Stem Cell Based Disease Modelling and Applications PDF

Stem Cell based Disease Modelling and Applications Induced Pluripotent Stem Cells Therapeutic applications of stem cells Organoid based Personalized Medicine Induced pluripotent stem cells The iPS cells are stem cells derived from somatic cells that have been genetically reprogrammed into a pluripot...

Stem Cell based Disease Modelling and Applications Induced Pluripotent Stem Cells Therapeutic applications of stem cells Organoid based Personalized Medicine Induced pluripotent stem cells The iPS cells are stem cells derived from somatic cells that have been genetically reprogrammed into a pluripotent state. iPS cells can differentiate into three germ layers, eliminating some of the hurdles in ES cell technology. Advances in iPS cell technology, from viral to non-viral systems and from integrating to non-integrating approaches of foreign genes into the host genome, have enhanced the existing technology, making it more feasible for clinical applications. Stage-by-stage hierarchy of stem cell development During development, the differentiation potency decreases, and the specialization increases at each stage. Generation of iPS cells from somatic cells using various approaches, including non-viral approaches (synthetic mRNA/ miRNA, proteins, and small molecules), Viral approaches (retrovirus, lentivirus, adenovirus, and Sendai virus), vectors (Episomal vectors), transposon (PiggyBac transposon), transient transfection and magnet-based Nanofection. Workflow to Characterize iPSC Lines Simple and cost-effective methods for determining heterogeneity, differentiation potential, and genome integrity of iPSC lines. Heterogeneity is assessed by flow cytometry on up to 60 iPSC lines simultaneously using barcoding optimization. In vitro differentiation potential is examined by qPCR using 12 marker genes on up to 96 samples. Digital karyotype is determined using Illumina genotyping BeadChips. Characterization of iPSC lines Pluripotency markers To characterize iPSCs and ESCs for expression of pluripotency markers with high resolution microscopy and imaging capabilities Immunocytochemistry (ICC) for key pluripotency markers: OCT4, SOX2, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase (AP staining); qPCR and RNA-seq Karyotyping/ Cytogenetics To identify and evaluate the stem cell genomic stability, chromosomal abnormalities, and copy number variants (CNVs) Differentiation potential Ability to form three germ layers and differentiate into somatic lineages Directed differentiation into specific lineages Embryoid body formation (EB) formation Microbiological contamination To ensure purity of the cells Mycoplasma testing Pathogen testing Cell authentication/ DNA fingerprinting To establish purity and identity of cell line Short tandem repeats (STR) profiling HLA typing To identify HLA markers for potential transplantation studies Sequencing of 11 HLA loci Genome Sequencing Using NGS to detect single nucleotide variants Whole genome sequencing (WGS) Exome sequencing cDNA expression analysis Residual programming factors To detect presence of reprogramming footprint if using nonintegrating methods such as plasmids and retroviruses PCR analysis Comparative genomic hybridization (CGH) analysis G-banding (high resolution) qPCR analysis Chromosomal microarray analysis (CMA) Assessing the differentiation potential into three germ layers Immunofluorescent staining for lineage-specific biomarkers of three germ layers are performed to confirm lineage commitment. Ectoderm markers: neuronal lineage markers, PAX6 (green), B-III Tubulin (red); Mesoderm markers: Brachyury (red) and GATA4 (green); Endoderm markers: SOX17 (green) and FOXA2 (red); DAPI (blue) was used to stain for nuclear localization. Various strategies are used to minimize the risk and possibility of neoplastic transformation: 1) Undifferentiated pluripotent stem cells (PSC), which are potentially tumorigenic, can be separated using antibodies that focus on specific surface-displayed biomarkers. Stem cell differentiation downregulates the expression of these biomarkers 2) Directed differentiation of iPSCs involves monitoring of the expression of the specific differentiation genes. Differentiated cells can be recognized and screened by using recombinant reporter proteins. 3) Antibody-guided toxins or toxic antibodies can kill the undifferentiated PSC through immune pathways. 4) Monoclonal antibodies against Claudin-6, a surface biomarker for undifferentiated pluripotent ESCs and iPSCs, can guide immune-toxins to these stem cells for targeted and selective killing. 5) Potentially tumorigenic pluripotent stem cells can be transformed with suicide genes for sensitization towards pro-drugs. For this purpose, the enzyme/pro-drug cancer therapy strategies can be improved to kill undifferentiated PSC 6) Tumorigenic PSC can be eradicated through self-induced transgenic expression of recombinant human Dnases. iPSCs is safer for clinical use than ESCs Unlike human ESCs, which must be prepared from human embryos, iPSCs can be reprogrammed from adult cells , thus avoiding ethical and pragmatic issues that arise with human ESCs. Reprogramming of adult somatic cells to iPSCs requires four transcription factors Oct4 , Sox2, Klf4, and c-Myc. Reprogramming of adult human cells generates iPSCs with unlimited potential to reconstruct genetically identical tissues. This offers opportunities to develop scalable, yet personalized cell-based therapy for patients with a variety of different diseases. Given that iPSCs do not have the ethical or immunogenic limitations associated with ESCs, they represent a stem cell technology that is more likely to be translatable to the clinic. It is not be feasible to make ESCs for every patient, and so the use of ESCs in regenerative medicine would be limited to allogeneic transplantation. Building on the knowledge obtained from human ESC studies, direct differentiation methods have been implemented to obtain highly specialized cell types from human iPSCs iPSCs is that they can be generated from various adult human tissues, including skin, hair, blood, and liver. These iPSCs can renew themselves indefinitely and can differentiate into many cell types, including pancreatic-β cells, liver hepatocytes, cardiomyocytes, hematopoietic cells, and dopaminergic neurons. A patient’s iPSCs be used to generate cells for transplantation to repair damaged tissue and the differentiated progeny of such cells could be used to screen candidate drugs to treat the disease. These iPSCs derived directly from the patient would be genetically identical to tissues of the patient enabling autologous transplantation. By focusing on diseases of unknown or unclear etiology, iPSC technology offers a robust platform to efficiently dissect molecular and cellular pathophysiology for the purposes of developing diagnostic, therapeutic, and preventive applications. iPSC technology and and P4 Medicine iPSC technology and Personalized Medicine iPS cell therapeutic approaches follow four steps: (1) iPSC generation from mutant donor somatic cells, (2) genetic correction of mutant genes with gene therapy, (3) in vitro differentiation of corrected iPS cells into mature cells (4) Transplantation. iPSC technology contributes to (A) disease modelling and drug screening, (B) cell transplantation, and (C) clinical trials. Experimental workflow of iPSC use for modelling diseases or for the derivation of cell products for cell therapy. Somatic cells from various sources (e.g. blood cells, fibroblasts) are isolated from the patient. They are then reprogrammed to iPSCs with reprogramming factors using various delivery methods. Isogenic pairs or iPSCs can be generated through gene editing to either correct the mutation in disease iPSCs or to introduce it in normal iPSCs. Upon in vitro differentiation into the appropriate cell type the cells can be used for cell-replacement therapy or disease modelling applications. Experimental strategies for genome-editing in iPSC In a patient-specific approach or mutation correction strategy , iPSCs are generated from a patient with a known mutation; genome-editing techniques are then used to correct the mutation thus providing a control iPSC clone. In a second strategy or mutation introduction strategy, a generic iPSC is used to introduce mutations of interest. Both approaches provide complementary information on the influence of the studied mutation and interactions with the genetic background. Applications of iPSCs in Cardiovascular Medicine When hiPSCs are derived from patients with a genetic disease caused by a mutation, such patient-derived iPSCs are called disease-specific hiPSCs. As disease-specific hiPSCs contain the same genetic information as the patient, including mutations corresponding to the altered gene function, disease-specific hiPSCs could potentially be a powerful tool for modelling human disease. In cardiovascular research, obtaining a sufficient number of cardiomyocytes (CMs) from patients is challenging due to the highly invasive procedures required to extract them. Further, the low proliferation capacity of CMs limits researchers’ ability to maintain these cells in culture. Being able to generate iPSC-derived CMs (hiPSC-CMs) from a specific patient overcomes this problem and enables identification of typical cellular responses to pathological stress and therapeutic agents because these cells potentially reflect the biological responses of an individual patient’s own CMs. CRISPR/Cas9-based gene editing enables the preparation of an isogenic control by normalizing a disease-relevant mutation in disease-specific hiPSCs or by inducing the mutation in wild-type hiPSCs so that diseased and control cells with the same genetic background are obtained. This method shows promise for the proper evaluation of the involvement of mutated genes in disease phenotype following in vitro differentiation. Polygenic diseases differ from monogenic inherited diseases in that more than one gene is involved in their dysfunction, impose limitation on the use of hiPSCs. Although gene editing has been used to edit multiple regions of the genome, a major challenge towards using hiPSCs to investigate polygenic diseases is identification of the corresponding mutations and understanding how each mutation contributes to the pathogenesis of these multifactorial diseases. Applications of iPSCs in Cardiovascular Medicine Applications of iPSCs in drug screening Standard drug discovery procedures have many limitations. Depending on the success rate of treating the disease in a disease model, the drugs may progress into clinical trials in humans. Disease-specific cells are not available for many diseases, especially degenerative diseases, including Alzheimer's disease Parkinson’s disease and amyotrophic lateral sclerosis (ALS), in which it is difficult to obtain cells or tissues with pathological phenotypes. it is difficult to prepare relevant cells for diseases occurring in sporadic forms and those affected by complicated unknown genetic aspects, such as autism spectrum disorders and type I diabetes. iPSC technology can resolve these issues by using its “disease modeling” potential. When iPS cells derived from a patient's fibroblasts are differentiated, mature cells express disease phenotypes and show disease progression. Disease modeling aids in both the understanding of disease mechanisms and the discovery of novel therapeutic compounds. Hepatotoxicity is the most common reason that many promising cancer drugs never make it into the clinic. Hepatocytes generated from human iPSCs of different genetic backgrounds will also aid efforts to test the toxicity of candidate cancer drugs. iPSC-derived hepatocytes and cardiomyocytes- Cardiotoxicity and hepatotoxicity screens are being developed for use in drug development, potentially increasing accuracy of safety testing, and bringing us closer to the reality of running a trial in a dish. In vitro Pharmacogenomics: Running a Trial in a Dish Flow Diagram of an iPSC-Supplemented Drug Trial for an Adipocyte Browning Therapy iPSC-driven Phase 0.5 and 1.5 trials in dishes might improve clinical trial accuracy and efficiency. Cytotoxic drugs or vulnerable patient populations might be screened out in Phase 0.5 cardio-, neuro-, and hepatotoxicity screens using iPSCs carrying representative and population-specific genotypes. Phase 1.5 trials might be used to stratify patients into responder and non-responder populations, thus increasing the relevance of further Phase 2 and 3 trials. The process of creating improved patient care using reprogramming and differentiation of donor tissue A biopsy is taken from a patient, reprogrammed ,before being differentiated to a cell type of interest. Disease-specific tissues can be used to improve current understanding of disease states and aid the drug development process. Validation is critical to the success of the process; there are six key steps which must be addressed. (A) Tissue from the patient must present the genetic traits of the disease state. (B) Reprogrammed cells must demonstrate pluripotency as assessed through a rigorous, standardized validation process. (C) Differentiated cells must demonstrate the key characteristics of the mature cell type as assessed by marker expression and functionality. (D) The differentiated cells should present the disease phenotype. (E) Genetic and drug interventions should be able to correct the phenotype. The cell model should predict the response of current therapies. This will lead to increased knowledge of the disease mechanism. (F) Patient benefit must be assessed through clinical trials. Therapeutic applications of stem cells Stem cells represent a great promise as a cell source for regenerative cell therapy. In addition to being useful tools for treating disease, stem cells are useful tools for investigating the disease mechanisms. Adult stem cells, ESCs, induced pluripotent stem cells (iPSCs), and cancer stem cells (CSCs) are widely used in basic science research and clinical application. Adult stem cells can be generated from patients’ own cells and there are no controversial issues in the aspects of immuno-rejection, ethics, and tumorigenesis. Thus, they are distinctly advantaged as being acceptable to all patients and widely used in clinical trials. The therapeutic effect and safe use of ESCs and iPSCs are validated in the treatment of multiple diseases such as myocardial infarction, spinal cord injury, and macular degeneration. iPSCs can be generated from diverse patient populations and differentiated into a disease-related specific cell types that can be either cultured as 2D monolayers or as stem cell-derived organoids, which can then be used as a tool to improve the understanding of disease mechanisms and to test therapeutic interventions. Cancer stem cells are tumor-initiating clonogenic cells. They may arise from normal stem cells that undergo gene mutations via complex mechanisms. Cancer stem cells play important roles in cancer growth, metastasis, and recurrence. Therefore, targeting these cells could provide a promising way to treat various types of solid tumors. Roadblocks to translating human iPSC technology to the clinic The main hurdles to using patient-specific iPSCs for disease modelling, drug screening, and transplantation purposes are a lack of effective differentiation protocols , little or no engraftment capability for the majority of human iPSC-derived specialized cells, difficulties in modelling multifactorial diseases, the need for GMP-compliant conditions at each step, safety concerns regarding the potential tumorigenicity of iPSCs associated with their pluripotent state or with insertional- or culture-driven mutagenesis. the c-Myc gene is known to increase the rate of tumor growth in some cases, which negatively affects iPSC efficiency in transplantation treatments. Overview of the workflow for development and preclinical testing of iPSC-based therapeutics The application of iPSC-derived products for therapy raises several issues that should be addressed to ensure safe and efficient treatment of human disease conditions. These challenges relate to the unique properties of the cells and will require development of novel technologies as well as assessment of additional risk factors, which are not addressed using current procedures for preclinical testing of biopharmaceuticals. The differentiated products of hESCs and iPSCs retain an immature phenotype even when terminally differentiated. The expression of pluripotency genes disappeared upon initiation of differentiation, other genes associated with embryonic tissue such as LIN28A, LIN28B, and DPPA4 were not silenced. Induced pluripotent stem cells (iPSCs) based approaches for cancer therapy There are three possibilities for tumors in which cancer stem cells play a critical role. 1) Mutations in the normal stem cells or progenitor cells transform them into cancerous stem cells which can lead to the growth of the primary tumour. 2) Many of the primary tumour cells may be killed during chemotherapy, but if the cancerous stem cells are not eliminated, they become cancer-resistant stem cells and may result in tumour recurrence. 3) The cancer stem cells may migrate to distal positions from the primary tumor and cause metastasis. Human iPSC technology potentially can be used 1) for screening new cancer drugs 2) for providing cells for transplant to treat cancer Genetic mutations can be corrected in patient-derived iPSCs by gene targeting approaches. iPSC-derived tissue can be used to replace or repair tissues of cancer patients that have been injured by radiation, chemotherapy, or the surgical treatment necessary to eliminate the tumors. Because most cancers involve acquired genetic mutations in a specific tissue, iPSCs derived from other healthy tissues of the same patient could be used to regenerate those tissues damaged by the tumors themselves or subsequent treatments. Human induced pluripotent stem cell approaches are developing as a hopeful strategy to improve our knowledge of genetic association studies and the underlying molecular mechanisms. Induced pluripotent stem cells (iPSCs) based approaches for cancer therapy iPSCs therapy may be a good alternative for damaging approaches such as chemotherapy, radiotherapy, or surgical treatment. iPSCs can be utilized to screen novel anticancer drugs. Derived iPSCs from differentiating cancer tissue generate cell types that may be more biologically linked to human tumors and be more suitable for drug screening methods. IPSCs are also better candidates than other stem cells for evaluation of the toxicities of antitumor drugs. Like most chemotherapies, stem cell therapy using a single agent cannot eliminate tumors. Therefore, a desired drug combination should be rationally generated and selected. Many combination therapies have been tested to improve treatment durability. For example, chemotherapy combined with interferon (IFN)-beta immunotherapy, by using a pro-drug/suicide gene system, has displayed synergistic therapeutic effects in human colorectal cancer. Induced pluripotent stem cells (iPSCs) based approaches for Hematopoietic Malignancies Most hematopoietic malignancies originate from cells that are functionally heterogeneous and few of them are responsible for maintaining tumour state. These cancer stem cells exhibit the quality characteristics of normal tissue stem cells, such as self-renewal, long term survival, and the ability to produce cells with more differentiated properties. Clinically, after treatment of malignancies with high-dose radiotherapy or chemotherapy, transplanting human stem cells has been generally used to facilitate lifelong haematological recovery iPSC technology for Neurodegenerative diseases Adult-onset neurodegenerative diseases are among the most difficult human health conditions to model for drug development. Most genetic or toxin-induced cell and animal models cannot faithfully recapitulate pathology in disease-relevant cells, making it excessively challenging to explore the potential mechanisms underlying sporadic disease. Patient-derived iPSCs can be differentiated into disease-relevant neurons, providing an unparalleled platform for in vitro modelling and development of therapeutic strategies. Patient iPSCs can provide a source of cells that harbour a precise constellation of genetic variants, which is associated with pathogenesis in the appropriate microenvironment. Cells derived from patient iPSCs have been shown to recapitulate phenotypes of various human neurodegenerative diseases, including Alzheimer’s disease, Amyotrophic lateral sclerosis, Huntington’s disease and fragile X syndrome. Expandable iPSCs can give rise to a large number of disease-related cells, providing an excellent opportunity for large-scale drug testing. Applications of human iPSC in neurodegenerative disease Efficient differentiation of patient-derived iPSCs into specific neuronal subtypes and 3D organoids, it is possible to recapitulate the cellular progression toward neurodegeneration in vitro. Region-specific brain organoids provide a platform to investigate early-stage phenotypes, which may potentially serve as biomarkers for early diagnosis and drug targets for preventing disease progression. Brain organoids lack vascularization, patterning cues and complex cell-cell interactions, such as those found in the nigrostriatal pathway, animal models will continue to provide essential readouts for drug evaluation, especially those related to physiological interactions and disease-associated behavioural phenotypes (e.g. cognitive impairment in AD and motor defects in PD and HD). The combination of iPSC technology with genome editing, organoid engineering will certainly accelerate the development of new medicines for human neurodegenerative disease and eventually may cure these devastating diseases by cell replacement therapy Organoid based Personalized Medicine Organoids are miniaturized versions of organs and tissues that are grown in vitro. Organoids are one of the most accessible and physiologically relevant models to study the dynamics of stem cells in a controlled environment that can be derived from a variety of sources. Organoids can be grown from tissue-specific adult stem cells or pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Organoids are in vitro 3D organ-like architectures that retain the specific structures and characteristics of the original organs in our body. 3D organoids would be more appropriate than traditional 2D culture systems for understanding organ development and morphogenesis and for exploring disease mechanism and drug discovery. In combination with genetic, transcriptome and proteomic profiling, both murine- and human-derived organoids have revealed crucial aspects of development, homeostasis and disease. The progress in generating organoids that faithfully recapitulate the human in vivo tissue composition has extended organoid applications from being just a basic research tool to a translational platform with a wide range of uses. Due to the heterogeneity of tissues and organs, the pathogens cause different clinical symptoms, and the drugs also show different toxicity and efficacy according to tissue or organ types. The capacity to indefinitely culture organoids, without introducing genetic variation, makes them a good model for high-throughput preclinical screenings, designing targeted and personalized therapies, and providing a source of fully functional tissue for regenerative medicine applications. Initiation of organoid culture requires the isolation of stem/progenitor cells, either pluripotent stem cells (PSCs) or tissueresident stem/progenitor/ differentiated cells isolated from embryonic stages or adult tissues. The cells of origin for PSC-derived organoids are ESCs or iPSCs, which are then cultured in media supplemented with growth factors in order to mimic the signals that cells are exposed to during embryonic patterning to give rise to the specific tissue. Pluripotent stem cells can be taken through the developmental steps that establish organs during embryogenesis. - CNS, kidney and GI organs. Tissue-specific organoids recapitulating the physiological features and functions of each organ can be used to conduct in-depth fundamental researches on human pathology and to develop advanced treatments to human diseases. Adult epithelial stem cells can be cultured under tissue-repair conditions and generate epithelial organoids directly from healthy and diseased organs such as the gut, the liver, the lung and the pancreas. Specifically, the methods use growth factors or nutrient combinations to drive the acquisition of organ precursor tissue identity. A permissive three-dimensional culture environment is applied, involves the use of extracellular matrix gels. This allows the tissue to self-organize through cell sorting out and stem cell lineage commitment in a spatially defined manner to recapitulate organization of different organ cell types. (A) Different cell types sort themselves because of different adhesive properties conferred by their differential expression of distinct cell adhesion molecules. (B) Progenitors give rise to more differentiated progeny, which are forced into a more superficial position that promotes their differentiation. These cells can sometimes further divide to give rise to more differentiated progeny, which are further displaced. Organoids have been generated for a number of organs from both mouse and human stem cells. To date, human pluripotent stem cells have been coaxed to generate intestinal, kidney, brain, and retinal organoids, as well as liver organoid-like tissues called liver buds. Derivation methods are specific to each of these systems, with a focus on recapitulation of endogenous developmental processes. Diverse types of organoids derived from pluripotent stem cells (PSCs) and tissue-specific stem cells. Epithelial organoids The intestinal epithelium is a tissue with an extreme self-renewal capacity fueled by Lgr5+ intestinal stem cells (ISC) These cells give rise to daughter or progenitor cells that can differentiate into mature epithelial cells required for normal gut function Homeostasis of the normal intestinal epithelium is ensured by continuous and rapid turnover of differentiated cells compensated by replication of ISCs expressing Lgr5, a seven-transmembrane receptor - a marker of Wnt-regulated adult stem cell populations in the intestine, stomach, pancreas, and prostate. In various pathological conditions, this renewal process can become substantially disordered, resulting in a loss of epithelial integrity, in local inflammation, or even carcinogenesis. Diseases of the intestinal epithelium include chronic disorders such as inflammatory bowel disease (IBD) and gastrointestinal (GI) cancers. Due to unavailability of effective drugs for treatment of these diseases, the mortality rates remain unacceptably high. Potential therapeutic and diagnostic uses for organoid technology in Personalized Medicine (a) Human iPSC- or tissue-derived organoids can contribute to the design of personalized treatment strategies. (b) The diseased and normal matched organoids can then be used to assess tissue function and prognosis in response to various treatments. A wide variety of active drugs and small compounds can be screened for targeting candidate signaling pathways (c) to design more effective drug regimens (d) in conjunction with other relevant diagnostic and prognostic factors. e) The organoid cultures can also provide an expansive source of tissue for regeneration and transplantation. f) Genetic manipulation of organoids can help restore functionality and normal physiology, and g) can be transplanted into donors for the re-establishment of physiological function. Applications of organoid technology for studying development, homeostasis and diseases a) Murine- and human-derived organoids from ESCs/iPSCs b) Tissue subunits contribute to the study of development and homeostasis. c) Genome, transcriptome and proteome profiling of the organoids enables delineation of the contribution of various signaling pathways in development and their dysregulation in disease. (d) Bacterial and viral infections can be studied by injecting infectious agents into the lumen of organoids. (e) Human disease can be modelled by biochemical and genetic manipulations to identify driver mutations and key signaling pathways. (f) Organoids represent a physiological model that can be used in highthroughput screens for effective drugs and small compounds at the preclinical stage. (g) human organoids are being collated and catalogued as open access biobanks. Applications of liver organoids Applications of liver organoids. Organoids derived from healthy donors or patients can be used as a model in basic research to investigate liver development and function in healthy conditions and to dissect the mechanisms of disease. Liver organoids are also a potential bridging tool towards personalized medicine, allowing for patient-specific drug screening and gene therapy. Liver organoids as liver disease modelling tools Advantages: Three-dimensional spatial organization (ASCs/iPSCs). Genetically stable (ASCs). Preservation of genetic and epigenetic signature of derived tissue (ASCs). Long-term culture (ASCs/iPSCs). Biobanks (ASCs/iPSCs). Safe for transplantation (ASCs). Unlimited source of patient-derived cells (iPSCs). Non-invasive derivation from a variety of cells (eg, skin/ fibroblasts/blood cells) (iPSCs). Recapitulate different aspects of liver development (iPSCs). Disease modelling (ASCs/iPSCs). High-throughput drug screening (ASCs/iPSCs). Personalized medicine (ASCs/iPSCs). Gene therapy (ASCs/iPSCs). Current limitations: Persistence of fetal markers (iPSCs). Limited cell maturation (ASCs/iPSCs). Restricted access to tissue and need for invasive methods(ASCs). Failure to recapitulate the multiple cell types of the liver (ASCs). Biobanking of Organoids Organoids can be expanded in vitro and cryopreserved enabling the establishment of biobanks. These can be used on a larger scale for regenerative medicine (including transplants), drug screening (patient-derived organoids can help identify drugs that a cohort of patients are most likely to respond to) and toxicology studies for predicting which potential therapies may induce drug-induced liver injury. Organoids and Regenerative Medicine Modern medicine can replace damaged and/or non-functional tissue with healthy tissue by allogenic transplantation, but a limited supply of healthy donor tissue and the inherent complication of tissue rejection highlight the need for additional tissue sources. Organoid technologies allow expansion of isogenic tissue from miniscule patient biopsies for transplantation use. Using iPSC technologies, it is also possible to generate isogenic or HLA-matched tissue-specific organoids from readily accessible tissue biopsies (for example, skin). Patient organoids harboring genetic defects can now be repaired using gene-editing technologies to generate healthy isogenic epithelia for use in orthotopic transplantation as an effective treatment regime. The feasibility of such an approach was demonstrated by CRISPR/Cas9 gene editing with patient derived colon organoids to correct germline CFTR mutations, thereby restoring enzymatic function to generate healthy epithelia capable of repopulating diseased tissue following transplantation. The capacity of in vitro cultured organoids to repair diseased or damaged tissue in vivo has been demonstrated by studies reporting functional engraftment of orthotopically transplanted organoids in the colon, pancreas and liver. Similarly, development of more physiological neural organoids would provide an excellent source of healthy tissue for treating a range of neurodegenerative diseases such as spinal cord injury and Parkinson’s disease. The use of mouse sarcoma-derived Matrigel as the artificial 3D matrix in organoid culture precludes the use of human organoids in clinical transplantation due to risks of unforeseen infection and immune/host rejection reactions. Hence, efforts are underway to design more defined ECMs that are compatible with clinical regulations for use in humans. The potential use of organoids in combination with gene editing for Personalized regenerative medicine Using genetic engineering techniques, diseased organoids derived from iPSCs or tissue biopsies from patients with genetic defects can be corrected to healthy organoids. Transplantation of genetically corrected organoids with functional biomaterials can increase the success rate of organoid engraftment to regenerate damaged organs or tissues in patients. Organoid-based disease modeling for drug testing, disease mechanisms and diagnostic tools Patient-derived organoids can be generated from pluripotent stem cells (ESCs, iPSCs) or tissue biopsies, which can provide modeling of various diseases, such as genetic diseases, cancer, infectious diseases, and injuries. Organoid models have been utilized for various biomedical applications to improve human healthcare. Drug Discovery via Human-Derived Stem Cell Organoids Patient-derived cell lines and animal models could be used for the understanding of human intestinal diseases and for drug development. Many genetically determined intestinal diseases occur in specific tissue microenvironments that are not adequately modelled by monolayer/2D cell culture. Animal models incompletely recapitulate the complex pathologies of intestinal diseases of humans and fall short in predicting the effects of candidate drugs. Patient-derived stem cell organoids are new and effective models for the development of novel targeted therapies. With the use of intestinal organoids from patients with inherited diseases, the potency and toxicity of drug candidates can be evaluated better. Using organoids as a platform to deconstruct organogenesis. Owing to the simplicity of the organoid system in comparison to in vivo organogenesis, it is a good choice for in silico-supported studies. First step is to observe the system properties and the parameters that affect them. The next step is to select an appropriate model that incorporates the observations of interest while effectively predicting the system. Finally, the in- silico predictions can be tested by manipulating the relevant medium or organoid elements. This leads to new observations that can be treated in the same manner, leading to either a model refinement or an entirely new model. Any Questions ?