PHOTORECEPTORS AND PHOTORECEPTOR PROGENITORS PRODUCED FROM PLURIPOTENT STEM CELLS
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.
1 . A substantially pure preparation of photoreceptor progenitor cells, comprising:
a plurality of photoreceptor progenitor cells, and
a medium suitable for maintaining the viability of the photoreceptor progenitor cells,
wherein greater than 90% of the cells in the preparation are immunocytochemically PAX6+ and CHX10−, and mRNA transcript positive for MASH1 as detected by qPCR.
2 . A preparation of photoreceptor progenitor cells, comprising:
a plurality of cells containing at least 50 percent photoreceptor progenitor cells, and
a medium suitable for maintaining the viability of the photoreceptor progenitor cells,
wherein the photoreceptor progenitor cells immunocytochemically PAX6(+) and CHX10(−), and mRNA transcript positive for MASH1 as detected by qPCR.
3 . A preparation of photoreceptor progenitor cells, comprising:
a plurality of photoreceptor progenitor cells substantially free of pluripotent stem cells, retinal ganglion cells, mature photoreceptors, and/or amacrine cells; and
a medium suitable for maintaining the viability of the photoreceptor progenitor cells,
wherein the photoreceptor progenitor cells are immunocytochemically PAX6(+) and CHX10(−), and mRNA transcript positive for MASH1 as detected by qPCR.
4 . A pharmaceutical preparation of photoreceptor progenitor cells that is suitable for use in a mammalian patient, comprising:
(a) a plurality of photoreceptor progenitor cells, wherein greater than 90% of the cells in the preparation are immunocytochemically PAX6+ and CHX10−, and mRNA transcript positive for MASH1 as detected by qPCR; and
(b) a pharmaceutically acceptable carrier for maintaining the viability of the photoreceptor progenitor cells for transplantation into a mammalian patient.
5 . A cryogenic cell preparation comprising at least 10 9 photoreceptor progenitor cells (PRPCs), comprising:
(a) a plurality of photoreceptor progenitor cells, wherein greater than 90% of the cells in the preparation are immunocytochemically PAX6+ and CHX10−, and mRNA transcript positive for MASH1 as detected by qPCR; and
(b) a cryopreservative system compatible with the viability of the photoreceptor progenitor cells upon thaw.
6 . The preparation of any one of claims 1 - 5 , wherein the photoreceptor progenitor cells are derived from pluripotent stem cells.
7 . The preparation of claim 6 , wherein pluripotent stem cells are selected from the group consisting of human embryonic stem cells and induced pluripotent stem cells.
8 . The preparation of any one of the preceding claims, wherein the photoreceptor progenitor cells are human cells.
9 . The preparation of any one of the preceding claims, wherein a majority of the photoreceptor progenitor cells are mRNA transcript positive for Nr2e3, Trβ2, RORβ and NRO as detected by qPCR.
10 . The preparation of any one of the preceding claims, wherein the photoreceptor progenitor cells express at least 2-fold more, relative to retinal neural progenitor cells, of one or more markers selected from uPA, Tenascin-C, CXCL16, CX3CL1 and Chitinase 3 like-1, as detected by immunoassay of secreted proteins or mRNA transcript levels by qPCR.
11 . The preparation of any one of the preceding claims, wherein the photoreceptor progenitor cells have replicative capacity to undergo at least 20 population doublings in cell culture with less than 25 percent of the cells undergoing cell death, senescing or differentiating into phenotypically non-photoreceptor cells by the 20 th doubling.
12 . The preparation of any one of the preceding claims, wherein the photoreceptor progenitor cells have transferrin protein and or transferrin mRNA levels that are at least 25 percent less than glyceraldehyde 3-phosphate dehydrogenase protein or mRNA levels respectively.
13 . The preparation of any one of the preceding claims, wherein the photoreceptor progenitor cells are HLA-genotypically identical.
14 . The preparation of any one of the preceding claims, wherein the photoreceptor progenitor cells are genomically identical.
15 . The preparation of any one of the preceding claims, wherein the photoreceptor progenitor cells have a mean terminal restriction fragment length (TRF) that is longer than 8 kb.
16 . The preparation of any one of the preceding claims, wherein the photoreceptor progenitor cells have a statistically significant decreased content and/or enzymatic activity, relative to fetal-derived photoreceptors, of proteins involved in one or more of (i) cell cycle regulation and cellular aging, (ii) cellular energy and/or lipid metabolism, and (iii) apoptosis.
17 . The preparation of any one of the preceding claims, wherein the photoreceptor progenitor cells have a statistically significant increased content and/or enzymatic activity of proteins involved in cytoskeleton structure and cellular dynamics relating thereto, relative to fetal derived photoreceptors.
18 . The preparation of claim 4 , which is suitable for administration to a human patient.
19 . The preparation of claim 4 , which is suitable for administration to a non-human veterinarian patient.
20 . The preparation of claim 6 or 7 , wherein the photoreceptor progenitor cells are differentiated from a common pluripotent stem cell source.
21 . The preparation of claim 1 , wherein the medium suitable for maintaining the viability of the photoreceptor progenitor cells is selected from the group consisting of a culture medium, a cryopreservative, and a biocompatible injection medium suitable for injection in a human patient.
22 . The preparation of any one of the preceding claims, wherein the photoreceptor progenitor cells maintain plasticity to differentiate into both rods and cones.
23 . The preparation of any one of the preceding claims, wherein 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.
24 . The pharmaceutical preparation of claim 4 , wherein the preparation is pyrogen- and mycogen-free.
25 . The preparation of any one of the preceding claims, wherein the photoreceptor progenitor cells have phagocytic activity, optionally the ability to phagocytose isolated photoreceptor outer segments, pHrodo™ Red E. coli BioParticles or both.
26 . The preparation of any one of the preceding claims, wherein the photoreceptor progenitor cells secrete one or more neuroprotective factors.
27 . A substantially pure preparation of pluripotent stem cell-derived photoreceptor cells comprising:
(a) pluripotent stem cell derived photoreceptor cells, wherein greater than 90% of the cells are immunocytochemically PAX6+, CHX10− and are rhodopsin+ and/or opsin+; and
(b) a medium suitable for maintaining the viability of the stem cell derived photoreceptor cells.
28 . A pharmaceutical preparation of photoreceptors that is suitable for use in a mammalian patient, comprising:
(a) pluripotent stem cell derived photoreceptor cells, wherein greater than 90% of the cells are immunocytochemically PAX6+, CHX10− and are rhodopsin+ and/or opsin+; and
(b) a pharmaceutically acceptable carrier for maintaining the viability of the photoreceptor cells for transplantation into a mammalian patient.
29 . A method of treating a disease or disorder caused by loss of photoreceptors in a patient, comprising administering the pharmaceutical preparation of photoreceptor progenitor cells of claim 4 or the pharmaceutical preparation of photoreceptor cells of claim 28 , or both.
30 . The method of claim 29 , wherein the preparation of cells are injected into the subretinal space of the patient.
31 . A method of producing photoreceptor progenitor cells, comprising the steps of
culturing eye field progenitor cells under culture conditions alternating between low adherence or non-adherent conditions for a period of time sufficient to form individual cell spheres, and then adherent conditions,
which alternating culture conditions are continued until a majority of the cells are photoreceptor progenitor cells, wherein the photoreceptor progenitor cells are characterized as PAX6(+) and CHX10(−), and wherein the photoreceptor progenitor cells differentiate into photoreceptor cells upon treatment with retinoic acid.
32 . A method of producing photoreceptor progenitor cells, comprising the steps of
(a) culturing eye field progenitor cells, preferably as cells clusters and preferably under low adherence or non-adherent conditions, in a neural differentiation media for a period of time sufficient for the cell clusters to form individual cell spheres, wherein the eye field progenitor cells are characterized as PAX6(+) and RX1(+) and OCT4(−) and NANOG(−), and preferably are also characterized as SIX3(+), SIX6(+), LHX2(+), TBX3(+), SOX2(+) and Nestin+, as determined by immunostaining and/or flow cytometry;
(b) culturing the cell spheres in a neural differentiation media under adherent conditions, preferably on a biomaterial scaffold such as gelatin, alginate, collagen type 1, Matrigel™, polyglycolide, collagen, fibrin, or self-assembling peptides, until a majority of cells in the culture are retinal neural progenitor cells characterized as PAX6(+), CHX10(+) and SOX2(−);
(c) thereafter, alternating culture conditions one or more times between low adherence or non-adherent conditions for a period of time sufficient for the retinal neural progenitor cells to form individual cell spheres, and then culturing the retinal neural progenitor cell containing cell spheres under adherent conditions, which alternating culture conditions are continued until a majority of the cells are photoreceptor progenitor cells, wherein the photoreceptor progenitor cells are characterized as PAX6(+) and CHX10(−), and preferably are also characterized as mRNA transcript positive for Mash1, Nr2e3, Trβ2, RORβ and NRO as detected by qPCR, and wherein the photoreceptor progenitor cells differentiate into photoreceptor cells upon treatment with retinoic acid.
33 . The method of any one of the claims 29 - 32 , wherein the photoreceptor progenitor cells are derived from pluripotent stem cells.
34 . The method of claim 33 , wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells, optionally human embryonic stem cells or human induced pluripotent stem cells.
35 . The method of claim 33 or 34 , wherein the photoreceptor progenitor cells are provided substantially free of pluripotent stem cells.
36 . The method of any one of the claims 29 - 35 , wherein the photoreceptor progenitor cells are at least 50% pure, at least 75%, at least 85%, at least 95%, at least 99% or about 100% pure with respect to other cell types.
37 . The method of any one of the claims 29 - 36 , including the further step of cryopreserving the photoreceptor progenitor cells.
38 . A method for preparing a substantially pure culture of pluripotent stem cell-derived photoreceptor progenitor cells comprising:
(a) culturing pluripotent stem cells in a feeder-free system to produce one or more eye field progenitor cells;
(b) culturing said one or more eye field progenitor cells to produce retinal neural progenitor cells that are PAX6+ and CHX10+;
(c) culturing said retinal neural progenitor cells to produce photoreceptor progenitor cells (PRPCs) that are PAX6+ and CHX10−.