Correction: Transcription factor induced conversion of human fibroblasts towards the hair cell lineage.

[This corrects the article DOI: 10.1371/journal.pone.0200210.].

Induced Pluripotent Stem Cells, a Stepping Stone to In Vitro Human Models of Hearing Loss.

Hearing loss is the most prevalent sensorineural impairment in humans. Yet despite very active research, no effective therapy other than the cochlear implant has reached the clinic. Main reasons for this failure are the multifactorial nature of the disorder, its heterogeneity, and a late onset that hinders the identification of etiological factors. Another problem is the lack of human samples such that practically all the work has been conducted on animals. Although highly valuable data have been obtained from such models, there is the risk that inter-species differences exist that may compromise the relevance of the gathered data. Human-based models are therefore direly needed. The irruption of human induced pluripotent stem cell technologies in the field of hearing research offers the possibility to generate an array of otic cell models of human origin; these may enable the identification of guiding signalling cues during inner ear development and of the mechanisms that lead from genetic alterations to pathology. These models will also be extremely valuable when conducting ototoxicity analyses and when exploring new avenues towards regeneration in the inner ear. This review summarises some of the work that has already been conducted with these cells and contemplates future possibilities.

Antiviral Activities of Halogenated Emodin Derivatives against Human Coronavirus NL63.

The current COVID-19 outbreak has highlighted the need for the development of new vaccines and drugs to combat Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2). Recently, various drugs have been proposed as potentially effective against COVID-19, such as remdesivir, infliximab and imatinib. Natural plants have been used as an alternative source of drugs for thousands of years, and some of them are effective for the treatment of various viral diseases. Emodin (1,3,8-trihydroxy-6-methylanthracene-9,10-dione) is a biologically active anthraquinone with antiviral activity that is found in various plants. We studied the selectivity of electrophilic aromatic substitution reactions on an emodin core (halogenation, nitration and sulfonation), which resulted in a library of emodin derivatives. The main aim of this work was to carry out an initial evaluation of the potential to improve the activity of emodin against human coronavirus NL63 (HCoV-NL63) and also to generate a set of initial SAR guidelines. We have prepared emodin derivatives which displayed significant anti-HCoV-NL63 activity. We observed that halogenation of emodin can improve its antiviral activity. The most active compound in this study was the iodinated emodin analogue E_3I, whose anti-HCoV-NL63 activity was comparable to that of remdesivir. Evaluation of the emodin analogues also revealed some unwanted toxicity to Vero cells. Since new synthetic routes are now available that allow modification of the emodin structure, it is reasonable to expect that analogues with significantly improved anti-HCoV-NL63 activity and lowered toxicity may thus be generated.

Meis2 Is Required for Inner Ear Formation and Proper Morphogenesis of the Cochlea.

Meis genes have been shown to control essential processes during development of the central and peripheral nervous system. Here we have explored the roles of the Meis2 gene during vertebrate inner ear induction and the formation of the cochlea. Meis2 is expressed in several tissues required for inner ear induction and in non-sensory tissue of the cochlear duct. Global inactivation of Meis2 in the mouse leads to a severely reduced size of the otic vesicle. Tissue-specific knock outs of Meis2 reveal that its expression in the hindbrain is essential for otic vesicle formation. Inactivation of Meis2 in the inner ear itself leads to an aberrant coiling of the cochlear duct. By analyzing transcriptomes obtained from Meis2 mutants and ChIPseq analysis of an otic cell line, we define candidate target genes for Meis2 which may be directly or indirectly involved in cochlear morphogenesis. Taken together, these data show that Meis2 is essential for inner ear formation and provide an entry point to unveil the network underlying proper coiling of the cochlear duct.

Stem cell-based approaches: Possible route to hearing restoration?

Disabling hearing loss is the most common sensorineural disability worldwide. It affects around 466 million people and its incidence is expected to rise to around 900 million people by 2050, according to World Health Organization estimates. Most cases of hearing impairment are due to the degeneration of hair cells (HCs) in the cochlea, mechano-receptors that transduce incoming sound information into electrical signals that are sent to the brain. Damage to these cells is mainly caused by exposure to aminoglycoside antibiotics and to some anti-cancer drugs such as cisplatin, loud sounds, age, infections and genetic mutations. Hearing deficits may also result from damage to the spiral ganglion neurons that innervate cochlear HCs. Differently from what is observed in avian and non-mammalian species, there is no regeneration of missing sensory cell types in the adult mammalian cochlea, what makes hearing loss an irreversible process. This review summarizes the research that has been conducted with the aim of developing cell-based strategies that lead to sensory cell replacement in the adult cochlea and, ultimately, to hearing restoration. Two main lines of research are discussed, one directed toward the transplantation of exogenous replacement cells into the damaged tissue, and another that aims at reactivating the regenerative potential of putative progenitor cells in the adult inner ear. Results from some of the studies that have been conducted are presented and the advantages and drawbacks of the various approaches discussed.

Differential deletion of GDNF in the auditory system leads to altered sound responsiveness.

Glial-derived neurotrophic factor (GDNF) has been proposed as a potent neurotrophic factor with the potential to cure neurodegenerative diseases. In the cochlea, GDNF has been detected in auditory neurons and sensory receptor cells and its expression is upregulated upon trauma. Moreover, the application of GDNF in different animal models of deafness has shown its capacity to prevent hearing loss and promoted its future use in therapeutic trials in humans. In the present study we have examined the endogenous requirement of GDNF during auditory development in mice. Using a lacZ knockin allele we have confirmed the expression of GDNF in the cochlea including its sensory regions during development. Global inactivation of GDNF throughout the hearing system using a Foxg1-Cre line causes perinatal lethality but reveals no apparent defects during formation of the cochlea. Using TrkC-Cre and Atoh1-Cre lines, we were able to generate viable mutants lacking GDNF in auditory neurons or both auditory neurons and sensory hair cells. These mutants show normal frequency-dependent auditory thresholds. However, mechanoelectrical response properties of outer hair cells (OHCs) in TrkC-Cre GDNF mutants are altered at low thresholds. Furthermore, auditory brainstem wave analysis shows an abnormal increase of wave I. On the other hand, Atoh1-Cre GDNF mutants show normal OHC function but their auditory brainstem wave pattern is reduced at the levels of wave I, III and IV. These results show that GDNF expression during the development is required to maintain functional hearing at different levels of the auditory system.

Transcription factor induced conversion of human fibroblasts towards the hair cell lineage.

Hearing loss is the most common sensorineural disorder, affecting over 5% of the population worldwide. Its most frequent cause is the loss of hair cells (HCs), the mechanosensory receptors of the cochlea. HCs transduce incoming sounds into electrical signals that activate auditory neurons, which in turn send this information to the brain. Although some spontaneous HC regeneration has been observed in neonatal mammals, the very small pool of putative progenitor cells that have been identified in the adult mammalian cochlea is not able to replace the damaged HCs, making any hearing impairment permanent. To date, guided differentiation of human cells to HC-like cells has only been achieved using either embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). However, use of such cell types suffers from a number of important disadvantages, such as the risk of tumourigenicity if transplanted into the host´s tissue. We have obtained cells expressing hair cell markers from cultures of human fibroblasts by overexpression of GFI1, Pou4f3 and ATOH1 (GPA), three genes that are known to play a critical role in the development of HCs. Immunocytochemical, qPCR and RNAseq analyses demonstrate the expression of genes typically expressed by HCs in the transdifferentiated cells. Our protocol represents a much faster approach than the methods applied to ESCs and iPSCs and validates the combination of GPA as a set of genes whose activation leads to the direct conversion of human somatic cells towards the hair cell lineage. Our observations are expected to contribute to the development of future therapies aimed at the regeneration of the auditory organ and the restoration of hearing.

Otx2 is a target of N-myc and acts as a suppressor of sensory development in the mammalian cochlea.

Transcriptional regulatory networks are essential during the formation and differentiation of organs. The transcription factor N-myc is required for proper morphogenesis of the cochlea and to control correct patterning of the organ of Corti. We show here that the Otx2 gene, a mammalian ortholog of the Drosophila orthodenticle homeobox gene, is a crucial target of N-myc during inner ear development. Otx2 expression is lost in N-myc mouse mutants, and N-myc misexpression in the chick inner ear leads to ectopic expression of Otx2. Furthermore, Otx2 enhancer activity is increased by N-myc misexpression, indicating that N-myc may directly regulate Otx2. Inactivation of Otx2 in the mouse inner ear leads to ectopic expression of prosensory markers in non-sensory regions of the cochlear duct. Upon further differentiation, these domains give rise to an ectopic organ of Corti, together with the re-specification of non-sensory areas into sensory epithelia, and the loss of Reissner's membrane. Therefore, the Otx2-positive domain of the cochlear duct shows a striking competence to develop into a mirror-image copy of the organ of Corti. Taken together, these data show that Otx2 acts downstream of N-myc and is essential for patterning and spatial restriction of the sensory domain of the mammalian cochlea.

Post-translational regulation of cathepsin B, but not of other cysteine cathepsins, contributes to increased glioblastoma cell invasiveness in vitro.

Cells that migrate away from a central tumour into brain tissue are responsible for inefficient glioblastoma treatment. This migratory behaviour depends partially on lysosomal cysteine cathepsins. Reportedly, the expression of cathepsins B, L and S gradually increases in the progression from benign astrocytoma to the malignant glioblastoma, although their specific roles in glioma progression have not been revealed. The aim of this study was to clarify their specific contribution to glioblastoma cell invasion. The differences between the matrix invading cells and non-invading core cells from spheroids derived from glioblastoma cell culture and from glioblastoma patients' biopsies, and embedded in type I collagen, have been studied at the mRNA, protein and cathepsin activity levels. Analyses of the two types of cells showed that the three cathepsins were up-regulated post-translationally, their specific activities increasing in the invading cells. The cystatin levels were also differentially altered, resulting in higher ratio of cathepsins B and L to stefin B in the invading cells. However, using specific synthetic inhibitors and silencing strategies revealed that only cathepsin B activity was involved in the invasion of glioblastoma cells, confirming previous notion of cathepsin B as tumour invasiveness biomarker. Our data support the concept of specific roles of cysteine cathepsins in cancer progression. Finally the study points out on the complexity of protease regulation and the need to include functional proteomics in the systems biology approaches to understand the processes associated with glioma invasion and progression.

Antiprotease therapy in cancer: hot or not?

The activity of a set of peptidases (proteases) involved in cancer progression is collectively known as the cancer 'degradome'. Invasion and metastasis were initially considered as late events in cancer development and the processes in which proteases were involved. However, recent studies indicate that invasion and metastasis are not late events, but can occur during early stages as well. Moreover, other processes occurring in various stages of cancer progression are also protease-dependent, such as (upregulation of) cell proliferation, (downregulation of) apoptosis, involvement of white blood cells, angiogenesis and induction of multi-drug resistance. Proteolytic activity in tumours is regulated in a complex manner, as both genetically unstable cancer cells and stable stromal cells, such as fibroblasts, endothelial cells and inflammatory cells, are involved. In vitro studies and studies using animal models have clearly shown protease dependency of many processes in carcinogenesis. However, clinical trials using protease inhibitors have thus far been unsuccessful except for a few applications of matrix metalloprotease (MMP) inhibitors when used in combination with cytostatic anticancer agents and/or in the early stages of cancer. Antithrombotics, such as low-molecular-weight heparin and warfarin, were also successful in clinical trials, probably by interfering with proteases of the coagulation cascade. The two-way association between cancer and thrombosis has long been recognised in the clinic. The poor outcome of other clinical trials of protease inhibitors is probably due to the late stages of cancer of the patient populations included, and the limited understanding of the complex regulation and effects of the activity of the various proteases in tumours depending on, among others, tumour type and stage, interactions between the cancer cells, other cells and the extracellular matrix in tumours. Therefore, a better fundamental understanding of the proteolytic complexity in tumours is essential before clinical trials can be rationally designed. At present, antithrombotics, the urokinase-type plasminogen activator system, the membrane-bound membrane-type 1-MMP, cathepsin L and the proteasome seem the most promising candidates as targets for anticancer strategies in early stages of cancer in combination with cytotoxic drugs. Moreover, metronomic therapy is an attractive approach using low doses of inhibitors for prolonged periods of time without interruption to specifically target endothelial cells that are involved in angiogenesis.