Sherry T. Hikita

Protective Effects of Human iPS-Derived Retinal Pigment Epithelium Cell Transplantation in the Retinal Dystrophic Rat

Transformation of somatic cells with a set of embryonic transcription factors produces cells with the pluripotent properties of embryonic stem cells (ESCs). These induced pluripotent stem (iPS) cells have the potential to differentiate into any cell type, making them a potential source from which to produce cells as a therapeutic platform for the treatment of a wide range of diseases. In many forms of human retinal disease, including age-related macular degeneration (AMD), the underlying pathogenesis resides within the support cells of the retina, the retinal pigment epithelium (RPE). As a monolayer of cells critical to photoreceptor function and survival, the RPE is an ideally accessible target for cellular therapy. Here we report the differentiation of human iPS cells into RPE. We found that differentiated iPS-RPE cells were morphologically similar to, and expressed numerous markers of developing and mature RPE cells. iPS-RPE are capable of phagocytosing photoreceptor material, in vitro and in vivo following transplantation into the Royal College of Surgeons (RCS) dystrophic rat. Our results demonstrate that iPS cells can be differentiated into functional iPS-RPE and that transplantation of these cells can facilitate the short-term maintenance of photoreceptors through phagocytosis of photoreceptor outer segments. Long-term visual function is maintained in this model of retinal disease even though the xenografted cells are eventually lost, suggesting a secondary protective host cellular response. These findings have identified an alternative source of replacement tissue for use in human retinal cellular therapies, and provide a new in vitro cellular model system in which to study RPE diseases affecting human patients.

Derivation of Functional Retinal Pigmented Epithelium from Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (iPSCs) have great promise for cellular therapy, but it is unclear if they have the same potential as human embryonic stem cells (hESCs) to differentiate into specialized cell types. Ocular cells such as the retinal pigmented epithelium (RPE) are of particular interest because they could be used to treat degenerative eye diseases, including age‐related macular degeneration and retinitis pigmentosa. We show here that iPSCs generated using Oct4, Sox2, Nanog, and Lin28 can spontaneously differentiate into RPE cells, which can then be isolated and cultured to form highly differentiated RPE monolayers. RPE derived from iPSCs (iPS‐RPE) were analyzed with respect to gene expression, protein expression, and rod outer segment phagocytosis, and compared with cultured fetal human RPE (fRPE) and RPE derived from hESCs (hESC‐RPE). iPS‐RPE expression of marker mRNAs was quantitatively similar to that of fRPE and hESC‐RPE, and marker proteins were appropriately expressed and localized in polarized monolayers. Levels of rod outer segment phagocytosis by iPS‐RPE, fRPE, and hESC‐RPE were likewise similar and dependent on integrin αvβ5. This work shows that iPSCs can differentiate into functional RPE that are quantitatively similar to fRPE and hESC‐RPE and further supports the finding that iPSCs are similar to hESCs in their differentiation potential. STEM CELLS 2009;27:2427–2434

A versatile approach to high-throughput microarrays using thiol-ene chemistry

Microarray technology has become extremely useful in expediting the investigation of large libraries of materials in a variety of biomedical applications, such as in DNA chips, protein and cellular microarrays. In the development of cellular microarrays, traditional high-throughput printing strategies on stiff, glass substrates and non-covalent attachment methods are limiting. We have developed a facile strategy to fabricate multifunctional high-throughput microarrays embedded at the surface of a hydrogel substrate using thiol-ene chemistry. This user-friendly method provides a platform for the immobilization of a combination of bioactive and diagnostic molecules, such as peptides and dyes, at the surface of poly(ethylene glycol)-based hydrogels. The robust and orthogonal nature of thiol-ene chemistry allows for a range of covalent attachment strategies in a fast and reliable manner, and two complementary strategies for the attachment of active molecules are demonstrated.

Roles of Integrins in Human Induced Pluripotent Stem Cell Growth on Matrigel and Vitronectin

Human induced pluripotent stem cells (iPSCs) hold promise as a source of adult-derived, patient-specific pluripotent cells for use in cell-based regenerative therapies. However, current methods of cell culture are tedious and expensive, and the mechanisms underlying cell proliferation are not understood. In this study, we investigated expression and function of iPSC integrin extracellular matrix receptors to better understand the molecular mechanisms of cell adhesion, survival, and proliferation. We show that iPSC lines generated using Oct-3/4, Sox-2, Nanog, and Lin-28 express a repertoire of integrins similar to that of hESCs, with prominent expression of subunits alpha5, alpha6, alphav, beta1, and beta5. Integrin function was investigated in iPSCs cultured without feeder layers on Matrigel or vitronectin, in comparison to human embryonic stem cells. beta1 integrins were required for adhesion and proliferation on Matrigel, as shown by immunological blockade experiments. On vitronectin, the integrin alphavbeta5 was required for initial attachment, but inhibition of both alphavbeta5 and beta1 was required to significantly decrease iPSC proliferation. Furthermore, iPSCs cultured on vitronectin for 9 passages retained normal karyotype, pluripotency marker expression, and capacity to differentiate in vitro. These studies suggest that vitronectin, or derivatives thereof, might substitute for Matrigel in a more defined system for iPSC culture.

Integrins in the development, function and dysfunction of the nervous system.

Integrin receptors mediate cell-cell and cell-extracellular matrix (ECM) interactions in many different cell types, including neuronal cells. Earlier studies have shown a clear role for integrins in axon extension and cell adhesion/migration in CNS inflammation. Here we summarize more recent work that shows integrin functions in many phases of neural development, from neuroblast migration to synapse formation. Integrins of the beta-1 and alpha-v family are widely expressed on neurons at many stages of development, and their activity is regulated. Integrins are also important in the adult nervous system, since they have been implicated in synaptic plasticity involved in memory and learning. In addition, several diseases of the nervous system appear to involve beta-1, beta-2, and alpha-v integrins on leukocytes and glial cells. Research challenges for the future include understanding functions of specific integrin heterodimers and identifying the relevant integrin ligands that function in the nervous system.

A Novel Approach for Subretinal Implantation of Ultrathin Substrates Containing Stem Cell-Derived Retinal Pigment Epithelium Monolayer

Objective: To evaluate the feasibility of a new technique for the implantation of ultrathin substrates containing stem cell-derived retinal pigment epithelium (RPE) cells into the subretinal space of retina-degenerate Royal College of Surgeon (RCS) rats. Methods: A platform device was used for the implantation of 4-µm-thick parylene substrates containing a monolayer of human embryonic stem cell-derived RPE (hESC-RPE). Normal Copenhagen rats (n = 6) and RCS rats (n = 5) were used for the study. Spectral-domain optical coherence tomography (SD-OCT) scanning and histological examinations were performed to confirm placement location of the implant. hESC-RPE cells attached to the substrate before and after implantation were evaluated using standard cell counting techniques. Results: SD-OCT scanning and histological examination revealed that the substrates were precisely placed in the rat’s subretinal space. The hESC-RPE cell monolayer that covered the surface of the substrate was found to be intact after implantation. Cell counting data showed that less than 2% of cells were lost from the substrate due to the implantation procedure (preimplantation count 2,792 ± 74.09 cells versus postimplantation count 2,741 ± 62.08 cells). Detailed microscopic examination suggested that the cell loss occurred mostly along the edges of the implant. Conclusion: With the help of this platform device, it is possible to implant ultrathin substrates containing an RPE monolayer into the rat’s subretinal space. This technique can be a useful approach for stem cell-based tissue bioengineering techniques in retinal transplantation research.

Synthetic surfaces for human embryonic stem cell culture

Human embryonic stem cells (hESCs) have numerous potential biomedical applications owing to their unique abilities for self-renewal and pluripotency. Successful clinical application of hESCs and derivatives necessitates the culture of these cells in a fully defined environment. We have developed a novel peptide-based surface that uses a high-affinity cyclic RGD peptide for culture of hESCs under chemically defined conditions.

Differentiation of human pluripotent stem cells to retinal pigmented epithelium in defined conditions using purified extracellular matrix proteins

A potential application of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is the generation of retinal pigmented epithelium (RPE) to treat age‐related macular degeneration (AMD), a common but incurable retinal disease. RPE cells derived from hESCs (hESC‐RPEs) and iPSCs (iPSC‐RPEs) express essential RPE markers and can rescue visual function in animal models. However, standard differentiation protocols yield RPE cells at low frequency, especially from iPSC lines, and the common use of Matrigel and xenogeneic feeder cells is not compatible with clinical applications. The extracellular matrix (ECM) can affect differentiation, and therefore changes in ECM composition may improve the frequency of stem cell‐RPE differentiation. We selected several purified ECM proteins and substrates, based on the in vivo RPE ECM environment, and tested their ability to support iPSC‐RPE differentiation and maintenance. iPSCs differentiated on nearly all tested substrates developed pigmented regions, with Matrigel and mouse laminin‐111 supporting the highest pigmentation frequencies. Although iPSC‐RPEs cultured on the majority of the tested substrates expressed key RPE genes, only six substrates supported development of confluent monolayers with normal RPE morphology, including Matrigel and mouse laminin‐111. iPSCs differentiated on mouse laminin‐111 produced iPSC‐RPEs expressing RPE proteins, and hESCs differentiated on mouse laminin‐111 resulted in high yields of functional hESC‐RPEs. This identification of key ECM proteins may assist with future scaffold designs and provide peptide sequences for use in synthetic, xeno‐free, GMP‐compliant generation of RPE from human pluripotent stem cells relevant to clinical translation. Copyright

MUC1* Mediates the Growth of Human Pluripotent Stem Cells

The MUC1 protein is aberrantly expressed on an estimated 75% of all human solid tumor cancers. We recently reported that a transmembrane cleavage product, MUC1*, is the predominant form of the protein on cancer cells [1]. Further, our evidence indicated that MUC1* functions as a growth factor receptor on tumor cells, while the full-length protein appeared to have no growth promoting activity. Here, we report that MUC1* acts as a growth factor receptor on undifferentiated human embryonic stem cells (hESCs). Cleavage of the full-length ectodomain to form MUC1*, a membrane receptor, appears to make binding to its ligand, NM23, possible. Unexpectedly, we found that newly differentiated cells no longer express the cleaved form, MUC1*, or its ligand, NM23. Newly differentiated stem cells exclusively present full-length MUC1. Antibody-induced dimerization of the MUC1* receptor on hESCs stimulated cell growth to a far greater degree than currently used methods that require the addition of exogenous basic fibroblast growth factor (bFGF) as well as factors secreted by fibroblast “feeder cells”. Further, MUC1* mediated growth was shown to be independent of growth stimulated by bFGF or the milieu of factors secreted by feeder cells. Stimulating the MUC1* receptor with either the cognate antibody or its ligand NM23 enabled hESC growth in a feeder cell-free system and produced pluripotent colonies that resisted spontaneous differentiation. These findings suggest that this primal growth mechanism could be utilized to propagate large numbers of pluripotent stem cells for therapeutic interventions.

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.