Investigation of Integrin Expression in Human Mesenchymal Stem Cells during Chondrogenesis in Different Culture Systems

Cartilage tissue engineering aims to develop a material that can be implanted with cells to repair damage in articular cartilage. The extracellular matrix is known to play a key role in regulating stem cell behaviour via cell-matrix interactions and integrins are major cell adhesion receptors that act as intermediates between cells and their surrounding matrix. Integrin-mediated signalling is known to be important in chondrocytes and to drive chondrogenesis. In our study, we investigated integrin expression during chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) between three different high density 3D culture methods: pellet culture, micromass culture and collagen type II 3D hydrogels. Integrins are [alpha][beta] heterodimeric transmembrane proteins, so we quantified the mRNA expression of every alpha and beta subunit using quantitative polymerase chain reaction over a period of 21 days. In all culture methods, alpha and beta integrin subunits were down-regulated in hMSCs undergoing chondrogenesis compared to the same cells in growth medium, with the exception for integrin subunits [alpha]11 and [alpha]V which were upregulated in cells undergoing chondrogenesis . Moreover, similar transcript expression was identified irrespective of the culture system for the alpha subunits related to collagen-binding integrins, while the expression of fibronectin- and laminin-binding receptors was greatly variable and dependent on the culture method. In conclusion, this work has led to an increased understanding of the expression of integrins in hMSCs and during chondrogenesis. This knowledge can be used to design materials that present the ligands for these integrins to further improve cartilage tissue engineering.

A novel cell culture approach for tissue engineering based on surface expansion

Hicham Majd

Most tissue engineering approaches include the delivery of reparative cells to a damaged host tissue. Generally these cells are sought to be of a stem cell character since they retain a high potential for proliferation and differentiation into diverse phenotypes. To restrict cell function to the desired phenotype and to avoid anti-immune responses, autologous stem cells are frequently isolated from intact regions of the same (damaged) organ, such as keratinocytes from the basal layer of the skin and hair follicles or from the same germ layer origin such as mesenchymal stem cells (MSC) that are obtained from the bone marrow. This approach faces two major problems: 1) The number of source cells obtained from biopsies is generally too low for direct implantation, which requires cell multiplication in culture. 2) The standard culture conditions, including growth in plastic dishes, passaging, and the composition of the growth medium, never correspond to the natural microenvironment and influence cell differentiation, often resulting in non-desired phenotypes. The objective of my thesis was to provide a solution for both problems that is applicable for the average cell culture laboratory. Our technique reduces traumatic enzymatic passaging, provides constant cell densities to keep cells in the exponential growth phase and at the same time avoids contact inhibition of cell growth. This novel approach comprises the development of a new elastic culture surface, produced from a high extension silicone rubber (HESR) that we here for the first time applied for cell culture. The HESR is enlarged by a novel mechanical approach permitting to visualize and control cells under expansion. Untreated silicone, the polymer component of the HESR, does not promote cell attachment and must therefore be functionalized. In the first part of my thesis (Chapter 2) we establish a surface treatment for silicone membranes that will resist large surface expansions. We compare established coating techniques that have originally been developed to provide static silicone surfaces with cell adhesive molecules by 1) hydrophobic, 2) electrostatic and 3) covalent interactions. Cell attachment, spreading and proliferation was evaluated between these three treatments as a function of the coating in static and stretched conditions. We show that covalent immobilization of collagen type I is most suitable to stretch cells cultured on elastic silicone substrates. We applied this technique to functionalize the HESR culture surface in all later applications. The second central part of my thesis (Chapter 3) develops a new culture method consisting of a motorized device that isotropically expands the coated HESR surface to yet unmatched 1000% of its initial surface. Proof-of-principle of this method is given by culturing human MSC, which are sensitive to passaging during expansion in vitro and that are therapeutically relevant cells to repair bone, cartilage, and cardiovascular tissue. MSC were cultured three consecutive passages on the culture surface that has been expanded in steps of 5% every 6 h for 20 days, respectively. After nine weeks of expansion culture, MSC exhibited consistently high proliferation rates, fully retained their pluripotent stem cell character and were inducible to differentiate into adipogenic, chondrogenic, osteogenic, and myogenic lineages. The cell surface expansion culture surface produced ∼10-times higher cell numbers compared with nine weeks of standard culture, which required nine events of passaging to attain the same culture surface. We propose our method to produce MSC for clinical cell therapy. In the third part of my thesis (Chapter 4) we implement our novel surface expansion method to culture fibroblasts with the aim to: 1) rapidly expand primary fibroblasts, 2) achieve higher cell yields than obtained with conventional culture, and 3) obtain homogeneous cell populations with non-fibrogenic character. The expansion of primary autologous dermal fibroblasts in culture is a pivotal step to obtain sufficiently high cell numbers for application in skin esthetics and for the treatment of large burn wounds. Dynamically enlarging the culture surface by 800% over 20 d, yielded ∼1.5 higher fibroblasts numbers than the equivalent three standard passages. By further optimizing the surface expansion speed according to the cell growth rates, the same high cell numbers were produced after only 9 d of culture. The highly compliant nature of the HESR surface had the beneficial effect of completely abolishing the formation of 'fibrotic' myofibroblasts. Hence, our novel method for fibroblast culture is faster and yields higher numbers of more homogeneous cells than standard culture passaging. It delivers a fibroblast quality and quantity that is appealing for cell therapeutic purposes. The aim of the last part of my thesis (Chapter 5) was to provide highly compliant silicone elastomers, such as the HESR, with a micropattern of cell adhesive proteins. In particular we aim to produce arrays of extracellular matrix proteins with a size and morphology to promote formation of single focal adhesions on highly elastic and expandable surfaces. This will allow future studies on cell mechanosensing through focal adhesions by discriminating between effects of focal adhesion size changes and stretch. An existing method to pattern rigid surfaces with proteins, so-called microcontact printing, is not suitable to transfer proteins onto surfaces that are softer than the stamp. We here establish a new transfer technique that is based on a stencil with micron-sized openings. The new stencil is used to expose the surface of compliant silicone substrates with Young's modulus of 10-100 kPa to a protein solution of fibronectin, collagen I and protein-repellent polymers, leaving behind the desired protein pattern. Fibroblasts were successfully attached onto the resulting focal islet arrays on compliant substrates.

EPFL2008

Rat bladder muscle regeneration induced by local delivery of an engineered insulin-like growth factor 1 in fibrin gels

Lirong Yang

Currently, both congenital abnormalities and developmental problems of the bladder in children, and other dysfunctions in adults, require reconstructive surgery. Such correction involves transplant action of native tissues (such as gastrointestinal segments, or mucosa), homologous tissues from a donor, heterologous tissues or substances, or artificial materials to act as a replacement for normal bladder tissue. However, such surgery does not entirely restore the function, as the replacement tissue is either rejected due to immune system, fibrosis, contraction or causes metabolic complications due to a mismatch in different functional parameters, such as gastrointestinal segment for absorbance versus bladder for excretion. Tissue engineering is emerging as a significant alternative potential treatment for bladder dysfunction. To achieve this goal, we have explored fibrin gels as a natural material based scaffold to seed infiltrating smooth muscle cells to mimic the ontogeny of the bladder tissue. Our interest in developing fibrin-based biomaterial technology is based on fibrin's central role in tissue binding and in the initiation of tissue repair and defence. It is well known that fibrinogen/fibrin binds to platelets as well as to different cells, growth factors, and extracellular matrix proteins, which is critical for the wound repair process. Insulin-like growth factor I (IGF1) is well known as a key regulator in carbohydrate metabolism and growth. It can promote smooth muscle cell growth. In this thesis it was also chosen due to its simple active form, namely a single chain of low molecular weight, 10kDa. A novel engineered insulin-like growth factor I (IGF1) – factor XIIIa substrate fusion protein was chosen as the bioactive macromolecule to be released from the scaffold in order to control cellular growth and differentiation. Therefore, we can achieve close to the natural biomechanical environment in the regenerated tissue. In this work, two generations of fibrin matrices for tissue engineering were developed. The first part of the work was devoted to preventing rapid diffusion of the growth factor from the matrix and to controlling its release. IGF1 was modified by inserting a factor XIIIa substrate sequence (denoted TG) based on the α2-plasmin inhibitor to cross-link into the fibrin gel during coagulation. This is so that the variant IGF1 will bind to the fibrin gel itself and will be released upon matrix degradation. In order to produce this recombinant protein TG-IGF1, IGF1 GST tag plasmid DNA was transformed into BL21 competent cells. After protein production, the recombinant TG-IGF1 protein was purified by GST affinity chromatography. The purified TG-IGF1 was confirmed via SDS-PAGE and western blot with an anti-hIGF1 antibody. The sequence was confirmed using MALDI-TOF mass spectrometry. The biological activity of TG-IGF1 was validated in vitro by receptor tyrosine phosphorylation and metabolic assay (MTT assay) with fibroblast 3T3 cells. The cell proliferation profile was similar for both TG-IGF1 and native recombinant IGF1. Although we were able to obtain relatively pure TG-IGF1 protein, the purification protocol may still require some development due to loss of protein via aggregation during protein production. Using a three-dimension in vitro model, neonatal human bladder smooth muscle cells were seeded in the fibrin gel only, in the fibrin gel either with TG-IGF1 or native recombinant IGF1 as a control. The morphology of the seeded cells was analysed using ultra-structural transmission electron microscopy after three days. Secretory vesicles had not been found in the cells without IGF1. The cells with the TG-IGF1 displayed larger and more numerous secretory vesicles than those with native recombinant IGF1, presumably due to the rapid diffusional loss of the native recombinant IGF1 from the gel. The bladder smooth muscle cell in the fibrin gel responses to either TG-IGF1 or native recombinant IGF1 at the genomic level were analyzed by qRT-PCR for extracellular matrix and adhesion molecules after 24 hours incubation. The cells in the fibrin gel only were used as control. We did not observe a difference in gene expression between the two groups. The bioactivity of TG-IGF1 was also investigated in vivo utilizing a rat bladder model. A wound was induced via resection on the rat bladder. Either the TG-IGF1 or native recombinant IGF1 containing fibrin gel was then applied to the wound site, with a second control group receiving only the fibrin gel alone. Three classic histological stainings were done using Hematoxylin & Erythrosine (HE), Masson's Trichrome (TM) and Prussian Blue (PB). It was found that TG-IGF1 greatly enhanced rat bladder muscle layer regeneration compared to native recombinant IGF1 and the control group of fibrin gel according to the histology staining and quantitative analysis of the ratio of detrusor muscle regenerated / normal detrusor muscle (0.27±0.10 for TG-IGF1 and 0.23±0.10 for native recombinant IGF1). The second part of the work was devoted to making a new generation of fibrin matrices. Fibrinogen (Fgn), a soluble plasma protein found in all vertebrates, is a covalent dimer composed of pairs of three polypeptide chains called Aα-, Bβ- and γ-chains. Fibrinogen Aα chain can be truncated to Bβ-and γ-chains while maintaining the capacity to be assembled into a secreted, detectable fibrinogen. It is feasible to produce recombinant chimeric fibrinogen in cell culture, which can then be used to incorporate growth factors into fibrin matrices as a fusion protein with fibrinogen at the genetic level. In this way, fibrin based natural material hydrogels can be formed which can bind and release signalling biomacromolecules for directed cellular growth and tissue regeneration. In order to produce this new fibrinogen recombinant protein, a novel stable cell line of Chinese Hamster Ovary cells, CHOfgn-hIGF1, needed to be developed. This was accomplished via transfection of an FGA-hIGF1 plasmid DNA into CHOβγ cells. After protein production, the recombinant chimeric fibrinogen variant hIGF1 fusion protein was purified by affinity chromatography. The successful production of these cell lines and the resultant variant IGF1 was confirmed via ELISA with fibrinogen and hIGF1 antibodies, and also by fluorescence activated cell sorting (FACS). Due to protein polymerisation in the column during purification, the protein purification step still requires additional development. Although the targeted fusion protein was successfully produced, overall protein yield was so low as to prevent further exploration. By harnessing the powerful technique of protein engineering and using the fibrinogen/fibrin hydrogels developed in our lab, we were able to demonstrate a new type of multifunctional gel capable of regenerating smooth muscle type tissue with the aim to repair bladder function. Here we were able to demonstrate the production of the variant IGF1 factor XIIIa substrate fusion protein, its biological activity in vitro, and its application in an in vivo rat model.

EPFL2010

Transient gene expression for rapid protein production

Natalie Muller

Recombinant proteins are important for biomedical research and for the treatment of human disease. Therefore it is necessary to develop reproducible bioprocesses to rapidly produce proteins of adequate quality and quantity. Expression in mammalian cells is preferred if the proteins are to be properly folded and post-translationally modified. For the rapid production of milligram to gram quantities of a protein in mammalian cells, large-scale transient gene expression is an attractive option. The two most important components of this technology are the cell cultivation system and the gene delivery method. For this reason the goal of this thesis was to develop novel cost-efficient cell cultivation systems and gene delivery methods for the large-scale transfection of mammalian cells in chemically defined media free of any animal-derived components including serum. Cultivation of mammalian cells in suspension is essential for large-scale transient gene expression. A superior system for the cultivation of mammalian cells based on the agitation of cells in "square-shaped" glass bottles (square bottles) was developed. It is an inexpensive but efficient means to grow cells to a high density without instrumentation. The growth and viability of cultures of both human embryo kidney (HEK) 293E and Chinese hamster ovary (CHO) DG44 cells in agitated one-liter square bottles exceeded that in spinner flasks, reaching cell densities 1.5 times higher on average. Furthermore, an efficient and reproducible method to transfect cells in agitated square bottles was developed for both HEK 293E and CHO DG44 cells. A novel gene delivery method termed calfection that relies on the addition of DNA and CaCl2 directly to a suspension culture was developed. Calfection has a number of advantages over existing gene delivery methods such as calcium phosphate-DNA coprecipitation in that there are no time-dependent steps. The DNA/CaCl2 solution can be stored for long periods and is filterable. It is suitable for both large-scale transfections in bioreactors and for high-throughput transfections in microtiter plates. One drawback, however, is its dependence on the presence of serum in the culture medium. A second gene delivery method, serum-free transfection with polyethylenimine (polyfection), proved to be highly efficient for the transfection of both HEK 293E and CHO DG44 cells. A reproducible method for polyfection in microtiter plates, 50 ml centrifuge tubes, agitated square and round bottles, spinner flasks, and bioreactors was optimized for these two cell lines. Polyfection proved to be a simple and successful gene transfer method in both instrumented and non-instrumented cultivation systems. Importantly, it could be performed in serum-free chemically defined media. With HEK 293E cells, 80 mg/l of antibody was produced in agitated square bottles and with CHO DG44 cells, more than 2 g of antibody were produced in one week in a 150-liter bioreactor.

EPFL2005