A facile method to generate cerebral organoids from human pluripotent stem cells.

Human cerebral organoids (COs) are self-organizing three-dimensional (3D) neural structures that provide a human-specific platform to study the cellular and molecular processes that underlie different neurological events. The first step of CO generation from human pluripotent stem cells (hPSCs) is neural induction, which is an in vitro simulation of neural ectoderm development. Several signaling pathways cooperate during neural ectoderm development and in vitro differentiation of hPSCs toward neural cell lineages is also affected by them. In this study, we considered some of the known sources of these variable signaling cues arising from cell culture media components and sought to modulate their effects by applying a comprehensive combination of small molecules and growth factors for CO generation. Histological analysis demonstrated that these COs recapitulate the neural progenitor zone and early cortical layer organization, containing different types of neuronal and glial cells which was in accordance with single-nucleus transcriptome profiling results. Moreover, patch clamp and intracellular Ca2+ dynamic studies demonstrated that the COs behave as a functional neural network. Thus, this method serves as a facile protocol for generating hPSC-derived COs that faithfully mimic the features of their in vivo counterparts in the developing human brain. See also Figure 1(Fig. 1).

Inner ear organoids: progress and outlook, with a focus on the vascularization.

The inner ear is a complex organ that encodes sound, motion, and orientation in space. Given the complexity of the inner ear, it is not surprising that treatments are relatively limited despite the fact that, in 2015, hearing loss was the fourth leading cause of years lived with disability worldwide. Inner ear organoid models are a promising tool to advance the study of multiple aspects of the inner ear to aid the development of new treatments and validate drug-based therapies. The blood supply of the inner ear plays a pivotal role in growth, maturation, and survival of inner ear tissues and their physiological functions. This vasculature cannot be ignored in order to achieve a truly in vivo-like model that mimics the microenvironment and niches of organ development. However, this aspect of organoid development has remained largely absent in the generation of inner ear organoids. The current review focuses on three-dimensional inner ear organoid and how recent technical progress in generating in vitro vasculature can enhance the next generation of these models.

Organoids: a novel modality in disease modeling.

Limitations of monolayer culture conditions have motivated scientists to explore new models that can recapitulate the architecture and function of human organs more accurately. Recent advances in the improvement of protocols have resulted in establishing three-dimensional (3D) organ-like architectures called 'organoids' that can display the characteristics of their corresponding real organs, including morphological features, functional activities, and personalized responses to specific pathogens. We discuss different organoid-based 3D models herein, which are classified based on their original germinal layer. Studies of organoids simulating the complexity of real tissues could provide novel platforms and opportunities for generating practical knowledge along with preclinical studies, including drug screening, toxicology, and molecular pathophysiology of diseases. This paper also outlines the key challenges, advantages, and prospects of current organoid systems.

The Dynamic Proteome of Oligodendrocyte Lineage Differentiation Features Planar Cell Polarity and Macroautophagy Pathways.

BACKGROUND: Generation of oligodendrocytes is a sophisticated multistep process, the mechanistic underpinnings of which are not fully understood and demand further investigation. To systematically profile proteome dynamics during human embryonic stem cell differentiation into oligodendrocytes, we applied in-depth quantitative proteomics at different developmental stages and monitored changes in protein abundance using a multiplexed tandem mass tag-based proteomics approach. FINDINGS: Our proteome data provided a comprehensive protein expression profile that highlighted specific expression clusters based on the protein abundances over the course of human oligodendrocyte lineage differentiation. We identified the eminence of the planar cell polarity signalling and autophagy (particularly macroautophagy) in the progression of oligodendrocyte lineage differentiation-the cooperation of which is assisted by 106 and 77 proteins, respectively, that showed significant expression changes in this differentiation process. Furthermore, differentially expressed protein analysis of the proteome profile of oligodendrocyte lineage cells revealed 378 proteins that were specifically upregulated only in 1 differentiation stage. In addition, comparative pairwise analysis of differentiation stages demonstrated that abundances of 352 proteins differentially changed between consecutive differentiation time points. CONCLUSIONS: Our study provides a comprehensive systematic proteomics profile of oligodendrocyte lineage cells that can serve as a resource for identifying novel biomarkers from these cells and for indicating numerous proteins that may contribute to regulating the development of myelinating oligodendrocytes and other cells of oligodendrocyte lineage. We showed the importance of planar cell polarity signalling in oligodendrocyte lineage differentiation and revealed the autophagy-related proteins that participate in oligodendrocyte lineage differentiation.

Chromosome-Centric Human Proteome Project Allies with Developmental Biology: A Case Study of the Role of Y Chromosome Genes in Organ Development.

One of the main goals of Chromosome-Centric Human Proteome Project is to identify protein evidence for missing proteins (MPs). Here, we present a case study of the role of Y chromosome genes in organ development and how to overcome the challenges facing MPs identification by employing human pluripotent stem cell differentiation into cells of different organs yielding unprecedented biological insight into adult silenced proteins. Y chromosome is a male-specific sex chromosome which escapes meiotic recombination. From an evolutionary perspective, Y chromosome has preserved 3% of ancestral genes compared to 98% preservation of the X chromosome based on Ohno's law. Male specific region of Y chromosome (MSY) contains genes that contribute to central dogma and govern the expression of various targets throughout the genome. One of the most well-known functions of MSY genes is to decide the male-specific characteristics including sex, testis formation, and spermatogenesis, which are majorly formed by ampliconic gene families. Beyond its role in sex-specific gonad development, MSY genes in coexpression with their X counterparts, as single copy and broadly expressed genes, inhibit haplolethality and play a key role in embryogenesis. The role of X-Y related gene mutations in the development of hereditary syndromes suggests an essential contribution of sex chromosome genes to development. MSY genes, solely and independent of their X counterparts and/or in association with sex hormones, have a considerable impact on organ development. In this Review, we present major recent findings on the contribution of MSY genes to gonad formation, spermatogenesis, and the brain, heart, and kidney development and discuss how Y chromosome proteome project may exploit developmental biology to find missing proteins.

Differentiation potential of human CD133 positive hematopoietic stem cells into motor neuron- like cells, in vitro.

Spinal cord injuries and motor neuron-related disorders impact on life of many patients around the world. Since pharmacotherapy and surgical approaches were not efficient to regenerate these types of defects; stem cell therapy as a good strategy to restore the lost cells has become the focus of interest among the scientists. Umbilical cord blood CD133+ hematopoietic stem cells (UCB- CD133+ HSCs) with self- renewal property and neural lineage differentiation capacity are ethically approved cell candidate for use in regenerative medicine. In this regard the aim of this study was to quantitatively evaluate the capability of these cells to differentiate into motor neuron-like cells (MNL), in vitro. CD133+ HSCs were isolated from human UCB using MACS system. After cell characterization using flow cytometry, the cells were treated with a combination of Retinoic acid, Sonic hedgehog, Brain derived neurotrophic factor, and B27 through a 2- step procedure for two weeks. The expression of MN-specific markers was examined using qRT- PCR, flow cytometry and immunocytochemistry. By the end of the two-week differentiation protocol, CD133+ cells acquired unipolar MNL morphology with thin and long neurites. The expression of Isl-1(62.15%), AChE (41.83%), SMI-32 (21.55%) and Nestin (17.46%) was detected using flow cytometry and immunocytochemistry. The analysis of the expression of PAX6, ISL-1, ACHE, CHAT and SMI-32 revealed that MNLs present these neural markers at levels comparable with undifferentiated cells. In Conclusion Human UCB- CD133+ HSCs are remarkably potent cell candidates to transdifferentiate into motor neuron-like cells, in vitro.

DDX3Y, a Male-Specific Region of Y Chromosome Gene, May Modulate Neuronal Differentiation.

Although it is apparent that chromosome complement mediates sexually dimorphic expression patterns of some proteins that lead to functional differences, there has been insufficient evidence following the manipulation of the male-specific region of the Y chromosome (MSY) gene expression during neural development. In this study, we profiled the expression of 23 MSY genes and 15 of their X-linked homologues during neural cell differentiation of NTERA-2 human embryonal carcinoma cell line (NT2) cells in three different developmental stages using qRT-PCR, Western blotting, and immunofluorescence. The expression level of 12 Y-linked genes significantly increased over neural differentiation, including RBMY1, EIF1AY, DDX3Y, HSFY1, BPY2, PCDH11Y, UTY, RPS4Y1, USP9Y, SRY, PRY, and ZFY. We showed that siRNA-mediated knockdown of DDX3Y, a DEAD box RNA helicase enzyme, in neural progenitor cells impaired cell cycle progression and increased apoptosis, consequently interrupting differentiation. Label-free quantitative shotgun proteomics based on a spectral counting approach was then used to characterize the proteomic profile of the cells after DDX3Y knockdown. Among 917 reproducibly identified proteins detected, 71 proteins were differentially expressed following DDX3Y siRNA treatment compared with mock treated cells. Functional grouping indicated that these proteins were involved in cell cycle, RNA splicing, and apoptosis, among other biological functions. Our results suggest that MSY genes may play an important role in neural differentiation and demonstrate that DDX3Y could play a multifunctional role in neural cell development, probably in a sexually dimorphic manner.