1. Field of the Invention
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.
2. Description of Related Art
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, including cellular proliferation, lineage commitment, and lineage progression, resulting in the formation of differentiated cell types. There are three main lineages that are derived from embryonic germ layers: ectoderm, mesoderm and endoderm. The ectoderm germ layer forms the epidermis of the skin, sense organs, nervous system, and spinal cord. The mesoderm germ layer forms smooth muscle, connective tissues, blood vessels, heart, blood cells and bone marrow, reproductive organ, excretory system, striated muscles, and skeletal muscles. Finally, the endoderm germ layer forms epithelial linings of respiratory and gastrointestinal tract, pharynx, esophagus, stomach, intestine, and other associated organs. ES cells are referred to as pluripotent stem cells because they can differentiate into almost all cell types in an adult organism.
During the last decade there has been ongoing research on the isolation and use of ES cells and cell lines, which in addition to having the ability to develop into most of the specialized cells in the human body also have the capacity to divide and proliferate indefinitely in culture. ES cells are often referred to as pluripotent stem cells because they are not fixed in their developmental potentialities and can differentiate into many different cell types in vitro. Cultured ES cells that are highly pluripotent can form clumps of cells in suspension culture referred to as embryoid bodies. ES cells were isolated from humans relatively recently (Thomson et al., (1998) Science 282:1145-1147; Gearhart, (1998) Science 282:1061-62). In embryoid bodies derived from human ES cells it is possible to discern differentiated cells bearing markers of a wide variety of cell types.
The isolation of human ES cells offers the promise of a remarkable array of novel therapeutics. Biologic therapies derived from such cells through tissue regeneration and repairs, as well as through targeted delivery of genetic material, are expected to be effective in the treatment of a wide range of medical conditions. 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. Use of autologous tissues is preferred for stem-cell-based therapies in order to avoid tissue rejection problems.
I. Adult Stem Cells
The problems associated with human ES cells led many researchers to turn their attention to adult tissues as a possible source of undifferentiated stem cells with properties similar to those of ES cells or germ cells derived from fetal tissue. It was known that after birth and throughout adulthood a small number of specialized stem cells are retained in an organism for the replacement of cells and the regeneration of tissues. Indeed, adult stem cells (also referred to as “tissue-specific stem cells”) have been found in very small numbers in various tissues of the adult body, including bone marrow, (Weissman, (2000) Science 287:1442-1446), neural tissue (Gage, (2000) Science 287:1433-1438), gastrointestinal tissue (Potten, (1998) Phil. Trans. R. Soc. Lond. B. 353:821-830), epidermal tissue (Watt, (1997) Phil. Trans. R. Soc. Lond. B. 353:831), hepatic tissue (Alison and Sarraf, (1998) J. Hepatol. 29:678-683), and mesenchymal tissue. (Pittenger et al., (1999) Science 284:143-147).
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. Third, adult stem cells are believed to be tissue specific and not pluripotent, generally capable of giving rise only to new cells of a few types closely related to their tissue of origin.
One particularly notable difference between ES cells and adult stem cells is that ES cells in suspension culture are capable of forming aggregates of cells known as embryoid bodies. These embryoid bodies usually contain germ cells of all three lineages that differentiate into various lineage-committed tissues. Therefore, embryoid bodies can be useful in the preparation of different types of differentiated cells in culture. To date, no other isolated adult stem cell lines have been reported that are capable of forming structures similar to embryoid-like bodies in culture.
Recently, however, it has been suggested that some adult stem cells have the capacity to be pluripotent. The most fully characterized are the hematopoetic stem cells known as bone marrow stromal cells or mesenchymal stem cells (Jiang et al., (2002) Nature 418:41-48). These were the first adult stem cells found to have pluripotent properties. Pluripotent adult stem cells have also been isolated from liver (U.S. Publ. No. 2003/0186439), mouse inner ear (Li and Heller, (2003) Nat. Med. 9:1293-1299), and amniotic fluid (Prusa et al., (2003) Hum. Reprod. 18:1489-1493). Pluripotent adult stem cells have also been recently described in many tissues such as skeletal muscle, brain, and intestinal epithelium (Howell et al., (2003) Ann. N.Y. Acad. Sci. 996:158-173). Still, while these papers report isolated or identified adult stem cells that are pluripotent, these “pluripotent” adult stem cells, unlike ES cells, differentiate into only a few lineages. 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.
II. Corneoscleral Limbus
Similar to the other sources of adult stem cells referenced above, it is known that adult stem cells are present in the corneoscleral limbus of the eye. These cells participate in the dynamic equilibrium of the corneal surface and replace superficial epithelial cells that are shed and sloughed off during eye-blinking. Severe damage to the limbal stem cells from chemical or thermal burns, contact lenses, severe microbial infection, multiple surgical procedures, cryotherapy, or diseases such as Steven-Johnson syndrome or ocular cicatrical pemphigoid can lead to destruction of limbal stem cells and limbal stem cell deficiency which can lead to an abnormal corneal surface, photophobia, and reduced vision (Anderson et al., (2001) Br. J. Opthalmol. 85:567-575). This damage cannot be repaired without the re-introduction of a source of limbal stem cells (Tseng et al., (1998) Arch. Opthalmol. 116:431-41; Tsai et al., (2000) N. Engl. J. Med. 343:86-93; Henderson et al., (2001) Br. J. Opthalmol. 85:604-609). Thus limbal stem cells, with their high proliferative capacity, are clearly crucial for the maintenance of a viable ocular surface because they provide an unbroken supply of corneal epithelial cells necessary to maintain the equilibrium of the corneal surface (Tseng, (1996) Mol. Biol. Rep. 23:47-58).
Experiments conducted in the 1980s first indicated the existence of limbal cells in the corneal epithelium (Schermer et al., (1986) J. Cell Biol. 103:49-62; Cotsarelis et al., (1989) Cell 57:201-209). Although it was later suggested that the transcription factor P-63 was a specific marker for human corneal stem cells, this marker is also expressed in other epithelial cells such as skin, and therefore is not specific to corneal stem cells. In addition, although P-63 expression has been shown to be principally limited to the basal limbal region in human corneas (Moore et al., (2002) DNA Cell Biol. 21:443-51), in mice expression of this transcription factor was maximal in paracentral cornea tissue rather than limbus (Moore et al., (2002) DNA Cell Biol. 21:443-451). Therefore, currently there is no known definitive stem cell marker for limbal epithelial stem cells.
It would be desirable to identify a source of adult stem cells that are capable of self-renewal in culture and that are pluripotent and ES cell-like in their ability to differentiate into cells of all three major lineages: ectoderm, mesoderm and endoderm. Further, it would be desirable to isolate and culture these adult stem cells, and to induce them to differentiate into various cell types.