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The development of 3D organoids

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The development of 3D organoids as in vitro experimental platform

3D organoids derive from different epithelial cell populations of the adult lung and iPSCs and impact the traditional system of primary culture. Some of the potential future uses of organoids include human disease modeling and gene editing. For example, airway epithelial cells on organoids could be used for observing the biological change under diseases, such as asthma or genetic disease. Organoids could also provide a reliable and reproducible source of human mutant cells by using CRISPR technology, which would overcome variability in the behavior of primary lung progenitor cells derived from even healthy individuals. For now, it is necessary to fully exploit the powerful technology of genome editing using CRISPR/Cas9, particularly for studying the role of specific genes or non-coding RNAs in the differentiation of stem cells and in generating models of human respiratory disease. Similarly, brain, liver, and retina organoids meet the same challenge. The advances in reprogramming technology have enabled the generation of induced pluripotent stem cells (iPSCs). iPSC has become an important tool and source of disease models for developing therapeutic drugs and understanding pathogenesis mechanisms through the differentiation process, allowing us to delineate the effects of specific disease-related genes in a disease model in vitro. Through established differentiation protocol, we are able to reconstruct human tissues as well as 3D organoids in vitro and to mimic the physiological condition of the organ and tissue in the real human body. 

Focus on three key diseases in lung, liver, and neuron

The core theme of the proposed research project will apply iPSC-derived 3D organoids and tissue as models to study respiratory lung disease, central nervous system dysfunction, and hepatic diseases. In lung disease, we focus on cystic fibrosis (CF), the genetically inherited autosomal-recessive disease. CF is characterized by abnormal fluid and electrolyte mobility across secretory epithelia. The first manifestations occur within hours of birth, later extending to other organs, generally affecting the respiratory tract. It is caused by the mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Despite the advanced healthcare system, no cure is currently available. In the present day, CF is mainly treated with modulators that exert only temporary effects on the patients. Gene editing, conversely, is a permanent solution with immense potential for treating CF, however, with unseen complications and boundaries to overcome. In liver disease, we focus on the non-alcoholic fatty liver disease (NAFLD) and CF-related liver steatohepatitis. We aim to provide an add-on value to explore the distinctive etiology of NAFLD and the pathogenesis in differentiation progress and 3D organoids of lung epithelial cells and hepatocytes. In nervous system dysfunction, we focus on Amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disorder caused by progressive loss of motor neurons (MNs) in the brain and spinal cord. The pathological mechanisms of ALS are not completed understood, and there is no cure for this disease. TDP43 mutation and the related lncRNA has been indicated in the pathogenesis pathway of the disease. We aim to gain further knowledge about the functions and the mechanisms by which lncRNAs contribute to disease.

Core Technology and Experimental Platforms

1. The use of single-cell NGS allows us to examine the sequence information from individual cells with optimized next-generation sequencing (NGS) technologies, providing a higher resolution of cellular differences and a better understanding of the function of an individual cell in the context of its microenvironment. Given that the differentiated tissue and 3D organoids are composed of more than one types of cells, normally heterogeneously varied in a different population of cells to mimic the real physiological status of real organ, it is important to apply single-cell NGS to specifically analyze gene expression profile of the different population of cells within the differentiated tissue or organoids. We will be able to observe the effects of therapeutic treatments or gene modifications on the cells of interest by referring cell type-specific gene signature. The crosstalk between different subpopulations of cells can also be investigated by the high resolution of single-cell sequencing analysis. This technology will advance our study to distinguish different responses from a variety of cell types against one single stimulation/stress/genomic modification.

2. Spatial transcriptomics is a technology used to spatially resolve RNA-seq data, thereby all mRNAs, in individual tissue sections. By adding spatial information to scRNA-seq data, spatial transcriptomics has transformed our understanding of functional tissue organization and cell-to-cell interactions in situ. Any combination of presence or absence of expression for a set of genes can be used to define a marker profile of interest for further analysis. Features were selected on the basis of the presence and/or absence of the target tissue-specific-marker genes. Compared to the RNA-sequencing of single cells, or the sequencing of bulk RNA extracted from tissue volumes, precise spatial information of spatial single RNA sequencing is kept. After obtaining human induced stem cells, they will differentiate into their target cells according to their main pathogenic factors. In the future, such gene correction technology can be used not only in cell gene therapy but also in the clinical application of regenerative medicine treatment of degenerative diseases. 

3. Application of CRISPR/CAS9 gene modification technology will allow us to specifically target the disease-related mutations/genes for recovery/therapeutic purposes. For the above-mentioned important clinical diseases, after finding important gene mutations, CRISPR/Cas9 genome editing will be used for the recompiled iPSC. The physical development of degenerative diseases will involve abnormal internal metabolism of cells, so each project will monitor the upstream and midstream of the cell’s metabolic pathway and analyze the accumulation of abnormal downstream caused by excessive abnormal metabolism in the pathway. Understand that, for example, sub-project three is aimed at studying liver disease models, conducting in vivo in vivo and in vitro in vitro iPSC culture of induced pluripotent stem cells, including the differentiation into liver cells and the establishment of animal models as mechanisms, and drug development. 

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