Direct Cell Reprogramming and Phenotypic Conversion: An Analysis of Experimental Attempts to Transform Astrocytes into Neurons in Adult Animals.

Central nervous system (CNS) repair after injury or disease remains an unresolved problem in neurobiology research and an unmet medical need. Directly reprogramming or converting astrocytes to neurons (AtN) in adult animals has been investigated as a potential strategy to facilitate brain and spinal cord recovery and advance fundamental biology. Conceptually, AtN strategies rely on forced expression or repression of lineage-specific transcription factors to make endogenous astrocytes become "induced neurons" (iNs), presumably without re-entering any pluripotent or multipotent states. The AtN-derived cells have been reported to manifest certain neuronal functions in vivo. However, this approach has raised many new questions and alternative explanations regarding the biological features of the end products (e.g., iNs versus neuron-like cells, neural functional changes, etc.), developmental biology underpinnings, and neurobiological essentials. For this paper per se, we proposed to draw an unconventional distinction between direct cell conversion and direct cell reprogramming, relative to somatic nuclear transfer, based on the experimental methods utilized to initiate the transformation process, aiming to promote a more in-depth mechanistic exploration. Moreover, we have summarized the current tactics employed for AtN induction, comparisons between the bench endeavors concerning outcome tangibility, and discussion of the issues of published AtN protocols. Lastly, the urgency to clearly define/devise the theoretical frameworks, cell biological bases, and bench specifics to experimentally validate primary data of AtN studies was highlighted.

TransSynW: A single-cell RNA-sequencing based web application to guide cell conversion experiments.

Generation of desired cell types by cell conversion remains a challenge. In particular, derivation of novel cell subtypes identified by single-cell technologies will open up new strategies for cell therapies. The recent increase in the generation of single-cell RNA-sequencing (scRNA-seq) data and the concomitant increase in the interest expressed by researchers in generating a wide range of functional cells prompted us to develop a computational tool for tackling this challenge. Here we introduce a web application, TransSynW, which uses scRNA-seq data for predicting cell conversion transcription factors (TFs) for user-specified cell populations. TransSynW prioritizes pioneer factors among predicted conversion TFs to facilitate chromatin opening often required for cell conversion. In addition, it predicts marker genes for assessing the performance of cell conversion experiments. Furthermore, TransSynW does not require users' knowledge of computer programming and computational resources. We applied TransSynW to different levels of cell conversion specificity, which recapitulated known conversion TFs at each level. We foresee that TransSynW will be a valuable tool for guiding experimentalists to design novel protocols for cell conversion in stem cell research and regenerative medicine.

Directed differentiation and direct reprogramming: Applying stem cell technologies to hearing research.

Hearing loss is the most widely spread sensory disorder in our society. In the majority of cases, it is caused by the loss or malfunctioning of cells in the cochlea: the mechanosensory hair cells, which act as primary sound receptors, and the connecting auditory neurons of the spiral ganglion, which relay the signal to upper brain centers. In contrast to other vertebrates, where damage to the hearing organ can be repaired through the activity of resident cells, acting as tissue progenitors, in mammals, sensory cell damage or loss is irreversible. The understanding of gene and cellular functions, through analysis of different animal models, has helped to identify causes of disease and possible targets for hearing restoration. Translation of these findings to novel therapeutics is, however, hindered by the lack of cellular assays, based on human sensory cells, to evaluate the conservation of molecular pathways across species and the efficacy of novel therapeutic strategies. In the last decade, stem cell technologies enabled to generate human sensory cell types in vitro, providing novel tools to study human inner ear biology, model disease, and validate therapeutics. This review focuses specifically on two technologies: directed differentiation of pluripotent stem cells and direct reprogramming of somatic cell types to sensory hair cells and neurons. Recent development in the field are discussed as well as how these tools could be implemented to become routinely adopted experimental models for hearing research.

Noncoding RNAs and Midbrain DA Neurons: Novel Molecular Mechanisms and Therapeutic Targets in Health and Disease.

Midbrain dopamine neurons have crucial functions in motor and emotional control and their degeneration leads to several neurological dysfunctions such as Parkinson's disease, addiction, depression, schizophrenia, and others. Despite advances in the understanding of specific altered proteins and coding genes, little is known about cumulative changes in the transcriptional landscape of noncoding genes in midbrain dopamine neurons. Noncoding RNAs-specifically microRNAs and long noncoding RNAs-are emerging as crucial post-transcriptional regulators of gene expression in the brain. The identification of noncoding RNA networks underlying all stages of dopamine neuron development and plasticity is an essential step to deeply understand their physiological role and also their involvement in the etiology of dopaminergic diseases. Here, we provide an update about noncoding RNAs involved in dopaminergic development and metabolism, and the related evidence of these biomolecules for applications in potential treatments for dopaminergic neurodegeneration.

CIRM tools and technologies: Breaking bottlenecks to the development of stem cell therapies.

The California Institute for Regenerative Medicine (CIRM) has a mission to accelerate stem cell treatments to patients with unmet medical needs. This perspective describes successful examples of work funded by CIRM's New Cell Lines and Tools and Technologies Initiatives, which were developed to address bottlenecks to stem cell research and translation. The tools developed through these programs evolved from more discovery-oriented technologies, such as disease models, differentiation processes, and assays, to more translation focused tools, including scalable good manufacturing processes, animal models, and tools for clinical cell delivery. These tools are available to the research community and many are facilitating translation of regenerative therapeutics today.

Notch Signaling Controls Transdifferentiation of Pulmonary Neuroendocrine Cells in Response to Lung Injury.

Production of an appropriate number of distinct cell types in precise locations during embryonic development is critical for proper tissue function. Homeostatic renewal or repair of damaged tissues in adults also requires cell expansion and transdifferentiation to replenish lost cells. However, the responses of diverse cell types to tissue injury are not fully elucidated. Moreover, the molecular mechanisms underlying transdifferentiation remain poorly understood. This knowledge is essential for harnessing the regenerative potential of individual cell types. This study investigated the fate of pulmonary neuroendocrine cells (PNECs) following lung damage to understand their plasticity and potential. PNECs are proposed to carry out diverse physiological functions in the lung and can also be the cells of origin of human small cell lung cancer. We found that Notch signaling is activated in proliferating PNECs in response to epithelial injury. Forced induction of high levels of Notch signaling in PNECs in conjunction with lung injury results in extensive proliferation and transdifferentiation of PNECs toward the fate of club cells, ciliated cells and goblet cells. Conversely, inactivating Notch signaling in PNECs abolishes their ability to switch cell fate following lung insult. We also established a connection between PNEC transdifferentiation and epigenetic modification mediated by the polycomb repressive complex 2 and inflammatory responses that involve the IL6-STAT3 pathway. These studies not only reveal a major pathway that controls PNEC fate change following lung injury but also provide tools to uncover the molecular basis of cell proliferation and fate determination in response to lung injury. Stem Cells 2018;36:377-391.

Concise Review: Application of In Vitro Transcribed Messenger RNA for Cellular Engineering and Reprogramming: Progress and Challenges.

Several diseases are caused by missing or defective synthesis of proteins due to genetic or acquired disorders. In recent years, in vitro transcribed (IVT) messenger RNA (mRNA)-based therapy for de novo protein expression in cells has increased in importance. Thereby, desired proteins can be produced in cells by exogenous delivery of IVT mRNA, which does not integrate into the host genome and results in transient production of target proteins. Due to the lack of genomic integration, the risk of mutation and tumor development is minimized. Different approaches using IVT mRNA have been applied to alter the expression profiles of cells by the production of proteins. IVT mRNAs encoding transcription factors have led to the highly efficient induction of pluripotency in somatic cells and generated induced pluripotent stem cells that are free of viral vector components. Furthermore, specific IVT mRNA cocktails containing more than one specific IVT mRNA can be used to directly induce the differentiation into a desired cell type. In theory, every desired mRNA can be produced in vitro and used to enable extrinsic biosynthesis of target proteins in each cell type. Cells can be engineered by IVT mRNA to express antigens on dendritic cells for vaccination and tumor treatment, surface receptors on stem cells for increased homing to distinct areas, and to produce industrial grade human growth factors. In this review, we focus on the progress and challenges in mRNA-based cell engineering approaches. Stem Cells 2017;35:68-79.

Rapid generation of functional dopaminergic neurons from human induced pluripotent stem cells through a single-step procedure using cell lineage transcription factors.

Current protocols for in vitro differentiation of human induced pluripotent stem cells (hiPSCs) to generate dopamine (DA) neurons are laborious and time-expensive. In order to accelerate the overall process, we have established a fast protocol by expressing the developmental transcription factors ASCL1, NURR1, and LMX1A. With this method, we were able to generate mature and functional dopaminergic neurons in as few as 21 days, skipping all the intermediate steps for inducting and selecting embryoid bodies and rosette-neural precursors. Strikingly, the resulting neuronal conversion process was very proficient, with an overall efficiency that was more than 93% of all the coinfected cells. hiPSC-derived DA neurons expressed all the critical molecular markers of the DA molecular machinery and exhibited sophisticated functional features including spontaneous electrical activity and dopamine release. This one-step protocol holds important implications for in vitro disease modeling and is particularly amenable for exploitation in high-throughput screening protocols.