1. Field of the Invention
The invention relates to the field of stem cell research, and more particularly to the isolation of stem cells from amniotic fluid. Stem cells derived from amniotic fluid can then be differentiated to many types of cells or tissues.
2. Description of the Related Art
Stem cells have the ability to divide for indefinite periods in culture and to give rise to specialized cells. Stem cells can give rise to many types of differentiated cells, and may be useful to treat many types of diseases. There are several types of stem cells, such as embryonic stem cells, which are undifferentiated cells from the embryo, and adult stem cells, which are undifferentiated cells derived from various mature tissues.
Embryonic stem cells have the potential to become a wide variety of specialized cell types. This ability of an embryonic stem cell to become a variety of cell types is termed “pluripotent.” Embryonic stem cells can be differentiated into a host of cell types and tissue types, which can be used for basic research, drug discovery, treatment and prevention of diseases. For example, U.S. Pat. No. 6,506,574 to Rambhatla, which is incorporated by reference herein in its entirety, discloses methods of differentiating embryonic stem cell cultures into hepatocyte lineage cells. Other methods for the preparation of embryonic stem cells are disclosed, for example, in U.S. Pat. No. 6,200,806 to Thomson; U.S. Pat. No. 5,670,372 to Hogan, and U.S. Pat. No. 6,432,711 to Dinsmore, each of which is incorporated by reference herein in its entirety.
Human Embryonic Stem cells (hES) are derived from the inner cell mass of the blastocyst, the earliest stage of embryonic development of the fertilized egg. The blastocyst is a preimplantation stage of the embryo, a stage before the embryo would implant in the uterine wall. When cultured on an inactivated feeder layer of cells according to conditions described by Thompson and colleagues (Thomson, et al., (1995) Proc. Natl. Acad. Sci. U.S.A. 92:7844-7848; Thomson, et al. (1998) Science 282:1145-1147; Marshall, et al., (2001) Methods Mol. Biol. 158:11-18), each of which is incorporated by reference herein in its entirety, the inner layer cells of the blastocyst can be grown in vitro indefinitely in an undifferentiated state. Properly propagated hES cells have unlimited potential to double while maintaining their capacity of differentiating into the three layers of the embryo, Ectoderm (Ec), Mesoderm (Me) and Endoderm (En); they are pluripotent. When grown as pluripotent hES, the cells maintain a euploid karyotype and are not prone to senescence. hES cells have been differentiated in vitro into skin and brain (Ec), heart, muscle, kidney and blood (Me), and into pancreatic, thyroid and lung cells (En) (Fraichard, et al., (1995) J. Cell Sci. 108:3181-3188; Itskovitz-Eldor, et al., (2000). Mol. Med. 6:88-95; Lee, et al., (2000) Nat. Biotechnol. 18:675-679; Liu, et al., (2000) Proc. Natl. Acad. Sci. U.S.A. 97:6126-6131; Lumelsky, et al., (2001) Science 292:1389-1394; Maltsev, et al., (1993). Mech. Dev. 44:41-50; Odorico, et al., (2001) Stem Cells 19:193-204. Potocnik, et al., EMBO. J. 13:5274-5283; Reubinoff, et al., (2000) Nat. Biotechnol. 18:399-404; Schuldiner, et al., (2001) Proc. Natl. Acad. Sci. USA 97:1997:11307-11312; Kim, et al., (2002) Nature 418:50-56; Wichterle, et al., (2002) Cell 110:385-397), each of which is incorporated by reference herein in its entirety.
Human embryonic stem cells display a distinct group of cell surface antigens, SSEA-3, SSEA-4, TRA-2-54 (alkaline phosphatase), TRA-1-60 and TRA-1-81, in addition to expressing specific transcription factors OCT-4, NANOG, SOX-2, FGF-4 and REX-1 (Henderson, et al., (2002) Stem Cells 20:329-337; Draper, et al., (2002). J. Anat. 200:249-258; Mitsui et al., (2003) Cell 113:631-642; Chambers et al., (2003) Cell 113:643-655), each of which is incorporated by reference herein in its entirety. Additionally, hES cells (i) are capable of symmetrical division in vitro without differentiating; (ii) can integrate into all fetal tissues during in vivo development; (iii) are capable of colonizing the germ line and give rise to egg or sperm cells; (iv) develop into teratocarcinomas in immunologically impaired adult mice—another measure of pluripotency, and lack the G1 checkpoint in the cell cycle like somatic cells but spend most of their time in S phase.
Stem cells can also be derived from nonembryonic sources. For example, an additional class of human stem cells are the mesenchymal or adult stem cells (MSC). Adult stem cells are undifferentiated, like embryonic stem cells, but are present in differentiated tissues. Adult stem cells are capable of differentiation into the cell types from the tissue that the adult stem cell originated. Adult stem cells (MSC) have been derived from the nervous system (McKay, R. (1997) Science 276:66-71. Shihabuddin, et al., (1999) Mol. Med. Today 5:474-480), bone marrow (Pittenger, et al., (1999) Science 284:143-147; Pittenger, M. F. and Marshak, D. R. (2001). In: Mesenchymal stem cells of human adult bone marrow. Marshak, D. R., Gardner, D. K., and Gottlieb, D. eds. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press) 349-374); adipose tissue (Gronthos, et al., (2001) J. Cell. Physiol. 189:54-63), dermis (Toma, et al., (2001) Nature cell Biol. 3:778-784) and pancreas and liver (Deutsch, et al., (2001) Development 128:871-881, each of which is incorporated by reference herein in its entirety, and other organs.
Several patents disclose various aspects of adult stem cells. For example, U.S. Pat. No. 5,486,359 to Caplan, which is incorporated by reference herein in its entirety, discloses methods of isolating human mesenchymal stem cells, and U.S. Pat. No. 5,556,783 to Lavker, which is incorporated by reference herein in its entirety, discloses methods of culturing hair follicle stem cells. Examples of patents disclosing haematopoietic stem cells include U.S. Pat. No. 4,714,680 to Civin, U.S. Pat. No. 5,061,620 to Tsukamoto, and U.S. Pat. No. 5,087,570 to Weissman, each of which is incorporated by reference herein in its entirety.
The mesenchymal/adult stem cells (MSC—or MAP cells) express a variety of cell surface proteins such as CD9, CD10, CD13, CD49b in addition to other markers. MAP cells have the potential to differentiate into different mesenchymal tissues including bone, cartilage, fat, tendon, muscle and bone marrow stroma: they are multipotent but not pluripotent. MAP cells do not, as a rule, express the SSEA-3, SSEA-4, TRA-2-54, TRA-1-60 and TRA-1-81 markers expressed by hES cells. Although adult stem cells can be expanded in culture, they do not appear to be immortal (McKay R. (2000) Nature 406:61-364; Vogel G. (2002) Science 292:820-1822; Donovan and Gearhart J. (2001) Nature 414:2-97, each of which is incorporated by reference herein in its entirety). It has been proposed that the mortality of mesenchymal/adult stem cells is due to the asymmetric kinetics of adult stem cell expansion (Sherley, J. L. (2002) Stem Cells 20:61-572, which is incorporated by reference herein in its entirety).
Unfortunately, researchers have found that non-embryonic types of stem cells (“adult stem cells”) are not as capable of differentiating into many different tissue types as are embryonic stem cells, so embryonic stem cells still have many advantages over the use of adult stem cells. However, one obstacle with the isolation of embryonic stem cells is that the cells are derived from embryos at the “blastocyst” stage. Human embryonic stem cell research is encumbered by an emotionally charged political and moral ethics debate and is likely to remain so for years to come.
Additionally, human embryonic stem cells (hES) have been found to be tumorigenic when injected into immunologically-impaired animals, i.e. in the context of post-natal tissues, whereas the MAP cells are not. The tumorigenic attributes of hES cells are not frequently addressed, though this issue may burden their use in replacement cell therapy in the future. The political, moral and ethical issues around hES cells and their tumorigenic properties, as well as the perceived difficulties of expanding undifferentiated adult stem cells in culture, while maintaining a genetically normal genome, are major barriers in the development of human cell replacement therapy.
To address the above-described obstacles, while still allowing research progress towards successful treatment of human diseases, methods of isolating novel sources of multipotent or pluripotent human stem cells that are not fraught by the ethical, tumorigenic, or mortality hurdles are needed.
USPTO Backgrounds
USPTO Backgrounds