Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-04-01
2002-01-08
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S377000, C435S455000, C536S023100, C536S024100
Reexamination Certificate
active
06337184
ABSTRACT:
BACKGROUND OF THE INVENTION
The field of the invention is molecular markers for muscle stem cells.
The development, growth, and repair of skeletal muscles all require mononucleate myoblasts that are committed to form multinucleate myofibers via intercellular fusion. Committed myoblasts are thought to be the progeny of uncommitted, self-renewing stem cells; however, molecular markers that would permit identification and study of muscle stem cells have not previously been described (Quinn et al., Exp. Cell Res. 154, 65-82 (1984); Baroffio et al., Differentiation 60, 47-57 (1996)). These and all other publications and patents cited herein are hereby incorporated by reference.
SUMMARY OF THE INVENTION
It has now been discovered that Bcl-2, an apoptosis-inhibiting protein, is expressed in muscle stem cells, but not in other myogenic cells (e.g., multinucleate myotubes and myofibers). Thus, Bcl-2 is a molecular marker for muscle stem cells (e.g., human muscle stem cells). This discovery suggests a number of methods for identifying and/or isolating muscle stem cells. For example, one method of the invention provides a means for identifying a muscle stem cell by providing a sample that includes a myogenic cell and detecting activity of a Bcl-2 promoter within the myogenic cell as an indication that the myogenic cell is a muscle stem cell. The invention can be used to detect muscle stem cells that produce skeletal muscle, smooth muscle, or cardiac muscle. The Bcl-2 marker also can be used to detect and characterize a stem cell component in muscle tumors (e.g., in methods of diagnosing or evaluating muscle tumors).
The activity of the Bcl-2 promoter can be detected by any of a variety of methods. For example, the activity of the Bcl-2 promoter can be detected by detecting a Bcl-2 protein in the myogenic cell. To this end, conventional methods, such as SDS-PAGE and/or immunoassays, can be employed. Antibodies that specifically bind Bcl-2 are known in the art and readily available for use in such immunoassays. If desired, the activity of the Bcl-2 promoter can be detected by detecting Bcl-2 mRNA in the myogenic cell. Art-known methods such as reverse transcription-PCR (RT-PCR), in situ hybridization, and Northern blots can be used to detect the Bcl-2 mRNA.
In a variation of the above methods, the activity of a Bcl-2 promoter is detected with the use of a heterologous reporter gene (e.g., a chloramphenicol acetyltransferase gene, an alkaline phosphatase gene, a luciferase gene, or a green fluorescent protein gene). In a typical method, the heterologous reporter gene is operably linked to a Bcl-2 promoter in a genetic construct (e.g., a viral-based vector or a plasmid). Conventional molecular biology techniques can be used to produce such a genetic construct. The genetic construct then is introduced into a population of cells containing myogenic cells and thought to contain muscle stem cells. Since no myogenic cells except muscle stem cells activate the Bcl-2 promoter, expression of the reporter gene is detected as an indication that a cell is a muscle stem cell. As above, conventional methods for detecting gene expression can be used to detect reporter gene expression (e.g., protein or mRNA assays).
Now that a molecular marker for identifying muscle stem cells has been discovered, several related methods are possible. Thus, the invention also provides a method for determining whether a test compound(s) modulates muscle stem cell differentiation. In this method, a cell is identified as a muscle stem cell (e.g., by using one of the abovedescribed methods). The muscle stem cell is contacted with the test compound (e.g., in vitro). A change in the differentiation of the stem cell, compared to control, is an indication that the compound modulates muscle stem cell differentiation. Any compound can be used as the test compound in this method. Both naturally-occurring and synthetic polypeptides and small organic molecules are suitable test compounds. Compounds and analogs of compounds that are known to affect the differentiation of other cells are particularly suitable for use in this method. Parameters such as the rate and pattern of cell differentiation can be measured using conventional means. In a related method, one can determine whether a test compound modulates muscle stem cell proliferation. This method is nearly identical to that described above, except that a change in cell proliferation, compared to control, is detected. Of particular interest are test compounds that modulate the rate of cell proliferation. Of course, the above-described methods for detecting compounds that modulate cell differentiation and proliferation can be combined into a single experiment using one or more test compounds.
The invention also provides a method for producing a population of cells that is enriched for muscle stem cells relative to a reference population of cells. The method entails providing a reference population of cells that includes a plurality of muscle stem cells and at least one cell (typically many cells) other than a muscle stem cell (e.g., myoblasts). Typically, the reference population is obtained by muscle biopsy. A genetic construct may be introduced into the reference population of cells. The genetic construct includes a Bcl-2 promoter that is operably linked to a gene encoding a marker protein. The marker protein is a protein that is heterologous to wild-type cells of the reference population. Cells that express the marker protein (i.e., cells in which the Bcl-2 promoter is active) are then isolated in order to produce a population of cells enriched for muscle stem cells. Of course, by removing the Bcl-2-expressing cells from the cell population, this method can be used to produce a population of cells depleted of muscle stem cells.
The heterologous marker protein can be viral, prokaryotic, eukaryotic, or synthetic in origin. Preferably, the marker protein is not naturally expressed in wild-type muscle stem cells or muscle cells in general. Typically, the marker protein is a polypeptide that is expressed on the cell surface. Examples of suitable marker proteins include CD8, &bgr;-galactosidase, green fluorescent protein, catechol 2,3-dioxygenase, aequorin, and influenza virus hemagglutinin (which can be detected using commercially available monoclonal antibodies); the genes encoding these and other suitable marker proteins are known in the art. Conventional cell sorting methods (e.g., fluorescence-activated cell sorting (FACS)) can be used to isolate those cells in which the Bcl-2 promoter directs the expression of the gene encoding the marker protein. Other techniques, such as the use of protein-conjugated magnetic beads that selectively bind particular cells, also can be used. For example, magnetic beads conjugated to anti-CD8 antibodies can be used to isolate muscle stem cells expressing CD8 under the control of the Bcl-2 promoter.
Included within the invention is a method for producing a population of living cells enriched for muscle stem cells relative to a reference population of cells (i.e., a starting population of cells). This method entails:
(a) providing a reference population of living cells that includes a plurality of muscle stem cells that express Bcl-2 and at least one cell other than a muscle stem cell (e.g., a myoblast); and
(b) treating the reference population of cells to induce apoptosis (i.e., programmed cell death) in cells that do not express Bcl-2, thereby producing a population of living cells enriched for muscle stem cells. The expression of Bcl-2 inhibits apoptosis of the muscle stem cells, thereby allowing the muscle stem cells to survive under conditions that result in the death of other cells. In this method, apoptosis can be induced by any of the art-known methods. In a preferred method, the cells are contacted with staurosporine (C
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The discovery of a molecular marker for muscle stem cells m
Fish & Richardson P.C.
McKelvey Terry
Sandals William
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