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The mammalian inner ear has very limited ability to regenerate lost sensory hair cells. This deficiency becomes apparent when hair cell loss leads to hearing loss as a result of either ototoxic insult or the aging process. Coincidently, with this inability to regenerate lost hair cells, the adult cochlea does not appear to harbor cells with a proliferative capacity that could serve as progenitor cells for lost cells. In contrast, adult mammalian vestibular sensory epithelia display a limited ability for hair cell regeneration, and sphere-forming cells with stem cell features can be isolated from the adult murine vestibular system. The neonatal inner ear, however, does harbor sphere-forming stem cells residing in cochlear and vestibular tissues. Here, we provide protocols to isolate sphere-forming stem cells from neonatal vestibular and cochlear sensory epithelia as well as from the spiral ganglion. We further describe procedures for sphere propagation, cell differentiation, and characterization of inner ear cell types derived from spheres. Sphere-forming stem cells from the mouse inner ear are an important tool for the development of cellular replacement strategies of damaged inner ears and are a bona fide progenitor cell source for transplantation studies.
The ability to form floating clonal colonies or “spheres” is not only a hallmark of certain stem and progenitor cell populations, but is also a useful feature for isolation of these cells from complex cell mixtures. Several laboratories have shown that sphere-forming cells reside in the neonatal and even in the adult inner ear (1–11). It has also been demonstrated that some of the inner ear–derived sphere-forming cells have the ability to self-renew, which is a characteristic feature of stem cells (2,7). Self-renewal has been reported for sphere-forming cells from the neonatal spiral ganglion, the organ of Corti (OC), and vestibular sensory epithelia, as well as from the adult utricular sensory epithelium. In this chapter, we refer to these sphere-forming and self-renewing cells as inner ear stem cells. Previous results show that different tissues of the neonatal inner ear harbor distinct populations of stem cells, each one displaying specific features. Spiral ganglion–derived spheres, for example, give rise to neurons and glial cell types and other unidentified cells after withdrawal of growth factors and attachment to a substrate (7, 8, 12). Only occasionally have we found cells positive for hair cell markers in cell populations differentiated from spiral ganglion–derived spheres. In contrast, we frequently observed the generation of hair cell marker expressing cells from spheres isolated from the OC or vestibular sensory epithelia. These spheres also readily gave rise to neurons and glial cell types, albeit with lower frequency when compared with spiral ganglion–derived spheres.
Here we provide detailed protocols for the isolation of sphere-forming cells from various parts of the neonatal inner ear. We also describe how to propagate spheres and how to initiate spontaneous differentiation of inner ear cell types. Spheres generated from inner ear stem cells can be used not only for in vitro studies but also for transplantation experiments, for example, in explorative studies aimed at development of cellular therapies.
Dissected tissues are dissociated by enzymatic digestion and mechanical trituration. Greiner six-well cell suspension culture plates are suitable for production of floating colonies because their non-stick surface does not allow adherent cell growth.
Total RNA is isolated from inner ear tissue samples, undifferentiated spheres, and differentiated sphere-derived cells using silica-gel-based membrane spin columns (Qiagen RNeasy Mini kit) (see Note 19 ). For the RT-PCR assay, at least 300–1,000 spheres should be analyzed directly or cultured for differentiation in six-well tissue culture plates (as described in steps 1–6, Section 3.3), since sufficient amount of RNA cannot be obtained in our hands from smaller samples.
Screening for up- or downregulation of mRNAs by comparative RT-PCR works only when equal amounts of total RNA are used from the beginning. Good results have been achieved when starting with 5–10 μg aliquots of total RNA with equal concentrations isolated from progenitor cells or differentiated cells. cDNA generated by reverse transcription can be used directly for PCR amplification or can be stored at −20 °C for future use.
Oligonucleotide primers for PCR should be carefully selected to discriminate between cDNA and genomic DNA, when possible. Using individual primers specific for different exons is a simple way to achieve this; amplification from genomic DNA with these primers will create a larger reaction product that often will not be efficiently amplified. Table 9.2 lists several primers that have been successfully used to compare marker gene expression of inner ear tissue, selected inner ear progenitor cells, and differentiated inner ear cell types (2, 7, 8, 12, 13). The following protocol is for one comparative (semi-quantitative) PCR using three samples, such as cDNA from inner ear tissue, selected progenitors, and differentiated cells.
The authors would like to thank the members of their research group for critically reading this manuscript. This work was supported by a McKnight Endowment Fund for Neuroscience Brain Disorders Award and grant DC006167 from the National Institutes of Health.
1All cell culture is done in a dedicated room, separated from the main laboratory by a closed door. Traffic in and out of the cell culture room has to be minimized. All supplies and instruments have to be dedicated only for cell culture use and should never be carried into the main laboratory and used for other experiments. To avoid contamination, all surfaces are wiped before and after use with 70% ethanol. Nothing inside the room should be touched without wearing gloves. Sterile technique and common sense are usually effective means to avoid loss of cell lines due to contamination. It is recommended that sterile plasticware be used instead of glassware.
250 μL of trypsin inhibitor is sufficient to completely deactivate 50 μL of 0.25% trypsin/EDTA solution. DnaseI helps to reduce viscosity caused by DNA released from damaged cells during trypsinization and trituration. We have successfully used inhibitor and DNaseI from Worthington, Lake-wood, NJ (cat. nos. LS003570 and LS002139).
3Attach a rubber bulb at the wide end of an autoclaved Pasteur pipette. Rotate the pipette tip in a flame (Bunsen burner) to fire polish it for a few seconds. From time to time, squeeze the rubber bulb to check for increasing resistance of airflow through the polished pipette tip. Airflow should be slightly but noticeably restricted. The edges of the pipettes should be nice and rounded. Freshly prepared fire polished pipettes are sterile and can be used directly after a 2-min cooling period.
4β-ME is toxic; dispense in a fume hood and wear protective clothing.
5Alternatively, leave the ampullae in contact with the utricle and take out the utricle with 2 ampullae (Fig. 9.1D inset). Grabbing one of the ampullae prevents the utricle from being damaged by forceps.
6It is not absolutely necessary to use pure sensory epithelium (Fig. 9.1F) for sphere generation. Spheres usually form from cells present in the sensory epithelia and not from the underlying non-sensory tissue (2).
7Make sure that there is no residue of neuronal fibers attached to the cochlear duct (Fig. 9.1J). Clean separation of the duct and the modiolus is also important for spiral ganglion preparation.
8The OC/GER region may contain adjacent mesenchymal tissue and some lesser epithelial ridge (LER) cells. Sphere-forming cells from the OC/GER region are likely to contain the proliferative cell populations previously reported to reside in the GER and LER (4, 11).
9Attempt to reduce the volume of HBSS, which contains calcium and magnesium ions, carried over into the DPBS drop since those ions will disturb the enzymatic activity of trypsin by obscuring the peptide bonds on which trypsin acts. A small amount of the ions will be chelated by the EDTA that is part of the trypsin solution. If this is not possible, rinse the organs briefly in DPBS, which is calcium and magnesium free.
10An Eppendorf pipette tip (cat. no. 022491245, 20–300 μL, Eppendorf) appears to be well suited for this specific kind of trituration. Fire-polished and silanized Pasteur pipettes can be used as a good substitute.
11Avoid bubbles because you will lose the majority of sphere-forming cells. Setting the dial of a 200-μL Pipetman® at 150–180 μL will help.
12If you want to ensure clonal sphere formation, you need to plate the cells at a much lower density (10 cells/cm2). The most stringent generation of clonal spheres can only be achieved with flow-cytometric placement of single cells into individual wells of 96-well non-stick plates. Keep in mind that 0.1–0.2% or less of the cells have the capacity for sphere formation.
13Tungsten needles can be sharpened by electrolytical erosion in a beaker of 1 M NaOH or KOH. The needle is connected to the positive terminal of a 9 V battery using a crocodile clip. A copper or carbon cathode attached to the negative terminal of the battery is immersed in a small beaker containing 1 M NaOH (or KOH). Slowly move the needle up and down in the electrolyte. Resharpening the needle can be done by repeating the same process.
14Most of the spheres will be attached within 10 h.
15Leukemia inhibitory factor (LIF) promotes neuronal differentiation from spiral ganglion–derived spheres. If you want to make use of this ability, plate the spheres and culture them in differentiation medium containing 1 ng/mL of LIF (recombinant rat LIF, cat. no. LIF3005 Chemicon, or similar product) for 10–14 days. The number of neurons is significantly increased up to sevenfold in a dose-dependent manner by the addition of LIF, reaching a maximum at 1 ng/mL (12).
16Throughout the procedure, do not allow the cells to dry. If you immunostain more than a few wells, consider working in staggered batches of four wells.
17Dilute primary antibody according to the supplier’s suggestion. If you incubate overnight, place the plates into a larger container and add a wet piece of tissue. Close the container tightly. This humidified chamber will help prevent the wells from drying.
18Dilute secondary antibody according to the supplier’s recommendation.
19Because of the high stability and high efficacy of Rnases, it is necessary to create an RNase-free environment by wiping the working area with RNaseZap. In addition, it is necessary to wear gloves while handling reagents and samples and to use RNase-free sterile, disposable plasticware for all experimental steps. For details on the abbreviated protocol described in this section, refer to the RNeasy kit protocol booklet (provided with the Qiagen kit).
20If the two RNA samples are not equally concentrated, use different volumes and reduce the amount of RNase-free water accordingly. We have used as little as 1 ng total RNA for successful RT-PCR experiments.
21Custom synthesized oligonucleotide primers are usually shipped lyophilized. Resuspend primers in sterile Milli-Q water at a concentration of 100 pM.
22It is important to establish specific PCR conditions (cycling parameters) for each of the different products so that they are optimized to generate products at the linear portion of the product accumulation curve. These parameters depend, in part, on the thermal cycler used in the experiment. It is recommended that a series of pilot experiments be conducted for each primer pair with all samples that will be compared (e.g., cDNA from tissue, selected progenitors, and differentiated cells). Cycling parameters need to be selected based on the sample that produces the highest amount of amplification product.
23It is convenient to program the thermal cycler to cool the reactions at 4 °C once the amplification is done. Because of the different cycle numbers of the different reactions, close monitoring of the reaction is required to ensure that samples are removed from the thermal cycler at the appropriate time points. The thermal cycler has to be programmed to run the program with the most cycles. Reactions that undergo less cycles have to be removed at appropriate time points at the end of the 72 °C extension period.