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. 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

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. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na+ and K+ currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for retinal replacement therapies.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140208/1/scd.2015.0144.pd

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 Prop1null (Prop1-/-) and the Ames dwarf (Prop1df/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. The wasting phenotype is associated with severe hypoglycemia. Circulating ACTH and corticosterone levels are elevated in juvenile and aged Prop1 mutants, indicating activation of the pituitary-adrenal axis. Despite this, young adult Prop1 deficient mice are capable of responding to restraint stress with further elevation of ACTH and corticosterone. Low blood glucose, an expected side effect of GH deficiency, is likely responsible for the elevated corticosterone level. These studies suggest that the mouse model differs from the human patients who display progressive hormone loss and hypocortisolism

Targeting prophet of PIT1 gene and designing mouse lines for cre -mediated gene targeting in somatotrophs.

Mouse models are invaluable for functional analysis of genes critical for pituitary development. Different alleles can be generated that vary in severity, allowing the dosage effect of genes to be ascertained, and alleles can be produced so that the genetic deficiency is induced at a particular time of development or in a specific tissue. Lesions in the prophet of PIT1 gene, PROP1, affect only the pituitary and are the most common cause of multiple pituitary hormone deficiencies in humans. To study the function of PROP1 in pituitary development, I generated a complete loss of function allele of Prop1 in mice using gene targeting. Homozygotes for the null allele exhibit respiratory distress and neonatal lethality that is secondary to pituitary hormone deficiency. These phenotypes are sensitive to genetic background. The animals that survive the perinatal period are dwarfs with hypogonadism, hypothyroidism, hypoglycemia, and alterations in the pituitary adrenal axis. An existing spontaneous allele has residual PROP1 function and is compatible with life but causes lack of GH, TSH, PRL and reduced gonadotropins. These studies help explain the variation in human patients with PROP1 deficiency and suggest that lesions in PROP1 may account for some cases of neonatal lethality with pituitary aplasia. Pitx2 is one of several genes that are critical for development of the pituitary and other organs required for viability. This makes it difficult to discover the role of these genes in specific mature pituitary cell types. To overcome this problem transgenic mouse lines were generated that express the site-specific DNA recombinase cre exclusively in the growth hormone producing cells of the pituitary gland. These lines are currently being used to evaluate the role of the homeobox gene Pitx2 in somatotrope development and function. They are an important resource for studying the role of other genes in growth hormone production.Ph.D.Animal PhysiologyBiological SciencesGeneticsMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/123246/2/3068933.pd

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. Modern biomedical research leveraging molecular and regenerative medicine approaches had a number of groundbreaking discoveries and proof-of-principle treatments of blindness in animals. However, these methods are slow to be standardized and commercialized as therapies to benefit people losing their eyesight due to retinal degenerative conditions. In this review, we will outline major regenerative medicine approaches, which are emerging as promising for preserving or/and restoring vision. We discuss the potential of each of these approaches to reach commercialization step and be converted to treatments, which could at least ameliorate blindness caused by retinal cell death

<i>Prop1</i>-defiency results in low blood glucose levels.

<p>Blood glucose levels were measured in normal and <i>Prop1</i> mutant mice at four ages. (A) Basal glucose levels in 3.5 to 5 week <i>Prop1^-/-</i> mice (n = 6) were lower than <i>Prop1^+/+</i> (n = 10) and <i>Prop1^+/−</i> (n = 10) mice from mixed genetic backgrounds, but the difference was not statistically significant at this age. (B) On mixed genetic backgrounds the blood-glucose measurements from 5 to 6.5 wk old <i>Prop1^+/+</i> (n = 6) and <i>Prop1</i>^df/df (n = 6) were normal, but <i>Prop1</i>^df/- (n = 3), <i>Prop1^-/-</i> healthy (n = 12) and <i>Prop1^-/-</i> wasting (n = 7) mice had significantly decreased blood-glucose levels. Values represent the mean blood glucose levels (mg glucose/dL blood) ± SE. *, <i>P</i><0.01; **, <i>P</i><0.005. (C) The low glucose levels in mutants shown in panel B are associated with elevated corticosterone levels (ng corticosterone/ml blood +/− SE.) (D) Blood-glucose levels were measured in 8 to 10 week old mice of the N4 B6 background prior to (white bars) and following restraint stress (black bars). <i>Prop1^-/-</i> (n = 8) mice had decreased basal and post-stress blood-glucose levels compared to <i>Prop1^+/+</i> (n = 9) and <i>Prop1^+/−</i> (n = 11). Values represent the mean blood glucose levels (mg glucose/dL blood) ± SE. *, <i>P</i><0.0001; **, <i>P</i><0.0005. (E) Blood-glucose levels in 34 to 52 wk old mice on mixed genetic background were decreased in all genotypes of <i>Prop1</i> mutants, <i>Prop1^df/df</i> (n = 4), <i>Prop1^df/-</i> (n = 11), <i>Prop1^-/-</i> (n = 7), compared to normals, <i>Prop1^+/+</i> (n = 4). Values represent the mean blood glucose levels (mg glucose/dL blood) ± SE. *, <i>P</i><0.005; **, <i>P</i><0.0005; ***, <i>P</i><0.0001.</p

Elevated basal corticosterone levels in young adult <i>Prop1</i> deficient mice become higher in response to restraint stress.

<p>RIA analysis of circulating corticosterone was carried out on serum from 8 to 10 week males (A) and females (B) of segregating the <i>Prop1</i> null allele at N4 B6 prior to (white bars) and following restraint stress (black bars). Male <i>Prop1</i>^-/- (n = 6) had significantly elevated basal and post-stress levels of corticosterone compared to <i>Prop1^+/−</i> (n = 7) and <i>Prop1^+/+</i> (n = 3). Values represent the mean corticosterone (ng/mL of blood) ± SE. *, <i>P</i><0.0001. Female <i>Prop1^-/-</i> (n = 3) mice had both elevated basal and post-stress levels of corticosterone compared to <i>Prop1^+/−</i> (n = 5) and <i>Prop1^+/+</i> (n = 6). Values represent the mean corticosterone (ng/mL of blood) ± SE. *, <i>P</i><0.005.</p

Adrenal glands of <i>Prop1</i> deficient mice are not hypotrophic.

<p>Adrenal glands were dissected from 5 and 8 week old female N4 B6 <i>Prop1^+/+</i> and <i>Prop1^-/-</i> mice, fixed, embedded, sectioned, and stained with hemotoxylin and eosin (Panels A, C, E, G) and immunostained for 20α-hydroxysteroid dehydrogenase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028355#pone.0028355-Hershkovitz1" target="_blank">[59]</a> and developed with diaminobenzidine (brown, Panels B, D, F, H) to visualize the X-zone (brackets). The ratio of adrenal weight to body weight (Panel I) was increased in <i>Prop1</i>^-/- (n = 5) compared to <i>Prop1^+/−</i> (n = 6) or <i>Prop1^+/+</i> (n = 3) N4 B6 male mice at 8 to 10 wks. Values represent the mean adrenal weight (mg) per body weight (g) ± SE. *, <i>P</i><0.0001; **, <i>P</i><0.0005.</p