Soraya Rasi Ghaemi

Exploring the mesenchymal stem cell niche using high throughput screening

In the field of stem cell technology, future advancements rely on the effective isolation, scale-up and maintenance of specific stem cell populations and robust procedures for their directed differentiation. The stem cell microenvironment - or niche - encompasses signal inputs from stem cells, supporting cells and from the extracellular matrix. In this context, the contribution of physicochemical surface variables is being increasingly recognised. This paradigm can be exploited to exert control over cellular behaviour. However, the number of parameters at play, and their complex interactions, presents a formidable challenge in delineating how the decisions of cell fate are orchestrated within the niche. Additionally, in the case of mesenchymal stem cells (MSC), more than one type of stem cell niche has been identified. By employing high throughput screening (HTS) strategies, common and specific attributes of each MSC niche can be probed. Here, we explore biological, chemical and physical parameters that are known to influence MSC self-renewal and differentiation. We then review techniques and strategies that allow the HTS of surface properties for conditions that direct stem cell fate, using MSC as a case study. Finally, challenges in recapturing the niche, particularly its three dimensional nature, in surface-based HTS formats are discussed.

Isolation and ex vivo expansion of human umbilical cord blood-derived CD34+ stem cells and their cotransplantation with or without mesenchymal stem cells

Abstract Umbilical cord blood (UCB) contains a high number of primitive progenitor cells, allowing UCB to be used as a source of hematopoietic progenitors for clinical transplantation. However the rate of UCB CD34+ stem cells graft is low. Mesenchymal stem cells (MSCs) have been implicated in playing an important role in hematopoietic stem cell engraftment. In this study we examined the effect of human MSC on engraftment of human UCB-derived CD34+ cells in irradiated Balb/c mice. Human UCB CD34+ cells were obtained from full-term normal deliveries by using an immunomagnetic separation technique and MSC were isolated by standard methodology from human bone marrow. Isolated CD34+ cells were cultured in Stemline Hematopoietic stem cell expansion medium supplemented with 100 ng/ml stem cell factor (SCF), and 100 ng/ml thrombopoietin (TPO) in 24-well plates and incubated at 37°C in a fully humidified atmosphere with 5% CO2, and maintained over 3 weeks and half the medium was exchanged twice a week. Irradiated (7 Gy) Balb/c mice were transplanted intravenously with 0·1 × 106 to 10 × 106 human UCB CD34+ cells in the presence or absence of 0·5 × 106 and 1 × 106 human bone marrow-derived MSC. After 11 days, in each group, the spleen was dissected and colony assay performed. Hematoxilin and eosin staining of the spleen colony was performed, and UCB CD34+ cells labeled with super paramagnetic iron oxide (SPIO). After establishing the presence of colonies in spleen, Prussian blue staining was performed. Flow cytometry assay showed that up to 90% purity of CD34+ cells and 96% for MSC. After 3 weeks the cell numbers showed a 1000-fold increase in CD34+. Cotransplantation of low doses of UCB CD34+ cells (0·2 × 106 and 0·3 × 106) and MSC (0·5 × 106 and 1 × 106) resulted in a significant increase in colony forming unit spleen, in comparison with engraftment of UCB CD34+ stem cells without MSC after 11 days (p<0·01). In conclusion the results showed that two cytokines (SCF, TPO) were sufficient for expansion of UCB CD34+ cells and cotransplantation of MSC with UCB CD34+ cells, promoting engraftment of UCB CD34+ cells.

Surface Engineering for Long-Term Culturing of Mesenchymal Stem Cell Microarrays

The cell microarray format can recreate a multitude of cell microenvironments on a single chip using only minimal amounts of reagent. In this study, we describe surface modifications to passivate cell microarrays, aiming to adapt the platform to the study of stem cell behavior over long-term culture periods. Functionalization of glass slides with (3-glycidyloxypropyl) trimethoxysilane enabled covalent anchoring of extracellular matrix proteins on microscale spots printed by a robotic contact printer. Subsequently, the surface was passivated by bovine serum albumin (BSA) or poly(ethylene glycol)bisamine (A-PEG) with molecular weights of 3000, 6000, and 10 000 Da. Cloud-point conditions for A-PEG grafting were attained that were compatible with protein deposition. Passivation strategies were assessed by culturing mesenchymal stem cells on the microarray platform. While both BSA and A-PEG passivation initially blocked cell adhesion between the printed spots, only A-PEG grafting was able to maintain cell pattern integrity over the entire culture period of 3 weeks.

Synergistic influence of collagen I and BMP 2 drives osteogenic differentiation of mesenchymal stem cells: a cell microarray analysis

Cell microarrays are a novel platform for the high throughput discovery of new biomaterials. By re-creating a multitude of cell microenvironments on a single slide, this approach can identify the optimal surface composition to drive a desired cell response. To systematically study the effects of molecular microenvironments on stem cell fate, we designed a cell microarray based on parallel exposure of mesenchymal stem cells (MSCs) to surface-immobilised collagen I (Coll I) and bone morphogenetic protein 2 (BMP 2). This was achieved by means of a reactive coating on a slide surface, enabling the covalent anchoring of Coll I and BMP 2 as microscale spots printed by a robotic contact printer. The surface between the printed protein spots was passivated using poly (ethylene glycol) bisamine 10,000Da (A-PEG). MSCs were then captured and cultured on array spots composed of binary mixtures of Coll I and BMP 2, followed by automated image acquisition and quantitative, multi-parameter analysis of cellular responses. Surface compositions that gave the highest osteogenic differentiation were determined using Runx2 expression and calcium deposition. Quantitative single cell analysis revealed subtle concentration-dependent effects of surface-immobilised proteins on the extent of osteogenic differentiation obscured using conventional analysis. In particular, the synergistic interaction of Coll I and BMP 2 in supporting osteogenic differentiation was confirmed. Our studies demonstrate the value of cell microarray platforms to decipher the combinatorial interactions at play in stem cell niche microenvironments.

A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations

Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response.

Identification and In Vitro Expansion of Buccal Epithelial Cells

Ex vivo-expanded buccal mucosal epithelial (BME) cell transplantation has been used to reconstruct the ocular surface. Methods for enrichment and maintenance of BME progenitor cells in ex vivo cultures may improve the outcome of BME cell transplantation. However, the parameter of cell seeding density in this context has largely been neglected. This study investigates how varying cell seeding density influences BME cell proliferation and differentiation on tissue culture polystyrene (TCPS). The highest cell proliferation activity was seen when cells were seeded at 5×104 cells/cm2. Both below and above this density, the cell proliferation rate decreased sharply. Differential immunofluorescence analysis of surface markers associated with the BME progenitor cell population (p63, CK19, and ABCG2), the differentiated cell marker CK10 and connexin 50 (Cx50) revealed that the initial cell seeding density also significantly affected the progenitor cell marker expression profile. Hence, this study demonstrates that seeding density has a profound effect on the proliferation and differentiation of BME stem cells in vitro, and this is relevant to downstream cell therapy applications.

High-Throughput Assessment and Modelling of a Polymer Library Regulating Human Dental Pulp-Derived Stem Cell Behavior

The identification of biomaterials that modulate cell responses is a crucial task for tissue engineering and cell therapy. The identification of novel materials is complicated by the immense number of synthesizable polymers and the time required for testing each material experimentally. In the current study, polymeric biomaterial-cell interactions were assessed rapidly using a microarray format. The attachment, proliferation, and differentiation of human dental pulp stem cells (hDPSCs) were investigated on 141 homopolymers and 400 diverse copolymers. The copolymer of isooctyl acrylate and 2-(methacryloyloxy)ethyl acetoacetate achieved the highest attachment and proliferation of hDPSC, whereas high cell attachment and differentiation of hDPSC were observed on the copolymer of isooctyl acrylate and trimethylolpropane ethoxylate triacrylate. Computational models were generated, relating polymer properties to cellular responses. These models could accurately predict cell behavior for up to 95% of materials within a test set. The models identified several functional groups as being important for supporting specific cell responses. In particular, oxygen-containing chemical moieties, including fragments from the acrylate/acrylamide backbone of the polymers, promoted cell attachment. Small hydrocarbon fragments originating from polymer pendant groups promoted cell proliferation and differentiation. These computational models constitute a key tool to direct the discovery of novel materials within the enormous chemical space available to researchers.