Pluripotent embryonic-like stem cells derived from corneal limbus, methods of isolation and uses thereof

Abstract

The present disclosure describes mammalian pluripotent embryonic-like stem cells (ELSCs) isolated from corneal limbal tissue, a non-embryonic tissue. The ELSCs of the present disclosure are capable of proliferating in an in vitro culture, maintain the potential to differentiate into cells of endoderm, mesoderm, and ectoderm lineage in culture, and are capable of forming embryoid-like bodies when placed in suspension culture. Thus, these cells possess multi-lineage differentiation potential and self-renewing capability. ELSCs may be a promising therapeutic tool, and may provide new therapeutic alternatives for various diseases, conditions, and injuries.

Description

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0001] Not applicable. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present disclosure relates to purified preparations of mammalian pluripotent stem cells, preferably human pluripotent stem cells, derived from corneal limbus tissue. In preferred embodiments, the pluripotent limbal stem cell lines are self-renewing and have the ability to differentiate into tissues derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm). Methods for isolating pluripotent limbal stem cell lines and methods of their use are also disclosed. [0004] 2. Description of Related Art [0005] In early development, the ultimate source of all tissues in a mammalian embryo or fetus are stem cells. In the embryonic stage embryonic stem cells (ES cells) are totipotent and therefore capable of developing into all the cells of a complete organism. Cellular development occurs through several phases, includi...

AI Technical Summary

Benefits of technology

[0031] In preferred embodiments the isolated population of pluripotent ELSCs are human ELSCs, which are more preferably SSEA-4 positive. In other embodiments, the donor of the corneal limbal tissue is human. In certain embodiments, the corneal limbal tissue is cultured in culture media such as DMEM or F12, further supplemented with a nutrient serum and one or more soluble factors selected from the group consisting of dimethyl sulphoxide (DMSO), recombinant human epidermal growth factor (rhEGF), insulin, sodium selenite, transferrin, basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF). Preferably, the corneal limbal tissue is cultured until the corneal limbal cells in the culture become confluent. In certain embodiments, the corneal limbal tissue is cultured on an extracellular matrix, for example Matrigel™, laminin, collagen-IV, poly-L-lysine, gelatin, poly-L-ornithin, fibronectin, and combinations thereof, or mammalian amniotic membrane. When the corneal limbal tissue is cultured on an extracellular matrix, the above methods preferably further comprise the step of dissociating the cultured corneal limbal cells from the extracellular matrix prior to isolating the pluripotent ELSCs.

[0032] In preferred embodiments, the corneal limbal cells are sorted using methods well known to those of skill in the art, for example magnetic-affinity cell sorting (MACS) or fluorescence-activated cell sorting (FACS) to isolate a population of pluripotent ELSCs. In other embodiments, the one or more undifferentiated cell-specific markers selected for to isolate pluripotent ELSCs include but are not limited to SSEA-4, SSEA-3, CD73, CD105, CD31, CD54, and CD117. In preferred embodiments, corneal limbal cells are sorted to select for SSEA-4 positive ELSCs. In certain embodiments, the sorted ELSCs comprise at least about 80%, 90%, 95%, 98%, or 99% pluripotent ELSCs that are SSEA-4 positive. In preferred embodiments, the isolated population of pluripotent ELSCs comprise at least about 70%, 80%, 90%, 95%, 98%, or 99% pluripotent ELSCs. Preferably the isolated population of pluripotent ELSCs are further cultured to produce an embryonic-like stem cell line. In certain embodiments, the pluripotent ELSCs are cultured in culture media such as DMEM or F12, further supplemented with a nutrient serum and one or more soluble factors selected from the group consisting of DMSO, rhEGF, insulin, sodium selenite, transferrin, bFGF, and LIF.

[0033] In alternate embodiments, pluripotent ELSCs isolated by the methods disclosed herein are capable of proliferating and maintaining the potential to differentiate in vitro or in vivo into cells or tissues of endoderm, mesoderm or endoderm lineage. Preferably, the isolated pluripotent ELSCs are also capable of forming embryoid-like bodies, for example when placed in suspension culture. In other preferred embodiments, the isolated ELSCs remain substantially undifferentiated in an in vitro culture for at least about 20 passages, more preferably at least about 50 passages, and most preferably at least about 100 passages in culture. Preferably, after multiple passages in culture the substantially undifferentiated ELSCs maintain normal karyotype and high telomerase activity. In further embodiments, the isolated ELSCs have the potential to terminally differentiate into cells or tissues of endoderm, mesoderm, or ectoderm lineage.

[0034] In further embodiments the isolated pluripotent ELSCs, preferably human ELSCs, are further differentiated in culture into endodermal lineage-committed cells or tissues, mesodermal lineage-committed cells or tissues, or ectodermal lineage-committed cells or tissues. Alternatively, the isolated pluripotent ELSCs are further differentiated into endodermal lineage-committed cells or tissues, mesodermal lineage-committed cells or tissues, or ectodermal lineage-committed cells or tissues in vivo. In other embodiments, these ELSCs are further differentiated by exposing the ELSCs to one or more agents known to induce differentiation of pluripotent embryonic stem (ES) cells, including but not limited to acidic fibroblast growth factor, bFGF, platelet-derived growth factor (PDGF), insulin, retinoic acid, transferrin, insulin-transferrin-selenious acid (ITS), dexamethasone, sodium butyrate, DMSO, nerve growth factor (NGF), Cytosine beta-d-Arabino Furanoside (Ara C), glial cell line-derived neurotrophic factor gene (GDNF), transforming growth factor β3 (TGF-β3), ascorbic acid, N-acetyl Cysteine, dibutaryl cyclic AMP, Neurturin, transforming growth factor β1 (TGF-β1), insulin-like growth factor I or II (IGF-I or IGF-II), epidermal growth factor (EGF), bone morphogenic proteins 2 (BMP-2), β glycerophosphate, ascorbic acid 2 phosphate, 5-Aza-deoxy-cytidine, oncostatin, hepatocyte growth factor (HGF), progesterone, nicotinamide, or any combination thereof.

[0035] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0036]FIG. 1. Limbal composite grafts (LCG): (a) H & E stained LCG (whole mount); (b) LCG probed using immunofluorescence for SSEA-4 antigen; (c) LCG analysis by RT-PCR for expression of the pluripotent stem cell markers Oct-4, Nanog, and Rex 1, as well as GAPDH as a positive control; and (d) SSEA-4 positive cells isolated from LCG by flow cytometry.

Problems solved by technology

However, despite the enormous potential of these materials, serious ethical issues related to the use of human pluripotent stem cells derived from human embryos or from fetal tissue obtained from terminated pregnancies make stem cell research and treatments problematic.

In addition, technical issues associated with the use of ES cells are problematic.

Tissues or cells derived from ES cells are not ideal for use in medical treatments because generally the ES cells will not be derived from the patient who will ultimately be receiving the treatment.

Nevertheless, while some potential sources of adult stem cells have been identified, to date adult stem cells have not been found to be an adequate replacement for ES cells.

First, adult stem cells can be difficult to isolate because they are usually present only in minute quantities in tissues that are often not easily accessible, and their numbers appear to decrease with age.

Second, adult stem cells appear to be a less desirable source of cultured tissue than ES cells because they have a shorter life span and less capacity for self-renewal.

In addition, none of the isolated adult stem cells reported to date appear to be capable of forming embryoid-like bodies in culture in a manner similar to ES cells.

Therefore, currently there is no known definitive stem cell marker for limbal epithelial stem cells.

Images