Generation and cryopreservation of pluripotent stem cell-derived clinical grade corneal endothelial cells

Abstract

To provide an alternative to donor corneas for corneal transplantation.SOLUTION: Human embryonic stem cell (hESC)- and induced pluripotent stem cell (iPSC)-derived clinical-grade corneal endothelial cell (CEC) generation and cryopreservation through the neural crest cell (NCC) lineage using peripheral blood as a donor source and hESCs maximizes the availability of these vital cells for treating the corneal endothelial disease as an alternative to donor corneas for corneal endothelium (CE) transplantation and other applications.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 778,862, filed December 12, 2018, the entire contents of which are incorporated herein by reference. Embodiments relate to the generation and cryopreservation of clinical-grade corneal endothelial cells from peripheral blood mononuclear cell (PBMC)-derived induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs), and uses thereof. [Background technology]

[0002] The cornea is the outermost transparent layer of the eye. It is composed of five layers: the epithelium, Bowman's membrane, stroma, Descemet's membrane and endothelium. 1 The corneal endothelium (CE) is a monolayer of hexagonal cells that is important in maintaining corneal transparency by mediating hydration via barrier and pump functions. 2 The CE cell density is approximately 2500 cells / mm in adult CE. 2 It is. 3 Corneal endothelial dystrophies and surgical trauma are major factors contributing to the loss of corneal endothelial cells (CECs) and the decrease in CE cell density. The physiological function of CEs is estimated to be below 500 cells / mm. 2 Less than cell density is compromised, resulting in corneal edema and loss of vision. 3

[0003] Fuchs endothelial corneal dystrophy (FECD) is the leading cause of corneal transplants performed annually in the United States and worldwide. 4、5 Although corneal transplants have been successful in restoring vision, graft rejection and the lack of suitable donor tissue for transplantation remain obstacles to reducing corneal blindness worldwide. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Delmonte DW,Kim T.Anatomy and physiology of the cornea.J Cataract Refract Surg.2011;37:588-598 [Non-Patent Document 2] Bonanno JA.Molecular mechanisms underlying the corneal endothelial pump. Exp Eye Res.2012;95:2-7 [Non-Patent Document 3] Claerhout I,Beele H,Kestelyn P.Graft failure:I.Endothelial cell loss.Int Ophthalmol.2008;28:165-173 [Non-Patent Document 4] Eye Bank Association of America. 2016 Eye banking Statistical Report. In: 2016 [Non-Patent Document 5] Gain P, Jullienne R, He Z, Aldossary M, Acquart S, Cognasse F, Thuret G. Global Survey of Corneal Transplantation and Eye Banking. JAMA Ophthalmol. 2016;134:167-173 Summary of the Invention [Problem to be solved by the invention]

[0005] Embodiments of the present invention relate, inter alia, to the generation and cryopreservation of clinical-grade corneal endothelial cells (CECs) derived from induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) of peripheral blood mononuclear cell (PBMC) origin, and their uses. [Means for solving the problem]

[0006] In certain embodiments, a method of producing corneal endothelial cells (CECs) comprises isolating peripheral blood mononuclear cells (PBMCs) from a biological sample, reprogramming the PBMCs to induce pluripotency to express pluripotency markers NANOG, OCT4, SOX2, SSEA4, TRA-1-60, and differentiating pluripotent stem cells, including induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs), to produce corneal endothelial cells (CECs). In certain embodiments, the step of culturing the cells comprises culturing iPSCs and hESCs on human recombinant protein, for example, on vitronectin (VTN-N) using Essential 8 medium. In certain embodiments, the differentiating cells (iPSCs and hESCs) express neural crest markers, NGFR, SOX10, or a combination thereof. In certain embodiments, the corneal endothelium (CE)-associated markers comprise AQP1, COL4A1, COL4A3, COL8A1, COL8A2, FOXC1, SLC16A3, or a combination thereof. In certain embodiments, mature CECs are identified by markers comprising AQP1, COL4A3, COL8A2, SLC16A3, or a combination thereof.

[0007] In certain embodiments, a method for preventing or treating a corneal-related disease or disorder includes producing corneal endothelial cells (CECs) from induced pluripotent stem cells (iPSCs) originating from human embryonic stem cells (hESCs) and peripheral blood mononuclear cells (PBMCs) and transplanting the CECs into the cornea of ​​a subject in need of a treatment. In certain embodiments, the corneal disease or disorder includes genetic disease or disorder, infection, wound, inflammatory disease, autoimmune disease, dry eye, dystrophy, or a combination thereof. In certain embodiments, the corneal disease or disorder includes Fuchs endothelial dystrophy (FECD), lattice dystrophy, keratoconus, herpes virus infection, varicella zoster virus infection, iridocorneal endothelial syndrome (ICE), pterygium, corneal ulcer, corneal abrasion, or bullous keratopathy.

[0008] In certain embodiments, the reprogramming method for producing induced pluripotent stem cells (iPSCs) includes a) infection of cells with reprogramming viral particles expressing c-MYC, KLF4, OCT3 / 4, SOX2 or a combination thereof, b) culturing the infected cells in a medium that supports the growth of the infected cells, and c) isolating iPSC colonies. In certain embodiments, iPSC colonies expressing pluripotency markers are selected and expanded. In certain embodiments, the pluripotency markers include NANOG, OCT4, SOX2, SSEA4, TRA-1-60 or a combination thereof. In certain embodiments, the cells expressing pluripotency markers are cultured on human recombinant vitronectin (VTN-N) using Essential 8 medium. In certain embodiments, iPSCs and human embryonic stem cells (hESCs) are confirmed by means including gene expression of pluripotency markers, protein expression of pluripotency markers, or a combination thereof.

[0009] In a particular embodiment, the present invention provides a method for stem cell therapy, comprising transplanting cells into a patient, the cells being obtained by inducing differentiation of induced pluripotent stem cells (iPSCs) obtained by the above-mentioned method using human embryonic stem cells (hESCs) and / or somatic cells isolated and harvested from the patient. The iPSCs prepared from somatic cells derived from healthy humans can be stored in an iPSC bank as a library of iPSCs, and one or more types of iPSCs in the library can be used for the preparation of somatic cells, tissues or organs that are not rejected by the patient undergoing stem cell therapy.

[0010] In certain embodiments, the present invention further provides methods for evaluating the physiological function or toxicity of compounds, medicines, toxicants, etc., by using various cells obtained by inducing differentiation of induced pluripotent stem cells (iPSCs) obtained by the above-mentioned methods.

[0011] In certain embodiments, the composition comprises cryopreserved corneal endothelial cells (CECs) produced from induced pluripotent stem cells (iPSCs) of human embryonic stem cells (hESCs) and peripheral blood mononuclear cells (PBMCs) origin.

[0012] In certain embodiments, the cryopreserved corneal endothelial cells (CECs) are placed in a vial. In certain embodiments, the cryopreserved corneal endothelial cells (CECs) are frozen in a cryopreservation composition or medium.

[0013] Other aspects are described below.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.In carrying out the present invention for testing, any method and material similar or equivalent to those described herein can be used, but the preferred materials and methods are described herein.In describing and claiming the present invention, the following terms are used.It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0015] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. Thus, the reference to "a cell," for example, includes a plurality of cells of the same type. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used anywhere in the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0016] As used herein, the terms "comprising," "comprise," or "comprised," and variations thereof, with respect to a defined or described element of an article, composition, apparatus, method, process, system, etc., are meant to be inclusive or open-ended and allow for additional elements, thereby indicating that the defined or described article, composition, apparatus, method, process, system, etc. includes those specified elements or their equivalents, as appropriate, and that other elements can be included and still fall within the scope / definition of the defined article, composition, apparatus, method, process, system, etc.

[0017] As used herein, "culture medium" or "cultivation medium" broadly refers to any liquid formulation designed to support the growth of cells. When referring to a formulation designed for the maintenance and growth of cells, the term "cell culture medium" is used. In the art, expressions such as induction medium, growth medium, differentiation medium, etc. can be considered as subspecies of the general expression culture medium. Those skilled in the art are familiar with the basic components required to maintain and nourish living subjects in or on culture medium, and commercially available basal media are widely available. Typically, such basic components are called "basal media" and contain the necessary amino acids, minerals, and organic compounds. In general, basal media can be obtained from biological sources such as serum, or can be combined from isolated pure components. If desired, the basal media can be supplemented with substances that contribute to the special characteristics or functions of the culture medium. Non-limiting examples of suitable basal media to be used in the present cell culture media of various embodiments include knockout serum replacement, Dulbecco's Modified Eagle Medium (DMEM) / F-12 medium, and any combination thereof.

[0018] As used herein, "differentiation" or "differentiate" or "differentiating" refers to the developmental process of lineage commitment. "Lineage" refers to a pathway of cell development in which precursor or progenitor cells undergo gradual physiological changes to become specific cell types with characteristic functions (e.g., nerve cells, muscle cells, T cells, B cells, red blood cells, or endothelial cells). Differentiation occurs in stages, whereby cells become progressively more specialized until they reach full maturity, also referred to as "terminal differentiation." Terminally differentiated cells are cells that have committed to a specific lineage and have reached the final stage of differentiation (i.e., fully mature cells).

[0019] The terms "patient" or "individual" or "subject" are used interchangeably herein and refer to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the present invention find use in laboratory animals, including but not limited to rodents, including mice, rats, rabbits, and primates, in veterinary applications, and in the development of animal models of disease.

[0020] "Prevent", "preventing", "prevention", and the like, generally in the context of a subject predisposed to having a particular disease, disorder, or condition, refer to a course of action (such as administering differentiated stem cells produced by the methods disclosed herein) that is initiated in such a way as to temporarily or permanently prevent, suppress, inhibit, or reduce the subject's risk of developing the disease, disorder, condition, and the like (e.g., as determined by the absence of clinical symptoms) or delay the onset of the disease, disorder, condition, and the like (e.g., prior to the onset of the disease, disorder, condition, or symptoms thereof). In certain instances, these terms also refer to slowing the progression of a disease, disorder, or condition, or inhibiting the progression of a disease, disorder, or condition to an adverse or otherwise undesirable state. As used herein, "in need of prevention" refers to a judgment made by a physician or other caregiver that a subject is in need of or would benefit from preventive care. This judgment is made based on a variety of factors that are within the physician's or caregiver's area of ​​expertise.

[0021] As used herein, "stem cell" refers to a mammalian cell that can undergo self-renewal and differentiate into multiple cell types. The term used herein includes natural and non-natural pluripotent (e.g., human embryonic stem cells and genetically modified induced pluripotent stem cells) and multipotent stem cells, unless otherwise specified. Thus, the term stem cell includes pluripotent stem cells, i.e., induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs); multipotent stem cells, i.e., hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), neural stem cells (NSCs) and epidermal stem cells; and genetically modified variants of each of the above stem cell types. iPSCs have an ESC-like morphology and express important pluripotency markers known to those skilled in the art, including, but not limited to, alkaline phosphatase, SSEA1, SSEA4, SOX2, OCT3 / 4, NANOG, TRA160, TRA181, TDGF1, DNMT3B, FOXD3, GDF3, CYP26A1, TERT and ZFP42.

[0022] As used herein, "TGFβ inhibitor" refers to an agent that reduces the activity of the TGFβ receptor by directly binding to the TGFβ receptor or by modulating the activity of any component of the TGFβ receptor pathway, such as SMAD, activin, nodal, bone morphogenetic protein (BMP), growth differentiation factor (GDF) or Müllerian inhibitory factor (MIF) or substances that promote the ubiquitination and degradation of the above proteins.

[0023] As used herein, "treating" a disease means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0024] As used herein, the term "xeno-free" refers to the absence of any foreign substances or components derived from animals.Thus, in the case of human cell culture, the term refers to conditions that do not contain non-human animal components.In other words, when xeno-free conditions are desired for the manufacture of ocular precursor cells or any cells matured therefrom for human use, embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) are selected to be of human origin. [Brief description of the drawings]

[0025] [Figure 1] Figure 1 is a series of images showing the generation of corneal endothelial cells (CECs) from induced pluripotent stem cells (iPSCs) of H9 human embryonic stem cells (hESCs) and human peripheral blood mononuclear cells (PBMC) origin. Phase contrast microscopy at various magnifications during CEC differentiation showing CEC-like hexagonal / polygonal morphology at day 20 (D20). [Diagram 2] Figure 2 is a series of images showing the characterization of corneal endothelial cells (CECs) derived from H9 human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) of human peripheral blood mononuclear cells (PBMCs) origin by immunocytochemistry. a-c, hESC-derived CECs immunostained for zona-tight junction-1 (ZO-1, a tight junction protein), d-f, human PBMC-origin iPSC-derived CECs immunostained for ZO-1, g-i, hESC-derived CECs immunostained for N-cadherin, j-l, PBMC-origin iPSC-derived CECs immunostained for N-cadherin. Cell nuclei were counterstained with DAPI. Images were captured using 60x magnification. Scale bar represents 10 μm. [Diagram 3]Figure 3 is a graph showing gene expression analysis of corneal endothelium (CE) and pluripotency-associated markers during differentiation of induced pluripotent stem cells (iPSCs) of human embryonic stem cell (hESC) and human peripheral blood mononuclear cell (PBMC) origin into corneal endothelial cells (CECs). At day 20 (D20), expression levels of seven CE-associated markers (AQP1, COL4A1, COL4A3, COL8A1, COL8A2, FOXC1, SLC16A3) and four pluripotency markers, namely NANOG, OCT4, SOX2, TRA-1-60, were analyzed by quantitative real-time PCR (qRT-PCR) in hESCs, hESC-derived CECs and iPSC-derived CECs of PBMC origin. Note: Expression of all markers is normalized to GAPDH and all values ​​are compared against their respective expression in hESCs. [Figure 4] Figure 4 is a series of images showing immunohistochemical characterization of H9 human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs) generated under feeder-free and xeno-free conditions. a-c, Immunostaining for zona junction-1 (ZO-1, a tight junction protein) of hESC-derived CECs. [Diagram 5] FIG. 5 shows the results of a comparative analysis of non-cryopreserved and cryopreserved H9 human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs) by next-generation RNA sequencing (RNA-Seq). a) Venn diagram showing the overlap between transcriptome datasets of non-cryopreserved and cryopreserved hESC-derived CECs. b) Pearson correlation (Pearson correlation R=0.975) showing normalized expression of non-cryopreserved and cryopreserved hESC-derived CEC datasets showing high correlation between both datasets. c) Box plot showing the range of variation within non-cryopreserved and cryopreserved hESC-derived CEC transcriptome datasets. d) Violin diagram showing the expression intensity distribution of genes within non-cryopreserved and cryopreserved hESC-derived CEC transcriptome datasets. [Figure 6]Figure 6 is a series of images showing regenerated corneal endothelium (CE) in rabbits after injection of cryopreserved human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs). Representative photographs of rabbit eyes injected with cryopreserved hESC-derived CECs at 1, 4, 7, 14, 21 days and 9 months. After injection of cryopreserved hESC-derived CECs into the anterior chamber, the right eye exhibits a clear cornea, similar to the left untreated control eye. [Figure 7] Figure 7 is a series of images showing the evaluation of regenerated corneal endothelium (CE) in rabbits after injection of cryopreserved human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs). The regenerated CEs were evaluated 6 and 9 months after injection using a confoscan4 scanning microscope. The injected eye exhibits a nearly similar CEC density and shape to the left untreated eye. [Figure 8] FIG. 8 is a series of images showing immunohistochemical characterization of regenerated corneal endothelium (CE) after injection of cryopreserved human embryonic stem cell (hESC)-derived corneal endothelial cells (CEC) in rabbits 9 months after injection. Immunostaining for junction of adhesions-1 (ZO-1), ATPase sodium / potassium subunit α1 (Na+ / K+ATPaseα1) and N-cadherin shows similar expression patterns in regenerated CE and left untreated control CE. Note: Cell nuclei were counterstained with DAPI. Images were captured using 60x magnification and scale bar represents 10 μm. [Figure 9] 9 is a series of images showing regenerated corneal endothelium (CE) in monkeys after injection of cryopreserved human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs). Representative photographs of monkey eyes injected with hESC-derived CECs at 1, 2, 4 days, and 1 week, 1 month, and 1 year. After injection of cryopreserved hESC-derived CECs into the anterior chamber, the right eye exhibits a clear cornea, similar to the left untreated control eye. [Figure 10]Figure 10 is a series of images showing the evaluation of regenerated corneal endothelium (CE) in monkeys after injection of cryopreserved human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs). The regenerated CEs were evaluated 6 months and 1 year after injection using a confoscan4 scanning microscope. The injected eye exhibits a nearly similar CEC density and shape to the left untreated eye. [Figure 11] FIG. 11 is an image showing immunohistochemical characterization of regenerated corneal endothelium (CE) after injection of cryopreserved human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs) one year after injection. Immunostaining for junction of tight junction-1 (ZO-1), ATPase sodium / potassium subunit α1 (Na+ / K+ATPase α1) and N-cadherin shows similar expression patterns in regenerated CE and left untreated control CE. Note: Cell nuclei were counterstained with DAPI. Images were captured using 60x magnification and scale bar represents 10 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention is based in part on the development of corneal endothelial cells (CECs) from induced pluripotent stem cells (iPSCs) of human embryonic stem cells (hESCs) and peripheral blood mononuclear cells (PBMCs) origin. Briefly, hESCs and iPSCs were differentiated into CECs via neural crest cells (NCCs). The morphology of differentiating hESCs and iPSCs was examined at regular intervals by phase contrast microscopy. In parallel, the expression of pluripotency and corneal endothelium (CE)-associated markers was investigated by quantitative real-time PCR (qRT-PCR). hESC- and iPSC-derived CECs were firmly adherent and showed a hexagon-like shape, one of the cardinal characteristics of CECs. CE-associated markers were expressed at significantly higher levels in hESCs and iPSC-derived CECs at day 20 compared to their respective levels in hESCs. Importantly, only residual expression levels of pluripotency markers were detected in hESC- and human induced pluripotent stem cell (iPSC)-derived CECs.

[0027] Human Embryonic Stem Cells (hESCs)

[0028] Human embryonic stem cells (also known as hESCs) are a type of pluripotent stem cell.

[0029] Currently, pluripotent stem cells hold great promise in the field of regenerative medicine. The aim is to repair tissues altered by accident, disease or aging. This represents a new therapeutic field with enormous medical impact, since it offers the possibility of treating and curing diseases for which no suitable treatment currently exists. Regenerative medicine is applicable in most medical fields and constitutes one of the most promising developments of the biotechnology industry. Ischemia, degeneration and / or age-related diseases are the main causes of death in the population of developed countries.

[0030] Human induced pluripotent stem cells (iPSCs)

[0031] Induced pluripotent stem cells (iPSCs) provide an autologous cell source for replacement cell therapy to replace or regenerate tissues by autologous transplantation. In addition, patient-specific iPSCs can serve as in vitro models for disease mechanism modeling and drug screening. The original set of reprogramming factors (also called Yamanaka factors) includes the genes OCT4, SOX2, cMYC and NANOG. More recently, it has been reported that some specific combinations including three of these factors also generate iPSCs. On the other hand, human embryonic stem cells (hESCs) provide an unlimited source of allogeneic cells for therapeutic purposes.

[0032] Thus, in certain embodiments, a method for producing corneal endothelial cells (CECs) includes isolating peripheral blood mononuclear cells (PBMCs) from a biological sample, reprogramming the PBMCs with pluripotency factors, selecting and culturing cells expressing pluripotency markers, and differentiating these cells to produce corneal endothelial cells (CECs). In certain embodiments, the reprogramming or pluripotency factors include c-MYC, KLF4, OCT3 / 4, SOX2, or combinations thereof. In certain embodiments, culturing the cells with one or more differentiation factors includes culturing the cells on a gelatinous protein coating.

[0033] In certain embodiments, the gelatinous protein comprises human recombinant vitronectin protein. In certain embodiments, the medium comprises Dulbecco's Modified Eagle Medium (DMEM) / F-12 medium and knockout serum replacement or a combination thereof. In certain embodiments, the medium comprises a TGF-β inhibitor, a fibroblast growth factor or a combination thereof. These induction supplements enhance the differentiation of pluripotent stem cells into ocular precursor cells and improve the efficiency of further differentiation of the precursor cells into clinically valuable ocular cells, such as corneal endothelial precursor cells. In certain embodiments, the differentiating cultured cells express neural crest markers, corneal endothelium (CE)-associated markers or a combination thereof. In certain embodiments, the neural crest markers include NGFR, SOX10 or a combination thereof. In certain embodiments, the CE-associated markers include AQP1, COL4A1, COL4A3, COL8A1, COL8A2, FOXC1, SLC16A3 or a combination thereof. In certain embodiments, mature CECs are identified by markers including AQP1, COL4A3, COL8A2, SLC16A3, or a combination thereof.

[0034] corneal endothelium

[0035] The cornea is the outermost tissue of the eye that covers the anterior portion of the ocular tissue. Corneal transparency is essential for clear vision, and lack of corneal transparency can result in corneal edema, impaired vision, and ultimately blindness. The endothelium is the innermost layer of the cornea and is essential for the dynamic regulation of corneal hydration. It is noteworthy that unlike other cells in the human body, corneal endothelial cells (CECs) damaged by disease and / or trauma are not repaired or replaced by the body. Nearly half of the corneal transplants performed in the United States each year are due to some form of corneal endothelial disease.

[0036] Thus, in certain embodiments, clinical grade corneal endothelial cells (CECs) derived from peripheral blood mononuclear cells (PBMCs)-derived induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) are produced and subsequently cryopreserved. In certain embodiments, CECs are generated from human embryonic stem cells. In certain embodiments, treatment of patients with autologous cells, e.g., PBMCs, allows for personalized medicine, establishing a "patient-specific" therapeutic strategy to generate a renewable supply of CECs for corneal endothelial dystrophies, reducing both donor cornea and corneal transplant dependency. Alternatively, the use of human embryonic stem cells provides an unlimited allogeneic supply of CECs for corneal endothelial dysfunction.

[0037] Generation of corneal endothelial cells (CECs) using human embryonic stem cells (hESCs) or peripheral blood-derived induced pluripotent stem cells (iPSCs) via the neural crest cell (NCC) lineage will maximize the availability of these important cells to treat corneal endothelial diseases, as a replacement for donor corneas for CE transplantation and other applications.

[0038] Corneal and Related Disorders

[0039] Corneal dystrophies. The term corneal dystrophies encompasses a heterogeneous group of bilateral, genetically determined, non-inflammatory corneal diseases that are confined to the cornea. Clinically, based on the sole or primary anatomical location of the abnormality, corneal dystrophies can be divided into three groups. Some primarily affect the corneal epithelium and its basement membrane or Bowman's layer and superficial corneal stroma (anterior corneal dystrophies), the corneal stroma (corneal stromal dystrophies), or Descemet's membrane and corneal endothelium (posterior corneal dystrophies). Most corneal dystrophies have no systemic symptoms and present with various forms of corneal opacity in a clear or cloudy cornea, affecting vision to various degrees. Corneal dystrophies may have a simple autosomal dominant, autosomal recessive or X-linked recessive Mendelian pattern of inheritance. Mutations in the CHST6, KRT3, KRT12, PIP5K3, SLC4A11, TACSTD2, TGFB1 and UBIAD1 genes cause different corneal dystrophies.

[0040] Like other corneal dystrophies, superficial corneal dystrophies are genetically determined and usually inherited as a Mendelian trait. The unique phenotypes of most corneal dystrophies are caused by mutations in different specific genes. Some corneal dystrophies phenotypes result from mutations in different genes, and different phenotypes can be the result of different mutations in the same gene. Mutations in four genes (KRT3, KRT12, TGFB1, and TACSTD2) are currently known to cause inherited diseases that are apparently restricted to the superficial cornea.

[0041] Corneal stromal dystrophies. This group of corneal dystrophies includes macular corneal dystrophy (MCD), granular corneal dystrophy (GCD) type I, lattice corneal dystrophy (LCD), Schneiderian corneal dystrophy (SCD), spotted corneal dystrophy (FCD), congenital corneal stromal dystrophy (CSCD), and posterior amorphic corneal dystrophy (PACD).

[0042] Posterior corneal dystrophies. The group of posterior corneal dystrophies includes Fuchs endothelial dystrophy (FECD), posterior polymorphous corneal dystrophy (PPCD), congenital hereditary endothelial corneal dystrophy (CHED) and X-linked endothelial corneal dystrophies (XECD). These diseases are characterized by abnormalities of the corneal endothelium and Descemet's membrane. In most of the posterior corneal dystrophies, defective active fluid transport by the corneal endothelium causes excessive edema of the corneal stroma, which impairs corneal transparency and reduces visual acuity.

[0043] The prevalence of Fuchs endothelial corneal dystrophy (FECD) varies significantly in different parts of the world. Fuchs endothelial corneal dystrophy is common and the most common corneal dystrophy in the United States, affecting approximately 4% of the population over the age of 40. In the United States, FECD is the main indication for penetrating corneal transplantation, and this surgical procedure accounted for 10-25% of all corneal transplants in different systems. This is a significant number considering that the annual number of corneal transplants in the United States is less than 32,000.

[0044] Fuchs endothelial corneal dystrophy (FECD) is a slowly progressing disease that usually affects both eyes and is slightly more common in women than men. FECD is caused by the gradual deterioration of cells in the corneal endothelium (CE) for a poorly understood cause. Normally, these endothelial cells maintain a healthy balance of fluid within the cornea. Healthy endothelial cells prevent the cornea from swelling and keep the cornea clear. In Fuchs dystrophy, endothelial cells slowly die, causing fluid buildup and swelling within the cornea. The cornea thickens and vision becomes blurred.

[0045] As the disease progresses, Fuchs endothelial corneal dystrophy (FECD) symptoms usually affect both eyes and include glare that affects vision in low light; blurred vision that occurs in the morning after waking and gradually improves during the day; astigmatism, sensitivity to light, night blindness, and seeing halos around lights at night; small, painful blisters on the surface of the cornea; and a cornea that appears cloudy or opaque.

[0046] Keratoconus. Keratoconus is the progressive thinning of the cornea. Keratoconus is the most common corneal dystrophy in the United States, affecting 1 in every 2,000 Americans. Keratoconus is most common in teenagers and adults in their 20s. Keratoconus causes the center of the cornea to thin and bulge outward, forming a rounded, conical shape. This abnormal curvature of the cornea can cause double or blurred vision, myopia, astigmatism, and increased sensitivity to light. A small number of people with keratoconus may develop severe corneal scarring or be unable to tolerate contact lenses. These people may require a corneal transplant.

[0047] Iridocorneal endothelial syndrome (ICE). ICE is more common in women and usually begins between the ages of 30 and 50. ICE has three major characteristics: visible changes in the iris, swelling of the cornea, and glaucoma.

[0048] Iridocorneal endothelial syndrome (ICE) is usually present in only one eye. Iridocorneal endothelial syndrome is caused by the migration of endothelial cells from the cornea to the iris. This loss of cells from the cornea results in swelling of the cornea and distortion of the iris and pupil. This migration of cells also blocks the eye's fluid outflow channels, causing glaucoma. There is no treatment to stop the progression of ICE, but glaucoma is treatable. If the cornea swells significantly and vision is significantly impaired, a corneal transplant may be necessary.

[0049] Bullous keratopathy. Bullous keratopathy is caused by edema of the cornea and results from the inability of the corneal endothelium to maintain the cornea's normal dehydrated state. Most frequently, bullous keratopathy results from Fuchs endothelial dystrophy (FECD) or trauma to the corneal endothelium. Another frequent cause of bullous keratopathy is trauma to the corneal endothelium, which can occur during intraocular surgery (e.g., cataract removal) or after placement of a poorly designed or placed intraocular lens implant. Bullous keratopathy after cataract removal is referred to as pseudophakic (if an intraocular lens implant is present) or aphakic (if an intraocular lens implant is not present) bullous keratopathy.

[0050] As the corneal stroma (the deeper layer of the cornea) swells, subepithelial fluid-filled blisters form on the corneal surface, resulting in ocular discomfort, decreased vision, loss of contrast, glare, and photophobia. Sometimes the blisters rupture, causing pain and a foreign body sensation. Bacteria can invade the ruptured blisters, resulting in corneal ulcers.

[0051] Treatment Method

[0052] In certain embodiments, a method for preventing or treating a corneal-related disease or disorder includes producing clinical grade corneal endothelial cells (CECs) from induced pluripotent stem cells (iPSCs) of human embryonic stem cells (hESCs) or peripheral blood mononuclear cells (PBMCs) origin, and transplanting the CECs into the cornea of ​​a subject in need of treatment. In certain embodiments, the corneal disease or disorder includes a genetic disease or disorder, an infection, a wound, an inflammatory disease, an autoimmune disease, dry eye, a dystrophy, or a combination thereof. In certain embodiments, the corneal disease or disorder includes Fuchs endothelial corneal dystrophy (FECD), keratoconus, keratoconjunctivitis sicca (KCS), iridocorneal endothelial syndrome (ICE), keratitis, corneal ulcer, corneal abrasion, or bullous keratopathy.

[0053] In certain embodiments, the composition comprises a therapeutically effective amount of corneal endothelial cells (CECs) produced from induced pluripotent stem cells (iPSCs) of human embryonic stem cell (hESC) or peripheral blood mononuclear cell (PBMC) origin.

[0054] In certain embodiments, the composition comprises a therapeutically effective amount of cells infected with pluripotency factors to generate corneal endothelial cells (CECs) derived from induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs) originating from peripheral blood mononuclear cells (PBMCs). In embodiments, the method of the present invention is applied ex vivo. That is, the subject's cells can be removed from the body, treated with the composition in culture, excised, and the treated cells can be returned to the subject's body. In some embodiments, the cells are human leukocyte antigen (HLA)-matched cell lines, autologous cell lines, allogeneic cell lines, or combinations thereof.

[0055] When using induced pluripotent stem cells (iPSCs) for therapeutic treatment of disease, it is desirable to use somatic cells isolated from patients. For example, somatic cells involved in disease, somatic cells involved in therapeutic treatment of disease, etc. can be used. Various media that can maintain the undifferentiated state and pluripotency of human embryonic stem cells (hESCs) are known in the art, and iPSCs can be efficiently isolated by using a combination of appropriate media. The differentiation and proliferation capabilities of isolated iPSCs and hESCs can be easily confirmed by those skilled in the art by using confirmation means that are widely applied to hESCs.

[0056] In certain embodiments, induced pluripotent stem cells (iPSCs) of peripheral blood mononuclear cell (PBMC) origin can include a population of stem cells such as embryonic stem cells (ESCs). ESCs have been established from many animal species, including humans. These types of pluripotent stem cells are useful cell sources for regenerative medicine, since these cells retain the ability to actively divide while maintaining their pluripotency, and can be differentiated into almost any organ by appropriate induction of differentiation.

[0057] In some aspects, the sample from which the cell is derived or isolated is blood or blood-derived sample.In certain embodiments, the cell is derived from peripheral blood mononuclear cell (PBMC).Samples include samples from autologous and allogeneic sources in the context of cell therapy, such as adoptive cell therapy.

[0058] In some embodiments, the isolation of cells includes one or more preparative and / or non-affinity-based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove undesirable components, enrich for desired components, and lyse or remove cells that are sensitive to a particular reagent. In some examples, cells are separated based on one or more properties, such as density, adhesive properties, size, sensitivity and / or resistance to a particular component.

[0059] In some examples, the sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some embodiments, contains cells other than red blood cells and platelets.

[0060] In some embodiments, blood cells taken from a subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution contains calcium and / or magnesium (Ca ++ / Mg ++ ) and / or many or all divalent cations. In some embodiments, the cells are, after washing, depleted of, for example, Ca ++ / Mg ++ The cells are resuspended in a variety of biocompatible buffers, such as PBS without IgG. In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0061] In some embodiments, the method comprises a density-based cell separation method, such as separation of leukocytes from peripheral blood by centrifugation through a Percoll or Ficoll gradient.

[0062] In some embodiments, the isolation method comprises the separation of different cell types based on the expression or presence in cells of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known separation method based on such markers can be used. In some embodiments, the separation is an affinity or immunoaffinity based separation. For example, the isolation in some aspects comprises the separation of cells and cell populations based on the expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, generally followed by a washing step and separation of the cells bound by the antibody or binding partner from the cells not bound by the antibody or binding partner.

[0063] Separation need not result in 100% enrichment or removal of a particular cell population or cells that express a particular marker. For example, positive selection or enrichment for a particular type of cell, such as cells that express a marker, refers to increasing the number or proportion of such cells, but not necessarily the complete elimination of cells that do not express the marker.

[0064] Once corneal endothelial cells (CECs) are generated from induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs) of peripheral blood mononuclear cells (PBMCs) origin, the cells are transplanted into the cornea. The cells embodied herein are prepared for transplantation according to established methods. After approximately 3-4 weeks of culture, the cells are cryopreserved. In this regard, the growth characteristics of the cells vary from patient to patient and from cell type to cell type. Approximately 72 hours prior to the transplantation of the cells, an aliquot is taken for analysis of the phenotype and the percentage of cells expressing corneal endothelium (CE)-associated markers. Administration can be achieved by a single dose.

[0065] However, it will be understood that the therapeutic amount and frequency of administration of corneal endothelial cells (CECs) for any particular subject may vary and will depend on a variety of factors, including the activity and viability of the particular cells used, as well as the age, weight, general health, sex, diet, mode and time of administration, engraftment rate, drug combinations, the severity of the particular condition, and the host being treated. In some embodiments, a therapeutically effective amount of CECs transplanted into a subject is an amount that allows such transplanted cells to engraft in the subject, whereby such cells ameliorate the symptoms of the disease by inducing a desired biological response.

[0066] For better clinical acceptance, all culture media used herein are preferably substantially xeno-free, substantially serum-free, or chemically defined, more preferably a combination thereof, and most preferably simultaneously xeno-free, serum-free, and chemically defined. As used herein, substantially means what is considered and accepted as xeno-free, serum-free, or chemically defined under clinical or research regulations, irrespective of unintentional traces, and applies here as well.

[0067] kit

[0068] In certain embodiments, a kit comprising a pharmaceutical composition and a therapeutically effective amount of clinical grade corneal endothelial cells (CECs) produced from induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs) of peripheral blood mononuclear cell (PBMC) origin.

[0069] All citations to sequences, patents, and publications in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference. EXAMPLES

[0070] [Example 1] Generation and cryopreservation of clinical-grade corneal endothelial cells derived from pluripotent stem cells

[0071] The derivation and cryopreservation of clinical-grade corneal endothelial cells (CECs) from peripheral blood mononuclear cell (PBMC)-origin induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) is reported herein. Proteomic characterization of iPSC-derived CECs under xenophilic conditions was achieved through mass spectrometry-based protein sequencing and was then compared with the proteome of human corneal endothelial (CE) specimens obtained from the Eye Bank. This analysis provides evidence that more than 90% of the proteins expressed in donor CEs are present in iPSC-derived CECs. Similarly, transcriptomic characterization of hESC- and iPSC-derived CECs under xenophilic conditions was completed by next-generation-based RNA sequencing (RNA-Seq). Comparative transcriptome analysis of hESC- and iPSC-derived CECs revealed 13,208 (>96%) genes common to both transcriptomes.

[0072] material and method

[0073] Subjects and Clinical Review. Institutional Review Board (IRB) approval for studies involving human subjects and pluripotent stem cells was obtained from the Johns Hopkins University School of Medicine, Baltimore, MD. Control subjects gave informed consent consistent with the tenets of the Declaration of Helsinki.

[0074] Isolation and cryopreservation of peripheral blood mononuclear cells (PBMCs). PBMCs were isolated according to the procedure published by Agu et al. 6 Briefly, 10 ml of human peripheral blood was collected in a 50 ml conical tube containing 0.5 M EDTA, and 2 volumes of phosphate-buffered saline (PBS) were added. The mixture was gently layered onto 15 ml of histopaque (Sigma, New York, NY) and centrifuged at 400 g for 30 min (no brake).

[0075] The mononuclear cell layer was gently harvested in a 50 ml tube and washed twice with phosphate-buffered saline (PBS) by centrifugation at 300 g for 10 min. Peripheral blood mononuclear cell (PBMC) pellets were cryopreserved in medium containing 10% dimethyl sulfoxide (DMSO; Sigma).

[0076] Human embryonic stem cells (hESCs). H9 hESCs (WiCell Research Institute, Madison, WI, USA) were cultured in Essential 8 medium (Life Technologies) in feeder-free conditions on vitronectin (VTN-N)-coated plates. Cells were passaged every 4–5 days with 0.5 mM EDTA and the culture medium was changed daily.

[0077] Generation of induced pluripotent stem cells (iPSCs) using the Sendai virus delivery system. Cryopreserved peripheral blood mononuclear cells (PBMCs) were reprogrammed using the Sendai virus delivery system according to the manufacturer's instructions (Life Technologies, Carlsbad, CA). Briefly, PBMC vials were removed from liquid nitrogen and thawed at 37°C in a water bath. PBMCs were then washed with StemSpan medium (Stemcell Technologies, Vancouver, Canada) and cultured at 37°C for 1 h in StemSpan medium supplemented with 100 ng / ml FMS-like tyrosine kinase 3 ligand (FLT-3L), 100 ng / ml stem cell factor (SCF), 20 ng / ml interleukin-3 (IL-3) and 20 ng / ml interleukin-6 (IL-6) (hereafter referred to as complete medium) at 37°C for 24 h at 5% CO. 2 The cells were cultured for 4 days in a humidified incubator at 37°C supplemented with

[0078] Approximately 5 × 10 cells in 1 ml of complete medium in a round-bottom tube 5Peripheral blood mononuclear cells (PBMCs) were collected at 1000 g / ml, followed by infection with reprogramming viral particles at a multiplicity of infection (MOI) of 5, 5 and 3 (KOS MOI=5, hc-Myc MOI=5, hKlf4 MOI=3). Infected cells were centrifuged at 1000 g for 30 min at room temperature. Cells were resuspended in 1 ml complete medium, transferred to a single well of a 12-well plate and cultured in complete medium for 3 days. Cells were then transferred to HEF (human embryonic fibroblast) coated plates containing complete medium for 3 days and finally transferred to Dulbecco's modified Eagle's medium (DMEM) / F-12 medium supplemented with 20% knockout serum substitute (KSR; Life Technologies, Carlsbad, CA) and 20 ng / ml basic fibroblast growth factor (bFGF; Life Technologies, Carlsbad, CA) until embryonic stem cell-like colonies were formed. Putative embryonic stem cell-like iPSC colonies were selected and cultured on vitronectin-coated plates in Essential 8 medium for characterization.

[0079] Phase-contrast microscopy. Phase-contrast microscopy was performed using a Zeiss Axio Observer A1 inverted microscope (Zeiss, Germany) equipped with Q-Capture software (Qlmaging, Surrey, Canada).

[0080] Characterization of induced pluripotent stem cells (iPSCs) by flow cytometry. iPSCs of peripheral blood mononuclear cell (PBMC) origin were characterized by flow cytometry. Approximately 2 × 10 5 Cells were labeled for 1 h at 4°C and subsequently treated with FITC-conjugated goat anti-mouse IgG antibodies (against SSEA4 and TRA-1-60; Millipore, Billerica, MA) for 40 min at 4°C. A total of 10,000 events were acquired for each sample using a Guava easyCyte flow cytometer (Millipore BDScience, Billerica, MA).

[0081] Characterization of induced pluripotent stem cells (iPSCs) by quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA from H9 human embryonic stem cells (hESCs) and iPSCs of peripheral blood mononuclear cell (PBMC) origin was extracted using TRIzol reagent (Invitrogen; Carlsbad, CA) according to the manufacturer's instructions. RNA was quantified with a NanoDrop Lite spectrophotometer (Thermo Scientific, Waltham, MA). First-strand cDNA was synthesized using the Superscript III kit (Invitrogen) according to the manufacturer's instructions.

[0082] Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on a STEP ONE ABI Real-Time PCR System. Expression of pluripotency markers (NANOG, OCT4, SOX2 and TRA-1-60) was quantified using Power SYBR Green PCR Master Mix (Life Technologies). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. Relative expression was determined using the delta-delta Ct method and normalized to GAPDH as previously reported. 7、8 All primers used in qRT-PCR analysis were designed using Real-time PCR tools (Integrated DNA Technologies).

[0083] Differentiation of induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) into corneal endothelial cells (CECs). iPSCs and hESCs of peripheral blood mononuclear cell (PBMC) origin were used to generate CECs by modifying a previously published procedure. 9Briefly, iPSCs and hESCs were plated on 35 mm vitronectin-coated plates (Thermo Fisher Scientific) at a 1:7 dilution (70% confluent plate split into 7 plates) using 0.5 mM EDTA (Life Technologies) on day 0. iPSCs and hESCs were grown for 4 days in Essential 8 medium (Life Technologies). On day 4, Essential 8 medium was replaced with dual Smad inhibitor medium containing 500 ng / ml human recombinant Noggin (R&D Systems, Minneapolis, MN) and 10 μM SB431542 (MilliporeSigma) in basal medium containing 80% Dulbecco's modified Eagle's medium (DMEM) / F-12 medium (Life Technologies), 20% knockout serum replacement (KSR; Life Technologies), 1% non-essential amino acids (Life Technologies), 1 mM L-glutamine (Life Technologies), 0.1 mM β-mercaptoethanol (Life Technologies) and 8 ng / ml basic fibroblast growth factor (bFGF; R&D Systems).

[0084] On day 6, the dual Smad inhibitor medium was replaced by corneal medium containing 0.1x B27 supplement (Life Technologies), 10ng / ml recombinant human platelet-derived growth factor-BB (PDGF-BB; R&D Systems) and 10ng / ml recombinant human Dickkopf-related protein-2 (Dkk-2; R&D Systems) in a basal medium of 80% Dulbecco's modified Eagle's medium (DMEM) / F-12 medium (Life Technologies), 20% knockout serum replacement (KSR; Life Technologies), 1% non-essential amino acids (Life Technologies), 1mM L-glutamine (Life Technologies), 0.1mM β-mercaptoethanol (Life Technologies) and 8ng / ml bFGF (R&D Systems). On day 7, the differentiating corneal endothelial cells (CECs) were transferred to new vitronectin-coated plates (35mm) and grown in corneal medium for an additional 13 days.

[0085] Characterization of human embryonic stem cell (hESC)- and induced pluripotent stem cell (iPSC)-derived corneal endothelial cells (CECs) by quantitative real-time PCR (qRT-PCR). Total RNA from day 20 H9 hESCs, iPSCs and differentiated CECs was extracted using TRIzol reagent (Invitrogen). First-strand cDNA was synthesized using the Superscript III kit (Invitrogen) as described above. Expression of pluripotency markers (NANOG, OCT4, SOX2 and TRA-1-60) and corneal endothelium (CE)-associated markers (AQP1, COL4A1, COL4A3, COL8A1, COL8A2, FOXC1 and SLC16A3) was quantified using qRT-PCR as described above. Primers were designed using a Real-Time PCR tool (Integrated DNA Technologies) and are available upon request.

[0086] Characterization of human embryonic stem cell (hESC)- and induced pluripotent stem cell (iPSC)-derived corneal endothelial cells (CECs) by immunocytochemistry. hESC- and iPSC-derived CECs were also analyzed by immunocytochemistry. Briefly, cells were fixed with 4% paraformaldehyde (PFA) for 15 min, followed by blocking with 5% bovine serum albumin (BSA; Sigma). First, cells were incubated overnight at 4°C with junction of adhesions-1 (ZO1, Cell Signaling Technology) and N-cadherin (Cell Signaling Technology) primary antibodies. Then, cells were treated with FITC-conjugated goat anti-rabbit IgG (against ZO1; MilliporeSigma) and FITC-conjugated goat anti-mouse IgG (against N-cadherin; MilliporeSigma) secondary antibodies for 2 h at room temperature. Nuclei were counterstained with 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI). Images of mounted cells were captured using an Olympus LX 81 microscope (Olympus, Tokyo, Japan) equipped with Slidebook Software (3i Denver, CO) and prepared using Adobe Photoshop CS5.

[0087] Cryopreservation of human embryonic stem cell (hESC)- and induced pluripotent stem cell (iPSC)-derived corneal endothelial cells (CECs) and their characterization. Differentiated CECs at day 20 were detached using cell dissociation buffer (Life Technologies), resuspended in CryoStor cell cryopreservation medium, and cryopreserved. Cryopreserved hESC- and iPSC-derived CECs were cultured after 1 week and maintained in corneal medium until they reached confluence. Expression of corneal endothelium (CE)-associated markers (AQP1, ATP1A1, ATP1A3, COL4A1, COL4A3, COL8A1, COL8A2, FOXC1, and SLC16A3) was quantified using quantitative real-time PCR (qRT-PCR) as described above. hESC- and iPSC-derived CECs expressed tight junction protein (ZO-1), N-cadherin, and CE pump function protein (Na + / K +They were also characterized by immunocytochemistry for the expression of ATPase α1).

[0088] RNA-Seq analysis of non-cryopreserved and cryopreserved hESC-derived CECs. Next-generation-based RNA-Seq analysis of day 20 hESC-derived CECs (non-cryopreserved) and cryopreserved hESC-derived CECs (cryopreserved in liquid nitrogen for 40 days) was performed. Briefly, three biological replicates for each of non-cryopreserved and cryopreserved hESC-derived CECs were used for RNA-Seq library preparation. Total RNA was isolated from each sample using TRIzol reagent (Invitrogen). Extracted RNA was examined using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific) and an RNA 6000 Pico Kit on an Agilent 2100 Bioanalyzer (Agilent; Palo Alto, CA).

[0089] Total RNA was subjected to RNA-Seq library preparation using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA). Approximately 1.0 μg of high-quality total RNA (>8.0 RIN score) was used for polyadenylated RNA selection using oligo-dT beads, followed by fragmentation of the selected polyadenylated RNA. The fragmented RNA was used as a template for cDNA synthesis by reverse transcriptase with random primers. The cDNA was further converted to double-stranded DNA, which was end-repaired to incorporate specific index adapters for multiplexing, followed by a purification step and 15 cycles of amplification. The amplified libraries were tested on an Agilent 2100 Bioanalyzer (Agilent) and quantitative PCR (qPCR) according to the manufacturer's instructions.

[0090] RNA-Seq libraries with unique index sequences were pooled in equimolar ratios and final size-selected to 400–500 bp. After quality control checks, the three RNA-Seq barcoded, pooled libraries were sequenced (2 × 100 bp) in a single lane on a HiSeq 2500 (rapid run mode) genome analyzer. Base calls were assigned through Illumina Real-Time Analysis (RTA) software (Version 1.17.20), and base call files were converted from binary format (BCL) to flat file format (qseq.txt) using Illumina BCL Converter software (Ver. 1.9.4). The qseq.txt files were demultiplexed into single-sample FASTQ files using the demultiplexing software.

[0091] Raw RNA-Seq reads (FASTQ) were processed and analyzed using Lasergene Genomics Suite (DNASTAR, Madison, WI, USA). Paired-end reads were assembled and aligned to the human reference genome (GRCh38.p11) with SeqMan NGen (Ver.12) using default parameters. ArrayStar (Ver, 12; DNASTAR) was used for normalization of mapped paired-end reads, differential gene expression analysis, and statistical analysis using default parameters. FPKM was used for gene expression normalization and quantification. 30 Spotfire DecisionSite with Functional Genomics (TIBCO Spotfire, Boston, MA) was used to estimate expression differences between non-cryopreserved and cryopreserved hESC-derived CECs. We selected a cutoff value of >2 standard deviations (Std.Dev.) from the mean of 0 to represent no change.

[0092] Injection of cryopreserved human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs) into the anterior chamber of rabbits. For injection of cryopreserved hESC-derived CECs, rabbits were anesthetized using acepromazine (subcutaneous), dexmedetomidine (intramuscular) and ketamine (intramuscular). To inhibit pain, inflammation and immunological responses, rabbits were also given buprenorphine (subcutaneous), dexamethasone (intramuscular) and Kenalog-40 (intramuscular). Tracheal intubation was performed to inhale a mixture of isoflurane and oxygen during the surgical procedure to keep the rabbit in an unconscious state. During the entire surgical procedure, rabbits were also injected with lactated Ringer's solution at a rate of 15 ml / h in the right ear using a 22-gauge catheter. Pupils were dilated using 2.5% phenylephrine solution and 1% tropicamide solution.

[0093] After application of 0.5% proparacaine hydrochloride ophthalmic solution, a 1.6 mm incision was introduced at the border zone of the 8 mm central corneal epithelial surface mark. A 20-gauge silicone needle was used to remove native CECs in an 8 mm diameter area of ​​the central cornea. A 30-gauge cannula was used to irrigate the anterior chamber and remove cellular debris from the anterior chamber. A 28-gauge needle was used to remove 7.5 × 10 6-cells in 200 μl of DMEM / F12 medium supplemented with 100 μM ROCK inhibitor Y-27632. 5 of cryopreserved hESC-derived CECs were injected into the anterior chamber. The incision was closed with three interrupted sutures (nylon 10-0). After cell injection, rabbits were also given topical Tobradex eye ointment to inhibit inflammation and immune responses. The rabbits were then placed in an eye-down position for 3 hours to enhance adhesion of the injected cells.

[0094] Following 3 hours of cell settling, the intermediate suture was removed and the anterior chamber was washed twice with sterile BSS. The anterior chamber was then reconstituted with BSS and the incision was closed using one interrupted suture (nylon 10-0). Tobradex eye ointment was applied three times a day for 10 days after injection. In preliminary experiments, we confirmed that the Descemet's membrane was intact using OCT and H&E staining and that there were no cells on the Descemet's membrane in the mechanically scraped area. The injection model rabbits were kept for 9 months after injection and then sacrificed for subsequent analysis.

[0095] Clinical evaluation of rabbit eyes. Clinical evaluation of postoperative and control eyes was performed in rabbits by evaluating intraocular pressure (IOP), corneal thickness and CEC density using Tono-Pen, ultrasonic pachymeter and confoscan4 scanning microscope, respectively, at different time points. Photographs of injected corneas were taken at different time points.

[0096] Histological evaluation of rabbit eyes. Rabbits (injection and injury injection models) were sacrificed by intravenous injection of euthanasia solution 9 months after injection. Postoperative and control eyes were removed, fixed in 10% formalin, and subjected to H&E and immunofluorescence staining. For H&E staining, after deparaffinization and hydration, hematoxylin was applied to the sections to stain the nuclei. The sections were then counterstained with eosin, followed by dehydration and mounting.

[0097] For immunohistochemical analysis, after deparaffinization, hydration, and antigen retrieval, sections were blocked with 5% bovine serum albumin (MilliporeSigma) for 45 min at room temperature. + / K +Sections were incubated overnight at 4°C with ATPase α1 (Thermo Fisher Scientific) and 1:250 N-cadherin (Thermo Fisher Scientific) primary antibodies. Then, sections were incubated overnight at 4°C with 1:100 FITC-conjugated goat anti-mouse IgG (against ZO-1; MillporeSigma), 1:200 Alexa Fluor 647 donkey anti-mouse IgG (Na + / K + Sections were treated with 1:100 FITC-conjugated goat anti-mouse IgG (against N-cadherin; MilliporeSigma) secondary antibodies for 2 h at room temperature. Nuclei were counterstained with DAPI (MilliporeSigma).

[0098] Images of mounted sections were captured of five random fields from the injected area of ​​at least two sections from each eye using a microscope (Olympus LX81; Olympus, Tokyo, Japan) equipped with software (Slidebook Software 3i; Denver, CO, USA) and prepared using image editing software (Adobe Photoshop CS5; Adobe Systems, Inc. San Jose, CA, USA).

[0099] Injection of cryopreserved human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs) into the anterior chamber of monkeys. In a non-human primate study, monkeys were used to evaluate the efficacy of cryopreserved hESC-derived CEC injection to form a functional CE monolayer. In the monkey injection model, monkeys were anesthetized and intubated using the same procedure as performed in the rabbit injection model.

[0100] After preparing the macaque corneal endothelial dysfunction model according to the procedure performed in the rabbit injection model described above, a 28-gauge needle was used to inject 1 × 10 cells suspended in 250 μl of DMEM / F12 medium supplemented with 100 μM ROCK inhibitor Y-27632. 6CECs were injected into the anterior chamber. The incision was closed with three interrupted sutures (nylon 10-0). The remaining procedures were the same as those performed in the rabbit injection model, except for the application of Tobradex eye ointment and Kenalog-40 (to inhibit immunological rejection), which were administered every other day for 12 days after injection using dexmedetomidine and ketamine sedation.

[0101] The injection model monkeys were kept for one year after injection and then sacrificed for subsequent analysis. In the monkey injury injection model, the monkeys were anesthetized and subjected to corneal endothelial injury as previously performed in the rabbit injury injection model. The anterior chamber was reconstituted with sterile BSS, and the incision was closed using 10-0 nylon sutures. After corneal endothelial injury by topical application of Tobradex ophthalmic ointment three times a day, the monkeys were kept for two days and then subjected to cryopreserved hESC-derived CEC injection using the same procedure as performed in the macaque injection model. The injury injection model monkeys are currently alive (3 months after injection), and we planned to keep two macaques for follow-up studies for at least two years after injection to further exclude any late offside effect of injected cryopreserved hESC-derived CEC.

[0102] Clinical evaluation of monkey eyes. Clinical evaluation of postoperative and control eyes was performed in monkeys by assessing IOP, corneal thickness and CEC density using a Tono-Pen, an ultrasound pachymeter and a confoscan4 scanning microscope, respectively, at different time points. Photographs of injected corneas were taken at different time points.

[0103] Histological evaluation of monkey eyes. Monkeys were sedated with dexmedetomidine and ketamine and sacrificed one year after injection by intravenous injection of euthanasia solution. Postoperative and control eyes were removed, fixed in 10% formalin, and subjected to H&E staining using the same procedure as performed in rabbits. For immunofluorescence staining, first, 1:200 ZO-1 (MilliporeSigma), 1:100 Na + / K +Sections were incubated overnight at 4°C with 1:200 Alexa Fluor 647 donkey anti-mouse IgG (ZO-1, Na + / K + Sections were treated with secondary antibodies against ATPase α1 and N-cadherin (Thermo Fisher Scientific) for 2 hours at room temperature. Nuclei were counterstained with DAPI (MilliporeSigma). Images of mounted sections were captured using the same procedure as performed in rabbits.

[0104] Necropsy and CBC / Blood Chemistry. Upon completion of the study, rabbits and macaques were subjected to detailed necropsy and CBC / Blood Chemistry. The following tissues were examined: eyes, brain, abdominal and thoracic viscera, kidneys, heart, liver, gastrointestinal tract, spleen, pituitary gland, femur with bone marrow, bladder, hip bone, femoral tibia, atlanto-occipital joint, and testes.

[0105] result

[0106] Generation and validation of human induced pluripotent stem cells (iPSCs). A 10 ml aliquot of blood was obtained from a healthy 67-year-old male with no history of corneal endothelial dystrophy. Peripheral blood mononuclear cells (PBMCs) were isolated from the 10 ml aliquot of blood by density gradient centrifugation and stored in liquid nitrogen until further use. PBMCs were reprogrammed with an integration-free Sendai virus gene delivery method by expressing pluripotency factors (c-MYC, KLF4, OCT3 / 4 and SOX2) according to the manufacturer's instructions. Putative embryonic stem cell-like iPSC colonies were selected and expanded for characterization. iPSC colonies were assessed for expression of pluripotency markers and revealed expression levels of pluripotency markers, namely NANOG, OCT4, SOX2 and TRA-1-60, comparable to human embryonic stem cells (hESCs) by quantitative real-time PCR (qRT-PCR) in PBMC-originating iPSCs. PBMC-derived iPSCs were also assessed for protein expression of pluripotency markers (SSEA4 and TRA-1-60) by flow cytometry. Flow cytometry analysis demonstrated that PBMC-derived iPSCs were highly positive for SSEA4 and TRA-1-60 (data not shown).

[0107] Differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into corneal endothelial cells (CECs). McCabe and colleagues reported the generation of CECs from hESCs. 9 Our group recently reported the generation and proteomic profiling of CECs from iPSCs of peripheral blood mononuclear cell (PBMC) origin, as well as comparative transcriptomic analysis of hESC- and iPSC-derived CECs. 10、11 The procedure is based on the differentiation of multipotent neural crest cells (NCCs) into CECs. To generate iPSC- and hESC-derived CECs of PBMC origin, we adopted a 20-day procedure that modifies the protocol reported by Ali and colleagues. 10、11The differentiation process was first characterized by examining the morphology of differentiating CECs. On day 7, prospective CECs were treated with cell dissociation buffer and transferred to new vitronectin-coated plates. Upon transfer to new vitronectin plates on day 7, iPSCs and hESCs were observed to differentiate into CECs. Morphological examination on day 20 revealed densely packed hexagonal / polygonal shaped CECs (Figure 1).

[0108] Gene expression and immunocytochemical analysis of differentiating corneal endothelial cells (CECs). To confirm the basic characteristics of CECs, we performed quantitative real-time PCR (qRT-PCR) analysis for pluripotency and corneal endothelium (CE)-associated markers at different time points during the differentiation procedure. We examined the expression of pluripotency markers (mentioned above) and CE-associated markers, namely AQP1, COL4A1, COL4A3, COL8A1, COL8A2, FOXC1 and SLC16A3, on days 4 and 20 during the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into CECs. On day 20, CECs expressed AQP1, COL4A3, COL8A2 and SLC16A3, indicating maturation of CECs. The expression of tight junction protein (ZO-1) and N-cadherin at the cell borders of hESC- and iPSC-derived CECs, which exhibited hexagonal / polygonal-like cells, confirms the structural integrity of hESC- and iPSC-derived CECs.

[0109] Validation of cryopreserved human embryonic stem cell (hESC)- and induced pluripotent stem cell (iPSC)-derived corneal endothelial cells (CECs) by quantitative real-time PCR (qRT-PCR) and immunocytochemistry. Differentiated CECs at day 20 were cryopreserved in liquid nitrogen, and after one week, the CECs were revived and cultured until they reached confluence. First, the effect of cryopreservation was evaluated by phase contrast microscopy. Cryopreserved CECs were tightly adherent with a hexagonal / polygonal shape (data not shown), suggesting that cryopreservation does not affect the morphology of hESC- and iPSC-derived CECs. It is noteworthy that the seeding density of cryopreserved CECs is important, and plating of CECs at low density results in a fibroblast-like morphology as the CECs expand into the open space.

[0110] Next, we investigated the expression of corneal endothelium (CE)-associated markers by quantitative real-time PCR (qRT-PCR). qRT-PCR analysis revealed that the expression levels of corneal endothelium (CE)-associated markers were similar before and after cryopreservation (data not shown), further supporting the idea that cryopreservation does not affect the expression of CE-associated markers in human embryonic stem cell (hESC)-derived and induced pluripotent stem cell (iPSC)-derived corneal endothelial cells (CECs). Finally, we found that tight junction protein (ZO-1), N-cadherin and CE pump function protein (Na + / K + The expression of ZO-1, N-cadherin and Na+ at the cell borders of cryopreserved hESC- and iPSC-derived CECs exhibiting hexagonal / polygonal-like cells was examined by immunocytochemistry. + / K + Immunostaining for ATPase α1 confirms the structural integrity of hESC- and iPSC-derived CECs after cryopreservation (data not shown).

[0111] Necropsy and CBC / Blood Chemistry. Detailed necropsy examination of the eyes, brain, abdominal and thoracic viscera, kidneys, heart, liver, gastrointestinal tract, spleen, pituitary gland, bladder, testes and femur with bone marrow was unremarkable. No abnormal masses were observed on detailed necropsy study. All CBC / Blood Chemistry parameters were within normal physiological ranges.

[0112] Consideration

[0113] Herein, the derivation of clinical-grade corneal endothelial cell (CEC)-like cells from induced pluripotent stem cells (iPSCs) of human embryonic stem cells (hESCs) and peripheral blood mononuclear cells (PBMCs) origin under xeno-free conditions is reported, and the effect of cryopreservation on hESC- and iPSC-derived CECs was examined, suggesting that cryopreservation does not affect the structural integrity of hESC- and iPSC-derived CECs. Previously, our group reported the generation and proteomic profiling of PBMC-origin iPSC-derived CECs as well as comparative transcriptomic analysis of hESC- and iPSC-derived CECs.

[0114] We employed a 20-day procedure modified from the protocol reported by Ali and colleagues. 10,11 High expression of corneal endothelium (CE)-associated markers, and more importantly, the lack of expression of pluripotency markers, strongly supports the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into the corneal endothelial cell (CEC) lineage.

[0115] The main safety concern regarding the use of cryopreserved hESC-derived CECs is the possibility of undifferentiated hESCs remaining throughout the differentiation process. Therefore, we examined detailed CBC / blood chemistry profiles and multiple organs of injected rabbits (9 months post-injection) and injected monkeys 1 year post-injection. All CBC / blood chemistry parameters were within normal physiological ranges, and no abnormalities were observed in any of the body organs as revealed by necropsy examination.

[0116] To our knowledge, this is the first report of the generation and cryopreservation of clinical-grade, human embryonic stem cell (hESC)-derived and peripheral blood-origin induced pluripotent stem cell (iPSC)-derived corneal endothelial cells (CECs).

[0117] References 1.Delmonte DW,Kim T.Anatomy and physiology of the cornea.J Cataract Refract Surg.2011;37:588-598. 2.Bonanno JA.Molecular mechanisms underlying the corneal endothelial pump.Exp Eye Res.2012;95:2-7. 3.Claerhout I,Beele H,Kestelyn P.Graft failure:I.Endothelial cell loss.Int Ophthalmol.2008;28:165-173. 4.Eye Bank Association of America.2016 Eye banking Statistical Report.In:2016. 5.Gain P, Jullienne R, He Z, Aldossary M, Acquart S, Cognasse F, Thuret G. Global Survey of Corneal Transplantation and Eye Banking. JAMA Ophthalmol.2016;134:167-173. 6.Agu CA,Soares FA,Alderton A,Patel M,Ansari R,Patel S,Forrest S,Yang F,Lineham J,et al.Successful Generation of Human Induced Pluripotent Stem Cell Lines from Blood Samples Held at Room Temperature for up to 48 hr.Stem Cell Reports.2015;5:660-671. 7.Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method.Methods.2001;25:402-408. 8.Khan SY,Vasanth S,Kabir F,Gottsch JD,Khan AO,Chaerkady R,Lee MC,Leitch CC,Ma Z,et al.FOXE3 contributes to Peters anomaly through transcriptional regulation of an autophagy-associated protein termed DNAJB1.Nat Commun.2016;7:10953. 9.McCabe KL,Kunzevitzky NJ,Chiswell BP,Xia X,Goldberg JL,Lanza R.Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation.PLoS One.2015;10:e0145266. 10.Ali M,Khan SY,Vasanth S,Ahmed MR,Chen R,Na CH,Thomson JJ,Qiu C,Gottsch JD,et al.Generation and Proteome Profiling of PBMC-Originated,iPSC-Derived Corneal Endothelial Cells.Invest Ophthalmol Vis Sci.2018;59:2437-2444. 11.Ali M, Khan SY, Kabir F, Gottsch JD, Riazuddin SA. Comparative transcriptome analysis of hESC-and iPSC-derived corneal endothelial cells.Exp Eye Res.2018. Equivalent

[0118] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A method for producing clinical grade corneal endothelial cells (CECs), comprising differentiation of peripheral blood mononuclear cell (PBMC)-derived induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) into clinical grade corneal endothelial cells (CECs) and cryopreservation thereof.

2. 10. The method of claim 1, wherein culturing the cells in a differentiation medium comprising one or more differentiation factors comprises culturing the cells on a protein coating.

3. The method of claim 2 , wherein the differentiation medium comprises one or more growth factors, inhibitors of transforming growth factors (TGF), or combinations thereof.

4. The method according to claim 2 or 3, wherein cells expressing pluripotency markers are cultured in a protein or gelatinous protein mixture together with one or more factors for inducing differentiation and cryopreservation of said cells.

5. The method of claim 4, wherein the cells cultured in the gelatinous protein mixture differentiate into corneal endothelial cells (CECs) as determined by expression of corneal endothelium (CE)-associated markers or a combination thereof.

6. The method according to any one of claims 1 to 5, wherein the cryopreserved clinical grade corneal endothelial cells (CECs) retain their viability and / or functionality when thawed.

7. 7. The method of any one of claims 1 to 6, wherein the medium comprises one or more of a cell culture medium, a cell differentiation medium, a gelatinous protein mixture, a cryopreservation medium, a pharmaceutical composition, a scaffold or a growth medium.

8. A method for treating a corneal-related disease or disorder, comprising producing clinical-grade corneal endothelial cells (CECs) from peripheral blood mononuclear cells (PBMCs)-derived induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) according to claim 1, and injecting the CECs into the anterior chamber of the eye of a subject in need of treatment.

9. 9. The method of claim 8, wherein the corneal disease or disorder comprises a genetic disease or disorder, an infection, a wound, an inflammatory disease, an autoimmune disease, dry eye, a dystrophy, or a combination thereof.

10. 9. The method of claim 8, wherein the corneal disease or disorder comprises Fuchs endothelial dystrophy (FECD), keratoconus, keratoconjunctivitis sicca (KCS), herpes virus infection, varicella zoster virus infection, iridocorneal endothelial syndrome (ICE), pterygium, Stevens-Johnson syndrome (SJS), corneal ulcer or bullous keratopathy.

11. A composition comprising a therapeutically effective amount of corneal endothelial cells (CECs) produced from induced pluripotent stem cells (iPSCs) of human embryonic stem cells (hESCs) and peripheral blood mononuclear cells (PBMCs) origin, obtainable or obtained by the method according to any one of claims 1 to 7.

12. A kit comprising a pharmaceutical composition and a therapeutically effective amount of corneal endothelial cells (CECs) produced from induced pluripotent stem cells (iPSCs) of human embryonic stem cells (hESCs) and peripheral blood mononuclear cells (PBMCs) origin, obtainable or obtained from the composition of claim 11 or the method of any one of claims 1 to 7.

13. A vial comprising cryopreserved corneal endothelial cells (CECs) of the composition according to claim 11 or obtainable or obtained from the method according to any one of claims 1 to 7.

14. A cryopreserved composition comprising cryopreserved corneal endothelial cells (CECs) of the composition of claim 11.

15. A cryopreserved composition comprising cryopreserved corneal endothelial cells (CECs) of a composition obtainable from the method according to any one of claims 1 to 7.

16. A cryopreserved composition comprising cryopreserved corneal endothelial cells (CECs) of the composition obtained from the method according to any one of claims 1 to 7.

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