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Progressive vision loss, caused by retinal degenerative (RD) diseases such as age-related macular degeneration, retinitis pigmentosa, and Leber congenital amaurosis, severely impacts quality of life and affects millions of people. Finding efficient treatment for blinding diseases is among the greatest unmet clinical needs. The evagination of optic vesicles from developing pluripotent stem cell-derived neuroepithelium and self-organization, lamination, and differentiation of retinal tissue in a dish generated considerable optimism for developing innovative approaches for treating RD diseases, which previously were not feasible. Retinal organoids may be a limitless source of multipotential retinal progenitors, photoreceptors (PRs), and the whole retinal tissue, which are productive approaches for developing RD disease therapies. In this study we compared the distribution and expression level of molecular markers (genetic and epigenetic) in human fetal retina (age 8-16 weeks) and human embryonic stem cell (hESC)-derived retinal tissue (organoids) by immunohistochemistry, RNA-seq, flow cytometry, and mass-spectrometry (to measure methylated and hydroxymethylated cytosine level), with a focus on PRs to evaluate the clinical application of hESC-retinal tissue for vision restoration. Our results revealed high correlation in gene expression profiles and histological profiles between human fetal retina (age 8-13 weeks) and hESC-derived retinal tissue (10-12 weeks). The transcriptome signature of hESC-derived retinal tissue from retinal organoids maintained for 24 weeks in culture resembled the transcriptome of human fetal retina of more advanced developmental stages. The histological profiles of 24 week-old hESC-derived retinal tissue displayed mature PR immunophenotypes and presence of developing inner and outer segments. Collectively, our work highlights the similarity of hESC-derived retinal tissue at early stages of development (10 weeks), and human fetal retina (age 8-13 weeks) and it supports the development of regenerative medicine therapies aimed at using tissue from hESC-derived retinal organoids (hESC-retinal implants) for mitigating vision loss.

Purpose: To determine the long-term biocompatibility of HyStem(®) hydrogel in the rabbit eye for use as a carrier for cell or drug delivery into the ocular space. Methods: HyStem hydrogel formulation solidifies ∼20 min after reconstitution, thus can potentially form a solid deposit after injection in situ. To study the ocular disposition of fluorescein-labeled HyStem, we delivered 50 μL/eye over 1 min into the vitreous space of the rabbit. We used 3 Dutch-Belted and 3 New Zealand-pigmented rabbits, all females, delivered the gel into the right eyes, and injected 50 μL BSS Plus into the left eyes as a control. Retinal morphology was assessed by optical coherence tomography (OCT) and white light fundus photography. Fluorescence fundus photography enabled measurement of the clearance of the labeled hydrogel from the posterior chamber. Visual function was evaluated using flash and flicker electroretinography (ERG) pre- and postinjection and at weekly intervals thereafter for 6 weeks. Retinal immunohistochemistry for microglial inflammatory markers was carried out with antiglial fibrillary acidic protein (GFAP) antibody, isolectin B4 (IB4), and 4′,6-diamidino-2-phenylindole (DAPI). Results: The gel was successfully delivered into the vitreous space without the formation of a discrete retinal deposit. Fundus imaging, OCT measurements of retinal thickness, and immunohistochemical data indicated an absence of retinal inflammation, and ERG indicated no impact on retinal function. The half-time of HyStem clearance calculated from the loss of fundus fluorescence was 3.9 days. Conclusions: HyStem hydrogel appears to be biocompatible in the ocular space of a large eye and safe for long-term intraocular application.

Retinal organoid technology enables generation of an inexhaustible supply of three-dimensional retinal tissue from human pluripotent stem cells (hPSCs) for regenerative medicine applications. The high similarity of organoid-derived retinal tissue and transplantable human fetal retina provides an opportunity for evaluating and modeling retinal tissue replacement strategies in relevant animal models in the effort to develop a functional retinal patch to restore vision in patients with profound blindness caused by retinal degeneration. Because of the complexity of this very promising approach requiring specialized stem cell and grafting techniques, the tasks of retinal tissue derivation and transplantation are frequently split between geographically distant teams. Delivery of delicate and perishable neural tissue such as retina to the surgical sites requires a reliable shipping protocol and also controlled temperature conditions with damage-reporting mechanisms in place to prevent transplantation of tissue damaged in transit into expensive animal models. We have developed a robust overnight tissue shipping protocol providing reliable temperature control, live monitoring of the shipment conditions and physical location of the package, and damage reporting at the time of delivery. This allows for shipping of viable (transplantation-competent) hPSC-derived retinal tissue over large distances, thus enabling stem cell and surgical teams from different parts of the country to work together and maximize successful engraftment of organoid-derived retinal tissue. Although this protocol was developed for preclinical in vivo studies in animal models, it is potentially translatable for clinical transplantation in the future and will contribute to developing clinical protocols for restoring vision in patients with retinal degeneration.

To develop biological approaches to restore vision, we developed a method of transplanting stem cell-derived retinal tissue into the subretinal space of a large-eye animal model (cat). Human embryonic stem cells (hESC) were differentiated to retinal organoids in a dish. hESC-derived retinal tissue was introduced into the subretinal space of wild-type cats following a pars plana vitrectomy. The cats were systemically immunosuppressed with either prednisolone or prednisolone plus cyclosporine A. The eyes were examined by fundoscopy and spectral-domain optical coherence tomography imaging for adverse effects due to the presence of the subretinal grafts. Immunohistochemistry was done with antibodies to retinal and human markers to delineate graft survival, differentiation, and integration into cat retina. We successfully delivered hESC-derived retinal tissue into the subretinal space of the cat eye. We observed strong infiltration of immune cells in the graft and surrounding tissue in the cats treated with prednisolone. In contrast, we showed better survival and low immune response to the graft in cats treated with prednisolone plus cyclosporine A. Immunohistochemistry with antibodies (STEM121, CALB2, DCX, and SMI-312) revealed large number of graft-derived fibers connecting the graft and the host. We also show presence of human-specific synaptophysin puncta in the cat retina. This work demonstrates feasibility of engrafting hESC-derived retinal tissue into the subretinal space of large-eye animal models. Transplanting retinal tissue in degenerating cat retina will enable rapid development of preclinical in vivo work focused on vision restoration.

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

The retina is a very fine and layered neural tissue, which vitally depends on the preservation of cells, structure, connectivity and vasculature to maintain vision. There is an urgent need to find technical and biological solutions to major challenges associated with functional replacement of retinal cells. The major unmet challenges include generating sufficient numbers of specific cell types, achieving functional integration of transplanted cells, especially photoreceptors, and surgical delivery of retinal cells or tissue without triggering immune responses, inflammation and/or remodeling. The advances of regenerative medicine enabled generation of three-dimensional tissues (organoids), partially recreating the anatomical structure, biological complexity and physiology of several tissues, which are important targets for stem cell replacement therapies. Derivation of retinal tissue in a dish creates new opportunities for cell replacement therapies of blindness and addresses the need to preserve retinal architecture to restore vision. Retinal cell therapies aimed at preserving and improving vision have achieved many improvements in the past ten years. Retinal organoid technologies provide a number of solutions to technical and biological challenges associated with functional replacement of retinal cells to achieve long-term vision restoration. Our review summarizes the progress in cell therapies of retina, with focus on human pluripotent stem cell-derived retinal tissue, and critically evaluates the potential of retinal organoid approaches to solve a major unmet clinical need-retinal repair and vision restoration in conditions caused by retinal degeneration and traumatic ocular injuries. We also analyze obstacles in commercialization of retinal organoid technology for clinical application.

Human somatic cells are mortal due in large part to telomere shortening associated with cell division. Limited proliferative capacity may, in turn, limit response to injury and may play an important role in the etiology of age-related pathology. Pluripotent stem cells cultured in vitro appear to maintain long telomere length through relatively high levels of telomerase activity. We propose that the induced reversal of cell aging by transcriptional reprogramming, or alternatively, human embryonic stem cells engineered to escape immune surveillance, are effective platforms for the industrial-scale manufacture of young cells for the treatment of age-related pathologies. Such cell-based regenerative therapies will require newer manufacturing and delivery technologies to insure highly pure, identified and potent pluripotency-based therapeutic formulations.

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the "open", transcriptionally active chromatin in developing and postmitotic mouse retina.

Aims: The transcriptome and fate potential of three diverse human embryonic stem cell-derived clonal embryonic progenitor cell lines with markers of cephalic neural crest are compared when differentiated in the presence of combinations of TGFβ3, BMP4, SCF and HyStem-C matrices. Materials & methods: The cell lines E69 and T42 were compared with MEL2, using gene expression microarrays, immunocytochemistry and ELISA. Results: In the undifferentiated progenitor state, each line displayed unique markers of cranial neural crest including TFAP2A and CD24; however, none expressed distal HOX genes including HOXA2 or HOXB2, or the mesenchymal stem cell marker CD74. The lines also showed diverse responses when differentiated in the presence of exogenous BMP4, BMP4 and TGFβ3, SCF, and SCF and TGFβ3. The clones E69 and T42 showed a profound capacity for expression of endochondral ossification markers when differentiated in the presence of BMP4 and TGFβ3, choroid plexus markers in the presence of BMP4 alone, and leptomeningeal markers when differentiated in SCF without TGFβ3. Conclusion: The clones E69 and T42 may represent a scalable source of primitive cranial neural crest cells useful in the study of cranial embryology, and potentially cell-based therapy.

The complexity of human pluripotent stem cell (hPSC) fate represents both opportunity and challenge. In theory, all somatic cell types can be differentiated from hPSCs, opening the door to many opportunities in transplant medicine. However, such clinical applications require high standards of purity and identity, that challenge many existing protocols. This underscores the need for increasing precision in the description of cell identity during hPSC differentiation. We highlight one salient example, namely, the numerous published reports of hPSC-derived mesenchymal stem cells (MSCs). We suggest that many of these reports likely represent an improper use of certain cluster of differentiation (CD) antigens in defining bone marrow-derived MSCs. Instead, most such hPSC-derived mesenchymal cells are likely a complex mixture of embryonic anlagen, primarily of diverse mesodermal and neural crest origins, making precise identification, reproducible manufacture, and uniform differentiation difficult to achieve. We describe a potential path forward that may provide more precision in nomenclature, and cells with higher purity and identity for potential therapeutic use.

Background: Adult articular cartilage has limited potential for healing. A new cell technology in which chondrocytes expanded in monolayer culture are transplanted into the cartilage defect of the knee has been introduced. A scaffold is used to hold tissue to the defect area while it heals. Methods: Sixteen paired, fresh-frozen cadaver knees were used. Full-thickness chondral defects of 2 cm × 3 cm were made on either medial or lateral femoral condyles. The scaffold, made of PGA/PLA foam reinforced by PDS mesh, was implanted into a prepared defect area using three or five PDS/PGA staples as the fixation approach. A custom-made fixation system was used to attach each knee to a material testing machine. Each knee was tested for 10 000 cycles at 1 Hz. Upon completion, knees were examined and rated on a semiquantitative scale for damage of the chondral and meniscal surfaces, and for wear and retention of the scaffold and staples. Findings: Implanted scaffolds were retained fully in defect spaces in all cases. No tearing or peeling of the scaffold was observed. The number of staples used for fixation did not influence the wear or retention of scaffolds and staples. Wearing to the adjacent cartilage or meniscal surfaces was undetectable. Results were similar, regardless of whether the scaffold was located in the medial or lateral condyle. No staple was found loose after 10 000 cycles. Interpretation: Three staples were adequate and five staples were feasible to hold the scaffold for a defect size of 2 cm × 3 cm during continuous passive motion.

Traumatic articular cartilage injuries heal poorly and may predispose patients to the early onset of osteoarthritis. One current treatment relies on surgical delivery of autologous chondrocytes that are prepared, prior to implantation, through ex vivo cell expansion of cartilage biopsy cells. The requirement for cell expansion, however, is both complex and expensive and has proven to be a major hurdle in achieving a widespread adoption of the treatment. This study presents evidence that autologous chondrocyte implantation can be delivered without requiring ex vivo cell expansion. The proposed improvement relies on mechanical fragmentation of cartilage tissue sufficient to mobilize embedded chondrocytes via increased tissue surface area. Our outgrowth study, which was used to demonstrate chondrocyte migration and growth, indicated that fragmented cartilage tissue is a rich source for chondrocyte redistribution. The chondrocytes outgrown into 3-D scaffolds also formed cartilage-like tissue when implanted in SCID mice. Direct treatment of full-thickness chondral defects in goats using cartilage fragments on a resorbable scaffold produced hyaline-like repair tissue at 6 months. Thus, delivery of chondrocytes in the form of cartilage tissue fragments in conjunction with appropriate polymeric scaffolds provides a novel intraoperative approach for cell-based cartilage repair.

Articular chondrocytes undergo a rapid change in phenotype and gene expression, termed dedifferentiation, when isolated from cartilage tissue and cultured on tissue culture plastic. On the other hand, “redifferentiation” of articular chondrocytes in suspension culture is characterized by decreased cellular proliferation and the reinitiation of synthesis of hyaline articular cartilage extracellular matrix molecules. The molecular triggers for these events have yet to be defined. Subtracted cDNA libraries representing genes involved in the early events of adult human articular chondrocyte redifferentiation were generated from human articular chondrocytes that were first cultured in monolayer, and subsequently transferred to suspension culture at 106 cells/ml for redifferentiation. Differential regulation of genes involved in cellular organization, nuclear structure, cellular growth regulation, and extracellular matrix deposition and remodeling were observed within 48 hr of this transfer. Many of these genes had not been previously identified in the chondrocyte differentiation pathway and a number of the isolated cDNAs did not have homologies to sequences in the public data banks. Genes involved in IL-6 signal transduction including acute phase response factor (APRF), Mn superoxide dismutase, and IL-6 itself were up-regulated in suspension culture. Membrane glycoprotein gp130, a component of the IL-6 receptor, was down-regulated. Other genes involved in cell polarity, cell adherence, apoptosis, and possibly TGF-beta signaling were differentially regulated. The differential regulation of the cytokine connective tissue growth factor (CTGF) during the early stages of articular chondrocyte redifferentiation, decreasing within 48 hours of transfer to suspension culture, was particularly interesting given its reported role in the stimulation of cellular proliferation. CTGF was highly expressed in proliferative monolayer culture, and then greatly reduced by redifferentiation in standard high-density suspension culture. When articular chondrocytes were seeded in suspension at low-density (104 cells/ml), however, high levels of CTGF were observed along with increased levels of mature articular cartilage extracellular matrix protein RNAs, such as type II collagen and aggrecan. Although the role of CTGF in articular cartilage biology remains to be elucidated, the results described here demonstrate the potential utility of subtractive hybridization in understanding the process of articular chondrocyte redifferentiation. Anat Rec 263:91–98, 2001. © 2001 Wiley-Liss, Inc.

Alkaline phosphatase activity is present in the mesoderm of embryonic chick skin and becomes spatially restricted to the dermal condensation of the developing feather germs. Inhibitors to tissue-nonspecific (liver/bone/kidney), but not intestinal alkaline phosphatase inhibit the establishment and growth of feather germs in cultured skins. A window of maximum sensitivity to the inhibitor was observed to be the first day of culture when early development and establishment of pattern takes place. The cDNA for the avian tissue-nonspecific alkaline phosphatase was cloned and sequenced, and Southern analysis revealed a single copy of this gene in the avian genome. Northern analysis revealed that a 2.8 kb transcript for this form of alkaline phosphatase is present in developing skin.

The disability and pain that result from damage to articular cartilage within the knee joint has stimulated the development of several approaches to facilitate the restoration of joint function (1-9). Recently, cultured autologous chondrocytes, isolated from an individual's own cartilage, have been expanded in vitro, and then implanted into the damaged site for repair of damaged knee cartilage (10). This remarkable process has been characterized by the modulation of gene expression during proliferation expansion and subsequent redifferentiation of cultured chondrocytes in vitro (11) and in vivo (12). Since the unique biomechanical properties of hyaline articular cartilage have been shown to be intimately linked with the biochemistry of the tissue (see Buckwalter and Mow ref. 13 for review), we have developed an in vitro system to verify that proliferatively expanded chondrocytes retain their ability to redifferentiate, or re-express their hyaline articular cartilage phenotype. Although the methods described herein were developed for specific application to chondrocytes, the principles for evaluation of biochemical and molecular biological properties of tissue-engineered materials, in vitro, may be applied to the development of any functional, high quality, tissue engineered implant.

Reexpression of aggrecan and type II collagen genes in dedifferentiated adult human articular chondrocytes (AHAC) in suspension culture varied widely depending on the specific lot of bovine serum used to supplement the culture medium. Some lots of serum provided strong induction of aggrecan and type II collagen expression by AHAC while others did not stimulate significant production of these hyaline cartilage extracellular matrix molecules even following several weeks in culture. Addition of 50 ng/ml insulin-like growth factor-I (IGF-I) to a deficient serum lot significantly enhanced its ability to induce aggrecan and type II collagen mRNA. Given this observation, IGF-I and other growth factors were tested in defined serum-free media for their effects on the expression of these genes. Neither IGF-I nor insulin nor transforming growth factor beta (TGF-beta) alone stimulated induction of aggrecan or type II collagen production by dedifferentiated AHAC. However, TGF-beta 1 or TGF-beta 2 combined with IGF-I or insulin provided a strong induction as demonstrated by RNase protection and immunohistochemical assays. Interestingly, type I collagen, previously shown to be downregulated in serum supplemented suspension cultures of articular chondrocytes, persisted for up to 12 weeks in AHAC cultured in defined medium supplemented with TGF-beta and IGF-I.

Link protein (LP) is an abundant protein of cartilage which stabilizes the interaction of aggrecan with hyaluronic acid (HA). In this study we report that LP is also present in a large number of embryonic non-cartilaginous tissues. We demonstrate, using RNase protection experiments, that the coding region of the LP mRNAs isolated from these tissues is identical to that present in cartilage. Furthermore, we show that the LP mRNAs are translated in non-cartilaginous tissues by the identification of LP polypeptides with monoclonal antibody 4B6/A5 in Western blots. LP is localized in the extracellular matrix of the mesoderm along the entire digestive tract and in the dermis of the embryonic skin as revealed by immunofluorescence analysis. Investigations on the interactions between LP and proteoglycans from skin and proventriculus demonstrate that LP can enhance the binding of proteoglycans from these tissues to HA. In addition, we find that the same proteoglycans bound to HA in the presence of LP are always more resistant to competition by soluble HA than in the absence of LP. Our results suggest that LP is involved in the stabilization of extracellular matrices of a wide variety of non-cartilaginous tissues.