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Photoreceptors and photoreceptor progenitors produced from pluripotent stem cells

a technology of photoreceptors and stem cells, which is applied in the direction of biocide, cardiovascular disorders, drug compositions, etc., can solve the problems of limited supply of donor-derived tissue from which photoreceptors and photoreceptor progenitors may be isolated, and achieve the effect of improving scotopic and photopic erg responses

Inactive Publication Date: 2016-02-04
ADVANCED CELL TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0023]In certain embodiments, the photoreceptor progenitor cells can be transplanted into the subretinal space of ELOVL4-TG2 mice, will migrate to the outer nucleated layer and will improve scotopic and photopic ERG responses in the ELOVL4-TG2 mice.
[0028]Another aspect of the present invention provides a pharmaceutical preparation of photoreceptors that is suitable for use in a mammalian patient, comprising pluripotent stem cell derived photoreceptor cells, wherein greater than 70%, 80%, 90%, 95% or even 98% of the cells are immunocytochemically PAX6+, CHX10− and are rhodopsin+ and / or opsin+; and a pharmaceutically acceptable carrier for maintaining the viability of the photoreceptor cells for transplantation into a mammalian patient.
[0040]In preferred embodiments, the photoreceptor progenitor cells, when transplanted into the subretinal space of ELOVL4-TG2 mice, migrate to the outer nucleated layer and improve scotopic and photopic ERG responses in the ELOVL4-TG2 mice relative to control (no cells injected) ELOVL4-TG2 mice.

Problems solved by technology

Retinal diseases often result in blindness due to loss of post-mitotic neuronal cells.
None of these approaches produced a homogeneous population of photoreceptor progenitor cells or photoreceptor cells for implantation.
Supplies of donor-derived tissue from which photoreceptors and photoreceptor progenitors may be isolated (such as cadavers, fetal tissue, and live animals) are limited.

Method used

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  • Photoreceptors and photoreceptor progenitors produced from pluripotent stem cells
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  • Photoreceptors and photoreceptor progenitors produced from pluripotent stem cells

Examples

Experimental program
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Effect test

example 1

Generation of Photoreceptor Progenitor Cells

[0267]Human embryonic stem cells were cultured under feeder free conditions in mTESR1 media (Stem Cell Technology) on a Matrigel™ (a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, BD Biosciences) surface. Upon 80-90% confluence, cells were passaged or frozen. Passaging of stem cells was performed using enzymatic (dispase) or non-enzymatic (EDTA-based cell dissociation buffer, Invitrogen) techniques.

[0268]Direct differentiation methods were used for generation of eye field progenitor cells, retinal neural progenitor cells, photoreceptor progenitor cells and retinal photoreceptor cells. Formation of embryoid bodies was not required.

[0269]The overall method used for photoreceptor development in these examples is schematically illustrated in FIG. 19, which further illustrates the media used at each step of the process.

[0270]Based on staining data it was determined that the cells become EFPC between day 7-day30 (indic...

example 2

Differentiation of Photoreceptor Progenitor Cells: Cell Treatment with Retinoic Acid and Taurine

[0293]Attached photoreceptor progenitors were treated with retinoic acid in the following conditions for two weeks: ND medium supplied with 100 ng / ml (or optionally 10-1000 ng / ml) retinoic acid and 100 μM (or optionally 20-500 μM) taurine. Half of the culture medium was changed every 2 days.

[0294]Differentiate cells in Photoreceptor differentiation media: The medium was changed to Photoreceptor Differentiation Medium comprising Neurobasal Medium (Invitrogen) supplied with 450 mg / ml D-glucose, 100 unit / ml of penicillin, 100 μg / ml of streptomycin, lx GlutaMAX™, 1% N2 supplement (Invitrogen), 2% B27 supplement (formula number 080085-SA), with the addition of 5 μM (or optionally 1-100 μM) Forskolin, 10 ng / ml (or optionally 1-100 ng / ml) BDNF, 10 ng / ml (or optionally 1-100 ng / ml) CNTF, 10 ng / ml (or optionally 5-50 ng / ml) LIF and 10 μM (or optionally 1-100 μM) DATP. Half of the medium was change...

example 3

Cryopreservation of Human ESC-Derived Retinal Neural Progenitors

[0296]Retinal neural progenitors of the invention, photoreceptor progenitors of the invention and retinoic acid treated photoreceptor progenitors of the invention can be frozen down in an animal-free cryopreservation buffer, such as Cryostor CS10, or another cryopreservation buffer such as 90% FBS and 10% DMSO. With respect to the photoreceptor progenitors, it was observed that freezing cells as neurospheres was beneficial, which may be due to the benefits of cell-cell contact. Preferably the neurospheres were frozen down at a size that was not too large, such as 50-250 cells.

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Abstract

Methods are provided for the production of photoreceptor cells and photoreceptor progenitor cells from pluripotent stem cells. Additionally provided are compositions of photoreceptor cells and photoreceptor cells, as well as methods for the therapeutic use thereof. Exemplary methods may produce substantially pure cultures of photoreceptor cells and / or photoreceptor cells.

Description

RELATED APPLICATIONS[0001]This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Ser. No. 61 / 793,168, entitled “PHOTORECEPTORS AND PHOTORECEPTOR PROGENITORS PRODUCED FROM PLURIPOTENT STEM CELLS” filed on Mar. 15, 2013, the entire contents of which are incorporated herein by reference.BACKGROUND[0002]Retinal diseases often result in blindness due to loss of post-mitotic neuronal cells. Among the retinal diseases are rod or cone dystrophies, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, Leber congenital amaurosis and Stargardt disease. In most retinal degenerations, cell loss is primarily in the outer nuclear layer which includes rod and cone photoreceptors. With the loss of post-mitotic neuronal cell populations, an exogenous source of new cells as a replacement for photoreceptor cells is needed.[0003]A potential replacement source of photoreceptor cells includes stem cells. Early studies incorporated the ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/545C12N5/0735C12N5/074
CPCA61K35/545C12N2506/02C12N5/0696C12N5/0606C12N5/062C12N2500/33C12N2501/105C12N2501/13C12N2501/155C12N2501/235C12N2501/33C12N2501/385A61K35/30A61P27/02A61P27/06A61P9/10Y02A50/30C12N5/0623A61K9/0051C12N2506/45
Inventor LANZA, ROBERT P.LU, SHI-JIANGWANG, WEI
Owner ADVANCED CELL TECH INC
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