Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2000-09-25
2004-09-07
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S378000, C435S375000, C435S383000, C435S325000
Reexamination Certificate
active
06787355
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to multipotent neural stem cells (MNSCs) purified from peripheral tissues containing sensory receptors such as the skin, olfactory epithelium, and tongue.
There are a number of diseases of the central nervous system (“CNS”) which have a devastating effect on patients. These diseases are debilitating, often incurable, and include, for example, Alzheimer's disease, Huntington's disease, Parkinson's disease, and Multiple Sclerosis.
By way of example, Parkinson's disease is a progressive degenerative disorder of unknown cause. In healthy brain tissue, dopaminergic neurons extend from the substantia nigra of the brain into the neighboring striatum. In Parkinson's disease, these dopaminergic neurons die.
There are a number of methods to treat Parkinson's disease. One method is to treat humans having Parkinson's disease with L-DOPA. A second method is to transplant cells into the substantia nigra or striatum. Transplanted cells replace endogenous cells that are lost as a consequence of disease progression. An animal model of Parkinson's disease is an MPTP-treated non-human primate. The MPTP-treated animals have been transplanted with dopamine-rich embryonic neurons with some success.
To date, the cells used for neural transplant have been collected from the developing brains of aborted fetuses. Aside from the ethical considerations, the method from a practical standpoint is unlikely to provide a sufficient amount of neural tissue to meet the demands. Thus, another source of cells for transplantation is desirable.
Stem cells are undifferentiated cells that exist in many tissues of embryos and adult mammals. In embryos, blastocyst stem cells are the source of cells which differentiate to form the specialized tissues and organs of the developing fetus. In adults, specialized stem cells in individual tissues are the source of new cells, replacing cells lost through cell death due to natural attrition, disease, or injury. Stem cells may be used as substrates for producing healthy tissue where a disease, disorder, or abnormal physical state has destroyed or damaged normal tissue.
MNSCs may be used as a source of cells for transplantation. The MNSCs may themselves be transplanted or, alternatively, they may be induced to produce differentiated cells (e.g., neurons, oligodendrocytes, Schwann cells, or astrocytes) for transplantation. Transplanted stem cells may also be used as vectors for the expression of therapeutic molecules, such as growth factors, cytokines, anti-apoptotic proteins, and the like. Thus, stem cells are a potential source of cells for alternative treatments of diseases involving loss of cells or tissues.
The safest type of tissue graft (using stem cells or otherwise) is one that comes from self (an autologous tissue source). Autologous tissue sources are widely used in procedures such as bone transplants and skin transplants because a source of healthy tissue is readily accessible for transplant to a damaged tissue site. In brain diseases, such as Parkinson's disease, healthy dopaminergic neuronal brain tissue may exist at other sites in the brain, but attempts to transplant these neurons may harm the site where the healthy neurons originate. MNSCs that can be differentiated into dopaminergic neurons may be available at other sites from which they may be transplanted, but the CNS, particularly the brain, is physically difficult to access.
In several tissues, stem cells have been purified and characterized. For example, MNSCs have been purified from the mammalian forebrain (Reynolds and Weiss, Science 255:1707-1710, 1992) and these cells were shown to be capable of differentiating into neurons, astrocytes, and oligodendrocytes. PCT publications WO 93/01275, WO 94/16718, WO 94/10292 and WO 94/09119 describe uses for these cells. It could be impractical or impossible, however, to first access brain or other CNS tissue for biopsy and then again for transplant in patients with weakened health. It would be very useful if there were accessible stem cells capable of differentiating into CNS cell types, such as dopaminergic neurons; such cells would be a source of cells for autologous transplants.
Thus, there is a clear need to develop methods for identifying from accessible tissues neural stem cells that can act as a source of cells that are transplantable to the CNS, PNS, or other tissues in vivo in order to replace damaged or diseased tissue.
SUMMARY OF THE INVENTION
We have substantially purified MNSCs from the peripheral tissue of postnatal mammals, including juvenile and adult mammals. Most importantly, we have identified skin as a source of MNSCs and provide methods for the purification of skin-derived MNSCs, thus simplifying the harvesting of cells for transplantation relative to previous methods. The MNSCs possess desirable features in that they are multipotent and self-renewing. The cells can be repeatedly passaged and differentiated into cell types of the CNS, including astrocytes, oligodendrocytes, and neurons. The MNSCs express nestin, an immunological marker of neural stem cells and progenitor cells. The cells are capable of differentiating as dopaminergic neurons, and thus are useful source of dopaminergic neurons for homotypic grafts into Parkinson's Disease patients. The MNSCs may also make non-neural cells such as cardiac muscle cells, pancreatic islet cells, smooth muscle cells, hematopoietic cells, adipocytes, hepatocytes, and the like. The cells may also be used for autologous or heterologous transplants to treat, for example, other neurodegenerative diseases, disorders, or abnormal physical states.
Accordingly, in a first aspect, the invention features a MNSC substantially purified from a peripheral tissue of a postnatal mammal, wherein the peripheral tissue includes a sensory receptor.
In a second aspect, the invention features a cell that is the progeny of a MNSC substantially purified from a peripheral tissue of a postnatal mammal. The cell may be a mitotic cell or a differentiated cell (e.g., a neuron, an astrocyte, an oligodendrocyte, a Schwann cell, or a non-neural cell). Preferred neurons include neurons expressing one or more of the following neurotransmitters: dopamine, GABA, glycine, acetylcholine, glutamate, and serotonin. Preferred non-neural cells include cardiac muscle cells, pancreatic cells (e.g., islet cells), chondrocytes, osteocytes, skeletal muscle cells, smooth muscle cells, hepatocytes, hematopoietic cells, and adipocytes.
In a third aspect, the invention features a population of at least ten cells, wherein at least 30% of the cells are MNSCs substantially purified from a peripheral tissue of a postnatal mammal or progeny of the MNSCs, wherein the peripheral tissue includes a sensory receptor.
Preferably, at least 50% of the cells are MNSCs substantially purified from the peripheral tissue or progeny of the MNSCs. More preferably, at least 75% of the cells are MNSCs substantially purified from the peripheral tissue or progeny of the MNSCs. Most preferably, at least 90%, 95%, or even 100% of the cells are MNSCs substantially purified from the peripheral tissue or progeny of the MNSCs. The MNSCs may be cultured for extended periods of time. Thus, the population of cells may have been in culture for at least thirty days, sixty days, ninety days, or longer (e.g., one year or more). Preferably, the population is at least twenty cells, and may be more than fifty cells, a thousand cells, or even a million cells or more.
In a fourth aspect, the invention features a pharmaceutical composition including (i) a mitotic or differentiated cell that is the progeny of a MNSC substantially purified from a peripheral tissue of a postnatal mammal, wherein the peripheral tissue includes a sensory receptor, and (ii) a pharmaceutically acceptable carrier, auxiliary or excipient.
In a fifth, related aspect, the invention features a pharmaceutical composition including (i) a MNSC substantially purified from a peripheral tissue of a postnatal mammal, where
Gloster Andrew
Miller Freda D.
Toma Jean
Bieker-Brady Kristina
Clark & Elbing LLP
Eyler Yvonne
McGill University
Murphy Joseph F.
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