Methods and materials for culturing, proliferating, and differentiating stem cells

Abstract

To provide compositions containing RPE cells or RPE monolayers, as well as methods and materials for making RPE cells or RPE monolayers from, for example, stem cells (e.g., iPSCs).SOLUTION: Disclosed is a method for making a retinal pigment epithelium monolayer, the method comprising, or consisting essentially of, culturing stem cells in a container having a surface coated with fibrinogen, wherein the surface is coated with greater than 3 μg / mL of fibrinogen, wherein the cells are in contact with the fibrinogen, and wherein the cells form the retinal pigment epithelium monolayer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 62 / 634,580, filed February 23, 2018, and U.S. Patent Application No. 62 / 515,286, filed June 5, 2017. The disclosures of these earlier applications are considered part of the disclosure of this application and are incorporated herein in their entireties. [Background technology]

[0002] 1. Technical Field This document relates to induced pluripotent stem cells (iPSCs) and retinal pigment epithelium. For example, this document relates to methods and materials for culturing, expanding, and differentiating stem cells (e.g., iPSCs). This document also relates to methods and materials for generating retinal pigment epithelium from stem cells (e.g., iPSCs).

[0003] 2. Background information One example of a disease target for iPSC-based regenerative medicine is macular degeneration, a disorder of the retinal pigment epithelium (RPE). Hereditary macular degeneration, including bestrophinopathy, is caused by mutations in proteins involved in RPE function. Bestrophinopathy, most commonly Best disease, is caused by mutations in the Best1 gene, causing RPE dysfunction in its role supporting photoreceptors and ultimately resulting in their death. The prevalence has previously been reported as 1 in 16,000–21,500 (Dalvin et al., Ophthalmic Genet., Epub:1–5 (2016)). While genetically induced macular degeneration is rare, age-related macular degeneration (AMD) is the leading cause of blindness in developed countries, with an estimated 5 million cases by 2050. AMD is a complex disease of immune and vascular function that directly impacts RPE function.

[0004] RPE replacement has recently become a popular focus for the treatment of macular degeneration. Modern advances in stem cell technology have made embryonic stem cells (ESCs) and iPSCs attractive transplant candidates. Several reports have demonstrated that both stem cell sources can be differentiated into the RPE lineage using various differentiation media (Sonoda et al., Nat. Protoc., 4:662-673 (2009); Johnson et al., Ophthalmology Vis. Sci., 56:4619 (2015); Brandl et al., NeuroMolecular Med., 16:551-564 (2014); Idelson et al., Cell Stem Cell., 5:396-408 (2009); Carr et al., Mol. Vis., 15:283-295 (2009)). Both ESC-RPE and iPSC-RPE have been shown to exhibit normal RPE functions, including cellular markers, phagocytosis, and pigmentation (Singh et al., Ophthalmol. Vis. Sci., 54:6767-6778 (2013)). Summary of the Invention

[0005] This document relates to iPSCs and RPE. For example, this document provides compositions containing RPE, as well as methods and materials for culturing, expanding, and differentiating stem cells (e.g., iPSCs). For example, this document provides compositions containing RPE, as well as methods and materials for generating RPE from stem cells (e.g., iPSCs). As described herein, human fibrinogen can be used as a xeno-free (non-xenogeneic), cGMP (current good manufacturing practice) compliant cell adhesion substrate for the culture, differentiation, and production of iPSC-RPE for human use. In some cases, fibrin (e.g., fibrin hydrogel) can be used in place of fibrinogen to practice the methods and materials provided herein.

[0006] Fibrinogen is the precursor polypeptide of fibrin, a protein involved in blood clot formation. Fibrinogen is a soluble 340 kDa polypeptide found in human blood at approximately 200–400 mg / dL. Upon activation of the coagulation cascade, the active enzyme thrombin cleaves fibrinogen into two fibrinopeptides, yielding fibrin monomers. Fibrin monomers have a very high affinity for each other and polymerize to form an insoluble 3D mesh hydrogel. While not an extracellular matrix protein, fibrinogen has been shown to promote the adhesion of primary platelets and endothelial cells in culture, as described elsewhere (Spectre et al., Thromb Haemost., 108: 328–37 (2012); Underwood et al., J. Biomater Sci Polym Ed., 13: 845–62 (2002)). Similarly, fibrinogen as a 3D mesh hydrogel has been used for tissue engineering applications. Fibrin gels exhibit very attractive properties, including three-dimensional (3D) mesh formation, non-xenogeneic origin, biocompatibility, and biodegradability. For example, fibrin has been used in angiogenesis and vascularization assays with cells such as human umbilical vein endothelial cells (HUVECs) (Mishra et al., Biomaterials, 77:255-66 (2016)).

[0007] In general, one aspect of this document features a method for producing a retinal pigment epithelial monolayer. The method includes, or consists essentially of, culturing stem cells in a vessel having a surface coated with fibrinogen, wherein the surface is coated with greater than 3 μg / mL of fibrinogen, the cells are in contact with the fibrinogen, and the cells form a retinal pigment epithelial monolayer. The stem cells can be induced pluripotent stem cells (e.g., human induced pluripotent stem cells). The vessel can be a culture dish (e.g., a culture flask). The surface can include polystyrene, polycarbonate, mixed cellulose, PTFE, PDMS, PET, glass, a poly-L-lysine coating, or a combination thereof. The fibrinogen can be human fibrinogen. The surface can be coated with about 3 to about 250 μg / mL of fibrinogen. The surface can be coated with about 15 to about 250 μg / mL of fibrinogen. The surface may be coated with about 25 to about 250 μg / mL of fibrinogen. The surface may be coated with about 50 to about 250 μg / mL of fibrinogen. The surface may be coated with about 75 to about 250 μg / mL of fibrinogen. The surface may be coated with the fibrinogen for about 1 to about 48 hours. The method may include culturing the cells for about 7 to about 90 days to form the retinal pigment epithelial monolayer. The method may be xeno-free.

[0008] In another aspect, this document features a retinal explant comprising a retinal pigment epithelial monolayer, wherein the retinal pigment epithelial monolayer is produced according to the methods described herein.

[0009] In another aspect, this document features a method for treating an ocular condition. The method includes, or consists essentially of, implanting a retinal implant comprising a retinal pigment epithelial monolayer into the eye of a mammal, wherein the retinal pigment epithelial monolayer is prepared according to the methods described herein. The ocular condition can be macular degeneration. The mammal can be a human.

[0010] In another aspect, this document features a method for maintaining stem cells in culture. The method includes, or consists essentially of, culturing stem cells in a vessel having a surface coated with fibrinogen, wherein the surface is coated with greater than 250 μg / mL of fibrinogen, and the stem cells contact the fibrinogen, and the stem cells maintain the ability to differentiate into cells of ectodermal, endodermal, and mesodermal origin after at least one passage. The stem cells can be induced pluripotent stem cells. The stem cells can be human induced pluripotent stem cells. The vessel can be a culture dish. The vessel can be a culture flask. The surface can include polystyrene, polycarbonate, mixed cellulose, PTFE, PDMS, PET, glass, a poly-L-lysine coating, or a combination thereof. The fibrinogen can be human fibrinogen. The surface can be a surface coated with about 250 to about 5000 μg / mL of fibrinogen. The surface may be coated with about 300 to about 900 μg / mL of fibrinogen. The surface may be coated with about 350 to about 750 μg / mL of fibrinogen. The surface may be coated with about 400 to about 600 μg / mL of fibrinogen. The surface may be coated with about 450 to about 550 μg / mL of fibrinogen. The surface may be coated with the fibrinogen for about 1 to about 48 hours. The method may include culturing the cells for about 2 to about 90 days. The method may be xeno-free. The stem cells may form endothelial cells. The stem cells may form epithelial cells (e.g., RPE cells). The fibrinogen may be obtained autologously.

[0011] In another aspect, this document features a method for maintaining stem cells in culture. The method includes, or consists essentially of, culturing stem cells in a vessel having a surface coated with fibrinogen, wherein the surface is coated with greater than 3 μg / mL of fibrinogen, and the stem cells contact the fibrinogen, and the stem cells maintain the ability to differentiate into cells of ectodermal, endodermal, and mesodermal origin after at least one passage. The stem cells can be induced pluripotent stem cells. The stem cells can be human induced pluripotent stem cells. The vessel can be a culture dish. The vessel can be a culture flask. The surface can include polystyrene, polycarbonate, mixed cellulose, PTFE, PDMS, PET, glass, a poly-L-lysine coating, or a combination thereof. The fibrinogen can be human fibrinogen. The surface can be a surface coated with about 100 to about 5000 μg / mL of fibrinogen. The surface may be coated with about 300 to about 900 μg / mL of fibrinogen. The surface may be coated with about 100 to about 250 μg / mL of fibrinogen. The surface may be coated with about 350 to about 750 μg / mL of fibrinogen. The surface may be coated with about 400 to about 600 μg / mL of fibrinogen. The surface may be coated with about 450 to about 550 μg / mL of fibrinogen. The surface may be coated with the fibrinogen for about 1 to about 48 hours. The method may include culturing the cells for about 2 to about 90 days. The method may be xeno-free. The stem cells may form endothelial cells. The stem cells may form epithelial cells (e.g., RPE cells). The fibrinogen may be obtained autologously.

[0012] In another aspect, this document features a method for maintaining stem cells in culture. The method includes, or consists essentially of, culturing stem cells in a vessel having a surface coated with a fibrin hydrogel, wherein the fibrin hydrogel is formed with greater than 0.5 mg / mL of fibrinogen, and the stem cells contact the fibrin hydrogel, and the stem cells maintain the ability to differentiate into cells of ectodermal, endodermal, and mesodermal origin after at least one passage. The stem cells can be induced pluripotent stem cells. The stem cells can be human induced pluripotent stem cells. The vessel can be a culture dish. The vessel can be a culture flask. The surface can include polystyrene, polycarbonate, mixed cellulose, PTFE, PDMS, PET, glass, a poly-L-lysine coating, or a combination thereof. The fibrinogen can be human fibrinogen. The fibrin hydrogel can be formed with a fibrinogen concentration of about 0.5 mg / mL to about 80 mg / mL. The fibrin hydrogel may be formed with a fibrinogen concentration of about 10 mg / mL to about 50 mg / mL. The fibrin hydrogel may be formed with a fibrinogen concentration of about 25 mg / mL to about 35 mg / mL. The fibrin hydrogel may be polymerized using 0.5 to 500 U / mL of thrombin. The fibrin hydrogel may contain an antifibrinolytic agent. The antifibrinolytic agent may be tranexamic acid at a concentration of about 0.5 mg / mL to about 50 mg / mL. The antifibrinolytic agent may be aprotinin at a concentration of about 0.1 U / mL to about 40 U / mL.

[0013] In another aspect, this document features a method for producing endothelial cells. The method includes, or consists essentially of, culturing stem cells in a vessel having a surface coated with fibrinogen, wherein the surface is coated with greater than 3 μg / mL of fibrinogen, and the stem cells contact the fibrinogen and form endothelial cells. The stem cells can be induced pluripotent stem cells. The stem cells can be human induced pluripotent stem cells. The vessel can be a culture dish. The vessel can be a culture flask. The surface can include polystyrene, polycarbonate, mixed cellulose, PTFE, PDMS, PET, glass, a poly-L-lysine coating, or a combination thereof. The fibrinogen can be human fibrinogen. The surface can be coated with about 3 to about 250 μg / mL of fibrinogen. The surface can be coated with about 15 to about 250 μg / mL of fibrinogen. The surface may be coated with about 25 to about 250 μg / mL of fibrinogen. The surface may be coated with about 50 to about 250 μg / mL of fibrinogen. The surface may be coated with about 75 to about 250 μg / mL of fibrinogen. The surface may be coated with the fibrinogen for about 1 to about 48 hours. The method may include culturing the cells for about 7 to about 90 days to form endothelial cells. The method may be xeno-free.

[0014] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are intended to be illustrative only and not limiting.

[0015] Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1 contains photographs of iPSC-RPE cells after 7 days of culture in differentiation medium. Initially, 1 x 10 cells were plated onto a 96-well plate coated with 0.1 μg / mL fibrinogen. The RPE cells formed organoids and did not attach; no monolayer was observed. [Figure 2] Figure 2 contains photographs of iPSC-RPE cells after 7 days of culture in differentiation medium. Initially, 1 x 10 cells were plated onto a 96-well plate coated with 0.5 μg / mL fibrinogen. The RPE cells formed organoids and did not attach; no monolayer was observed. [Figure 3] Figure 3 contains photographs of iPSC-RPE cells after 7 days of culture in differentiation medium. Initially, 1 x 10 cells were plated onto a 96-well plate coated with 1 μg / mL fibrinogen. The RPE cells formed a patchy monolayer that was approximately 70% confluent. [Figure 4] Figure 4 contains photographs of iPSC-RPE cells after 7 days of culture in differentiation medium. Initially, 1 x 10 cells were plated onto a 96-well plate coated with 5 μg / mL fibrinogen. The RPE cells formed a largely confluent monolayer with large patches (arrows). [Figure 5] Figure 5 contains photographs of iPSC-RPE cells after 7 days of culture in differentiation medium. Initially, 1 x 10 cells were plated onto a 96-well plate coated with 10 μg / mL fibrinogen. The RPE cells formed a largely confluent monolayer with large patches (arrows). [Figure 6]Figure 6 contains photographs of iPSC-RPE cells after 7 days of culture in differentiation medium. Initially, 1 x 10 cells were plated onto a 96-well plate coated with 15 μg / mL fibrinogen. The RPE cells formed a confluent monolayer with occasional patches (arrows). [Figure 7] Figure 7 contains photographs of iPSC-RPE cells after 7 days of culture in differentiation medium. Initially, 1 x 10 cells were plated onto a 96-well plate coated with 25 μg / mL fibrinogen. The RPE cells formed a confluent monolayer. [Figure 8] Figure 8 contains photographs of iPSC-RPE cells after 7 days of culture in differentiation medium. Initially, 1 x 10 cells were plated onto a 96-well plate coated with 50 μg / mL fibrinogen. The RPE cells formed a confluent monolayer. [Figure 9] Figure 9 includes photographs of iPSC-RPE cells after 7 days of culture in differentiation medium. Initially, 1 x 10 cells were plated onto a 96-well plate coated with 75 μg / mL fibrinogen. The RPE cells formed a confluent monolayer. [Figure 10] Figure 10 includes photographs of iPSC-RPE cells after 7 days of culture in differentiation medium. Initially, 1 x 10 cells were plated onto a 96-well plate coated with 100 μg / mL fibrinogen. The RPE cells formed a confluent monolayer. [Figure 11] Figure 11 shows photographs of iPSC-RPE cells after 7 days of culture in differentiation medium. Initially, 1 x 10 cells were plated onto a 96-well plate coated with 200 μg / mL Matrigel as a positive control. RPE cells formed a confluent monolayer. [Figure 12]Figure 12 is a graph plotting the percent confluency of plated iPSC-RPE cells after 7 days of culture against the concentration (μg / mL) of fibrinogen used to coat the plates. A confluent monolayer was achieved with 25-100 μg / mL of fibrinogen, a confluent monolayer with few patches was achieved with 5-15 μg / mL of fibrinogen, a poor monolayer was achieved with 1 μg / mL of fibrinogen, and no significant adhesion was achieved with 0.1-0.5 μg / mL of fibrinogen. [Figure 13] Figure 13 includes photographs of iPSC-RPE cells after one week of culture in differentiation medium. 5 x 10 cells were initially plated onto a 6-well plate coated with 100 μg / mL fibrinogen (right panel) or 200 μg / mL Matrigel (left panel) as a positive control. The RPE cells formed a confluent monolayer. 4x objective. [Figure 14] Figure 14 includes photographs of iPSC-RPE cells after one week of culture in differentiation medium. 5 x 10 cells were initially plated onto a 6-well plate coated with 100 μg / mL fibrinogen (right panel) or 200 μg / mL Matrigel (left panel) as a positive control. The RPE cells formed a confluent monolayer. The RPE cells form a cobblestone appearance with hexagonal patterning. 20x objective. [Figure 15] Figure 15 contains photographs of iPSC-RPE cells after 6 weeks of culture in differentiation medium. 5 x 10 cells were initially plated onto a 6-well plate coated with 100 μg / mL fibrinogen (right panel) or 200 μg / mL Matrigel (left panel) as a positive control. The RPE cells formed a confluent monolayer. Large areas of pigmented cells are visible in both groups. 4x objective. [Figure 16]Figure 16 includes photographs of iPSC-RPE cells after 6 weeks of culture in differentiation medium. Initially, 5 x 10 cells were plated onto a 6-well plate coated with 100 μg / mL fibrinogen (right panel) or 200 μg / mL Matrigel (left panel) as a positive control. The RPE cells formed a confluent monolayer. The characteristic pigmented, hexagonally patterned RPE cells are visible in both groups. 20x objective. [Figure 17] Figure 17 includes photographs of iPSC-RPE cells after 12 weeks of culture in differentiation medium. 5 x 106 cells were initially plated onto a 6-well plate coated with 100 μg / mL fibrinogen (right panel) or 200 μg / mL Matrigel (left panel) as a positive control. The RPE cells formed a confluent monolayer. In both groups, the RPE appeared pigmented and hexagonal in shape. 20x objective. [Figure 18] Figure 18 shows a Western blot of iPSC-RPE cells after 12 weeks of culture in differentiation medium. 5 × 10 cells were initially plated onto plates coated with 100 μg / mL fibrinogen (fibrinogen-coated) or 200 μg / mL Matrigel (Matrigel-coated) as a positive control. MERTK represents the proto-oncogene tyrosine-protein kinase MER; RPE65 represents retinoid isomerohydrolase; Best1 represents Bestrophin 1; CRALBP represents retinaldehyde-binding protein 1; and B-actin represents the β-actin polypeptide. [Figure 19] Figure 19 contains bar graphs plotting the concentration of VEGF and PEDF secreted into the culture medium over a 24-hour period. 5 x 10 cells were initially plated onto plates coated with 100 μg / mL fibrinogen (Fg) or 200 μg / mL Matrigel (MG) as a positive control and cultured for 12 weeks. Concentrations were determined via ELISA. n=1. [Figure 20]Day 60 culture of iPSC-RPE grown on human fibrinogen. A) A T25 flask of hiPSC-RPE cells grown on a human fibrinogen-coated plate. B) Micrograph of hiPSC-RPE in the flask shown in A. The cells have a cobblestone appearance and pigmentation. The out-of-focus areas are domes resulting from fluid transport through the monolayer. C) Western blot using Proteinsimple WES for RPE markers. Arrows indicate the location of bands for the indicated markers. [Figure 21] Retention of pluripotency in iPSC lines grown on plates coated with human fibrinogen. A) An example of pluripotency marker expression assessed by flow cytometry for cells grown on human fibrinogen. Each panel represents staining with two markers. B) Comparative gating % of iPSCs assessed by flow cytometry (Geltrex vs. three human fibrinogen replicates). C) Expression of pluripotency markers by immunofluorescence. D) Demonstration that iPSCs grown on human fibrinogen retain the ability to differentiate into all three lineages using the STEMDIFF Trilineage differentiation kit (Stemcell Technologies). [Figure 22] Effect of Tris-HCl concentration on the clottability of human fibrinogen. A) 2 mg / mL fibrinogen was diluted in Tris-HCl, pH 8.0, to the indicated concentrations, and clotting was stimulated by the addition of thrombin. Transmittance was determined using a plate reader. B) Clotted fibrin has a lower transmittance than the fibrinogen solution. No clotting occurred at Tris-HCl concentrations ≥ 250 mM. [Figure 23]Figure 23 contains graphs plotting the percent internalized rod outer segments (ROS) and total ROS binding using a phagocytosis assay in iPSC-RPE. 1 x 10 cells were plated onto each well of a 96-well plate coated with 100 μg / mL fibrinogen (three different preparations: Evicel (evi), cryol (Aneg), and cryo2 (Bpos)) or 200 μg / mL Matrigel (MG) as a positive control, and cultured for 8 weeks. N=3. [Figure 24] Figure 24 contains graphs plotting relative RPE marker expression using Western blot analysis. 1 x 10 cells were plated onto each well of a 96-well plate coated with 100 μg / mL fibrinogen (three different preparations: Evicel (evi), cryol (Aneg), and cryo2 (Bpos)) or 200 μg / mL Matrigel (MG) as a positive control, and cultured for 8 weeks. N=3. [Figure 25] Figure 25 contains a photograph of a Western blot of iPSC-RPE cells after two weeks of culture in differentiation medium. 1 x 10 cells were plated onto each well of a 96-well plate coated with 5, 25, or 100 μg / mL fibrinogen (Fg5, Fg25, and Fg100, respectively). [Figure 26] Figure 26 contains a graph of Western blot quantification of RPE markers in iPSC-RPE after culture in differentiation medium from week 2 to week 14. 1 x 10 cells were plated onto each well of a 96-well plate coated with 5, 25, or 100 μg / mL fibrinogen (Fg5, Fg25, and Fg100, respectively). [Figure 27] Figure 27 contains graphs plotting the concentration of PEDF and VEGF secreted into the culture medium over 48 hours. From week 2 to week 14, 1 x 10 cells were plated onto each well of a 96-well plate coated with 5, 25, or 100 μg / mL fibrinogen (Fg5, Fg25, and Fg100, respectively). Concentrations were determined by ELISA. N=1. [Figure 28]Figure 28 contains a graph of Western blot quantification of RPE markers in iPSC-RPE after 8 weeks of culture in differentiation medium. 1 x 10 cells were plated onto each well of a 96-well plate coated with 100 μg / mL fibrinogen (four different fibrinogen sources: Evicel, stemCOAT, Tisseel, and Millipore). n=1. [Figure 29] Figure 29 contains photographs of iPSC-RPE after 12 weeks of culture in differentiation medium. 1 x 10 cells (WiCell clone 4) were plated onto each well of a 96-well plate coated with various concentrations of fibrinogen (six fibrinogen sources: Tisseel, stemCOAT, Ethanol Precipitate 1, Sigma, Millipore, and Ethanol Precipitate 2). The concentrations listed represent the lowest concentration with the best monolayer appearance. [Figure 30] Figure 30 includes photographs of iPSC cells cultured on fibrin in mTESR medium without aprotinin supplementation after two days of culture. iPSC colonies were plated at a moderate concentration (1:10 dilution from a confluent plate) onto 12-well plates containing fibrin hydrogel formed with 30 mg / mL fibrinogen and 100 U / mL thrombin. iPSCs appear as colonies. [Figure 31] Figure 31 includes photographs of iPSC cells cultured on fibrin in mTESR medium supplemented with 50 U / mL aprotinin after two days of culture. iPSC colonies were plated at a moderate concentration (1:10 dilution from a confluent plate) onto 12-well plates containing fibrin hydrogel formed with 30 mg / mL fibrinogen and 100 U / mL thrombin. iPSCs appear as colonies. [Figure 32]FIG. 32 contains a photograph of an SDS-PAGE gel of various preparations of fibrinogen. Lane 1 is a molecular weight ladder; lane 2 is 1:40 diluted ethanol precipitated fibrinogen; lane 3 is 1:30 diluted ethanol precipitated fibrinogen; lane 4 is 1:110 diluted Evicel (cryoprecipitated fibrinogen); lane 5 is 1:100 diluted Evicel; lane 6 is 1:6 diluted second batch of ethanol precipitated fibrinogen; lane 7 is 1:8 diluted second batch of ethanol precipitated fibrinogen; lane 8 is 1:20 diluted cryoprecipitated fibrinogen; lane 9 is 1:25 diluted cryoprecipitated fibrinogen stripped of plasminogen, von Willebrand factor, and fibronectin; lane 10 is 1:22 diluted stripped cryoprecipitated fibrinogen. [Figure 33] Figure 33A-B contains photographs of pluripotency staining of iPSCs grown on various substrates. A) Immunofluorescence staining of pluripotency factors Oct4, Ssea4, Nanog, and Tra1-60 in iPSCs cultured on evicel (cryoprecipitated fibrinogen), ethanol precipitated fibrinogen (EPF), and geltrex as a positive control. B) FACS analysis of pluripotency factors among the three groups of culture coatings. [Figure 34] Figure 34 contains photographs of pluripotency markers using a different iPSC line, WiCell clone 4, grown on fibrinogen-coated plates. Immunofluorescence was performed for the pluripotency factors Oct4, Ssea4, Nanog, and Tra1-60 in iPSCs cultured on EPF. [Figure 35]Figure 35 contains graphs of three-lineage differentiation analysis of iPSCs (WiCell clone 4) cultured on fibrinogen. Endoderm differentiation was successful in 73.91 ± 8.58% of iPSCs on fibrinogen-coated plates. Mesoderm differentiation was successful in 42.17 ± 3.91% of iPSCs on fibrinogen-coated plates. Ectoderm differentiation was successful in 56.69 ± 8.15% of iPSCs on fibrinogen-coated plates. [Figure 36] Figure 36 includes photographs of cultured undifferentiated iPSC-ECs after one day of culture in Endothelial Growth Media (EGM-2). iPSC-ECs were differentiated on Matrigel, and then 2.5 x 10 iPSC-ECs were plated onto a T25 flask coated with 100 μg / mL fibrinogen. iPSC-ECs appear with a characteristic spindle shape and round nuclei. [Figure 37] Figure 37 includes photographs of iPSC-ECs cultured in EGM-2 for 1 day and then differentiated directly on fibrinogen-coated plates. 2.5 x 10 iPSC-ECs were plated onto a T25 flask coated with 100 μg / mL fibrinogen. iPSC-ECs appear with a characteristic spindle shape and round nuclei. [Figure 38] Figure 38 contains photographs of iPSC-ECs differentiated on fibrinogen-coated plates. Immunofluorescence staining was performed for endothelial markers, including CD31 and UEA-lectin. CD31 staining appears on the cell surface, in the cytoplasm, and around the nucleus. UEA-lectin staining appears uniformly throughout the cell surface. [Figure 39] Figure 39 includes photographs of iPSC cells cultured on fibrinogen-coated plates in mTESR medium without supplements after two days of culture. iPSC colonies were plated at moderate density (1:10 dilution from a confluent plate) onto 12-well plates coated with 100 μg / mL or 750 μg / mL fibrinogen. The fibrinogen used here was obtained by ethanol precipitation of frozen human plasma. iPSCs appear as colonies. [Figure 40] Figure 40 includes photographs of iPSC cells cultured on fibrinogen-coated plates in mTESR medium without supplements after 3 days of culture. iPSC colonies were plated at high density (1:3 dilution from a confluent plate) onto 12-well plates coated with 1 mg / mL fibrinogen or geltrex as a positive control. The fibrinogen (Evicel) used here was obtained by cryoprecipitation. iPSCs appear as a confluent monolayer. [Figure 41] Figure 41 contains photographs of immunofluorescent staining of iPSCs after trilineage differentiation. For both EPF and geltrex cultured cells, endodermal differentiation was confirmed using FoxA2 and Sox17 staining. For both EPF and geltrex cultured cells, mesodermal differentiation was confirmed using CD31 and NCAM staining. For both EPF and geltrex cultured cells, ectodermal differentiation was confirmed using Nestin and Pax6 staining. DETAILED DESCRIPTION OF THE INVENTION

[0017] This document relates to iPSCs and RPE. In some cases, this document provides methods and materials for culturing, expanding, and differentiating stem cells (e.g., iPSCs). For example, this document provides methods and materials for creating a surface for culturing, expanding, and differentiating stem cells using a fibrinogen coating. In some cases, stem cells can expand and differentiate (e.g., into RPE). In some cases, stem cells can expand without differentiating. In some cases, this document provides compositions containing RPE, as well as methods and materials for culturing, expanding, and differentiating stem cells (e.g., iPSCs) into RPE cells. For example, this document provides methods and materials for creating a surface for stem cells to form an RPE monolayer using a fibrinogen coating. As described herein, fibrinogen can be used as a substrate for RPE monolayer formation in a xeno-free (non-xenogeneic) manner. For example, all animal-derived components used to create a human RPE monolayer can be derived from humans. Furthermore, RPE monolayers formed as described herein can be used to create RPE grafts. RPE transplants can be used to treat ocular conditions such as retinal degeneration or macular degeneration. In some cases, the RPE monolayers or RPE transplants provided herein can be designed such that the RPE is a flat, wrinkle-free monolayer.

[0018] Any suitable method can be used to produce fibrinogen for creating an RPE monolayer. For example, fibrinogen can be isolated from a blood product (e.g., isolated from human blood) or produced using recombinant technology. In some cases, fibrinogen is commercially available, for example, from Baxter International (Tisseel), Ethicon Inc. (Evicel), or CSL Behring (RiaSTAP). For example, fibrinogen can be formed as a hydrogel. In some cases, a fibrinogen hydrogel can be formed by mixing fibrinogen (e.g., a fibrinogen solution) with thrombin (e.g., a thrombin solution). In some cases, the fibrinogen hydrogel can also include an antifibrinolytic agent (e.g., aprotinin and tranexamic acid).

[0019] Any suitable method can be used to coat a surface with fibrinogen. For example, the surface of a cell culture vessel can be coated with fibrinogen by exposing the surface to a solution containing fibrinogen for a certain period of time. Any suitable concentration of fibrinogen can be used to coat a surface with fibrinogen. For example, a solution containing about 3 μg / mL to about 1000 μg / mL of fibrinogen (e.g., about 5 μg / mL to about 500 μg / mL, about 15 μg / mL to about 500 μg / mL, about 25 μg / mL to about 500 μg / mL, about 5 μg / mL to about 250 μg / mL, about 5 μg / mL to about 150 μg / mL, about 5 μg / mL to about 100 μg / mL, about 15 μg / mL to about 100 μg / mL, about 25 μg / mL to about 100 μg / mL, or about 50 μg / mL to about 100 μg / mL) can be used to coat a surface with fibrinogen. In some cases, the fibrinogen-containing solution may be exposed to the surface being coated for about 1 hour to about 72 hours (e.g., 2 hours to about 72 hours, 4 hours to about 72 hours, 6 hours to about 72 hours, 2 hours to about 48 hours, 2 hours to about 24 hours, 6 hours to about 48 hours, or 6 hours to about 24 hours). In some cases, spray coating, sputter coating, spin coating, or dip coating methods may be used to coat the surface with fibrinogen.

[0020] For example, the surface of a cell culture vessel can be coated with fibrinogen by forming a fibrin hydrogel on the surface. Any suitable concentration of fibrinogen hydrogel can be used to coat the surface with fibrinogen. In some cases, the fibrin hydrogel can be formed by mixing a fibrinogen-containing solution with a thrombin-containing solution. For example, concentrations of fibrinogen ranging from about 1 mg / mL to about 100 mg / mL (e.g., about 1 mg / mL to about 80 mg / mL, about 1 mg / mL to about 75 mg / mL, about 1 mg / mL to about 60 mg / mL, about 1 mg / mL to about 50 mg / mL, about 1 mg / mL to about 35 mg / mL, about 1 mg / mL to about 25 mg / mL, about 1 mg / mL to about 10 mg / mL, about 15 mg / mL to about 100 mg / mL, about 25 mg / mL to about 100 mg / mL, about 40 mg / mL to about 100 mg / mL, A solution containing fibrinogen at about 50 mg / mL to about 100 mg / mL, about 60 mg / mL to about 100 mg / mL, about 70 mg / mL to about 100 mg / mL, about 85 mg / mL to about 100 mg / mL, about 10 mg / mL to about 85 mg / mL, about 25 mg / mL to about 75 mg / mL, about 40 mg / mL to about 60 mg / mL, about 20 mg / mL to about 40 mg / mL, about 30 mg / mL to about 50 mg / mL, or about 60 mg / mL to about 80 mg / mL. However, the concentration is about 1 U / mL to about 1,000 U / mL (e.g., about 15 U / mL to about 1,000 U / mL, about 25 U / mL to about 1,000 U / mL, about 50 U / mL to about 1,000 U / mL, about 100 U / mL to about 1,000 U / mL, about 250 U / mL to about 1,000 U / mL, about 500 U / mL to about 1,000 U / mL, about 750 U / mL to about 1,000 U / mL, about 900 U / mL to about 1,000 U / mL, about 1 U / mL to about 750 U / mL, about 1 U / mL It can be mixed with a solution containing thrombin at a concentration of about 100 U / mL to about 500 U / mL, about 1 U / mL to about 250 U / mL, about 1 U / mL to about 100 U / mL, about 1 U / mL to about 75 U / mL, about 1 U / mL to about 50 U / mL, about 50 U / mL to about 800 U / mL, about 100 U / mL to about 600 U / mL, about 200 U / mL to about 500 U / mL, about 300 U / mL to about 400 U / mL, about 100 U / mL to about 300 U / mL, or about 500 U / mL to about 750 U / mL.For example, the solution containing fibrinogen may be diluted at a ratio of about 1:0.25 to about 1:200 (e.g., about 1:0.25 to about 1:150, about 1:0.25 to about 1:100, about 1:0.25 to about 1:75, about 1:0.25 to about 1:50, about 1:0.25 to about 1:25, about 1:0.25 to about 1:10, about 1:0.25 to about 1:5, about 1:0.25 to about 1:1, about 1:0.25 to about 1:0.5, about 1:0.5 to about 1:200, about 1:1 to about 1: The fibrin hydrogel may be mixed with a solution containing thrombin at a ratio of about 1:200, about 1:10 to about 1:200, about 1:25 to about 1:200, about 1:50 to about 1:200, about 1:75 to about 1:200, about 1:100 to about 1:200, about 1:150 to about 1:200, about 1:175 to about 1:200, about 1:1 to about 1:150, about 1:25 to about 1:100, about 1:50 to about 1:100, about 1:25 to about 1:75, or about 1:100 to about 1:150. In some cases, the fibrin hydrogel formed on the surface may be rehydrated. For example, fibrin hydrogel can be formed on the coated surface about 10 minutes to about 24 hours (e.g., about 30 minutes to about 24 hours, about 1 hour to about 24 hours, about 2 hours to about 24 hours, about 6 hours to about 24 hours, about 12 hours to about 24 hours, or about 18 hours to about 24 hours) before rehydration. In some cases, the fibrinogen-containing solution and / or the thrombin-containing solution can also contain an antifibrinolytic agent (e.g., aprotinin and tranexamic acid). In some cases, spray coating, sputter coating, spin coating, or dip coating methods can be used to coat the surface with the fibrinogen-containing solution and the thrombin-containing solution.

[0021] In some cases, the surface of the cell culture vessel may also be coated with one or more additional molecules, such as extracellular matrix proteins. For example, collagen, vitronectin, fibronectin, gelatin, elastin, laminin, or any combination thereof may be used to coat the surface of the cell culture vessel.

[0022] Once the surface of a cell culture vessel is coated with fibrinogen as described herein, stem cells can be placed in contact with the fibrinogen and cultured. Any suitable stem cells can be used. For example, embryonic stem cells (e.g., human embryonic stem cells), induced pluripotent stem cells (e.g., human induced pluripotent stem cells), or adult stem cells (e.g., mesenchymal stem cells and adipose-derived stem cells) can be used. Stem cells can be placed in contact with the fibrinogen as single cells (e.g., single-cell suspensions), colonies, or spheroids. In some cases, stem cells can be passaged onto a fresh surface of a cell culture vessel coated with fibrinogen as described herein. For example, stem cells can be dissociated from the surface of the cell culture vessel and replated onto a fresh surface of a cell culture vessel coated with fibrinogen and, optionally, one or more additional molecules (e.g., collagen, vitronectin, fibronectin, gelatin, elastin, or any combination thereof) as described herein. Passaging stem cells can be effective in expanding the number of stem cells (e.g., iPSCs). For example, passage of stem cells can be effective in increasing the number of stem cells that can differentiate (e.g., into RPE cells that can form an RPE monolayer). In some cases, when stem cells (e.g., iPSCs) differentiate, the stem cells can differentiate into any suitable type of cell. The stem cells can differentiate into cells of any germ layer (e.g., endodermal cells, mesodermal cells, or ectodermal cells). The stem cells can differentiate into any suitable type of cell. For example, the stem cells can differentiate into vascular endothelium. For example, the stem cells can differentiate into. For example, the stem cells can differentiate into epithelial cells (e.g., RPE cells). Any suitable differentiation protocol can be used to differentiate the stem cells (e.g., into RPE cells that can form an RPE monolayer).Examples of differentiation protocols that can be used to differentiate stem cells (e.g., iPSCs) into RPE cells capable of forming an RPE monolayer include, but are not limited to, techniques described elsewhere (see, e.g., Sonoda et al., Nat. Protoc., 4:662-673 (2009); Johnson et al., Ophthalmology Vis. Sci., 56:4619 (2015); Brandl et al., NeuroMolecular Med., 16:551-564 (2014); Idelson et al., Cell Stem Cell., 5:396-408 (2009); and Carr et al., Mol. Vis., 15:283-295 (2009)). Once an RPE monolayer is formed, it can be used to create an RPE graft for treating ocular conditions.

[0023] This document also provides methods for using the RPE monolayers or RPE grafts provided herein to treat ocular conditions such as high myopia, angioid streaks, and macular degeneration. Some of the diseases that are classified as macular degeneration and can be treated as described herein include, but are not limited to, age-related macular degeneration (AMD), central geographic atrophy, bestrophinopathy, Leber's congenital amaurosis, choroideremia, gyrate atrophy, Thorsby's macular degeneration, mitochondrial-inherited diabetes and deafness (MIDD), chloroquine-associated retinopathy, malattia leventinese, North Carolina dystrophy, hyperornithinememia, central serous chorioretinopathy, adult-onset foveomacular dystrophy, and Stargardt's disease. For example, a mammal (e.g., a human) may be prepared for ocular surgery, and a sub-retinal detachment may be performed to expose the damaged RPE area. At this point, a transplantation device may be used to deliver an RPE graft provided herein onto the desired area. In some cases, a cannula may be used to gain access to the eye. In some cases, an air-phase bubble may be used to press the RPE graft into place. A laser tool (e.g., a laser tool used for diabetic retinopathy) may be used to secure the graft and prevent slippage via laser photocoagulation. At this point, a transplantation device may be used to deliver a second RPE graft provided herein onto the desired area inside the eye. The second graft may be placed adjacent to the first graft, preferably through the initial incision or a cannula. A laser tool may be used to secure the second graft and prevent slippage. A transplantation device may be used to deliver a third RPE graft provided herein onto the desired area inside the eye. The third graft may be The second graft may be placed adjacent to the first, preferably through the initial incision or cannula. A laser tool may be used to secure the third graft and prevent slippage. While this section describes implanting three RPE grafts, any appropriate number may be used to cover the area to be treated. For example, one, two, three, four, five, six, or more RPE monolayer / fibrin grafts provided herein may be implanted into a single eye to be treated. Overall, this modular tiling approach allows clinicians to customize grafts to suit the patient's needs, is scalable to a wide range of areas, can be applied to any region of the retina, and reduces the number of incisions required.

[0024] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0025] Example 1 - Formation of iPSC-RPE Monolayers Using Fibrinogen <Chemical products> Fibrinogen was obtained from three sources: Ethicon as Evicel (60 mg / mL), Baxter as Tisseel (95 mg / mL), and research-grade material from Sigma-Aldrich (57 mg / mL) and Millipore (44 mg / mL). Final working concentrations were made up in PBS to a concentration range of 0–1 mg / mL.

[0026] <Covering Protocol> Matrigel-coated plates were used as a positive control as described elsewhere (Johnson et al., Investig. Ophthalmology Vis. Sci., 56:4619 (2015)). Various concentrations of fibrinogen were prepared by diluting stock fibrinogen in PBS and plated onto wells of various sizes at 4–37°C for 1–24 hours. After subsequent washing with PBS, cells were cultured at various concentrations (0.1 × 10 6 ~1×10 6 cells / cm 2 ) and incubated at 37°C under 5% CO2. Cells were monitored over time for adhesion and viability.

[0027] <cell> Partially differentiated iPSC-RPE cells at passage 1 or 2 were obtained from LAgen Laboratories LLC (Rochester, MN). The initial differentiation process was performed as described elsewhere (Johnson et al., Investig. Ophthalmology Vis. Sci., 56:4619 (2015)), with modifications. Cells were first dissociated from the plate by digestion with collagenase, then cell suspensions were prepared in Accumax and plated at various concentrations (0.1 × 10) onto tissue culture polystyrene (TCPS) or polycarbonate plates coated with fibrinogen or Matrigel. 6 ~1×10 6 cells / cm 2 ) and replated with differentiation medium as described elsewhere (Johnson et al., Investig. Ophthalmology Vis. Sci., 56:4619 (2015)).

[0028] <Differentiation> Brightfield images of plated cells were taken and cell morphology was assessed at various time points during the differentiation process. After 2 weeks of culture, complete differentiation of RPE cells was assessed by Western blot analysis and growth factor secretion by ELISA at various time points.

[0029] Western blot analysis was performed using a ProteinSimple Wes instrument (ProteinSimple, San Jose, CA) for specific RPE markers, including RPE65, Best1, CRALBP, MERTK, and β-actin. Growth factor secretion was quantified by ELISA. After 6–12 weeks of culture, spent medium was collected 24–48 hours before testing and frozen at −20°C. ELISA was performed on the collected medium for VEGF and PEDF using commercially available kits (DuoLISA, RND Systems).

[0030] <Result> Cells cultured on plates coated with 0.1-0.5 μg / mL fibrinogen for 1 week did not adhere or adhered poorly to the plates (Figures 1 and 2). Some cells cultured on plates coated with 1 μg / mL fibrinogen for 1 week attached, but monolayer formation was poor (Figure 3). Cells cultured on plates coated with 5-15 μg / mL fibrinogen for 1 week resulted in the formation of patchy monolayers (Figures 4-6). Cells cultured on plates coated with 25 μg / mL or more fibrinogen for 1 week formed confluent monolayers (Figures 7-10). The monolayers shown in Figures 7-10 were similar to those obtained using the positive control (Figure 11). See also Figure 12.

[0031] Monolayers formed using partially differentiated iPSC-RPE cells and cultured on plates coated with 100 μg / mL fibrinogen or 200 μg / mL Matrigel (i.e., positive control) for 1 week (Figures 13-14), 6 weeks (Figures 15-16), or 12 weeks (Figure 17) resulted in monolayers indistinguishable from each other. These monolayers exhibited characteristic RPE morphology (pigmentation and a "cobblestone" appearance) within 6 weeks. Furthermore, cells cultured on plates coated with 100 μg / mL fibrinogen or 200 μg / mL Matrigel (i.e., positive control) exhibited comparable protein expression profiles after 12 weeks of culture (Figure 18). 24-48-hour culture supernatants from both cultures also contained high levels of secreted VEGF and pigment epithelium-derived factor (PEDF), characteristic of RPE cells (Figure 19). These results demonstrate that fibrinogen can be used as a substrate to form a healthy, viable iPSC-RPE monolayer.

[0032] Fibrinogen preparations from the following sources were tested: Ethicon (Evicel), Baxter (Tisseel), Sigma-Aldrich, Millipore, and an extract made from cryoprecipitate and ethanol. Differences were observed with respect to the efficiency of plate coating and the minimum concentration required (Table 1). Furthermore, fibrinogen from Sigma-Aldrich or Millipore / EMD did not support proper attachment of iPSCs. These products were obtained as lyophilized powders and reconstituted before use. This may suggest that lyophilization may affect the ability to use fibrinogen as a coating material. However, Tisseel, similarly reconstituted from lyophilization, did allow proper attachment of iPSC-RPE. No correlation was observed between absolute fibrinogen levels and the effectiveness of the various commercially available preparations. Protein denaturation can occur during the lyophilization process / preparation or after prolonged storage, especially in the absence of an appropriate freeze-drying process and / or cryo-lyoprotectant. Therefore, it would be beneficial to optimize commercially available human fibrinogen preparations specifically formulated for use as surface coatings for tissue culture and for use in the manufacture of hydrogels that can be used in 3D cell culture, or to develop scaffolds for cell therapy applications.

[0033] [Table 1]

[0034] <phagocytosis results> Phagocytosis assays were performed as described elsewhere (Marmorstein et al., Sci. Rep., 8:4487 (2018)). iPSC-RPE were grown on various fibrinogen sources: evicel and two distinct cryoprecipitates (Aneg and Bpos). Matrigel coating was used as a positive control. RPE cultured on evicel (Evi) exhibited similar total OS binding as Matrigel, whereas Aneg and Bpos showed an approximately two-fold increase in total OS binding (Figure 23). Upon binding, Aneg (67%) and Bpos (57%) showed similar internalization rates to MG (55%), whereas Evi (14%) was significantly lower than MG (p=0.009).

[0035] <Western blot analysis results> Western blot comparison of RPE65, CRALBP, and BEST1 expression was performed on RPE grown on various coating reagents (Figure 24). Fibrinogen-based substrates showed increased expression of RPE65 and Best1 compared to Matrigel controls when normalized to the internal β-actin signal. CRALBP showed no difference between all groups.

[0036] iPSC-RPE grown on fibrinogen-coated plates expressed characteristic RPE markers, including RPE65, Best1, CRALBP, and MERTK, as early as 2 weeks after plating (Figure 25). Matrigel-coated plates typically required 6–8 weeks before RPE65 and Best1 expression. Western blots were analyzed over time for three different fibrinogen coating concentrations (5, 25, and 100 μg / mL) (Figure 26). Each of the four markers peaked around week 8 and appeared stable after week 10. There were no normalized differences between the three coating concentrations, although the 5 μg / mL condition did not consistently result in confluent monolayers.

[0037] Similarly, the time course of PEDF and VEGF secretion was measured using ELISA (Figure 27). PEDF showed an overall trend of increasing concentrations from weeks 2 to 14, reaching a maximum at week 10. Differences between coating concentrations were not significant. VEGF showed an overall increase in secretion over time from weeks 6 to 14. VEGF release at the 5 μg / mL coating concentration appeared to be reduced compared to the 25 and 100 μg / mL conditions.

[0038] We compared RPE marker expression in RPE cultured on various commercially available fibrinogen sources at 100 μg / mL (Figure 28). Evicel and stemCOAT outperformed all other sources tested, including Tisseel and Millipore. Sigma did not allow iPSC-RPE to attach at 100 μg / mL and was not included in the Western blot analysis.

[0039] Similarly, transmitted light photographs were taken of WiCell clone 4-derived iPSC-RPE cells cultured on various fibrinogen sources (Figure 29). Tisseel, stemCOAT, EPF1, and EP2 demonstrated the formation of confluent pigmented monolayers. Notably, EPF2 demonstrated complete monolayer adhesion at only 4 μg / mL. Neither Sigma nor Millipore fibrinogen resulted in confluent monolayers, as voids were detected in all samples, even at concentrations as high as 2 mg / mL.

[0040] Example 2 - Protocol for Retinal Pigment Epithelial Monolayer Formation Dilute human fibrinogen (e.g., Evicel, 60 mg / mL) to 100 μg / mL with DPBS. Plate 2 mL onto a well of a 6-well plate. Incubate the plate overnight at 4°C. Aspirate the fibrinogen solution and wash the plate three times with DPBS. Add 1 x 10 human partially differentiated iPSC-RPE (passage 2) to the wells. 6 cells / cm 2The cells are plated onto wells at a concentration of 0.1%. The cells are incubated overnight at 37°C, 5% CO2 to allow for attachment. The cells are plated in differentiation medium as described elsewhere (Johnson et al., Investig. Ophthalmology Vis. Sci., 56:4619 (2015)). Medium changes are performed every other day for up to approximately 8 weeks. The cells are harvested and prepared for analysis or use.

[0041] This protocol allows the production of RPE cells and RPE monolayers suitable for transplantation into human eyes using xeno-free covering materials. For example, the RPE monolayer is placed on a surgical delivery device, and the human eye is prepared for surgery. After preparation, the RPE monolayer is transplanted into the subretinal space of the eye.

[0042] Example 3 - Use of fibrinogen coating to culture and differentiate iPSCs To develop a reproducible, quality-controlled product for coating tissue culture plasticware with human fibrinogen and for fabricating 3D human fibrin hydrogels, the following was done: The product was evaluated for its ability to support iPSC cultures and to be used to differentiate iPSCs into various cell types, such as RPE, endothelial, and cardiomyocytes.

[0043] As described herein, human fibrinogen served as an effective coating for the differentiation of iPSCs into RPE cells. See also Figures 20A-C. To verify the extent to which human fibrinogen can be used as a coating for tissue culture plasticware during iPSC differentiation into RPE cells, we performed the following. Human iPSC line 006-BIOTR-0001, clone 1 (Cl1), grown on plates coated with human fibrinogen obtained from both ethanol precipitation and cryoprecipitation, maintained its pluripotency, as determined by continued expression of pluripotency markers at levels comparable to those of Cl1 cells grown on Geltrex® (Figures 21A-C). Furthermore, the cells retained the ability to undergo differentiation into endoderm, mesoderm, and ectoderm using a trilineage differentiation endpoint assay (Figure 21D). These results demonstrate that human fibrinogen can be used as a broadly applicable, non-xenogeneic coating for tissue culture plasticware used in the growth, proliferation and differentiation of human iPSCs.

[0044] Research-grade human fibrinogen is typically sold as a lyophilized product. Material denaturation due to manufacturing variations among manufacturers affects the ratio of clottable to total fibrinogen. There are also differences among manufacturers in residual buffer and salt concentrations and absolute purity. Finally, there is no guarantee of sterility or that the product is free of mycoplasma or other pathogens. These variables affect the properties of fibrin hydrogels formed from these materials, making consistent results difficult to obtain and resulting in significant differences in concentration and efficiency when used to coat surfaces for tissue culture. To develop a reproducible, quality-controlled human fibrinogen product for generating reproducible human fibrin hydrogels and for reproducibly coating tissue culture plasticware, we performed the following:

[0045] A highly concentrated fibrinogen concentrate is produced using human plasma cryoprecipitate as the starting material. The fibrinogen concentrate is evaluated for purity, clotting ability, sterility, and other criteria. After lot testing, the fibrinogen is used to generate fibrin gels that are expected to support the growth of iPSC-RPE cells as described herein. If successful, the material is titrated for use in coating plates.

[0046] Human plasma cryoprecipitate is prepared by slowly thawing frozen plasma at 4°C. Fibrinogen, clotting factors, and fibronectin precipitate in the blood bag, forming a white "pellet" that settles in a centrifuge. The material is obtained directly from a blood bank and is typically supplied frozen with approximately 10-20 mL of plasma, still containing the pellet. Clinically, the plasma is used to reconstitute pellets for therapeutic administration (e.g., to hemophilia patients). The procedure is as follows: the cryoprecipitate is thawed overnight at 4°C and excess plasma is removed. The pellet is washed twice in ice-cold sterile saline to remove excess plasma proteins. The pellet is then solubilized in 5 mL of 250 mM Tris-HCl, pH 8.0. One unit of cryoprecipitated pellet, with a volume of approximately 5 mL, is designed to contain approximately 500 to 1500 mg of fibrinogen. After solubilization, the fibrinogen is dissolved in a 10 mL volume at a final concentration of 50 to 150 mg / mL in 125 mM Tris-HCl, pH 8.0. This concentration of Tris-HCl at pH 8.0 was confirmed to dissolve fibrinogen to very high concentrations without inhibiting clotting ability (Figure 22A-B). This concentration is desirable for the fabrication of fibrin hydrogels. A more dilute concentration is desired for coating surfaces for cell culture, and this determination is needed. After determining clottable fibrinogen using the von Clauss method (Mackie et al., Br. J. Haematol., 121(3):396-404 (2003); and Machin et al., BMJ, 307(6909):882-3 (1993)) on a Stago Start Coagulation Analyzer (Stago, France), the fibrinogen is diluted to the appropriate concentration, filtered through a 0.22 μm filter, and aseptically dispensed into 10 and 50 mL bottles. The manufacturing process is scaled up as needed.

[0047] Additional lot testing is performed. The blood bank screens donors in accordance with FDA CFR 21-1271 and directly tests donated blood for HIV, HTLV, Zika, West Nile virus, hepatitis B, and hepatitis C. In addition to these tests for human pathogens, manufactured fibrinogen lots are assayed in-house for pH, total protein concentration using a biuret assay, albumin concentration using bromcresol purple, and mycoplasma using the AMP® Mycoplasma Detection Kit (Sartorius, Gottingen, Germany). Sterility is assessed by Steris (Mentor, OH) according to USP <71> Each lot is assayed according to standard endotoxin testing.

[0048] Geltrex® and Matrigel® contain primarily mouse laminin concentrated to approximately 80% purity. Fibrinogen preparations manufactured as described herein are designed to achieve similar fibrinogen purity. Raw cryoprecipitate typically consists of approximately 65% ​​fibrinogen. For further concentration, precipitation steps with protamine, glycine, and / or cold ethanol are incorporated into the manufacturing process.

[0049] The starting material is clinical-grade cryoprecipitate. It is delivered sterile and likely mycoplasma-free. The preparation process is carried out in a clean room. Finally, the diluted fibrinogen preparation used for plate coating is sterile filtered through a 0.22 μm filter and bottled.

[0050] A successful fibrinogen preparation is determined and a lot of human fibrinogen with an initial clottable fibrinogen concentration of >5 mg / mL and purity of >70% is manufactured. The material is designed to pass sterility testing, be mycoplasma-free, have a pH of 7.9-8.1, and form a gel that supports the growth and differentiation of iPSCs, including iPSC-RPE cells.

[0051] As described elsewhere (Gandhi et al., Acta Biomater., 67:134-146 (2018)), the manufactured fibrinogen will be tested for its ability to form a gel to determine whether iPSC-RPE can adhere to the manufactured gel. In particular, the manufactured fibrinogen will be compared side-by-side with Geltrex®, Matrigel®, and recombinant human laminin 521 as a coating for passaging iPSCs and differentiating them into RPE, endothelial, and cardiomyocytes. The experiments will be used to demonstrate that the manufactured fibrinogen product can function to replace laminin in the culture of iPSCs and iPSC-derived cell types.

[0052] As a titration for plate coverage for iPSC attachment and growth, the following is performed. As described herein, data obtained demonstrated that plate coverage can be achieved by simply adding fibrinogen solution to the plate and incubating at 37°C for 1 hour. In completed studies, 100 μg / mL of fibrinogen was used for iPSC-RPE culture, but this was not sufficient for iPSC adhesion. As shown in Figures 21A-D, plates were coated with a solution containing 500 μg fibrinogen / mL at 250 μL / cm. 2 This worked for the first fibrinogen preparation prepared from human plasma presented here, and for fibrinogen obtained from clinical fibrin tissue "glue." It did not work for research-grade fibrinogen obtained from Sigma-Aldrich or Millipore / EMD, supplied as a lyophilized powder.

[0053] To determine the minimum concentration of clottable fibrinogen required for the culture of iPSCs and cells differentiated from iPSCs, the following was done: Fibrinogen was prepared as described herein and diluted to a concentration of 2 mg / mL. A series of two-fold serial dilutions was made down to a concentration of 0.03125 mg / mL, and nine wells of each of three 12-well multiwell plates were coated with these solutions. The remaining three wells of each plate were coated with Geltrex® (42 μg / mL), Matrigel® (2 mg / mL), or human laminin 521 (30 μg / mL) using dilutions / concentrations previously shown to work. After coating, iPSCs were plated into each well of each plate. The entire well was photographed daily, and colonies were counted using a Spectramax 3 with a Molecular Devices Minimax 300 cytometer attachment. iPSC colonies that appear to be spontaneously differentiating each day could be manually cleaned or removed. However, for this experiment, the wells were not cleaned. Instead, blinded observers are instructed to count the number of colonies that appear to be spontaneously differentiating from the photographs according to a uniform set of criteria. At the end of day 7, the number of colonies is graphed relative to the number of colonies that are spontaneously differentiating for each well of each plate. The results are used to determine the minimum fibrinogen concentration required to support iPSC attachment and proliferation without a rate of spontaneous differentiation exceeding that observed when laminin is used to coat the plates. To control for lot-to-lot variation, three independently produced lots of human fibrinogen are tested and used to define a threshold as the minimum concentration that is valid for all three lots tested.

[0054] To determine whether fibrinogen preserves iPSC pluripotency, pluripotency is tested using iPSCs after multiple passages on fibrinogen. 60 mm dishes are coated with the optimal concentration of fibrinogen determined as described herein, and iPSCs from five iPSC lines derived from different donors are plated onto them. Cells are serially passaged five times, and cells from each passage are examined using flow cytometry as described for Figures 21A-D to determine whether they retain expression of pluripotency markers. Similarly, Figure 41 shows qualitative staining of pluripotency markers for iPSCs cultured on EPF and geltrex. After the fifth passage, cells are also stained by immunofluorescence for pluripotency markers, karyotyped, and assayed for their ability to undergo differentiation into ectoderm, mesoderm, and endoderm using the STEMDIFF® Tri-Lineage Differentiation Kit (Stemcell Technologies).

[0055] To titrate the plate coating for attachment and growth of cells differentiated from iPSCs, we performed the experiments described above, substituting iPSCs with iPSC-derived RPE, endothelial cells, or cardiomyocytes. These cells represent differentiated cells from three lineages: ectoderm (RPE), endoderm (endothelium), and mesoderm (cardiomyocytes). The results were used to identify the minimum concentration of fibrinogen required for attachment and support of each cell type. RPE, endothelial cells, and cardiomyocytes were obtained from two iPSC lines (cl1 and IMP90 clone 4) using established differentiation protocols, except for substituting fibrinogen as the plate coating at each step. These two cell lines were chosen because they generate all three cell types on laminin, are derived from different sources, and are produced using different reprogramming systems (Sendai virus and retrovirus, respectively).

[0056] Successful results would be achieved if human fibrinogen were demonstrated to be equivalent to or superior to laminin for iPSC attachment and growth, and equivalent to or superior for iPSC differentiation into cells of ectodermal, endodermal, and mesodermal origin.

[0057] Example 4 - Fibrinogen coating for iPSC culture and differentiation <cell> The CLR-0001-BIOTR iPSC line was used at passages 10–20 (Johnson et al., Investig. Ophthalmology Vis. Sci., 56:4619 (2015); and Marmorstein et al., Sci. Rep., 8:4487 (2018)). An additional line, CLR-0004 from WiCell, was also used (Johnson et al., Investig. Ophthalmology Vis. Sci., 56:4619 (2015)). mTESR (Stem Cell Technologies) was used for iPSC growth, and mRESLR (Stem Cell Technologies) was used to dissociate cells for passaging. iPSC clusters were passaged and replated at a density of approximately 20–40% confluency to maintain pluripotency and expansion.

[0058] <Fibrinogen extraction> Fibrinogen was extracted using standard methods, including ethanol precipitation (Dietrich et al., Tissue Eng. Part C Methods., 19:216-226 (2013)) and cryoprecipitation (Sparrow et al., Methods Mol. Biol. Clifton NJ., 728:259-265 (2011)). After precipitation, fibrinogen was reconstituted in various molarities of Tris-HCl, TBS, PBS, and citrate-buffered saline. Samples were sterile filtered and aliquoted as stock solutions to prevent repeated freeze-thaw cycles. Commercially available fibrinogen (Evicel; Ethicon) was also used for comparison.

[0059] The clottable fibrinogen concentration was confirmed using the Clauss method. Total protein concentration was determined using a commercially available BCA assay (Pierce Technologies) with Evicel's known total protein concentration as a standard. For purity, SDS-PAGE gels were run using a 10% mini protean gel (Bio-Rad) at 120 V and 0.1 A for 1.5 hours. The gels were removed from the cartridge, stained overnight with Coomassie blue solution (Bio-Rad), and washed multiple times in destaining solution (Bio-Rad). The gels were then imaged using GelDOC (Bio-Rad).

[0060] <Plate coating> Fibrinogen stock solutions from various sources were thawed at 37°C and diluted to working concentrations in each buffer. Evicel was diluted in PBS. 0.3125 mL / cm 2 The plating density of the surface area was used. Plates were incubated at 37°C for a minimum of 2 hours before use. After incubation, plates were washed three times with PBS before plating iPSCs.

[0061] Geltrex (Thermo Fisher) was used as a positive control. A frozen aliquot of geltrex was thawed on ice and diluted 1:240 in DMEM / F12 medium at 0.3125 mL / cm 2 The cells were plated at a density that matched the surface area of ​​the plate. The plates were incubated at 37°C for at least 2 hours. After incubation, the plates were aspirated and the cells were immediately plated.

[0062] <facs> Cells cultured on various coated surfaces were cultured for at least 48 hours before flow cytometry. Cells were dissociated using TrypLE (Life Technologies) at 37°C for a maximum of 5 minutes, centrifuged at 800g for 4 minutes, resuspended in PBS, split into two tubes for unstained controls, and centrifuged again. Cells were fixed in PerFix-nc (Beckman Coulter) according to the manufacturer's protocol. Cells to be stained were mixed with a staining solution consisting of permeabilizing reagent, 1:10 Alexa 488 anti-human Nanog (BD), 1:10 Alex 647 anti-OCT 3 / 4 (BD), 1:10 PE anti-SSEA4 (BD), and 1:10 PerCP-Cy5.5 anti-human TRA1-60 (BD). Cell clumps were removed by allowing the cell slurry to settle before running. Samples were run on a Gallios (Beckman Coulter) using four channels. A total of 1000 cells were counted with double positive cells required to confirm expression.

[0063] <Differentiation> iPSCs were passaged from 60 mm plates containing each coating material onto 6-well (Ecto) or 24-well (Endo, Meso) plates containing each coating material using Accutase (Innovative Cell Tech; San Diego, CA). Ectodermal differentiation was performed using STEMdiff Neural Induction Medium (Stem Cell Tech) according to the manufacturer's protocol. Y-27632 (RND Systems) was added only to the day 0 medium. After 9 days of culture, cells were passaged from the 6-well plates using Accutase and replated onto 24-well plates. Differentiation was completed using Neural Induction Medium until the cells reached approximately 70% confluence. Endoderm differentiation was performed using the STEMdiff Definitive Endoderm Kit (Stem Cell Tech) according to the manufacturer's protocol. After 5 days, cells were fixed in 4% PFA. Mesoderm differentiation was performed using StemDiff Mesoderm Induction Medium according to the manufacturer's protocol. After 5 days, cells were fixed in 4% PFA.

[0064] <Immunofluorescence staining> Fixed iPSCs were stained for pluripotency markers to assess clonal variation between culture substrates. Fixed cells were permeabilized in 0.2% Triton-X 100 (Sigma-Aldrich) for 30 minutes at room temperature and then incubated in blocking solution (DAKO). Each well was incubated for 1 hour at room temperature with one of the following primary antibody combinations: (A) 1:200 rabbit anti-Oct 3 / 4 (Abcam) and 1:100 mouse anti-SSea4 (Abcam) or (B) 1:100 rabbit anti-Nanog (Cell Signaling) and 1:100 mouse anti-Tra1-60 (Abcam). Wells were washed three times with washing solution (DAKO). A secondary antibody mixture was then incubated for 30 minutes at room temperature: 1:200 anti-rabbit Alexa 594 and 1:300 anti-mouse Alexa 488. Wells were washed again, stained with DAPI for 5 min, and imaged using a Cytation 5 imager (BioTek).

[0065] Differentiated cells were stained using a similar protocol, but modified to include the following primary antibodies: (external) 1:20 sheep anti-Pax6 (RND Systems), (internal) 1:200 rabbit anti-Fox A2 (Cell Signaling), or (internal) 1:200 rabbit anti-MixL1 (Millipore). Images were analyzed using Gen5 Imaging Prism (BioTek; Winooski, VT) software, and differentiation efficiency was calculated as total double-stained positive cells divided by total DAPI-positive cells.

[0066] <Gel culture> Fibrin gels were prepared as described elsewhere (Gandhi et al., Acta Biomater., 67:134-146 (2018)). Briefly, a mixture of 30 mg / mL fibrinogen and 100 U / mL thrombin (final concentrations) was mixed in the wells of a 12-well plate, and the gel was flattened within the well using a custom-made polycarbonate mold with parafilm. The gel was allowed to fully polymerize for 2 hours at 37°C, washed with PBS, and iPSCs were seeded onto the gel.

[0067] <Result> <iPSCs on fibrin> iPSCs were successfully cultured on fibrin hydrogels. Figure 30 shows iPSC colonies cultured on fibrin hydrogels made with 30 mg / mL fibrinogen and 100 U / mL thrombin without supplementation with aprotinin. The iPSC colonies expanded over time. When plated in larger numbers, the iPSCs formed a confluent monolayer. Without aprotinin, there were numerous strain lines within the gel, suggesting mechanical forces exerted by the iPSCs. Without aprotinin, the gels did not appear to be significantly degraded upon reaching confluency.

[0068] Figure 31 shows iPSC colonies cultured on a similar fibrin hydrogel supplemented with 50 U / mL aprotinin. Similarly, iPSC colonies expanded over time and formed monolayers within the sample when plated in larger quantities. The addition of aprotinin did not appear to prevent strain lines within the gel. With aprotinin, the gel did not degrade for at least one month.

[0069] <SDS-PAGE gel of various fibrinogens> To determine the qualitative purity of the various fibrinogen preparations, SDS-PAGE gels were run and stained with Coomassie blue. Each fibrinogen preparation was successfully used to culture iPSCs. Lanes 9 and 10 represent a positive control, cryoprecipitated fibrinogen, from which plasminogen, von Willebrand factor, and fibronectin were removed (Figure 32). Fibrinogen characteristically appears as three bands: α (67 kDa), β (54 kDa), and γ (47 kDa) (Figure 32). All experimental samples showed a similar characteristic fibrinogen profile. Lanes 2 and 3 represent an ethanol-precipitated fibrinogen preparation (EPF1) (Figure 32). It contains multiple bands at approximately 260 kDa, 150 kDa, and 120 kDa. Fibronectin is a common component of plasma precipitates and is found as a band around 260 kDa. Lanes 4 and 5 represent commercially available cryoprecipitated fibronectin (Evicel, EVI) (Figure 32). Lanes 6 and 7 are for a second different preparation of ethanol-precipitated fibrinogen (EPF2) (Figure 32). This sample appeared to have a much lower fibronectin concentration. This was also the sample that allowed for successful plating of iPSCs at 100 μg / mL. Finally, lane 9 represents cryoprecipitated fibrinogen (CPF1) (Figure 32).

[0070] <Maintaining pluripotency of iPSCs on fibrinogen> iPSCs were cultured on various preparations of fibrinogen. Using EPF2, iPSC adhesion was successful at the lowest concentration. For example, Figure 39 shows iPSCs adhered to plates coated with 100 μg / mL fibrinogen using EPF2 conditions. A fibrinogen concentration of 700 μg / mL showed similar adhesion.

[0071] We confirmed that the use of fibrinogen as a coating material for iPSC colonies maintained pluripotency markers. Immunofluorescence staining of iPSC colonies on EVI, EPF1, and geltrex (GT) showed positive staining for Oct4, SSea4, Nanog, and Tra1-60 throughout individual colonies (Figure 33A). Individual cell expression of pluripotency markers was quantified using FACS analysis on a minimum of 1,000 cells (Figure 33B). A marker was considered positive only if it was double-positive with all other markers. Oct4 expression was positive in 97.4 ± 4.1% of cells on EVI, 98.9 ± 1.8% of cells on EPF1, and 98.4 ± 2.4% of cells on GT. SSea4 expression was positive in 99.3 ± 1.2% of cells on EVI, 98.4 ± 2.3% of cells on EPF1, and 97.1 ± 4.3% of cells on GT. NANOG expression was positive in 97.5±3.3% of EVI, 94.1±7.6% of EPF1, and 94.9±4.5% of GT samples. TRA1 60 expression was positive in 92.2±6.1% of EVI, 91.4±5.5% of EPF1, and 88.6±5.5% of GT samples.

[0072] <iPSC differentiation on fibrinogen> Prior to differentiation, the ability to generate iPSC monolayers on fibrinogen-coated plates was determined. Figure 40 shows that iPSCs were able to form confluent monolayers when cultured on fibrinogen compared to the geltrex positive control.

[0073] iPSCs cultured on fibrinogen were differentiated into the three germ lines using a commercially available kit (STEMdiff 3-lineage Differentiation Kit, StemCell Technologies). After induction in endoderm, mesoderm, and ectoderm differentiation media, the iPSCs were fixed and stained for each marker. iPSCs cultured on EPF showed expression for FoxA2 and Sox17 after endoderm induction, CD31 and NCAM after mesoderm induction, and Nestin and Pax6 after ectoderm induction. iPSCs cultured on geltrex were used as a positive control and were positive for all markers.

[0074] <Different iPSC clones> To demonstrate that the ability to culture iPSCs on fibrinogen is not cell line-specific, another commercially available iPSC cell line (WISCi004-A-1) (Srikanth et al., Cell Rep., 12:1414-1429 (2015); and Zeng et al., PloS One, 5:e11853 (2010)) was used. WISCi004-A-1 successfully maintained pluripotency when cultured on fibrinogen-coated plates. Immunofluorescence revealed positive staining for Oct4, SSea4, Nanog, and Tra1-60 (Figure 34). Furthermore, the iPSCs differentiated into the three germ lines. In the WISCi004-A-! line cultured on fibrinogen, endoderm differentiation was achieved in 73.9 ± 8.6% of the iPSCs, mesoderm in 42.2 ± 3.9% of the iPSCs, and ectoderm in 56.7 ± 8.2% of the iPSCs (Figure 35).

[0075] [Example 5 - iPSC Endothelial Cell Differentiation and Culture on Fibrinogen-Coated Plates] <iPSC-EC Differentiation> The CLR-0001-BIOTR iPSC line was used at passages 10–15. Six-well plates were coated with 1 mg / mL fibrinogen reagent, 2 mL per well, and incubated at 37°C for 2 hours before use. iPSC-EC differentiation was performed as described elsewhere (Orlova et al., Nat. Protoc., 9:1514–1531 (2014)). Briefly, iPSC colonies were split into 0.5–1 mm diameter fragments and 5–8 colonies were plated per cell. iPSCs were cultured in mTeSR1 medium for 2 days and then replaced with mesoderm induction medium (BPEL (Orlova et al., Nat. Protoc., 9:1514-1531 (2014)) based on BPEL supplemented with 25 ng / mL activin A (RND Systems), 30 ng / mL BMP4 (Miltenyi Biotec), 50 ng / mL VEGF (RND Systems), and 1.5 μM CHIR (RND Systems)). After 2 days, the medium was replaced with vascular-specific medium (BPEL based on BPEL supplemented with 50 ng / mL VEGF and 10 μM SB431542 (RND Systems)). After 4 days, the vascular-specific medium was replenished and then again after another 2 days.

[0076] After EC islands appeared, iPSC-ECs were purified using magnetic beads. CD31-labeled beads (Thermo Fisher) were suspended in 0.1% BSA in DMEM, using 21 μL of beads per well. After washing the cells with PBS, the beads were incubated with the cells for 30 minutes at room temperature with slight agitation. The beads were then washed, and the cells were dissociated with TrypLE (Thermo Fisher) and incubated for 5 minutes at room temperature. The reaction was stopped with FACSB-10 solution (10% FBS in FACS buffer). The cell solution was strained using a 100 μm cell strainer. The cells were placed in a magnetized column and washed twice with FACSB-10 and twice with 0.1% BSA in DMEM. After removal from the magnetic column, the remaining cells were resuspended in endothelial growth medium (EGM2) (Lonza) and plated onto T75 flasks coated with 100 μg / mL fibrinogen.

[0077] <iPSC-EC Culture and Subculture> iPSC-ECs were subcultured and grown up to passage 4. After aspirating the medium, iPSC-ECs were washed in PBS and dissociated using TrypLE at room temperature for 5 minutes. The cells were resuspended in EGM2 medium and centrifuged at 300 g for 10 minutes. The cells were plated at 1×10 4 cells / cm 2 onto a T25 flask coated with 100 μg / mL fibrinogen or a 4-chamber culture slide (BD) coated with 100 μg / mL fibrinogen. The medium was changed every 2 days and the cells were grown until 6 days before subculture.

[0078] <iPSC-EC Staining> Fixed iPSC-ECs were stained for endothelial markers. iPSC-ECs cultured on a 4-well culture slide coated with 100 μg / mL fibrinogen were fixed with ice-cold methanol for 5 minutes. The methanol was washed away with PBS three times. The fixed cells were blocked with a blocking solution (6% normal goat serum in PBS, 0.3% Triton-X (triton-X) 100 (Sigma-Aldrich)) at room temperature for 45 minutes. The cells were incubated overnight at 4°C with one of the following primary antibodies: (A) 10 μg / mL anti-CD31 (BBA7, RND Systems) or (B) 10 μg / mL FITC-labeled UEA-lectin (Vector Labs). The wells were washed three times with PBS. Then, for only CD31 staining, 10 μg / mL of FITC-anti-mouse secondary antibody in 0.3% TX-PBS was incubated with the cells at room temperature for 1 hour. The wells were washed again, fluorescent dye (fluromount) was dropped onto the slide, and a coverslip was placed. The slide was imaged using a fluorescence microscope (Nikon).

[0079] <Results> <iPSC-EC Culture><G iPSC-ECs were differentiated as previously described elsewhere (Orlova et al., Nat. Protoc., 9:1514-1531 (2014)). This protocol used Matrigel-coated 6-well plates to initiate differentiation of iPSCs into ECs. After successful purification and culture on Matrigel, iPSC-ECs were passaged onto fibrinogen-coated plates using TrypLE. iPSC-ECs successfully attached to the fibrinogen-coated plates and appeared with a typical spindle-shaped phenotype (Figure 36).

[0080] To test whether fibrinogen-coated plates could be used to differentiate iPSCs, we modified a previously mentioned protocol to use fibrinogen-coated 6-well plates for initial iPSC adhesion. After differentiation, iPSC-ECs were purified and replated onto fibrinogen-coated T75 plates. iPSC-ECs appeared with a characteristic spindle shape and round nuclei (Figure 37). Immunofluorescence staining of iPSC-ECs revealed positive staining for CD31 and UEA-lectin (Figure 38).

[0081] [Other embodiments] While the present invention has been described in connection with its detailed description, it should be understood that the above description is intended to be illustrative and not limiting of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.< / facs>

Claims

1. A method for producing a retinal pigment epithelial monolayer, comprising culturing stem cells in a container having a surface coated with fibrinogen, wherein the surface is coated with more than 3 μg / mL of fibrinogen, and wherein the cells contact the fibrinogen and form the retinal pigment epithelial monolayer.

2. The method of claim 1, wherein the stem cells are induced pluripotent stem cells.

3. The method of claim 1 or 2, wherein the stem cells are human induced pluripotent stem cells.

4. The method according to any one of claims 1 to 3, wherein the container is a culture dish.

5. The method according to any one of claims 1 to 4, wherein the container is a culture flask.

6. 6. The method of any one of claims 1 to 5, wherein the surface comprises polystyrene, polycarbonate, mixed cellulose, PTFE, PDMS, PET, glass, a poly-L-lysine coating, or a combination thereof.

7. The method according to any one of claims 1 to 6, wherein the fibrinogen is human fibrinogen.

8. The method of any one of claims 1 to 7, wherein the surface is coated with about 3 to about 250 µg / mL of fibrinogen.

9. The method of any one of claims 1 to 8, wherein the surface is coated with about 15 to about 250 µg / mL of fibrinogen.

10. The method of any one of claims 1 to 9, wherein the surface is coated with about 25 to about 250 µg / mL of fibrinogen.

11. The method of any one of claims 1 to 10, wherein the surface is coated with about 50 to about 250 µg / mL of fibrinogen.

12. The method of any one of claims 1 to 11, wherein the surface is coated with about 75 to about 250 µg / mL of fibrinogen.

13. The method of any one of claims 1 to 12, wherein the surface is coated with the fibrinogen for about 1 to about 48 hours.

14. 14. The method of any one of claims 1 to 13, comprising culturing the cells for about 7 to about 90 days to form the retinal pigment epithelial monolayer.

15. The method of any one of claims 1 to 14, which is xeno-free.

16. A retinal graft comprising a retinal pigment epithelial monolayer, wherein the retinal pigment epithelial monolayer is produced according to the method of any one of claims 1 to 15.

17. 16. A method for treating an ocular condition, comprising transplanting into a mammalian eye a retinal graft comprising a retinal pigment epithelial monolayer, wherein the retinal pigment epithelial monolayer is produced according to the method of any one of claims 1 to 15.

18. 18. The method of claim 17, wherein the eye condition is macular degeneration.

19. 19. The method of claim 17 or 18, wherein the mammal is a human.

20. 1. A method for maintaining stem cells in culture, comprising culturing the stem cells in a container having a surface coated with fibrinogen, the surface being coated with greater than 250 μg / mL of fibrinogen, wherein the stem cells contact the fibrinogen and the stem cells maintain their ability to differentiate into cells of ectodermal, endodermal, and mesodermal origin after at least one passage.

21. 21. The method of claim 20, wherein the stem cells are induced pluripotent stem cells.

22. 22. The method of claim 20 or 21, wherein the stem cells are human induced pluripotent stem cells.

23. The method according to any one of claims 20 to 22, wherein the container is a culture dish.

24. The method according to any one of claims 20 to 23, wherein the container is a culture flask.

25. 25. The method of any one of claims 20 to 24, wherein the surface comprises polystyrene, polycarbonate, mixed cellulose, PTFE, PDMS, PET, glass, a poly-L-lysine coating, or a combination thereof.

26. The method of any one of claims 20 to 25, wherein the fibrinogen is human fibrinogen.

27. The method of any one of claims 20 to 26, wherein the surface is coated with about 250 to about 5000 μg / mL of fibrinogen.

28. The method of any one of claims 20 to 27, wherein the surface is coated with about 300 to about 900 μg / mL of fibrinogen.

29. The method of any one of claims 20 to 28, wherein the surface is coated with about 350 to about 750 μg / mL of fibrinogen.

30. 30. The method of any one of claims 20 to 29, wherein the surface is coated with about 400 to about 600 μg / mL of fibrinogen.

31. The method of any one of claims 20 to 30, wherein the surface is coated with about 450 to about 550 μg / mL of fibrinogen.

32. 32. The method of any one of claims 20 to 31, wherein the surface is coated with the fibrinogen for about 1 to about 48 hours.

33. 33. The method of any one of claims 20 to 32, comprising culturing the cells for about 2 days to about 90 days.

34. The method of any one of claims 20 to 33, which is xeno-free.

35. 1. A method for maintaining stem cells in culture, comprising culturing the stem cells in a vessel having a surface coated with fibrinogen, the surface being coated with greater than 3 μg / mL of fibrinogen, wherein the stem cells contact the fibrinogen and the stem cells maintain their ability to differentiate into cells of ectodermal, endodermal, and mesodermal origin after at least one passage.

36. 36. The method of claim 35, wherein the stem cells are induced pluripotent stem cells.

37. 37. The method of claim 35 or 36, wherein the stem cells are human induced pluripotent stem cells.

38. The method according to any one of claims 35 to 37, wherein the container is a culture dish.

39. The method of any one of claims 35 to 38, wherein the container is a culture flask.

40. 40. The method of any one of claims 35 to 39, wherein the surface comprises polystyrene, polycarbonate, mixed cellulose, PTFE, PDMS, PET, glass, a poly-L-lysine coating, or a combination thereof.

41. The method of any one of claims 35 to 40, wherein the fibrinogen is human fibrinogen.

42. The method of any one of claims 35 to 41, wherein the surface is coated with about 100 to about 5000 μg / mL of fibrinogen.

43. The method of any one of claims 35 to 42, wherein the surface is coated with about 300 to about 900 μg / mL of fibrinogen.

44. The method of any one of claims 35 to 42, wherein the surface is coated with about 100 to about 250 μg / mL of fibrinogen.

45. 43. The method of any one of claims 35 to 42, wherein the surface is coated with about 350 to about 750 μg / mL of fibrinogen.

46. The method of any one of claims 35 to 42, wherein the surface is coated with about 400 to about 600 μg / mL of fibrinogen.

47. 43. The method of any one of claims 35 to 42, wherein the surface is coated with about 450 to about 550 μg / mL of fibrinogen.

48. 48. The method of any one of claims 35 to 47, wherein the surface is coated with the fibrinogen for about 1 to about 48 hours.

49. 49. The method of any one of claims 35 to 48, comprising culturing the cells for about 2 days to about 90 days.

50. 50. The method of any one of claims 35 to 49, which is xeno-free.

51. The method of any one of claims 20 to 50, wherein the stem cells form endothelial cells.

52. 52. The method of any one of claims 20 to 51, wherein the fibrinogen is autologously obtained.

53. 1. A method for maintaining stem cells in culture, comprising culturing the stem cells in a container having a surface coated with a fibrin hydrogel, the fibrin hydrogel being formed with greater than 0.5 mg / mL of fibrinogen, wherein the stem cells contact the fibrin hydrogel and the stem cells maintain their ability to differentiate into cells of ectodermal, endodermal, and mesodermal origin after at least one passage.

54. 54. The method of claim 53, wherein the stem cells are induced pluripotent stem cells.

55. 55. The method of claim 53 or 54, wherein the stem cells are human induced pluripotent stem cells.

56. The method of any one of claims 53 to 55, wherein the container is a culture dish.

57. 57. The method of any one of claims 53 to 56, wherein the container is a culture flask.

58. 58. The method of any one of claims 53 to 57, wherein the surface comprises polystyrene, polycarbonate, mixed cellulose, PTFE, PDMS, PET, glass, a poly-L-lysine coating, or a combination thereof.

59. 59. The method of any one of claims 53 to 58, wherein the fibrinogen is human fibrinogen.

60. 60. The method of any one of claims 53 to 59, wherein the fibrin hydrogel is formed at a fibrinogen concentration of about 0.5 mg / mL to about 80 mg / mL.

61. 61. The method of any one of claims 53 to 60, wherein the fibrin hydrogel is formed at a fibrinogen concentration of about 10 mg / mL to about 50 mg / mL.

62. 62. The method of any one of claims 53 to 61, wherein the fibrin hydrogel is formed at a fibrinogen concentration of about 25 mg / mL to about 35 mg / mL.

63. 63. The method of any one of claims 53 to 62, wherein the fibrin hydrogel is polymerized using 0.5 to 500 U / mL of thrombin.

64. 64. The method of any one of claims 53 to 63, wherein the fibrin hydrogel comprises an antifibrinolytic agent.

65. 65. The method of claim 64, wherein the antifibrinolytic agent is tranexamic acid at a concentration of about 0.5 mg / mL to about 50 mg / mL.

66. 51. The method of any one of claims 38 to 50, wherein the antifibrinolytic agent is aprotinin at a concentration of about 0.1 U / mL to about 40 U / mL.

67. A method for producing endothelial cells, comprising culturing stem cells in a container having a surface coated with fibrinogen, wherein the surface is coated with more than 3 μg / mL of fibrinogen, and wherein the stem cells contact the fibrinogen and form endothelial cells.

68. 68. The method of claim 67, wherein the stem cells are induced pluripotent stem cells.

69. 69. The method of claim 67 or 68, wherein the stem cells are human induced pluripotent stem cells.

70. 70. The method of any one of claims 67 to 69, wherein the container is a culture dish.

71. 71. The method of any one of claims 67 to 70, wherein the container is a culture flask.

72. 72. The method of any one of claims 67-71, wherein the surface comprises polystyrene, polycarbonate, mixed cellulose, PTFE, PDMS, PET, glass, a poly-L-lysine coating, or a combination thereof.

73. 73. The method of any one of claims 67 to 72, wherein the fibrinogen is human fibrinogen.

74. 74. The method of any one of claims 67 to 73, wherein the surface is coated with about 3 to about 250 μg / mL of fibrinogen.

75. 75. The method of any one of claims 67 to 74, wherein the surface is coated with about 15 to about 250 μg / mL of fibrinogen.

76. 76. The method of any one of claims 67 to 75, wherein the surface is coated with about 25 to about 250 μg / mL of fibrinogen.

77. 77. The method of any one of claims 67 to 76, wherein the surface is coated with about 50 to about 250 μg / mL of fibrinogen.

78. 78. The method of any one of claims 67 to 77, wherein the surface is coated with about 75 to about 250 μg / mL of fibrinogen.

79. 79. The method of any one of claims 67 to 78, wherein the surface is coated with the fibrinogen for about 1 to about 48 hours.

80. 80. The method of any one of claims 67 to 79, comprising culturing the cells for about 7 to about 90 days to form endothelial cells.

81. 81. The method of any one of claims 67 to 80, wherein the method is xeno-free.

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