Zara Melkoumian

Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells

Human embryonic stem cells (hESCs) have two properties of interest for the development of cell therapies: self-renewal and the potential to differentiate into all major lineages of somatic cells in the human body. Widespread clinical application of hESC-derived cells will require culture methods that are low-cost, robust, scalable and use chemically defined raw materials. Here we describe synthetic peptide-acrylate surfaces (PAS) that support self-renewal of hESCs in chemically defined, xeno-free medium. H1 and H7 hESCs were successfully maintained on PAS for over ten passages. Cell morphology and phenotypic marker expression were similar for cells cultured on PAS or Matrigel. Cells on PAS retained normal karyotype and pluripotency and were able to differentiate to functional cardiomyocytes on PAS. Finally, PAS were scaled up to large culture-vessel formats. Synthetic, xeno-free, scalable surfaces that support the self-renewal and differentiation of hESCs will be useful for both research purposes and development of cell therapies.

A Synthetic, Xeno-Free Peptide Surface for Expansion and Directed Differentiation of Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells have the potential to become an unlimited cell source for cell replacement therapy. The realization of this potential, however, depends on the availability of culture methods that are robust, scalable, and use chemically defined materials. Despite significant advances in hiPSC technologies, the expansion of hiPSCs relies upon the use of animal-derived extracellular matrix extracts, such as Matrigel, which raises safety concerns over the use of these products. In this work, we investigated the feasibility of expanding and differentiating hiPSCs on a chemically defined, xeno-free synthetic peptide substrate, i.e. Corning Synthemax® Surface. We demonstrated that the Synthemax Surface supports the attachment, spreading, and proliferation of hiPSCs, as well as hiPSCs’ lineage-specific differentiation. hiPSCs colonies grown on Synthemax Surfaces exhibit less spread and more compact morphology compared to cells grown on Matrigel™. The cytoskeleton characterization of hiPSCs grown on the Synthemax Surface revealed formation of denser actin filaments in the cell-cell interface. The down-regulation of vinculin and up-regulation of zyxin expression were also observed in hiPSCs grown on the Synthemax Surface. Further examination of cell-ECM interaction revealed that hiPSCs grown on the Synthemax Surface primarily utilize αvβ5 integrins to mediate attachment to the substrate, whereas multiple integrins are involved in cell attachment to Matrigel. Finally, hiPSCs can be maintained undifferentiated on the Synthemax Surface for more than ten passages. These studies provide a novel approach for expansion of hiPSCs using synthetic peptide engineered surface as a substrate to avoid a potential risk of contamination and lot-to-lot variability with animal derived materials.

Long term expansion of bone marrow-derived hMSCs on novel synthetic microcarriers in xeno-free, defined conditions.

Human mesenchymal stem cells (hMSCs) present an attractive target for cell therapy given their wide availability, immunomodulatory properties, and multipotent nature for differentiation into chondrocytes, osteocytes, and adipocytes. With the progression of hMSC clinical studies, there is an increasing demand for development of technologies that enable efficient cell scale-up into clinically relevant quantities. Commercial scale manufacturing of hMSCs will require a large surface area which is not cost effective with available two-dimensional culture vessels. Recent studies showed that microcarriers provide a three-dimensional culture environment suitable for hMSC expansion. Traditionally, biological coatings and/or serum-containing medium are required to facilitate hMSC attachment and expansion in dynamic conditions. These limitations may hinder the use of microcarriers as a scale-up technology for hMSC therapeutics, where cell products, and therefore patient safety, are more controlled with the use of xeno-free, defined culture conditions. Here we report the long term culture of hMSCs on novel synthetic Synthemax II microcarriers in two different xeno-free media. Cells were maintained over 40 days on sterile, ready-to-use microcarriers in spinner flasks with programmed agitation. hMSC expansion was obtained by addition of fresh beads without the need for enzymatic dissociation. We achieved a cumulative cell expansion of >10,000 fold, and cells retained normal hMSC phenotype, karyotype, and tri-lineage differentiation potential. To our knowledge, this report is the first example of long term culture of hMSCs on synthetic microcarriers in xeno-free, defined conditions.

Differentiation of oligodendrocyte progenitor cells from human embryonic stem cells on vitronectin-derived synthetic peptide acrylate surface.

Human embryonic stem cell (hESC)-derived oligodendrocyte progenitor cells (OPCs) are being studied for cell replacement therapies, including the treatment of acute spinal cord injury. Current methods of differentiating OPCs from hESCs require complex, animal-derived biological extracellular matrices (ECMs). Defined, low-cost, robust, and scalable culture methods will need to be developed for the widespread deployment and commercialization of hESC-derived cell therapies. Here we describe a defined culture system that uses a vitronectin-derived synthetic peptide acrylate surface (VN-PAS; commercially available as Corning(®) Synthemax(®) surface) in combination with a defined culture medium for hESC growth and differentiation to OPCs. We show that synthetic VN-PAS supports OPC attachment and differentiation, and that hESCs grown on VN-PAS are able to differentiate into OPCs on VN-PAS. Compared to OPCs derived from hESCs grown on ECM of animal origin, higher levels of NG2, a chondroitin sulfate proteoglycan expressed by OPCs, were observed in OPCs differentiated from H1 hESCs grown on VN-PAS, while the expression levels of Nestin and PDGFRα were comparable. In summary, this study demonstrates that synthetic VN-PAS can replace complex, animal-origin ECM to support OPC differentiation from hESCs.

Defined Culture of Human Embryonic Stem Cells and Xeno-Free Derivation of Retinal Pigmented Epithelial Cells on a Novel, Synthetic Substrate

Age‐related macular degeneration (AMD), a leading cause of blindness, is characterized by the death of the retinal pigmented epithelium (RPE), which is a monolayer posterior to the retina that supports the photoreceptors. Human embryonic stem cells (hESCs) can generate an unlimited source of RPE for cellular therapies, and clinical trials have been initiated. However, protocols for RPE derivation using defined conditions free of nonhuman derivatives (xeno‐free) are preferred for clinical translation. This avoids exposing AMD patients to animal‐derived products, which could incite an immune response. In this study, we investigated the maintenance of hESCs and their differentiation into RPE using Synthemax II‐SC, which is a novel, synthetic animal‐derived component‐free, RGD peptide‐containing copolymer compliant with good manufacturing practices designed for xeno‐free stem cell culture. Cells on Synthemax II‐SC were compared with cultures grown with xenogeneic and xeno‐free control substrates. This report demonstrates that Synthemax II‐SC supports long‐term culture of H9 and H14 hESC lines and permits efficient differentiation of hESCs into functional RPE. Expression of RPE‐specific markers was assessed by flow cytometry, quantitative polymerase chain reaction, and immunocytochemistry, and RPE function was determined by phagocytosis of rod outer segments and secretion of pigment epithelium‐derived factor. Both hESCs and hESC‐RPE maintained normal karyotypes after long‐term culture on Synthemax II‐SC. Furthermore, RPE generated on Synthemax II‐SC are functional when seeded onto parylene‐C scaffolds designed for clinical use. These experiments suggest that Synthemax II‐SC is a suitable, defined substrate for hESC culture and the xeno‐free derivation of RPE for cellular therapies.

Synthetic Surface for Expansion of Human Mesenchymal Stem Cells in Xeno-Free, Chemically Defined Culture Conditions

Human mesenchymal stem cells (hMSCs) possess three properties of great interest for the development of cell therapies and tissue engineering: multilineage differentiation, immunomodulation, and production of trophic factors. Efficient ex vivo expansion of hMSCs is a challenging requirement for large scale production of clinical grade cells. Low-cost, robust, scalable culture methods using chemically defined materials need to be developed to address this need. This study describes the use of a xeno-free synthetic peptide acrylate surface, the Corning® Synthemax® Surface, for culture of hMSCs in serum-free, defined medium. Cell performance on the Corning Synthemax Surface was compared to cells cultured on biological extracellular matrix (ECM) coatings in xeno-free defined medium and in traditional conditions on tissue culture treated (TCT) plastic in fetal bovine serum (FBS) supplemented medium. Our results show successful maintenance of hMSCs on Corning Synthemax Surface for eight passages, with cell expansion rate comparable to cells cultured on ECM and significantly higher than for cells in TCT/FBS condition. Importantly, on the Corning Synthemax Surface, cells maintained elongated, spindle-like morphology, typical hMSC marker profile and in vitro multilineage differentiation potential. We believe the Corning Synthemax Surface, in combination with defined media, provides a complete synthetic, xeno-free, cell culture system for scalable production of hMSCs.

Synthetic Surfaces for Human Embryonic Stem Cell Culture

Human embryonic stem cells (hESCs) have two properties that distinguish them from other cell types: self-renewal, the ability to propagate indefinitely in culture, and pluripotency, the ability to differentiate into any type of specialized cells found in the human body. These properties provide the foundation for the development of hESC-derived cell-based therapeutics, where specific cell types derived by differentiation of hESCs become a therapeutic agent that cures the disease or restores the function of damaged organs or tissue. To make this a reality, several technologies must be developed to provide an unlimited and consistent supply of hESC-derived cells for clinical use. These include robust and scalable methods for production of undifferentiated hESCs, differentiation of the hESCs into desirable cell types, recovery, purification, storage and transportation of the derived cells to the location of use, and methods and techniques for delivery of the therapeutic cells to a human body to provide health benefits. Since the derivation of the first hESC lines by Thomson, J. et al. (Thomson, 1998) and Reubinoff, B. et al. (Reubinoff et al., 2000), hundreds of new lines have been established and propagated under various cell culture conditions. Historically, hESCs were maintained in complex culture systems under poorly defined conditions comprising mouse or human feeder cell layers and medium containing fetal bovine serum (FBS) or serum replacement to provide an extracellular matrix (ECM)-rich environment for cell adhesion, as well as soluble growth factors for self-renewal. It is highly desirable that the cell culture systems utilized for therapeutic cells, including cell culture surfaces and the media, are well defined (all components are known and characterized and their abundance is controlled) and of nonanimal origin or xeno-free (do not contain biological materials of a non-human nature). Establishment of the first human embryonic stem cell line (Thomson, 1998) was accomplished by extending to hESCs a cell culture system developed for culturing mouse embryonic stem cells that is based on inactivated mouse embryonic fibroblasts (MEF) as a feeder layer. Soon after the first reports on isolation of human pluripotent cells came realization that feeder-free cell culture is essential for production of cells for transplantation (Donovan & Gearhart, 2001; Pera et al., 2000); (Pedersen, 2002). Back in 2000 this looked like a challenge that would require a very long time to overcome, as 19 prior years of using MEFs to support stem cell culture of non-human cells did not result in significant understanding of what exactly MEFs provide for stem cells. To make matters worse, there was experimental evidence showing that neither MEF conditioned medium nor ECM

Chemical identity and mechanism of action and formation of a cell growth inhibitory compound from polycarbonate flasks

This paper reports the chemical identity and mechanism of action and formation of a cell growth inhibitory compound leached from some single-use Erlenmeyer polycarbonate shaker flasks under routine cell culture conditions. Single-use cell culture vessels have been increasingly used for the production of biopharmaceuticals; however, they often suffer from issues associated with leachables that may interfere with cell growth and protein stability. Here, high-performance liquid-chromatography preparations and cell proliferation assays led to identification of a compound from the water extracts of some polycarbonate flasks, which exhibited subline- and seeding density-dependent growth inhibition of CHO cells in suspension culture. Mass spectroscopy, nuclear magnetic resonance spectroscopy, and chemical synthesis confirmed that this compound is 3,5-dinitro-bisphenol A. Cell cycle analysis suggests that 3,5-dinitro-bisphenol A arrests CHO-S cells at the G1/Go phase. Dynamic mass redistribution assays showed that 3,5-dinitro-bisphenol A is a weak GPR35 agonist. Analysis of the flask manufacturing process suggests that 3,5-dinitro-bisphenol A is formed via the combination of molding process with γ-sterilization. This is the first report of a cell culture/assay interfering leachable compound that is formed through γ-irradiation-mediated nitric oxide free radical reaction.