesion to each spot is identified using PLS regression. This identifies a role for both the surface chemistry of the bare polymer and the pre-adsorbed Fn, as-represented in the ToF SIMS spectra, in controlling cellular adhesion. In contrast, no relationship is found between cell adhesion and wettability, surface roughness, elemental or functional surface composition. The correlation between ToF SIMS data of the surfaces and the cell adhesion demonstrates the ability to identify surface moieties that control protein adsorption and subsequent cell adhesion using ToF SIMS and multivariate analysis.

Combinatorial Extracellular Matrices for Human Embryonic Stem Cell Differentiation in 3D

Embryonic stem cells (ESCs) are promising cell sources for tissue engineering and regenerative medicine. Scaffolds for ESC-based tissue regeneration should provide not only structural support, but also signals capable of supporting appropriate cell differentiation and tissue development. Extracellular matrix (ECM) is a key component of the stem cell niche in vivo and can influence stem cell fate via mediating cell attachment and migration, presenting chemical and physical cues, as well as binding soluble factors. Here we investigated the effects of combinatorial extracellular matrix proteins on controlled human ESC (hESC) differentiation. Varying ECM compositions in 3D markedly affects cell behavior, and optimal compositions of ECM hydrogels are identified that facilitate specific-lineage differentiation of stem cells. To our knowledge, this is the first combinatorial analysis of ECM hydrogels for their effects on hESC differentiation in 3D. The 3D matrices described herein may provide a useful platform for studying the interactive ECM signaling in influencing stem cell differentiation.

Mapping the Interactions among Biomaterials, Adsorbed Proteins, and Human Embryonic Stem Cells

An integrated high-throughput polymer synthesis and rapid material/protein/cell interaction assays were developed to optimize stem cell microenvironments. Microarrayed polymers were synthesized and studied for the ability to support the growth of partially differentiated human embryonic stem cells. In parallel, a programmed laser scanning cytometry system was developed to allow for rapid quantification of cell material interaction.

Cell-Compatible, Multicomponent Protein Arrays with Subcellular Feature Resolution†

Recent developments in micro/nano-scale technology have enabled the generation of extracellular matrix (ECM) protein microarrays with well-defined geometries. These patterned surfaces have shown utility for the study and control of a variety of cellular behaviors.[1–7] In particular, the patterning of proteins with feature sizes smaller than a single cell have demonstrated potential application for use as tools to control cellular activity.[4, 8] To date, most research has been limited to studies with single protein factors due to technical limitations of existing printing methods. Herein, we describe the development of a microscale direct writing (MDW) technology for the generation of complex ECM protein arrays at subcellular feature size with multiple components. Automated printing techniques based on atomic force microscopy were developed to allow programmable generation of cell-compatible surfaces with multiple ECM proteins, at a subcellular feature size of 6–9 microns. Cell-compatible, two component ECM protein arrays were systematically generated with varying spacing and composition. These arrays were then studied for their effects on cellular attachment and spreading of a model cell line, human myofibroblasts. Interestingly, the precise tuning of spacing and placement two components at subcellular resolution can lead to an increase in cellular alignment. Given the complexity of the in vivo cellular microenvironment, we believe the MDW methods described here could prove generally applicable for the study and optimization of biomaterial surfaces.

Spatiotemporal effects of a controlled-release anti-inflammatory drug on the cellular dynamics of host response

In general, biomaterials induce a non-specific host response when implanted in the body. This reaction has the potential to interfere with the function of the implanted materials. One method for controlling the host response is through local, controlled-release of anti-inflammatory agents. Herein, we investigate the spatial and temporal effects of an anti-inflammatory drug on the cellular dynamics of the innate immune response to subcutaneously implanted poly(lactic-co-glycolic) microparticles. Noninvasive fluorescence imaging was used to investigate the influence of dexamethasone drug loading and release kinetics on the local and systemic inhibition of inflammatory cellular activities. Temporal monitoring of host response showed that inhibition of inflammatory proteases in the early phase was correlated with decreased cellular infiltration in the later phase of the foreign body response. We believe that using controlled-release anti-inflammatory platforms to modulate early cellular dynamics will be useful in reducing the foreign body response to implanted biomaterials and medical devices.