CLAIM OF PRIORITY
This application is continuation of International Application No. PCT/US2007/084654, filed on Nov. 14, 2007, which claims the benefit of U.S. Provisional Patent Application Ser. No. 60/859,041, filed on Nov. 15, 2006; the entire contents of the foregoing applications are hereby incorporated by reference.
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government support under Grant No. F33 DC006789, RO1 DC007174, and P30 DC05209 from the National Institute on Deafness and other Communicative Disorders (NIDCD) of the National Institutes of Health. The Government has certain rights in the invention.
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This invention relates to methods using bone marrow mesenchymal stem cells to regenerate inner ear cells, e.g., hair cells and supporting cells, to treat inner ear damage.
A source of sensory cells and neurons for regeneration of inner ear cells would provide a valuable tool for clinical application because neurons and hair cells could be employed in cell replacement therapy for hearing loss. Recent work has shown that hair cells and neurons can be differentiated from endogenous stem cells of the inner ear (Li et al., Nat Med 9, 1293-1299 (2003); Rask-Andersen et al., Hear Res 203, 180-191 (2005)) and other work has shown that endogenous cells of the sensory epithelium can be converted to hair cells when the proneural transcription factor, Atoh1, is expressed exogenously (Izumikawa et al., Nat Med 11, 271-276 (2005); Zheng and Gao, Nat Neurosci 3, 580-586 (2000)) and yet the endogenous stem cells of the inner ear do not spontaneously generate hair cells. Injection of whole bone marrow to reconstitute a lethally irradiated mouse resulted in engraftment of these cells in areas occupied by inner ear mesenchymal cells and fibrocytes but did not yield hair cells (Lang et al., J Comp Neurol 496, 187-201 (2006)).
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The present invention is based, at least in part, on the discovery of methods that can be used to induce stem cells to differentiate into hair cells and supporting cells. Thus, described herein are methods for providing populations of hair cells and/or supporting cells, compositions comprising said cells, and methods of use thereof, e.g., for the treatment of subjects who have or are at risk of developing a hearing loss.
In one aspect, the invention provides methods for providing populations of hair cells and/or supporting cells. The methods include:
obtaining a population of stem cells with neurogenic potential;
culturing the stem cells under conditions sufficient to induce the differentiation of at least some of the stem cells into inner ear progenitor cells, and doing one (or more) of the following:
(i) inducing the expression of Atoh1 in the inner ear progenitor cells, in an amount and for a time sufficient to induce at least some of the inner ear progenitor cells to differentiate into hair cells;
(ii) contacting the inner ear progenitor cells with an inhibitor of Notch signalling (e.g., a gamma-secretase inhibitor or inhibitory nucleic acid), in an amount and for a time sufficient to induce at least some of the inner ear progenitor cells to differentiate into hair cells; or
(iii) culturing the inner ear progenitor cells in the presence of chick otocyst cells for a time and under conditions sufficient for at least some of the
inner ear progenitor cells to differentiate into hair cells, thereby providing populations of hair cells and/or supporting cells.
In some embodiments, the methods include isolating the inner ear progenitor cells, hair cells, and/or supporting cells, e.g., to provide a purified population thereof.
In some embodiments, the inner ear progenitor cells express nestin, sox2, musashi, Brn3C, Pax2, and Atoh1.
In some embodiments, the hair cells express one or more genes selected from the group consisting of Atoh1, jagged 2, Bm3c, p27Kip, Ngn1, NeuroD, myosin VIIa and espin. In some embodiments, the hair cells express jagged 2, Brn3c, myosin VIIa and espin. In some embodiments, the hair cells express F-actin in a V pattern on the apical surface of the cells.
In some embodiments, the supporting cells express one or more of claudin14, connexin 26, p75Trk, Notch 1, and S100A.
In some embodiments, the methods further include transplanting the hair cells or supporting cells into a subject in need thereof, e.g., into or near the sensory epithelium of the subject. In some embodiments, the population of stem cells is obtained from a subject in need of the transplant.
Also described herein are isolated populations of hair cells, supporting cells, and inner ear progenitor cells obtained by a method described herein.
In another aspect, the invention features methods for treating a subject who has or is at risk for developing a disorder, e.g., a hearing disorder or vestibular disorder, wherein the disorder is treatable with a transplant of hair cells and/or supporting cells, the method comprising transplanting cells obtained by a method described herein into the cochlea of the subject, thereby treating the subject. In these embodiments, it is preferable if the population of stem cells was obtained from the subject in need of the transplant.
In some embodiments, inducing the expression of Atoh1 in the cells comprises inducing the expression of exogenous Atoh1 in the cells, e.g., by transducing the cells with a vector encoding a Atoh1 polypeptide, e.g., a plasmid vector or a viral vector, e.g., an adenovirus, lentivirus, or retrovirus.
In some embodiments, inducing the expression of exogenous Atoh1 in the stem cells comprises increasing expression of endogenous Atoh1, e.g., by increasing activity of the Atoh1 promoter or by replacing the endogenous Atoh1 promoter with a more highly active promoter.
In some embodiments, culturing the stem cells in the presence of chick otocyst cells for a time and under conditions sufficient for at least some of the stem cells to differentiate into hair cells comprises culturing the stem cells in medium comprising IGF, EGF, and FGF.
In some embodiments, the stem cells used in the methods described herein are mesenchymal stem cells. In some embodiments, the stem cells used in the methods described herein are human stem cells.
As noted, the invention also features cells isolated by a method described herein, as well as compositions containing them.
Methods for treating subjects (e.g., mammals such as humans) who have, or who are at risk for developing, a hearing loss, are also described herein. These methods include administering a cell or population of cells (as described herein; e.g., a population of hair cells obtained by differentiating a population of stem cells) to the ear of the patient, e.g., to the cochlea. The administered cells may be obtained by the methods described herein, and the starting material may be stem cells obtained from the patient to be treated.
There may be certain advantages to the use of the cells described herein for the treatment of hearing loss. For example, the stem cells can be obtained from humans for clinical applications. Because the stem cells can be harvested from a human, and in particular can be harvested from the human in need of treatment, the immunological hurdles common in xeno- and allotransplantation experiments can be largely avoided.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
DESCRIPTION OF DRAWINGS
FIG. 1A is a row of four photomicrographs of bone marrow MSCs from passage 3 immunostained with antibodies against CD44, CD45, CD34 and Sca-1 followed by secondary antibodies against mouse immunoglobulins labeled with TRITC (medium gray, shown in red in the original). Staining for CD34 and CD45 was negative, but CD44 and Sca-1 were expressed. Nuclei were stained with DAPI (darker gray, blue in the original).
FIG. 1B is a row of four photomicrographs of bone marrow MSCs from passage 3 immunolabeled for CD44 (first panel, medium gray, shown in red in the original) and nestin (second panel, lighter gray, shown in green). The third panel is a DAPI nuclear stain (blue in original). The merged image in the right-most panel shows co-staining of a population of cells with both markers (lightest gray, yellow/orange in the original)
FIG. 1C is a row of four photomicrographs of bone marrow MSCs from passage 3 stained for co-expression of Sca-1 (first panel, red in the original) and nestin (second panel, green in the original). Merged image in the right-most panel shows co-staining.
FIG. 1D is a row of four plots showing the results of analysis of bone marrow MSCs by chip flow cytometry indicating the ratio of immunopositive cells for each of the listed antibodies (CD44, first panel; Sca-1, second panel; CD34, third panel; and CD45, last panel); axes are “Fluorescence” and “No. of events.”
FIG. 1E is a pair of photomicrographs showing the potential for lineage differentiation, as demonstrated by formation of chondrocytes and extracellular matrix after treatment of bone marrow MSCs with TGF-β. Cells that grew out from a micro-aggregate (left) were stained for type II collagen (right).
FIG. 1F is a pair of photomicrographs showing the differentiation of bone marrow MSCs to neurons by differentiation in serum-free medium containing neuronal growth supplements and bFGF. Staining for neurofilament (NF-M) is shown in these cells.
FIG. 2A is a gel showing the results of genetic analysis for neural progenitor markers by RT-PCR of MSCs treated with IGF-1, EGF and bFGF for 14 days followed by analysis. MSC (bone marrow MSCs), NP (neural progenitors at 2 wks after induction of progenitor formation). The genes analyzed are shown to the left of the gel.
FIGS. 2B-C are two sets of four photomicrographs showing that the neural progenitor marker, nestin, visualized by immunohistochemistry using a secondary antibody labeled with FITC (top right panel in 2B and 2C, shown in green in the original), was co-expressed with CD44 (2B, top left panel, shown in red in the original) and with Sca-1 (2C, top left panel, shown in red in the original). DAPI is shown in blue (lower left panel in each figure). Scale bars are 50 μm. Merged images in the lower right panel of each figure show coexpression of nestin and CD44 (2B) or Sca1 (2C) (all of the cells appeared green in the original, indicating coexpression).
FIG. 3A is a gel showing the results of genetic analysis by RT-PCR of precursor cells incubated in NT3, FGF and BDNF (which support neuronal and sensory cell progenitors in the inner ear). The gene profiles included expression of Oct4, nestin, Otx2, and Musashi, as well as proneural transcription factors, GATA3, NeuroD, Ngn1, Atoh1, Brn3c, and Zic2. These cells did not express hair cells genes, myosin VIIa and espin.
FIG. 3B is a gel showing the results of genetic analysis by RT-PCR of the cells obtained after induction with NT3, FGF, and BDNF. Genes characteristic of supporting cells (claudin14, connexin 26, p75Trk, Notch 1, and S100A) were also observed. These progenitor cells thus had expression profiles characteristic of neuronal or sensory progenitors. Genes analyzed are shown to the left of the gels.
FIG. 4A is a photomicrograph showing exogenous expression of Atoh1 in bone marrow MSCs; expression was observed in cells and nuclei (green in the original) due to the expression of GFP from the vector.
FIG. 4B is a gel showing the results of gene expression in cells transfected with Atoh1 followed by treatment of the cells with NT3, FGF and BDNF. The results indicate that this protocol gave rise to progenitor cells that subsequently matured into cells expressing hair cell genes, including espin, myosin VIIa, jagged 2, and Brn3c, and p27Kip, in addition to the proneural genes, Ngn1 and NeuroD.
FIG. 4C is a gel showing the results of further genetic analysis of the cells under the differentiating conditions described in 4B; the results showed that the cells also expressed S100A, p75Trk, claudin 14, connexin 26, and Notch1, consistent with some cells having a supporting cell phenotype.
FIG. 4D is a photomicrograph of an MSC cell line selected in Zeocin; the cells had a high percentage of GFP expression when cultured in serum (green in original).
FIG. 4E is a row of 4 photomicrographs of cells stained for Myo7a (first panel), Math1/Atoh1 (second panel), or DAPI (third panel); the last panel is a merged image. After differentiation, the number of hair cell-like cells per DAPI nucleus rose and these cells stained for myosin VIIa (shown in red in the first panel) and Atoh1 (shown in green in the second panel; arrows in the second and last panels).
FIG. 4F is two rows of 4 photomicrographs of an Atoh1 expressing cell line differentiated to cells with nuclei that were immunopositive for Bm3c (second column, green in original; indicated by arrowheads) and cytoplasm positive for myosin VIIa (first column, red in original; indicated by arrows). Nuclei were stained with DAPI (third column, blue in original).
FIG. 4G is a row of three photomicrographs showing that the differentiated cells were positive for F-actin which protruded from the apex of the cell in the shape of a stereocilia bundle (arrow).
FIG. 4H is a row of three photomicrographs showing that F-actin staining was arranged in a characteristic V pattern on the apical surface.
FIG. 5A is a gel showing the results of genetic analysis of bone marrow MSC derived progenitors were co-cultured for 21 days with chick otocyst cells that had been treated with mitomycin C (Mito C); the results showed that expression of jagged 2, p27Kip, Atoh1, Brn3c, myosin VIIa and espin was increased, whereas the expression of these genes in chick cells was undetectable. Chick otocyst cells that had been fixed by incubation with paraformaldehyde were less effective (PFA) than the unfixed cells but did cause differentiation of the progenitors. Conditioned medium from the chick cells (Cnd Med) had no effect (levels of expression of these markers similar to previously shown data for differentiating conditions).
FIG. 5B is a set of three photomicrographs showing that expression of Atoh1 (Math-1, middle panel, green in original) and myosin VIIa (top panel, red in original) in cells from a Atoh1-GFP mouse showed green fluorescence corresponding to the induction of this marker in the nucleus and had expression of myosin VIIa in the cytoplasm.
FIG. 6A is a set of four photomicrographs showing an increase in fluorescence (green in original) indicating the conversion of bone marrow cells to cells expressing Atoh1. The cells stained for Atoh1 (Math1, bottom left, green in original), myosin VIIa (top left, red in original) and DAPI (top right, blue in original). A merged image is shown in on the bottom right panel.
FIG. 6B is a photomicrograph showing that Atoh1-expressing cells were found incorporated into the tissue of the chick otic epithelium. The hair cells of the chick were stained with the chick-specific marker, HCA (white in original) and myosin VIIa (red in original), whereas the Atoh-1 expressing mouse cells were green due to expression of GFP (arrows).
FIG. 6C is a set of four photomicrographs showing a lack of cell fusion, demonstrated by the presence of HCA (arrowhead, lower panels) in cells that did not have green fluorescence and of Atoh1-GFP (arrow, right column) exclusively in cells that did not stain for HCA, a marker for chicken cells. No cells with both GFP and HCA were observed in these experiments. Scale bars are 100 μm.
FIG. 7 is a gel showing the results of genetic analysis of cells after inhibition of Notch signaling with an inhibitor of γ-secretase increases expression of hair cell markers. Gene expression in MSCs treated with a γ-secretase inhibitor showed that loss of Notch signaling increased Atoh1 expression. The timing of inhibition was critical: γ-secretase inhibitor added at d1 of differentiation in vitro for a total of 10 days led to an increase in hair cell markers, myosin VIIa and espin, whereas inhibitor added at d3 did not induce hair cell markers.
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Although stem cells are present in the inner ear (Li et al., Trends Mol Med 10, 309-315 (2004); Li et al., Nat Med 9, 1293-1299 (2003); Rask-Andersen et al., Hear Res 203, 180-191 (2005)), hair cells do not regenerate after damage, and, therefore, a source of cells that could potentially be used for cell transplantation in a therapeutic replacement of these sensory cells has important implications for treatment of sensorineural hearing loss. Bone marrow has been harvested and used extensively in clinical applications and is a highly desirable source, because cells from a patient\'s bone marrow could potentially be transplanted without the problem of immune rejection. The present methods include a treatment regimen for hearing loss including transplantation of hair cells obtained by methods described herein.
By a combination of growth factor stimulation and expression of the transcription factor, Atoh1, that is required for hair cell formation in the inner ear, the present inventors demonstrate herein that stem cells, e.g., mesenchymal stem cells derived from bone marrow, can be induced to differentiate into hair cells. In addition, the neurosensory progenitors obtained from bone marrow can be converted to sensory cells by co-culture with cells of the developing sensory epithelium, even in the absence of Atoh1 expression.
Stem cells in bone marrow are known to be the precursors for all lymphoid and erythroid cells, but mesenchymal stem cells in bone marrow also act as precursors to bone, cartilage, and fat cells (Colter et al., Proc Natl Acad Sci USA 97, 3213-3218 (2000); Pittenger et al., Science 284, 143-147 (1999)). In addition to mesenchymal tissues, these stem cells have been shown to give rise to cells of other lineages including pancreatic cells (Hess et al., Nat Biotechnol 21, 763-770 (2003)), muscle cells (Doyonnas et al., Proc Natl Acad Sci USA 101, 13507-13512 (2004)) and neurons (Dezawa et al., J Clin Invest 113, 1701-1710 (2004); Hermann et al., J Cell Sci 117, 4411-4422 (2004); Jiang et al., Proc Natl Acad Sci USA 100 Suppl 1, 11854-11860 (2003)). The evidence provided herein demonstrates an extended range of cell fates available for these bone marrow-derived cells that includes cells of the neurosensory lineage, even including differentiation to inner ear hair cells.
Methods for Generating Cells of the Inner Ear
Methods of generating cells of the inner ear are provided, including progenitor cells and differentiated inner ear cells including hair cells and supporting cells. Stem cells are unspecialized cells capable of extensive proliferation. Stem cells are pluripotent and are believed to have the capacity to differentiate into most cell types in the body (Pedersen, Scientif. Am. 280:68 (1999)), including neural cells, muscle cells, blood cells, epithelial cells, skin cells, and cells of the inner ear (e.g., hair cells and cells of the spiral ganglion). Stem cells are capable of ongoing proliferation in vitro without differentiating. As they divide, they retain a normal karyotype, and they retain the capacity to differentiate to produce adult cell types.
Hematopoietic stem cells resident in bone marrow are the source of blood cells, but in addition to these hematopoietic stem cells, the bone marrow contains mesenchymal stem cells (MSCs) that can differentiate into cell types of all three embryonic germ layers (Colter et al., Proc Natl Acad Sci USA 97, 3213-3218 (2000); Doyonnas et al., Proc Natl Acad Sci USA 101, 13507-13512 (2004); Herzog et al., Blood 102, 3483-3493 (2003); Hess et al., Nat Biotechnol 21, 763-770 (2003); Jiang et al., Nature 418, 41-49 (2002); Pittenger et al., Science 284, 143-147 (1999)). This has been demonstrated in vivo in studies that track transplanted bone marrow cells to specific tissues where they differentiate into the resident tissue type (Mezey et al., Proc Natl Acad Sci USA 100, 1364-1369 (2003); Weimann et al., Proc Natl Acad Sci USA 100, 2088-2093 (2003)).
Many of these cells have been used for transplantation and are a preferred source of new cells for therapies because the transplanted cells are immunologically matched when harvested from a patient to be treated and because they have been extensively used in clinical applications so that their safety is known.
Stem cells can differentiate to varying degrees. For example, stem cells can form cell aggregates called embryoid bodies in hanging drop cultures. The embryoid bodies contain neural progenitor cells that can be selected by their expression of an early marker gene such as Sox1 and the nestin gene, which encodes an intermediate filament protein (Lee et al., Nat. Biotech. 18:675-9, 2000).
Neurogenic Stem Cells
Inner ear cells or inner ear cell progenitors can be generated from mammalian stem cells. As described herein, stem cells suitable for use in the present methods can be any stem cell that has neurogenic potential, i.e., any stem cell that has the potential to differentiate into a neural cell, e.g., neurons, glia, astrocytes, retinal photoreceptors, oligodendrocytes, olfactory cells, hair cells, supporting cells, and the like. Neurogenic stem cells, including human adult stem cells such as bone marrow mesenchymal stem cells, can be induced to differentiate into inner ear progenitor cells that are capable of giving rise to mature inner ear cells including hair cells and supporting cells. Neurogenic stem cells useful in the methods described herein can be identified by the expression of certain neurogenic stem cell markers, such as nestin, sox1, sox2, and musashi. Alternatively or in addition, these cells express high levels of helix-loop-helix transcription factors NeuroD, Atoh1, and neurogenin1.
Examples of neurogenic stem cells include embryonic stem cells or stem cells derived from mature (e.g., adult) tissue, such as the ear (e.g., inner ear), central nervous system, blood, skin, eye or bone marrow. In some embodiments, the stem cells are mesenchymal stem cells. Any of the methods described herein for culturing stem cells and inducing differentiation into inner ear cells (e.g., hair cells or supporting cells) can be used.
Stem cells useful for generating cells of the inner ear can be derived from a mammal, such as a human, mouse, rat, pig, sheep, goat, or non-human primate. For example, stem cells have been identified and isolated from the mouse utricular macula (Li et al., Nature Medicine 9:1293-1299, 2003).
Generation of Neural Progenitor Cells
There are a number of induction protocols known in the art for inducing differentiation of stem cells with neurogenic potential into neural progenitor cells, including growth factor treatment (e.g., treatment with EGF, FGF, and IGF, as described herein) and neurotrophin treatment (e.g., treatment with NT3 and BDNF, as described herein). Other differentiation protocols are known in the art; see, e.g., Corrales et al., J. Neurobiol. 66(13):1489-500 (2006); Kim et al., Nature 418, 50-6 (2002); Lee et al., Nat Biotechnol 18, 675-9 (2000); and Li et al., Nat Biotechnol 23, 215-21 (2005).
As one example of an induction protocol, the stem cells are grown in the presence of supplemental growth factors that induce differentiation into progenitor cells. These supplemental growth factors are added to the culture medium. The type and concentration of the supplemental growth factors is be adjusted to modulate the growth characteristics of the cells (e.g., to stimulate or sensitize the cells to differentiate) and to permit the survival of the differentiated cells such as neurons, glial cells, supporting cells or hair cells.
Exemplary supplementary growth factors are discussed in detail below, and include, but are not limited to basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), and epidermal growth factor (EGF). Alternatively, the supplemental growth factors can include the neurotrophic factors neurotrophin-3 (NT3) and brain derived neurotrophic factor (BDNF). Concentrations of growth factors can range from about 100 ng/mL to about 0.5 ng/mL (e.g., from about 80 ng/mL to about 3 ng/mL, such as about 60 ng/mL, about 50 ng/mL, about 40 ng/mL, about 30 ng/mL, about 20 ng/mL, about 10 ng/mL, or about 5 ng/mL).
Neural progenitor cells produced by these methods include inner ear progenitor cells, i.e., cells that can give rise to inner ear cells such as hair cells and supporting cells. Inner ear progenitor cells can be identified by the expression of marker genes such as nestin, sox2, and musashi, in addition to certain inner-ear specific marker genes Brn3C, Pax2, and Atoh1. The invention includes purified populations of inner ear progenitor cells expressing nestin, sox2, musashi, Brn3C, Pax2, and Atoh1. These inner ear progenitor cells are lineage committed, and can be induced to further differentiate into hair cells and supporting cells by a method described herein.
Progenitor cells prepared by a method described herein can optionally be frozen for future use.
Cell Culture Methods
In general, standard culture methods are used in the methods described herein. Appropriate culture medium is described in the art, such as in Li et al. (supra). For example, stem cells can be cultured in serum free DMEM/high-glucose and F12 media (mixed 1:1), and supplemented with N2 and B27 solutions and growth factors. Growth factors such as EGF, IGF-1, and bFGF have been demonstrated to augment sphere formation in culture. In vitro, stem cells often show a distinct potential for forming spheres by proliferation of single cells. Thus, the identification and isolation of spheres can aid in the process of isolating stem cells from mature tissue for use in making differentiated cells of the inner ear. The growth medium for cultured stem cells can contain one or more or any combination of growth factors. This includes leukemia inhibitory factor (LIF) which prevents the stem cells from differentiating. To induce the cells (and the cells of the spheres) to differentiate, the medium can be exchanged for medium lacking growth factors. For example, the medium can be serum-free DMEM/high glucose and F12 media (mixed 1:1) supplemented with N2 and B27 solutions. Equivalent alternative media and nutrients can also be used. Culture conditions can be optimized using methods known in the art.
Differentiation by Expression of Atoh1
As described herein, expression of Atoh1 in stem-cell derived progenitor cells was sufficient to drive them into adopting hair cell markers. Studies of Atoh1 expression in the ear have indicated that this helix-loop-helix transcription factor occupies a key place in the hierarchy of inner ear transcription factors for differentiation of hair cells.