Compositions, Systems, and Methods for Generating Inner Ear Hair Cells for Treatment of Hearing Loss
a technology of hair cells, which is applied in the field of compositions, systems and methods for generating inner ear hair cells for hearing loss treatment, can solve the problems of potential exhaustion of stem cell pool, lack of mother cell capacity to divide into hair cells, etc., and achieves the effects of avoiding variability in drug exposure, and prolonging the life of stem cells
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example 1
[0963]Cell culture: Heterozygous Lgr5-EGFP-IRES-CreERT2 mice were obtained from Jackson Labs, and neonatal P2-P5 mice were used for cell isolation. Organ of Corti was isolated from Lgr5-GFP mice and further dissociated into single cells using trypsin. Cells were then cultured as previously described (Yin et al, 2014). Briefly, cells were entrapped in Matrigel and plated at the center of wells in a 24-well plate. Following polymerization of Matrigel, 500 μl of culture media (Advanced DMEM / F12 with N2 and B27) was added, containing growth factors including EGF (50 ng / ml) (epidermal growth factor), bFGF (20 ng / ml) (fibroblast growth factor), and IGF1 (50 ng / ml) (insulin-like growth factor 1), and small molecules including CHIR99021 (5 μM), valproic acid (1 mM), 2-phospho-L-ascorbic acid (280 μM), and 616452 (2 μM). Y-27632 (10 μM) was added for the first 2 days. In some experiments, bFGF was used at 50 ng / ml.
[0964]For single cell isolation, the organs of Corti were then treated with Ce...
example 2
Differentiation of Expanded Lgr5-Positive Cells to Hair Cells
[0972]Materials and Methods
[0973]Differentiation protocol: For differentiation of expanded Lgr5-positive cells, following 10 days of culture in the cell expansion condition (EFICVP6), the cell colonies were transferred to fresh Matrigel and further cultured in differentiation media. Differentiation media contains Notch pathway inhibitor (e.g. DAPT, D, 5 μM, or LY411575, LY, 5 μM), with or without Gsk3β inhibitor (e.g. CHIR99021). Media were changed every other day. Following another 6-10 days of incubation with differentiation media, the colonies were harvested for qPCR analysis or fixed with 4% PFA and immunostained with hair cell markers Myo7a and Prestin.
[0974]RNA extraction and quantitative real-time PCR (qPCR): RNA was isolated from cultured cells (RNeasy Mini Kit; Qiagen) according to the manufacturer's protocol. Quantitative real-time PCR was performed with QuantiTect Probe PCR kit (Qiagen) using commercially availa...
example 3
Expansion and Hair Cell Differentiation of Lgr5-Expressing Cells from Adult Inner Ear Tissue
[0980]Materials and Methods
[0981]For adult tissue, the stria vascularis was removed but the epithelium was not removed from the underlying mesenchyme due to the limited amount of intact cochlea that could be extracted. For adult cells, additional small molecule TTNPB (2 μM, Tocris) was added and the complete media contain EGF, bFGF, IGF-1, CHIR99021, VPA, pVc, 616452 and TTNPB.
[0982]Results
[0983]We found that although the condition EFICVP6 support the survival and growth of Lgr5 inner ear cells from adult mice (FIGS. 13A-C), the proliferation was very slow. We thus performed addition screening for addition factors that can promote the proliferation of Lgr5 cells from adult mice. We found that a small molecule RA signaling pathway agonist, TTNPB, significantly promoted the proliferation of cultured cells (FIG. 14). Thus it was further incorporated in the expansion media for Lgr5 inner ear cell...
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