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Extracellular, Stem Cells and Regenerative Ophthalmology

Wang, Yifeng MSc*,†; Xie, Ting PhD*,†

doi: 10.1097/IJG.0000000000000112
The Extracellular Matrix

Retinal degenerative diseases, including retinitis pigmentosa, age-related macular degeneration, and glaucoma, still lack effective medical treatments. The stem cell–based regenerative approach has been proposed to treat these degenerative diseases. The major challenge for regenerative ophthalmology is to produce enough desirable retinal neurons in vitro from various stem cell types. Extracellular matrix proteins are important for stem cell self-renewal and differentiation in various systems. They have also been used in combination with various growth factors to expand retinal stem cells and produce desirable retinal neuronal types. This review summarizes our current understanding of how extracellular matrix proteins regulate stem cell function and discusses their application in regenerative ophthalmology.

*Stowers Institute for Medical Research, Kansas City, MO

Department of Cell Biology and Anatomy, University of Kansas School of Medicine, Kansas City, KS

Disclosure: The authors declare no conflict of interest.

Reprints: Ting Xie, PhD, Stowers Institute for Medical Research, 1000 East 50th street, Kansas City, MO 64110 (e-mail:

Received August 1, 2014

Accepted August 2, 2014

Extracellular matrix (ECM) proteins are secreted into the extracellular space and form a 3-dimensional architecture to support cells and tissues structurally and functionally. They are composed of various structural proteins, including laminins, collagens, fibronectins, and proteoglycans.1 In addition to providing structural support, these protein can also bind to integrin receptor complexes and regulate cell fate determination, differentiation, proliferation, polarity, survival, and migration.2 Besides major structural proteins, ECM also contains proteases and protease inhibitors, which help remodel ECM and its cellular functions. In addition, ECM proteins are known to modulate the stability, diffusion, or receptor binding of growth factors including, epidermal growth factor (EGF), basic fibroblast growth factor, transforming growth factor-β, bone morphogenetic proteins, and Wnts.2 Finally, they can also modulate cadherin-mediated cell-cell interactions by acting together with extracellular domains of cadherin molecules. Therefore, ECM proteins have broad biological functions during normal development, and dysfunctional ECM proteins lead to cancer formation, tissue dysfunction, and even degeneration.

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Stem cells in adult tissues can continuously self-renew and generate differentiated cells that replenish lost cells and thus maintain tissue homeostasis. In addition to intrinsic factors, microenvironments or niches play an instructive role in controlling self-renewal and differentiation in various organisms and tissue types.3,4 ECM proteins are present in many different stem cell niches, and provide structural support for stem cells and their niche.5 More importantly, they can regulate stem cell behaviors via integrin-mediated cell adhesion and signaling and also by modulating cadherin-mediated cell adhesion and growth factor signaling (Fig. 1A).



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ECM-Integrin-mediated Stem Cell Anchorage

Transmembrane integrin proteins, which are composed of α and β subunits, mediate cell-ECM interactions by binding directly to ECM proteins.6 In addition, they can also bind to other cell-surface adhesion molecules, such as intercellular adhesion molecule 1 (Icam1, also known as CD54) and vascular cell adhesion molecule 1 (Vcam1, also known as CD106). Many different stem cell types express various integrin proteins and also directly contact ECM proteins or the ECM-rich basal membrane.5 For instance, spermatognonial stem cells in the mouse testis highly express α6β1 integrin complexes, which are known to bind laminin proteins, and directly contact the basal membrane.7 Importantly, β1 integrin is crucial for the homing of spermatognonial stem cells to their testicular niche.8 In the immune system, β1 integrin-deficient adult hematopoietic stem cells (HSCs) exhibit a severe defect in homing to the bone marrow niche, indicating its important role in HSC interaction with the niche.9 Similarly, stem cells in the brain, the skin, and the muscle also require integrin proteins for their interactions with the niche.10,11

In addition to ECM/integrin-mediated cell adhesion, ECM proteins and integrin have 2 additional roles in the regulation of stem cell-niche interaction.12 Cadherin-mediated cell adhesion has also been shown to be required for anchoring some stem cell types in the niche.5 ECM proteins can modulate cadherin-mediated cell adhesion by binding to cadherin extracellular domains to regulate their interactions.12 In addition, integrin signaling modulates cadherin-mediated cell adhesion by activating focal adhesion kinases, which are known to be important to modulate cadherin-mediated cell adhesion.12 Therefore, cadherin-mediated cell adhesion and ECM/integrin-mediated cell adhesion are coupled together to regulate stem cell-niche interactions.

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ECM-mediated Signaling in the Regulation of Stem Cell Self-Renewal, Proliferation, and Survival

Integrin proteins can directly signal via phosphoinositide 3-kinase (PI3K) activation.6 PI3K signaling is known to be important for stem cell self-renewal, proliferation, and survival.13–16 Indeed, integrin proteins can modulate HSC migration and proliferation.17 The laminin/integrin-mediated interaction contributes to the development and maintenance of the neural stem cell (NSC) niche.11 Astrocyte-secreted Netrin 4 and stem cell–secreted laminin can activate α6β1-mediated signaling to regulate NSC proliferation.18 In addition, integrin signaling also promotes NSC self-renewal by facilitating Notch and epidermal growth factor receptor signaling.19 Furthermore, integrin signaling has recently been shown to regulate thrombopoietin-mediated HSC maintenance.20 In addition, ECM-mediated cell adhesion is critical for stem cell polarity and asymmetric cell division, and might be also involved in stem cell aging.5 Therefore, ECM-activated integrin signaling controls self-renewal, proliferation, and survival directly via PI3K signaling activation and also by modulating the activities of other signaling pathways.

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Retinal degenerative diseases, including glaucoma and age-related macular degeneration, cause a complete or partial loss of vision, and affect millions of people worldwide. Currently, there is no cure for these diseases. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been directed to differentiate into photoreceptors and retinal epithelial cell (RPE) cells in vitro and regain functions in retinal disease models.21 More amazingly, both ESCs and iPSCs can develop into the optic vesicle capable of forming a retina-like structure in vitro.22–24 hESC-derived photoreceptors can regain light response in the crx mutant mouse eye lacking photoreceptors.25 Excitingly, hESC-derived RPE cells have shown great promise in the treatment of macular degeneration in the first phase of a clinical trial.26 Adult retina-derived stem cell–like cells can also be used to generate functional photoreceptors, which help restore light response of rd1 mutant eyes.27 These results demonstrate that transplantation of stem cell–derived retinal cells into retinal degenerative eyes may become a practical strategy to treat blinding diseases in the future.

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Application of ECM Proteins in Generation of Functional Retinal Cells for Transplantation

On the basis of critical roles of ECM proteins in the regulation of stem cell self-renewal and differentiation in other tissues, they are also expected to play important roles in the modulation of retinal stem/progenitor cells and the generation of differentiated retinal cell types for transplantation in vitro (Fig. 1B). Indeed, laminin and fibronectin proteins are often used to promote the differentiation of ESCs and iPSCs into photoreceptors.28,29 In addition, generation of RPEs from ESCs and iPSCs also require fibronectin or laminin ECM proteins.26,30,31 Cultured Müller cells exhibit many stem cell–like properties in vitro.32,33 ECM proteins, fibronectin, or laminin, are used to expand Müller cells and maintain their stem cell–like properties in vitro in combination with EGF and FGF growth factors.34–36 ECM proteins are also used to differentiate Müller stem cells into various retinal cell types along with other differentiation-promoting growth factors.34–36 For adult retina-derived stem cells, ECM proteins, laminin, and fibronectin, are also used to generate various retinal cell types.27 Therefore, it will be important to optimize different combinations of ECM proteins in the production of various retinal cell types from stem cells in the future.

In the adult mammalian retina, inhibitory ECM and cell adhesion molecules, such as CD44 and neurocan, prevent integration of transplanted photoreceptors into the retina.37 In the MRL/MpJ (healer) mouse, an established tissue regeneration model, transplanted photoreceptors can be efficiently integrated into the retina due to elevated matrix metalloproteinase proteins (MMPs). Cotransplantation of biodegradable MMP2 microspheres with retinal progenitors offers robust integration of differentiated retinal cells into the retina.38 Consistent with the notion that ECM proteins have an inhibitory role in the integration of transplanted photoreceptor cells, the degradation of chondroitin sulfate proteoglycans by chondroitinase ABC (ChABC) can also increase the integration efficiency of transplanted photoreceptors.39,40 These studies have confirmed the inhibitory role of ECM proteins in the integration of transplanted retinal cells (Fig. 1B).

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Application of ECM Proteins in Engineering Eye Tissues

Recently, ESCs have been shown to produce optic cup-like or retina-like structures in suspension culture, which consist of RPE and neural retinal layers.22,24 In addition, patient-specific iPSCs can also form optic cup-like structures on the plate coated with ECM proteins, collagen, laminin, and fibronectin.41 These optic cup-like structures can further develop into retinal structures, which are composed of different layers, including the photoreceptor layer. These in vitro ESC-derived or iPSC-derived optic cup structures offer great opportunities for studying human retinal development and retinal disease mechanisms.22,41 This approach also represents an effective strategy for producing photoreceptor precursor cells.42 In a diseased retina, where the photoreceptor layer is completely absent, transplanted stem cell–derived photoreceptors often fail to adopt normal photoreceptor-like morphology and form the normal outer nuclear layer.25,27 For effective treatments of photoreceptor degenerative diseases, it is important to engineer the entire photoreceptor layer, which exhibits normal polarity and uniform morphology. ECM proteins could play a critical role in orienting photoreceptor cells in the same direction and keeping them intact in the same layer. Therefore, ECM proteins should play critical roles in engineering retinal tissues or other eye-specific structures for transplantation (Fig. 1B).

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As ECM proteins have important roles in the regulation of stem cell self-renewal and differentiation, they have been used to expand Müller stem cells and retinal stem cells in combination with EGF and FGF growth factors. In addition, ECM proteins have also been applied to facilitate the differentiation of ESCs and iPSCs into photoreceptors cells in combination with various differentiation-promoting growth factors. In regenerative ophthalmology, ECM proteins have a bright future in efficient production of a specific retinal cell type and in engineering the entire photoreceptor layer for transplantation (Fig. 1B). Moreover, ECMs can also provide structural support and instructive microenvironments for using stem cells to engineer artificial lens and cornea for transplantation. Therefore, more research is urgently needed to investigate how various ECM proteins affect the production of various eye cell types for transplantation and for engineering the photoreceptor layer, artificial corneas, and lenses for treating eye diseases.

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exfoliation syndrome; extracellular matrix; stem cells

© 2014 by Lippincott Williams & Wilkins.