Survival and Functional Integration of Human Embryonic Stem Cell-Derived Retinal Organoids After Shipping and Transplantation into Retinal Degeneration Rats.

Because derivation of retinal organoids (ROs) and transplantation are frequently split between geographically distant locations, we developed a special shipping device and protocol capable of the organoids' delivery to any location. Human embryonic stem cell (hESC)-derived ROs were differentiated from the hESC line H1 (WA01), shipped overnight to another location, and then transplanted into the subretinal space of blind immunodeficient retinal degeneration (RD) rats. Development of transplants was monitored by spectral-domain optical coherence tomography.

Retinal Cell Transplantation, Biomaterials, and In Vitro Models for Developing Next-generation Therapies of Age-related Macular Degeneration.

Retinal pigment epithelium (RPE) cells grown on a scaffold, an RPE patch, have potential to ameliorate visual impairment in a limited number of retinal degenerative conditions. This tissue-replacement therapy is suited for age-related macular degeneration (AMD), and related diseases. RPE cells must be transplanted before the disease reaches a point of no return, represented by the loss of photoreceptors.

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model.

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.

Comparison of Developmental Dynamics in Human Fetal Retina and Human Pluripotent Stem Cell-Derived Retinal Tissue.

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.

Intraocular Injection of HyStem Hydrogel Is Tolerated Well in the Rabbit Eye.

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. HyStem hydrogel formulation solidifies ∼20 min after reconstitution, thus can potentially form a solid deposit after injection . To study the ocular disposition of fluorescein-labeled HyStem, we delivered 50 μL/eye over 1 min into the vitreous space of the rabbit.

Limitations and Promise of Retinal Tissue From Human Pluripotent Stem Cells for Developing Therapies of Blindness.

The self-formation of retinal tissue from pluripotent stem cells generated a tremendous promise for developing new therapies of retinal degenerative diseases, which previously seemed unattainable. Together with use of induced pluripotent stem cells or/and CRISPR-based recombineering the retinal organoid technology provided an avenue for developing models of human retinal degenerative diseases "in a dish" for studying the pathology, delineating the mechanisms and also establishing a platform for large-scale drug screening. At the same time, retinal organoids, highly resembling developing human fetal retinal tissue, are viewed as source of multipotential retinal progenitors, young photoreceptors and just the whole retinal tissue, which may be transplanted into the subretinal space with a goal of replacing patient's degenerated retina with a new retinal "patch.

Pluripotent Stem Cell-Based Organoid Technologies for Developing Next-Generation Vision Restoration Therapies of Blindness.

Blindness, associated with death of retinal cells at the back of the eye, is caused by a number of conditions with high prevalence such as glaucoma, age-related macular degeneration, and diabetic retinopathy. In addition, a large number of orphan inherited (mostly monogenic) conditions, such as retinitis pigmentosa and Leber Congenital Amaurosis, add to the overall number of patients with blinding retinal degenerative diseases. Blindness caused by deterioration and loss of retina is so far incurable.

Development of a protocol for maintaining viability while shipping organoid-derived retinal tissue.

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.

Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in the Subretinal Space of the Cat Eye.

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.

Clonal derivation of white and brown adipocyte progenitor cell lines from human pluripotent stem cells.

Background: The role of brown fat in non-shivering thermogenesis and the discovery of brown fat depots in adult humans has made it the subject of intense research interest. A renewable source of brown adipocyte (BA) progenitors would be highly valuable for research and therapy. Directed differentiation of human pluripotent stem (hPS) cells to white or brown adipocytes is limited by lack of cell purity and scalability.

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina.

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.

Pluripotent Stem Cells for Retinal Tissue Engineering: Current Status and Future Prospects.

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.

Use of deep neural network ensembles to identify embryonic-fetal transition markers: repression of in embryonic and cancer cells.

Here we present the application of deep neural network (DNN) ensembles trained on transcriptomic data to identify the novel markers associated with the mammalian embryonic-fetal transition (EFT). Molecular markers of this process could provide important insights into regulatory mechanisms of normal development, epimorphic tissue regeneration and cancer. Subsequent analysis of the most significant genes behind the DNNs classifier on an independent dataset of adult-derived and human embryonic stem cell (hESC)-derived progenitor cell lines led to the identification of gene as a potential EFT marker.

Dnmt1, Dnmt3a and Dnmt3b cooperate in photoreceptor and outer plexiform layer development in the mammalian retina.

Characterizing the role of epigenetic regulation in the mammalian retina is critical for understanding fundamental mechanisms of retinal development and disease. DNA methylation, an epigenetic modifier of genomic DNA, plays an important role in modulating networks of tissue and cell-specific gene expression. However, the impact of DNA methylation on retinal development and homeostasis of retinal neurons remains unclear.

Adult Versus Pluripotent Stem Cell-Derived Mesenchymal Stem Cells: The Need for More Precise Nomenclature.

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.

Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures.

Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue.

Conditional knockdown of DNA methyltransferase 1 reveals a key role of retinal pigment epithelium integrity in photoreceptor outer segment morphogenesis.

Dysfunction or death of photoreceptors is the primary cause of vision loss in retinal and macular degenerative diseases. As photoreceptors have an intimate relationship with the retinal pigment epithelium (RPE) for exchange of macromolecules, removal of shed membrane discs and retinoid recycling, an improved understanding of the development of the photoreceptor-RPE complex will allow better design of gene- and cell-based therapies. To explore the epigenetic contribution to retinal development we generated conditional knockout alleles of DNA methyltransferase 1 (Dnmt1) in mice.

Long-term survival and differentiation of retinal neurons derived from human embryonic stem cell lines in un-immunosuppressed mouse retina.

Purpose: To examine the potential of NIH-maintained human embryonic stem cell (hESC) lines TE03 and UC06 to differentiate into retinal progenitor cells (hESC-RPCs) using the noggin/Dkk-1/IGF-1/FGF9 protocol. An additional goal is to examine the in vivo dynamics of maturation and retinal integration of subretinal and epiretinal (vitreous space) hESC-RPC grafts without immunosuppression.

Methods: hESCs were neuralized in vitro with noggin for 2 weeks and expanded to derive neuroepithelial cells (hESC-neural precursors, NPs).

Aged PROP1 deficient dwarf mice maintain ACTH production.

Humans with PROP1 mutations have multiple pituitary hormone deficiencies (MPHD) that typically advance from growth insufficiency diagnosed in infancy to include more severe growth hormone (GH) deficiency and progressive reduction in other anterior pituitary hormones, eventually including adrenocorticotropic hormone (ACTH) deficiency and hypocortisolism. Congenital deficiencies of GH, prolactin, and thyroid stimulating hormone have been reported in the Prop1(null) (Prop1(-/-)) and the Ames dwarf (Prop1(df/df)) mouse models, but corticotroph and pituitary adrenal axis function have not been thoroughly investigated. Here we report that the C57BL6 background sensitizes mutants to a wasting phenotype that causes approximately one third to die precipitously between weaning and adulthood, while remaining homozygotes live with no signs of illness.

Distinct nuclear localization patterns of DNA methyltransferases in developing and mature mammalian retina.

DNA methyltransferases--DNMT1, DNMT3a, and DNMT3b--produce methylation patterns that dynamically regulate chromatin remodeling and gene expression. The vertebrate retina provides an ideal model to elucidate molecular control of neurogenesis as all neuronal cell types and Müller glia are generated in a conserved order from common pools of progenitor cells. As a prelude to exploring epigenetic regulation of mammalian retinal development, we investigated the expression of Dnmt1, Dnmt3a, and Dnmt3b in the mouse retina from embryonic day (E) 10.

Nanofiber matrices promote the neuronal differentiation of human embryonic stem cell-derived neural precursors in vitro.

The potential of human embryonic stem (ES) cells as experimental therapies for neuronal replacement has recently received considerable attention. In view of the organization of the mature nervous system into distinct neural circuits, key challenges of such therapies are the directed differentiation of human ES cell-derived neural precursors (NPs) into specific neuronal types and the directional growth of axons along specified trajectories. In the present study, we cultured human NPs derived from the NIH-approved ES line BGO1 on polycaprolactone fiber matrices of different diameter (i.

Long-term, stable differentiation of human embryonic stem cell-derived neural precursors grafted into the adult mammalian neostriatum.

Stem cell grafts have been advocated as experimental treatments for neurological diseases by virtue of their ability to offer trophic support for injured neurons and, theoretically, to replace dead neurons. Human embryonic stem cells (HESCs) are a rich source of neural precursors (NPs) for grafting, but have been questioned for their tendency to form tumors. Here we studied the ability of HESC-derived NP grafts optimized for cell number and differentiation stage prior to transplantation, to survive and stably differentiate and integrate in the basal forebrain (neostriatum) of young adult nude rats over long periods of time (6 months).

Cre-mediated recombination in pituitary somatotropes.

We report a transgenic line with highly penetrant cre recombinase activity in the somatotrope cells of the anterior pituitary gland. Expression of the cre transgene is under the control of the locus control region of the human growth hormone gene cluster and the rat growth hormone promoter. Cre recombinase activity was assessed with two different lacZ reporter genes that require excision of a floxed stop sequence for expression: a chick beta-actin promoter with the CMV enhancer transgene and a ROSA26 knock-in.

Genetic variation in C57BL/6 ES cell lines and genetic instability in the Bruce4 C57BL/6 ES cell line.

Genetically modified mouse strains derived from embryonic stem (ES) cells are powerful tools for gene function analysis. ES cells from the C57BL/6 mouse strain are not widely used to generate mouse models despite the advantage of a defined genetic background. We assessed genetic variation in six such ES cell lines with 275 SSLP markers.