Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
2001-04-12
2004-01-06
Reynolds, Deborah J. (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S325000, C435S366000, C435S368000, C435S377000, C424S093100
Reexamination Certificate
active
06673606
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to preparing and using different types of cells to ameliorate pathologies of the central nervous system (CNS) which are associated with the dysfunction or loss of neurons and oligodendrocytes, respectively.
DESCRIPTION OF THE RELATED ART
Two components of the mammalian CNS, oligodendrocytes and neurons, do not readily undergo mitotic division and, hence, are not replaced in vivo upon their loss, occasioned by disease or trauma. For example, Parkinson's disease involves a loss of neurons in a portion of the brain that produces dopamine. The resultant decline in dopamine levels is manifested in the development of muscle tremors, a stiffening of muscles and joints, and an overall lack of coordination. Another neurodegenerative disease, multiple sclerosis (MS), is marked by a breakdown in the axonal sheathing of myelin, constituted by oligodendrocytes, during early post-natal development. This demyelination disrupts signal transmission along the axon, causing vision impairment, loss of coordination, and even memory loss.
The devastating effects of such diseases have motivated research into cell lines and culture conditions that can accommodate in vitro differentiation of cells into neurons and the supporting cells of the CNS, including oligodendrocytes. It has been determined, for example, that rat O-2A cells, the progenitors of oligodendrocytes, can be prompted to differentiate into mature oligodendrocytes by the removal of mitogens from the growth medium. Barres et al.,
Development
120: 1097 (1994). Alternatively, rat O-2A cells will differentiate into mature oligodendrocytes when cultured in a serum-free medium, in the presence of mitogens, if ligands for hydrophobic cell-surface receptors are present, such as thyroid hormones, glucocorticoids and retinoic acid. ld. A human teracarcinoma cell line, designated “NT2,” can be differentiated into post-mitotic CNS neurons, by means of a complex regimen of in vitro manipulations, over a period of 6 to 10 weeks. Younkin et al.,
Proc. Nat'l Acad. Sci. USA
90: 2174 (1993).
The differentiation of multipotent neural stem cells is another possible route to obtaining neurons. Thus, U.S. Pat. No. 5,851,832 discloses a subpopulation of constitutively proliferating cells, from the subependymal region lining the lateral ventricles in the forebrain, that are said to be capable of differentiating into a neuronal lineage.
While capable of differentiating into oligodendrocytes or neurons, neural stem cells are difficult to obtain in quantities sufficient for potential therapeutic uses. This fact and the drawbacks associated with known cell lines, such as teracarcinoma-derived NT2 line, have meant that few therapeutic alternatives were available for treating disorders characterized by reduced levels of oligodendrocytes or neurons.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a source for differentiated oligodendrocytes and neurons, respectively, that is independent of neural stem cells and conventional cell lines.
It is another object of the present invention to provide a workable therapeutic approach toward pathologies, affecting the CNS, that are characterized by the loss of neurons and/or oligodendrocytes.
In accomplishing these and other objects, there has been provided, according to one aspect of the present invention, a method for treating a pathology, characterized by damaged myelin or neurological deterioration, that comprises (A) providing a composition in vitro that consists essentially of (i) mesenchymal stromal cells, or “MSCs,” and (ii) a physiologically compatible carrier therefor, (B) exposing the composition to conditions such that the MSCs differentiate into neurons and/or oligodendrocytes, and then (C) allowing the differentiated cells to compensate for neurological deterioration or damaged myelin in a subject suffering from the pathology in question. In a preferred embodiment, the composition is introduced into the nervous system of the subject.
Pursuant to another aspect of the present invention, differentiated cells are prepared by exposing an MSC-containing composition, as described above, to conditions such that the MSCs differentiate in vitro into neurons and/or oligodendrocytes.
In a related vein, the present invention further provides a composition that consists essentially of immortalized mesenchymal stromal cells and a physiologically compatible carrier. Additionally, the invention encompasses a composition that consists essentially of immortalized mesenchymal stromal cells, one or more exogenous genes, and a physiological compatible carrier.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
It has been found that mesenchymal stromal cells, stem-like precursors of non-hematopoietic cells, can differentiate into oligodendrocytes and neurons, respectively. Prior to this invention, there was no indication that MSCs could differentiate into oligodendrocytes or neurons. It had been suggested that at least a subset of cells isolated from bone marrow could follow the developmental pathway of astrocytes, another CNS cell type. See Azizi et al.,
Proc. Nat'l Acad. Sci. USA
95: 3908 (1999). But there was no reason to predict heretofore that MSCs would be able to differentiate along a lineage leading to an oligodendrocytic or to a neuronal morphology.
This surprising capability, discovered by the inventor, makes MSCs and MSC-differentiated cells, in accordance with the present invention, a therapeutic resource for treating (A) CNS pathologies characterized by neuron loss, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and stroke, and (B) metabolic lipid-storage diseases, such as Tay-Sachs, GM1 gangliosidosis, adrenoleukodystrophy, Krabbe's disease, metachromatic leukodystrophy and multiple sclerosis, which involve oligodendrocytic loss. By the same token, the present invention also provides for the use of MSCs and MSC-differentiated cells to ameliorate the neuronal loss brought on by head injury or other trauma.
To these ends, the invention contemplates, in one of its aspects, administering a therapeutically effective amount of MSC or MSC-differentiated cells to a patient in need of either oligodendrocytes or neurons. In this regard, “MSC-differentiated cells” refers to any cell that has differentiated from an MSC, including both immature and mature oligodendrocytes and neurons, respectively. “Therapeutically effective” connotes an amount of MSCs or MSC-differentiated cells that lessens the detrimental effect of (“ameliorates”) the CNS disorder in a patient receiving the therapy.
As described below in more detail, MSCs are relatively easy to isolate from aspirates of bone marrow, which can be obtained under local anesthesia. They also are relatively easy to expand in culture, and to transfect with exogenous DNA, as reported in Prockop,
Science
276: 71 (1997). These advantages, combined with their stem cell-like qualities of in situ migration and pluripotency, recommend MSCs for use, pursuant to the present invention, as vehicles for therapeutic DNA delivery in the context, for example, of treating CNS disorders, such as brain cancer.
Although MSCs themselves can be administered therapeutically, according to the present invention, in many instances it may be more practical to administer MSC-differentiated cells to a patient suffering from a CNS disorder. To this end, MSCs can be differentiated into oligodendrocyte and neurons, respectively, under appropriate culture conditions determined empirically, by adding and removing various trophic factors known to effect neuronal or oligodendrocytal differentiation, thereby mimicking in vivo physiological conditions.
Isolating MSCs From Bone Marrow
For purposes of the present invention, MSCs can be obtained by known methodologies disclosed, for example, by Azizi et al. (1999), supra. To this end, bone marrow can be aspirated from the iliac crest of a donor, who can be the patient to be treated in accordance with the present inventio
Beesley Jackie S.
Coyle Andrew J.
Grinspan Judith
Tennekoon Gihan
Dann Dorfman Herrell and Skillman
Reynolds Deborah J.
The Children's Hospital of Philadelphia
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