Peter A. George

Directing osteogenic and myogenic differentiation of MSCs: interplay of stiffness and adhesive ligand presentation

The mechanical properties of the extracellular matrix (ECM) can exert significant influence in determining cell fate. Human mesenchymal stem cells (MSCs) grown on substrates with varying stiffness have been shown to express various cell lineage markers, without the use of toxic DNA demethylation agents or complex cocktails of expensive growth factors. Here we investigated the myogenic and osteogenic potential of various polyacrylamide gel substrates that were coated with covalently bound tissue-specific ECM proteins (collagen I, collagen IV, laminin, or fibronectin). The gel-protein substrates were shown to support the growth and proliferation of MSCs in a stiffness-dependent manner. Higher stiffness substrates encouraged up to a 10-fold increase in cell number over lower stiffness gels. There appears to be definitive interplay between substrate stiffness and ECM protein with regard to the expression of both osteogenic and myogenic transcription factors by MSCs. Of the 16 gel-protein combinations investigated, osteogenic differentiation was found to occur significantly only on collagen I-coated gels with the highest modulus gel tested (80 kPa). Myogenic differentiation occurred on all gel-protein combinations that had stiffnesses >9 kPa but to varying extents as ascertained by MyoD1 expression. Peak MyoD1 expression was seen on gels with a modulus of 25 kPa coated in fibronectin, with similar levels of expression observed on 80-kPa collagen I-coated gels. The modulation of myogenic and osteogenic transcription factors by various ECM proteins demonstrates that substrate stiffness alone does not direct stem cell lineage specification. This has important implications in the development of tailored biomaterial systems that more closely mimic the microenvironment found in native tissues.

Self-assembling polystyrene-block-poly(ethylene oxide) copolymer surface coatings: Resistance to protein and cell adhesion

In this paper we report a method for biomaterial surface modification that utilizes the self-assembly of block copolymers of poly(styrene-block-ethylene oxide) (PS-PEO) to generate micro-phase separated surfaces with varying density PEO domains. These PS-PEO self-assembled surfaces showed a significant reduction in protein adsorption compared to control polystyrene surfaces. The adhesion of NIH-3T3 fibroblast cells was shown to be significantly affected by the surface coverage of PEO nano-domains formed by copolymer self-assembly. These nano-domains, when presented at high number density (almost 1000 domains per square micron), were shown to completely prevent cellular attachment, even though small amounts of protein were able to bind to the surface.

Hierarchical scaffolds via combined macro- and micro-phase separation

Recent advances in biomaterial surface engineering have shown that surface biomechanical, spatial and topographical properties can elicit control over fundamental biological processes such as cell shape, proliferation, differentiation and apoptosis. Along these lines, we have very recently shown that the self-assembly of block copolymers into thin films can be used as an extremely labile method to precisely position cellular adhesion molecules, at nanometre lateral spacings, to effect control over cell attachment and morphology. Here, we extend our work in 2-dimensional block copolymer films into the production of 3-dimensional porous block copolymer scaffolds. The reported method combines macro-scale temperature induced phase separation and micro-phase separation of block copolymers to produce highly porous scaffolds with surfaces comprised of nano-scale self-assembled block copolymer domains, representing a significant advance in currently available scaffold engineering technologies. The phase behaviour of these polymer-solvent systems is described and potential mechanisms leading to the observed structure formation are presented. The nano-domains have thereafter been functionalised with CGRGDS peptides throughout the scaffold and shown to effect changes in cell attachment and spreading, in agreement with previous 2-dimensional studies. These multi-scale, functional scaffolds are easy to manufacture and scaleable, making them ideal candidates for tissue engineering applications.

Nanoscale presentation of cell adhesive molecules via block copolymer self-assembly.

Precise control over the nanoscale presentation of adhesion molecules and other biological factors represents a new frontier for biomaterials science. Recently, the control of integrin spacing and cellular shape has been shown to affect fundamental biological processes, such as differentiation and apoptosis. Here, we present the self-assembly of maleimide functionalised polystyrene-block-poly (ethylene oxide) copolymers as a simple, yet highly precise method for controlling the position of cellular adhesion molecules. By manipulating the phase separation of the functional PS-PEO block copolymer used in this study, via a simple blending technique, we alter the nanoscale (on PEO domains of 8-14 nm in size) presentation of the adhesion peptide, GRGDS, decreasing lateral spacing from 62 nm to 44 nm and increasing the number density from approximately 450 to approximately 900 islands per microm2. The results indicate that the spreading of NIH-3T3 fibroblasts increases as the spacing between domains of RGD binding peptides decreases. Further, the same functional PS-PEO surfaces have been utilised to immobilise, via a zinc chelating peptide sequence, poly-histidine tagged proteins and extracellular matrix (ECM) fragments. This method is seen as an ideal platform for investigations into the role of spatial arrangements of cell adhesion molecules and ECM molecules on cell function and, in particular, control of cell phenotype.

Surface-bound stem cell factor and the promotion of hematopoietic cell expansion.

In vivo, stem cell factor (SCF) exists in both a bound and soluble isoform. It is believed that the bound form is more potent and fundamentally required for the maintenance of hematopoietic stem cells (HSCs). This theory is supported by the observation that steel-Dickie mice lacking the bound isoform of SCF are unable to maintain hematopoiesis and by the fact that bound SCF displayed on the surface of transgenic cells is better able to maintain c-kit activation than soluble SCF. Further work has shown that recombinant SCF molecules, which include a surface-binding domain, are more potent than their soluble equivalent. It is generally assumed that such an elegant approach is necessary to provide the correct molecular orientation and avoid the pitfalls of random cross-linking or the denaturation associated with the adsorption of proteins to surfaces. However, in this work we demonstrate that SCF physisorbed to tissue culture plastic (TCP) is not only bioactive, but more potent than the soluble equivalent. By contrast, cross-linking of SCF via free amines is shown to compromise its bioactivity. These observations demonstrate that simple surface modification solutions cannot be discounted and with the advent of low-cost pharmaceutical grade proteins, they should not be.

Modeling the adhesion of human embryonic stem cells to poly(lactic-co-glycolic acid) surfaces in a 3D environment

Human embryonic stem cells (hESCs) have previously been cultured on three dimensional (3D) biodegradable polymer scaffolds. Although complex structures were formed from the hESCs, very little is known about the mechanism of adhesion of these cells to the surfaces of the scaffolds. In this study, we achieved the efficient adhesion of pluripotent hESCs to 3D poly(lactic-co-glycolic acid) (PLGA) scaffolds based on our data from a novel two dimensional (2D) model that imitates the surface properties of the scaffolds. In the 2D model, single cell preparations of pluripotent hESCs adhered efficiently and predominantly to PLGA surfaces coated with laminin in comparison to collagen I, collagen IV, or fibronectin-coated surfaces. Flow cytometry analysis revealed that almost all of the pluripotent single cells expressed the integrin alpha 6, with a small percentage also expressing alpha 3ss1, which facilitates adhesion to laminin. This data was then translated into the 3D environment, with the efficient binding of single pluripotent hESCs to PLGA scaffolds coated with laminin. The utility of this system was shown by the directed differentiation of single hESCs seeded within laminin-coated scaffolds toward the endoderm lineage.

The Generation of Definitive Endoderm from Human Embryonic Stem Cells on 3D Biodegradable Poly(lactic-co-glycolic Acid) Scaffolds and its Comparison to those Generated on 2D Monolayer Cultures

The generation of insulin producing cells from human embryonic stem cells (hESCs) has shown great promise as a cellular replacement therapy for the treatment of Type 1 Diabetes. Mature functional β-cell surrogates however, have yet to be successfully generated in vivo. One approach to potentially improve current differentiation protocols is the use of 3 dimensional (3D) scaffolds, which has been shown to enhance cellular function and differentiation potential. The present study aimed to explore the feasibility of using single cell preparations of pluripotent hESCs seeded onto laminin or Matrigel coated 3D poly(lactic-co-glycolic) acid (PLGA) scaffolds to derive definitive endoderm, the first vital stage of endoderm tissue differentiation. Our results demonstrated that hESCs which were induced to differentiate on laminin or Matrigel coated 3D scaffolds can be successfully coaxed to differentiate into definitive endoderm. The cells that were cultured on laminin or Matrigel coated 3D scaffolds expressed significantly higher levels of the key endoderm transcription factors SOX17 and FOXA2 in comparison to those differentiated on 2D monolayers. On Matrigel coated 3D scaffolds, the differentiated cells expressed lower levels of the endoderm surface marker CXCR4 and anterior endoderm marker CER in comparison to its monolayer counterpart. Together, the results of this study demonstrated the positive effect of 3D cultures on endoderm commitment from hESCs over traditional monolayer cultures. Furthermore, the definitive endoderm produced on Matrigel coated scaffolds may have a more posterior phenotype in comparison to those derived from monolayers. This may have an effect on later stages of pancreatic differentiation and warrants further detailed investigations.