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Link:  Pharm/Biotech Resources


Title:  Pharmaceuticals containing multipotential precursor cells from tissues containing sensory receptors

United States Patent:  6,969,608

Issued:  November 29, 2005

Inventors:  Miller; Freda (Montreal, CA); Gloster; Andrew (Saskatoon, CA)

Assignee:  McGill University (Montreal, CA)

Appl. No.:  920272

Filed:  August 22, 1997

Abstract

Current sources of neural stem and progenitor cells for neural transplantation are essentially inaccessible in living animals. This invention relates to neural precursor cells (stem cells, progenitor cells or a combination of both types of cells) isolated from the olfactory epithelium of mammals that can be passaged and expanded, and that will differentiate into cell types of the central nervous system (CNS), including astrocytes, oligodendrocytes, and tyrosine-hydroxylase-positive neurons. These precursor cells provide an accessible source for autologous transplantation in CNS, PNS, spinal cord and other damaged tissues.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates to multipotential precursor cells isolated from peripheral tissues containing sensory receptors such as the olfactory epithelium and tongue. The invention also relates to cells differentiated from the precursor cells. The invention includes pharmaceutical compositions containing precursor cells. The invention also includes cells differentiated from precursor cells and uses for those cells.

BACKGROUND OF THE INVENTION

There are a number of diseases of the central nervous system ("CNS") which have a devastating effect on patients. These diseases are incurable and debilitating. They include Alzheimer's disease, Huntington's disease, Parkinson's disease and Multiple Sclerosis, to name a few.

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 striatum. Parkinson's disease occurs when these dopaminergic neurons die. There are a number of methods to treat Parkinson's disease.

One method is to treat humans having parkinsonism with L-DOPA. Another method is to transplant cells into the substantia nigra or striatum. Transplanted cells replace endogenous cells that are lost as a consequence of damage. Transplanted cells may also be used as vectors for the expression of therapeutic molecules. Another method is to implant fetal brain grafts containing dopaminergic neurons. This method is experimental (Widner et al., 1993; Callahan et al., 1992). An animal model of Parkinson's disease is an MPTP-treated non-human primate. The animal models have been transplanted with dopamine-rich embryonic neurons with some success (Dunnett et al., 1991). (MPTP is a selective dopaminergic toxicant that produces parkinsonian symptoms in humans and in primates after a one-hit lesion to the neurons in the substantia nigra (Langston et al., 1983; Burns et al., 1983)).

Investigators studying other neurodegenerative diseases, such as Alzheimer's disease and Huntington's disease, are exploring the possible usefulness of fetal-tissue implants in the treatment of these diseases.

Current approaches to transplantation suffer from a number of serious limitations. First, many investigators are utilizing non-neural cells such as fibroblasts or transformed cell lines for transplantation. Second, the safety of transplantation of immortalized cell sources into the human brain is a concern. These cells may become unregulated and develop into tumors. Third, transplants of dopaminergic neuron fetal tissue to Parkinson's disease patients have a number of difficulties:

bulletthe fate of implanted dopaminergic neurons in patients with Parkinson's disease is uncertain—whatever caused the loss of endogenous dopaminergic neurons may also eventually injure the implanted ones,
bulletin many cases, implants provide only temporary relief as the symptoms associated with the disease often return after a number of years,
bulletthe patient may reject foreign fetal tissue,
bulletthere are adverse reactions associated with immunosuppression (immunosuppression is needed to try to help the patient accept the foreign fetal tissue, even though the brain is, to some degree, immunologically privileged),
bulleta sufficient number of cells in the fetal tissue being implanted are unable to survive during and after implantation,
bulletthe implants may not be regulated by the host brain,
bulletother diseases or disorders may be transmitted to the patient via the implant,
bulletthe cost and effort associated with implanting fetal tissue may not be justified by the results, and
bulletthere are objections to the ethics associated with implanting fetal tissue.

Many of these problems are encountered with transplants used to treat other neurodegenerative diseases, disorders or abnormal physical states.

In some tissues, stem cells and progenitor cells are proposed as a source for alternative treatments of disease or injury to tissues. The proposed treatments involve transplants of healthy tissue or endogenous stimulation of stem cells or progenitor cells to produce healthy tissue.

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 specialised tissues and organs of the developing fetus. In adults, specialised stem cells in individual tissues are the source of new cells which replace cells lost through cell death due to natural attrition, disease or injury. No stem cell is common to all tissues in adults. Rather, the term "stem cell" in adults describes different groups of cells in different tissues and organs with common characteristics.

Stem cells are capable of producing either new stem cells or cells called progenitor cells. A progenitor cell differentiates to produce the mature specialized cells of mammalian organs. In contrast, stem cells never terminally differentiate (i.e. they never differentiate into specialized tissue cells). Progenitor cells and stem cells are referred to collectively as "precursor cells". This term is often used when it is unclear whether a researcher is dealing with stem cells or progenitor cells or a combination of both cells.

Progenitor cells may differentiate in a manner which is unipotential or multipotential. A unipotential progenitor cell is one which can form only one particular type of cell when it is terminally differentiated. A multipotential progenitor cell has the potential to differentiate to form more than one type of tissue cell. Which type of cell it ultimately becomes depends on conditions in the local environment such as the presence or absence of particular peptide growth factors, cell—cell communication, amino acids and steroids. For example, it has been determined that the hematopoietic stem cells of the bone marrow produce all of the mature lymphocytes and erythrocytes present in fetuses and adult mammals. There are several well-studied progenitor cells produced by these stem cells, including three unipotential and one multipotential tissue cell. The multipotential progenitor cell may divide to form one of several types of differentiated cells depending on circumstances such as which hormones or factors act upon it and cell—cell contact.

Weiss et al, 1996, summarises the five defining characteristics of stem cells as the ability to:
 
bulletProliferate: Stem cells are capable of dividing to produce daughter cells.
bulletExhibit self-maintenance or renewal over the lifetime of the organism: Stem cells are capable of reproducing by dividing symmetrically or asymmetrically to produce new stem cells. Symmetric division occurs where one stem cell divides into two daughter stem cells. Asymmetric division occurs where one stem cell forms one new stem cell and one progenitor cell. Symmetric division is a source of renewal of stem cells. This permits stem cells to maintain a consistent level of stem cells in an embryo or adult mammal.
bulletGenerate large number of progeny: Stem cells may produce a large number of progeny through the transient amplification of a population of progenitor cells.
bulletRetain their multilineage potential over time: Stem cells are the ultimate source of differentiated tissue cells, so they retain their ability to produce multiple types of progenitor cells, which will in turn develop into specialized tissue cells.
bulletGenerate new cells in response to injury or disease: This is essential in tissues which have a high turnover rate or which are more likely to be subject to injury or disease, such as the epithelium or blood cells.

Thus, the key features of stem cells are that they are multipotential cells which are capable of long-term self-renewal over the lifetime of a mammal.

There has been much effort to isolate stem cells and determine which peptide growth factors, hormones and other metabolites influence stem cell renewal and production of progenitor cells, which conditions control and influence the differentiation of progenitor cells into specialized tissue cells, and which conditions cause a multipotential progenitor cell to develop into a particular type of cell.

Stem cells or progenitor cells may be used as substrates for producing healthy tissue where a disease, disorder or abnormal physical state has destroyed or damaged normal tissue. For example, stem cells and progenitor cells may be used as a target for in vivo stimulation with growth factors or they may be used as a source of cells for transplantation. The stem cells or progenitor cells may be transplanted or they may be induced to produce healthy differentiated cells for transplant.

In several tissues, stem cells have been isolated and characterised in an attempt to develop new therapies to repair or replace damaged tissues. For example, neural stem cells have been isolated from the mammalian brain (Reynolds and Weiss, Science 255:107 (1992)) and these cells were shown to be multipotential and able to differentiate into neurons, astrocytes and oligodendrocytes. WO 93/01275, WO 94/16718, WO 94/10292 and WO 94/09119 describe uses for these cells.

WO 95/13364 reports the delivery of growth factors to the ventricles of the CNS in order to stimulate neural stem cells to proliferate and produce neural progenitor cells which will develop into neurons, oligodendrocytes or astrocytes. This procedure has many complications which must be addressed before it may be used clinically. Differentiating the target neural stem cells or neural progenitor cells into a desired type of tissue which is functional is one complication. Another complication is choosing a growth factor which does not cause side effects in other areas of the brain.

These publications are limited to isolating or using adult stem cells from the brain (in particular, the tissue around the brain ventricles, the ventricle ependyma, which is the remnant of the embryonic brain germinal zone). Although these publications suggest that progenitor cells may be isolated from the adult peripheral nervous system ("PNS"), the publications define the PNS as the system which originates from the neural crest. There is no reported isolation of a stem cell from the PNS which does not originate from the neural crest.

There are no clinical treatments involving transplants of neural stem cells or neural progenitor cells isolated from the brain nor are there clinical treatments using differentiated cells produced from the neural stem cells or neural progenitor stem cells isolated from the brain. There are also no clinical treatments to endogenously stimulate the neural stem cells or neural progenitor cells of the brain in vivo to produce differentiated cells. Even if there were clinical procedures to transplant fetal neural stem cells or neural progenitor cells from the brain, or to transplant cells differentiated from these stem cells or progenitor cells (e.g. dopaminergic neurons into Parkinson's disease patients), this would not overcome the many problems of transplants from one human to another. As mentioned above, the only current, accessible human source for these neural stem cells and neural progenitor cells is aborted human fetuses, raising serious ethical concerns. Heterologous transplants are also very risky and complicated because of problems with graft rejection, immunosuppression, and the potential for donor grafts transferring diseases or disorders to a recipient. Encapsulation of cells in microspheres has the potential to decrease the likelihood of graft rejection, but this effect is lost if the integrity of the microsphere is disrupted. There is a clear need for safer tissue grafts which can be transplanted to a recipient without being rejected.

The safest type of tissue graft would be 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 would harm the site where the healthy neurons originate. Neural stem cells or neural precursor cells that can be differentiated into dopaminergic neurons may be available at the damaged site or at other sites from which they may be transplanted, but the CNS, particularly the brain, is physically difficult to access. It would be impractical or impossible to 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 or progenitor cells that could be differentiated into CNS cell types, such as dopaminergic neurons, to provide a source of cells for autologous transplants.

It would be useful if neural stem cells or progenitor cells could be identified and isolated outside the CNS and outside the PNS which originates from the neural crest. Medical treatments could then be developed using those neural stem cells, neural progenitor cells or cells differentiated from those cells. It is clear that despite the work that has been done to attempt to treat neurodegenerative diseases by tissue transplant, a need still exists for a pharmaceutical composition in which (1) the composition is accepted by the patient, thus avoiding the difficulties associated with immunosuppression, (2) the composition is safe and effective, thus justifying the cost and effort associated with treatment, (3) the composition provides long term relief of the symptoms associated with the disease, (4) the composition is efficacious during and after transplantation and (5) there are no objections to the ethics of the composition's use.

Thus, there is a clear need to develop neural stem cell cultures or neural progenitor cell cultures from accessible tissues of the PNS which can act as a source of cells that are transplantable to the CNS, PNS, spinal cord or other tissues in vivo in order to replace damaged tissue.

SUMMARY OF THE INVENTION

This invention relates to the isolation of "precursor cells" (which may be neural stem cells or neural progenitor cells or a combination of both types of cells) from peripheral tissue with sensory receptors, specifically olfactory epithelium and tongue, of the PNS. The olfactory epithelium is part of the PNS, but does not originate from the neural crest. Rather, it is of placodal origin. Hence, peripheral sensory neurons of the olfactory epithelium are developmentally distinct from the neurons of the neural crest derived PNS. Olfactory precursor cells have been isolated, determined to be multipotential and capable of generating CNS cell types. Thus, they are a useful source of tissue for autologous or heterologous transplant to the CNS, PNS, spinal cord and other damaged tissues.

The invention also includes isolated and purified precursor cells of a mammal from peripheral tissue containing sensory receptors, wherein the precursor cells are selected from a group consisting of neural stem cells, neural progenitor cells and a combination of neural stems cells and neural progenitor cells. The cells can be isolated from tongue.

The inventors have isolated precursor cells from the olfactory epithelium of mammals (juvenile and adult mice, adult rat and humans). The precursor cells of the olfactory epithelium possess the two key characterising features of stem cells: they are mutipotential and are self-renewing. They can be passaged and differentiated into cell types of the CNS, including astrocytes, oligodendrocytes, and dopaminergic neurons. Precursor cells isolated from the olfactory epithelium of neonatal mice express the immunological marker of neural stem and progenitor cells, nestin. These cells are not restricted to assuming an olfactory phenotype, but instead can differentiate into astrocytes, oligodendrocytes, and dopaminergic neurons. This shows that the olfactory epithelium is a useful source of dopaminergic neurons for homotypic grafts into Parkinson's Disease patients. The precursor cells of the olfactory epithelium may also be used for autologous or homologous transplants to treat other neurodegenerative diseases, disorders or abnormal physical states.

Precursor cells were also isolated from tongue and these may also be used for autologous or homologous transplants to treat neurotrauma or neurodegenerative diseases, disorders or abnormal physical states.

The stem cells or progenitor cells can be taken from an individual suffering from a neurodegenerative disease and then differentiated into neurons, astrocytes, oligodedrocytes for implantation into the nervous system of the individual. In a preferred mode of the invention, cells may be transplanted into the CNS, PNS, spinal cord or other damaged tissues.

Thus, this invention overcomes the needs outlined above in that the precursor cells of this invention (1) are accepted by the patient because they can be taken from the patient's own olfactory epithelium or tongue, (2) are safe in that the patient is not receiving cells or tissue from another source, (3) are effective in that the cells are of neural tissue origin and can be differentiated into neurons, astrocytes and oligodendrocytes for implantation and the cells survive during and after implantation, (4) offer the potential to provide long term relief of the symptoms associated with neurodegenerative diseases, and (5) would not raise objections to the ethics of their use.

Therefore, this invention relates to isolated and purified precursor cells of peripheral tissues with sensory receptors, such as the olfactory epithelium of a mammal (juvenile or adult). Under appropriate conditions, the precursor cells can differentiate into neurons, astrocytes or oligodendrocytes. The precursor cells may be transfected with a heterologous gene encoding, for example, a trophic factor. The precursor cells may then be implanted into the CNS, PNS, spinal cord or other damaged tissues of a patient and the heterologous gene expressed.

This invention also relates to neurons, astrocytes and oligodendrocytes differentiated from the precursor cells of this invention.

The invention also includes a pharmaceutical composition for use in implant therapy. The composition includes the precursor cells of this invention or neurons, astrocytes or oligodendrocytes differentiated from the precursor cells of this invention, in a pharmaceutically acceptable carrier, auxiliary or excipient. The composition may include one or more types of cells selected from a group consisting of precursor cells, neurons, oligodendrocytes and astrocytes.

A method of treating an individual suffering from a neurodegenerative disease is included within this invention. The method includes implanting the precursor cells of this invention, or the neurons, astrocytes or oligodendrocytes derived from the precursor cells of this invention, into the CNS, PNS, spinal cord or other damaged tissues of the individual. Another method consists of treating an individual suffering from a neurodegenerative disease by administering the pharmaceutical composition of this invention to the individual.

This invention also includes a method for isolating and purifying precursor cells from the olfactory epithelium of a mammal. The method includes (1) taking a sample of the olfactory epithelium from the mammal, (2) dissociating the sample into single cells, (3) placing the cells in culture, (4) isolating the cells which survive in culture. These isolated cells may be differentiated into neurons, astrocytes or oligodendrocytes. The precursor cells which survive in culture are spherical aggregates. The step of placing the cells in culture includes placing the cells in a tissue culture incubator in an appropriate medium. We isolate precursor cells from the tongue and other peripheral tissues with sensory receptors using a similar technique.

In this method, the mammal may be a human who is suffering from a neurodegenerative disease, disorder (such as neurotrauma) or abnormal physical state. The method may further include implanting the precursor cells or the neurons, astrocytes or oligodendrocytes differentiated from the neural stem cells, into the CNS, PNS, spinal cord or other damaged tissues of the human. In another case, the mammal is a human and is not suffering from a neurodegenerative disease or neurotrauma. Then, the method includes implanting the precursor cells or the neurons, astrocytes or oligodendrocytes differentiated from the precursor cells, into a second human who is suffering from the neurodegenerative disease or neurotrauma. The neurodegenerative disease may be one selected from a group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease and Multiple Sclerosis, while types of neurotrauma include stroke and spinal cord injury.

This invention also includes a kit for the treatment of a disease, disorder or abnormal physical state. The kit includes one or more types of cells including the precursor cells of this invention, or the neurons differentiated from these precursor cells, the astrocytes differentiated from these precursor cells and the neurons, astroycytes and oligodendrocytes differentiated from these precursor cells.

The invention also provides precursor cell cultures which may be used in toxicity testing, drug development testing or studies of genes and proteins. Precursor cell cultures may also be induced to produce healthy differentiated cells which may be used for toxicity testing or drug development testing. Toxicity testing is done by culturing precursor cells or cells differentiated from precursor cells in a suitable medium and introducing a substance, such as a pharmaceutical or chemical, to the culture. The precursor cells or differentiated cells are examined to determine if the substance has had an adverse effect on the culture. Drug development testing may be done by developing derivative cell lines, for example a pathogenic cell line, which may be used to test the efficacy of new drugs. Affinity assays for new drugs may also be developed from the precursor cells, differentiated cells or cell lines derived from the precursor cells or differentiated cells. The methods of performing toxicity testing and drug development testing are well known to those skilled in the art.

Precursor cells also provide a culture system from which genes, proteins and other metabolites involved in cell development can be isolated and identified. The composition of stem cells may be compared with that of progenitor cells and differentiated cells in order to determine the mechanisms and compounds which stimulate production of stem cells, progenitor cells or differentiated cells. Methods of isolating proteins and genes from cells are well known to those skilled in the art.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have isolated multipotential precursor cells from the olfactory epithelium of mammals juvenile and adult mice, adult rat and humans). The isolated cells proliferate in culture, so that large numbers of precursor cells can be generated. In culture, these cells form floating spheres which are named "olfballs". These cells can be induced to differentiate into neurons, astrocytes, and oligodendrocytes by altering the culture conditions. The precursor cells can generate differentiated cells for use in autologous transplants for the treatment of certain neurodegenerative disorders or neurotrauma. For example, precursor cells may be differentiated into dopaminergic neurons and implanted in the substantia nigra or striatum of Parkinson's disease patients. They can also be used to generate oligodendrocytes for use in autologous transplants for multiple sclerosis. The precursor cells are easily accessible by biopsy from the olfactory epithelium, so they are a ready source of cells for autologous transplants. Finally, they could be used as autologous cellular vectors to introduce growth factors into the diseased or traumatized CNS, PNS, spinal cord and other damaged tissues.

The olfballs display some similarities to forebrain stem cells, but also possess some distinctive differences. In particular, (i) when olfballs differentiate in the presence of serum, almost half of the differentiated cells express neuronal markers, whereas differentiated forebrain stem cell neurospheres generate only a small percentage of neurons, (ii) significant numbers of dopaminergic neurons are found in all differentiated cultures of olfballs, whereas they are never found in cultures of forebrain stem cell neurospheres differentiated in serum, and (iii) many of the undifferentiated progenitor cells that are found in olfball cultures express glutamic acid-decarboxylase (GAD), a neurotransmitter enzyme that is expressed transiently in many neuroepithelial cells in vivo; in contrast, the only GAD-positive cells that derive from forebrain stem cell neurosphere cultures are neurons.

The precursor cells of this invention may be used to prepare pharmaceutical compositions which can be administered to humans or animals. Dosages to be administered depend on patient needs, on the desired effect and on the chosen route of administration.

The invention also relates to the use of the cells of this invention to introduce growth factors into the diseased, damaged or physically abnormal CNS, PNS, spinal cord or other damaged tissues. The precursor cells act as a vector to transport a recombinant molecule, for example, or to transport a sense or antisense sequence of a nucleic acid molecule. In the case of a recombinant molecule, the molecule would contain suitable transcriptional or translational regulatory elements.

Suitable regulatory elements may be derived from a variety of sources, and they may be readily selected by one with ordinary skill in the art. If one were to upregulate the expression of the gene, one would insert the sense sequence and the appropriate promoter into the vehicle. If one were to downregulate the expression of the gene, one would insert the antisense sequence and the appropriate promoter into the vehicle. These techniques are known to those skilled in the art.

Examples of regulatory elements include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the vector employed, other genetic elements, such as selectable markers, may be incorporated into the recombinant molecule. The recombinant molecule may be introduced into the precursor cells or the cells differentiated from the precursor cells using in vitro delivery vehicles such as retroviral vectors, adenoviral vectors, DNA virus vectors and liposomes. They may also be introduced into such cells in vivo using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of DNA into liposomes. The genetically altered cells may be encapsulated in microspheres and implanted in the CNS, PNS, spinal cord and other damaged tissues.
 

Claim 1 of 11 Claims

1. A composition consisting of an isolated population of neural stem cells of a postnatal mammal and a carrier, wherein said neural stem cells form non-adherent clusters in culture, are self renewing, proliferate in an EGF-independent manner, express nestin, and differentiate, in the presence of serum, into neurons expressing tyrosine hydroxylase, said stem cells produced by a method comprising the steps of:

(a) providing a culture of peripheral tissue containing sensory receptors from said mammal;

(b) isolating neural stem cells from said peripheral tissue, based on the tendency of said neural stem cells to aggregate and form non-adherent clusters in culture, wherein said neural stem cells form non-adherent clusters in culture, are self renewing, proliferate in an EGF-independent manner, express nestin, and differentiate, in the presence of serum, into neurons expressing tyrosine hydroxylase.

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

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