Use Of iPSCs Research Articles

IPS cell generation-associated point mutations include many C > T substitutions via different cytosine modification mechanisms

Genomic aberrations are a critical impediment for the safe medical use of iPSCs and their origin and developmental mechanisms remain unknown. Here we find through WGS analysis of human and mouse iPSC lines that genomic mutations are de novo events and that, in addition to unmodified cytosine base prone to deamination, the DNA methylation sequence CpG represents a significant mutation-prone site. CGI and TSS regions show increased mutations in iPSCs and elevated mutations are observed in retrotransposons, especially in the AluY subfamily. Furthermore, increased cytosine to thymine mutations are observed in differentially methylated regions. These results indicate that in addition to deamination of cytosine, demethylation of methylated cytosine, which plays a central role in genome reprogramming, may act mutagenically during iPSC generation.

Characterization of the human induced pluripotent stem cell (iPSC) SZGJMSi004-A line from a 28-year-old Han male patient with depression

Peripheral blood mononuclear cells (PBMCs) from a 28-year-old male patient with unipolar depression were reprogrammed with reprogramming factors by electroporation. The pluripotency of transgene-free induced pluripotent stem cells (iPSCs) was verified by immunofluorescence staining for pluripotency markers, and these iPSCs were able to differentiate into the 3 germ layers in vitro. These iPSCs also showed normal karyotypes. Thus, we believe that these iPSCs could be valuable models for exploring the underlying biological mechanism of depression and the safety of antidepressants through the use of iPSCs differentiated into different kinds of neurons or brain organoids.

Evaluation of the impact of iPSC differentiation protocols on transcriptomic signatures

Human induced pluripotent stem cells (iPSC) have the potential to produce desired target cell types in vitro and allow for the high-throughput screening of drugs/chemicals at population level thereby minimising the cost of drug discovery and drug withdrawals after clinical trials. There is a substantial need for the characterisation of the iPSC derived models to better understand and utilise them for toxicological relevant applications. In our study, iPSC (SBAD2 or SBAD3 lines obtained from StemBANCC project) were differentiated towards toxicologically relevant cell types: alveolar macrophages, brain capillary endothelial cells, brain cells, endothelial cells, hepatocytes, lung airway epithelium, monocytes, podocytes and renal proximal tubular cells. A targeted transcriptomic approach was employed to understand the effects of differentiation protocols on these cell types. Pearson correlation and principal component analysis (PCA) separated most of the intended target cell types and undifferentiated iPSC models as distinct groups with a high correlation among replicates from the same model. Based on PCA, the intended target cell types could also be separated into the three germ layer groups (ectoderm, endoderm and mesoderm). Differential expression analysis (DESeq2) presented the upregulated genes in each intended target cell types that allowed the evaluation of the differentiation to certain degree and the selection of key differentiation markers. In conclusion, these data confirm the versatile use of iPSC differentiated cell types as standardizable and relevant model systems for in vitro toxicology.

Biallelic EPCAM deletions induce tissue-specific DNA repair deficiency and cancer predisposition

We report a case of Mismatch Repair Deficiency (MMRD) caused by germline homozygous EPCAM deletion leading to tissue-specific loss of MSH2. Through the use of patient-derived cells and organoid technologies, we performed stepwise in vitro differentiation of colonic and brain organoids from reprogrammed EPCAMdel iPSC derived from patient fibroblasts. Differentiation of iPSC to epithelial-colonic organoids exhibited continuous increased EPCAM expression and hypermethylation of the MSH2 promoter. This was associated with loss of MSH2 expression, increased mutational burden, MMRD signatures and MS-indel accumulation, the hallmarks of MMRD. In contrast, maturation into brain organoids and examination of blood and fibroblasts failed to show similar processes, preserving MMR proficiency. The combined use of iPSC, organoid technologies and functional genomics analyses highlights the potential of cutting-edge cellular and molecular analysis techniques to define processes controlling tumorigenesis and uncovers a new paradigm of tissue-specific MMRD, which affects the clinical management of these patients.

Unraveling Tumorigenicity in iPSC-Derived Immune Cells: The Impact of Chromosomal Abnormalities and Proliferative Intermediates

Response to tumor cells by the immune system can determine the course of a cancer. This guiding principle and the dynamic, adaptable nature of our immune system underlies the drive to generate immune cells for cancer immunotherapy. Induced pluripotent stem cells (iPSCs) provide an unlimited resource for the generation of immune cells such as T cells and dendritic cells (DCs) for cancer immunotherapy. However, the use of iPSC-derived immune cells in clinical applications has been limited due to concerns of safety. In vitro passage of iPSCs can introduce selective pressures that promote the accumulation of mutations that confer a growth advantage. Over several passages, this can lead to the acquisition of chromosomal abnormalities. Moreover, differentiation protocols of varying efficiency permit the presence of immature cells, contaminating the differentiated cell population. However, the impact of chromosomal abnormalities and intermediate products with proliferation potential on tumorigenicity of iPSC-derived immune cells remains unclear. Here, we investigated the tumorigenic potential of iPSC-derived T cells and DCs in immunocompetent and immunodeficient mice. Pmel-1 T-cell receptor (TCR) transgenic mouse T cells were reprogrammed using Sendai virus vectors carrying Oct3/4, Sox2, Klf4 and cMyc. While our iPSCs were phenotypically normal, even at early passage numbers, various chromosomal abnormalities were found in the resulting cell population. These abnormalities included trisomy 8 and 11 and loss of X chromosome with high frequency. Furthermore, within a population of iPSCs determined to be karyotypically normal by G-banding, unbalanced translocations were found by spectral karyotyping. These findings are consistent with those reported in the literature and highlight the prevalence of chromosomal abnormalities in iPSC populations. As iPSCs can harbor chromosomal abnormalities that are undetectable by standard methods, we assessed the tumorigenic potential of iPSC-derived cells, similar to what may be used in clinical applications. Mouse iPSCs were differentiated into CD8αβ T cells by co-culture with murine OP9 bone marrow stromal cells expressing the Notch ligand Delta-like 1 (OP9-DL1) and were enriched by CD8 positive selection. Activated iPSC-derived T cells were adoptively transferred to immunocompetent C57BL/6 and immunodeficient NOD/SCID, IL-2 receptor γ chain knockout mice. Immunodeficient but not immunocompetent mice developed hepatic tumors. Reverse transcription (RT)-PCR identified Pme1-l TCR gene in tumor cells, confirming tumor origin. Moreover, histology of liver tumors is suggestive of lymphoma or lymphosarcoma. Within the population of CD8+ iPSC-derived cells, a small subpopulation co-expressing CD8 and SSEA-1 was present. Sorting the cells by SSEA-1 positivity eliminated tumor formation in vivo. Mouse iPSCs were also differentiated into DCs by co-culture with OP9-DL1 cells and granulocyte macrophage colony-stimulating factor. Despite similar use of iPSCs with chromosomal abnormalities and prolonged passaging in vitro, SSEA-1+ cells were not identified when they were differentiated to DCs, and iPSC-derived DCs did not form tumors in immunodeficient mice. Taken together, this study revealed the potential risk of tumorigenicity of iPSC-derived immune cells in our preclinical model. The presence of chromosomal abnormalities did not definitively determine risk of tumorigenesis upon transplantation. The relationship between SSEA-1 positivity and tumor formation suggests contaminating cells may carry tumorigenic characteristics. Further work is required to develop differentiation methods that generate pure populations, free of tumorigenic intermediates, to potentiate the clinical use of iPSC-derived immunotherapy.

Induced pluripotent stem cells in periodontal reconstructive therapy: A narrative review of pre-clinical studies

Background: Regenerative periodontal surgical therapy faces significant challenges due to the limited ability of the body to regenerate damaged periodontal tissue. One of the primary goals in regenerative periodontal therapy is regaining periodontal tissue attachment after destruction by periodontal disease. Currently, stem cells, harnessing three pivotal components—cells, biomaterials, and growth factors—are widely used in periodontal regeneration. Stem cells can be obtained from various sources, either by isolating cells from bone marrow, teeth, and muscles or through the somatic cell programming method (reprogramming) known as induced pluripotent stem cells (iPSCs). Purpose: This review aims to describe the potential use of iPSCs in the treatment of periodontal defects. Review: Search strategies were developed using the PubMed, LILACS, Scielo, and Wiley online databases during the period of 2012–2022. Ten articles met the inclusion criteria. iPSCs were obtained by inducing somatic cells from both dental and non-dental sources with factors Oct3/4, Sox2, Klf4, and c-Myc. Periodontal tissue regeneration procedures can be augmented with iPSCs. Unlike tooth-based stem cells, iPSCs offer several advantages, such as unlimited cell sources and the capability to differentiate into any cell type, including periodontal tissue. The potential of iPSCs extends to correcting periodontal bone defects and forming new periodontal tissues, such as alveolar bone, cementum, and periodontal ligament. However, iPSCs do have limitations, including the need for clinical trials, cell programming production facilities, and optimization of differentiated-cell functionality. Conclusion: The combined use of iPSCs in cell-based tissue engineering holds vast potential for future periodontal treatment strategies.

On the utilization of the induced pluripotent stem cell (iPSC) model to study substance use disorders: A scoping review protocol.

Induced pluripotent stem cells (iPSCs) are cells derived from somatic cells via reprogramming techniques. The iPSC approach has been increasingly used in neuropsychiatric research in the last decade. Though substance use disorders (SUDs) are a commonly occurring psychiatric disorder, the application of iPSC model in addiction research has been limited. No comprehensive review has been reported. We conducted a scoping review to collate existing evidence on the iPSC technologies applied to SUD research. We aim to identify current knowledge gaps and limitations in order to advance the use of iPSCs in the SUD field. We employed a scoping review using the methodological framework first created by Arksey and O'Malley and further updated by Levac et al. and the Joanna Briggs Institute (JBI). We adopted the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Protocols (PRISMA-P) to report items for the protocol. We searched evidence from four electronic databases: PubMed®, Embase®, Web of Science™, and Scopus®. Primary research, systematic reviews, and meta-analyses were included and limited to studies published in English, at the time from 2007 to March 2022. This is an "ongoing" scoping review. Searched studies will be independently screened, selected, and extracted by two reviewers. Disagreement will be solved by the third reviewer and discussion. Extracted data will be analyzed in descriptive and quantitative approaches, then summarized and presented in appropriate formats. Results will be reported following the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guideline and disseminated through a peer-reviewed publication and conference presentations. To our best knowledge, this is the first comprehensive scoping review of iPSC methods specifically applied to a broad range of addictive drugs/substances that lead to SUDs or misuse behavior. This protocol is registered on Zenodo repository (https://zenodo.org/) with doi:10.5281/zenodo.7915252.

Recent advances in the application of induced pluripotent stem cell technology to the study of myeloid malignancies

Acquired myeloid malignancies are a spectrum of clonal disorders known to be caused by sequential acquisition of genetic lesions in hematopoietic stem and progenitor cells, leading to their aberrant self-renewal and differentiation. The increasing use of induced pluripotent stem cell (iPSC) technology to study myeloid malignancies has helped usher a paradigm shift in approaches to disease modeling and drug discovery, especially when combined with gene-editing technology. The process of reprogramming allows for the capture of the diversity of genetic lesions and mutational burden found in primary patient samples into individual stable iPSC lines. Patient-derived iPSC lines, owing to their self-renewal and differentiation capacity, can thus be a homogenous source of disease relevant material that allow for the study of disease pathogenesis using various functional read-outs. Furthermore, genome editing technologies like CRISPR/Cas9 enable the study of the stepwise progression from normal to malignant hematopoiesis through the introduction of specific driver mutations, individually or in combination, to create isogenic lines for comparison. In this review, we survey the current use of iPSCs to model acquired myeloid malignancies including myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), acute myeloid leukemia and MDS/MPN overlap syndromes. The use of iPSCs has enabled the interrogation of the underlying mechanism of initiation and progression driving these diseases. It has also made drug testing, repurposing, and the discovery of novel therapies for these diseases possible in a high throughput setting.

Induced pluripotent stem cells-podocytes promote repair in acute kidney injury is dependent on Mafb/CCR5/Nampt axis-mediated M2 macrophage polarization

Induced pluripotent stem cells (iPSCs) have been the focus of cellular therapy studies. The use of iPSCs in regenerative medicine is limited by their tumorigenic potential. This study sought to determine whether iPSCs-derived podocytes attenuate acute kidney injury (AKI) and the molecular mechanism. Inoculation of iPSCs-podocytes significantly promoted the repair of kidney injury in AKI mice, reduced the levels of kidney injury factors Scr, BUN, and urinary NAG, and alleviated the inflammatory response. Histological analysis revealed a significant increase in the number of M2 macrophages and a significant decrease in M1 macrophages in the kidney tissues. Subsequently, the genes and signaling pathways that may be associated with kidney injury repair in mice were analyzed by RNA-seq and bioinformatics prediction. The polarization of M2 macrophages was promoted by MAF bZIP transcription factor B (Mafb)-mediated activation of C-C motif chemokine receptor 5 (Ccr5) and nicotinamide phosphoribosyltransferase (Nampt) signaling pathway. Taken together, these results show that iPSCs-podocytes depend on Mafb to activate the Nampt signaling pathway through transcriptional activation of Ccr5, thereby promoting the repair of AKI caused by ischemia-reperfusion.

IPSC-derived retinal pigmented epithelial cells from patients with macular telangiectasia show decreased mitochondrial function.

Patient-derived induced pluripotent stem cells (iPSCs) provide a powerful tool for identifying cellular and molecular mechanisms of disease. Macular telangiectasia type 2 (MacTel) is a rare, late-onset degenerative retinal disease with an extremely heterogeneous genetic architecture, lending itself to the use of iPSCs. Whole-exome sequencing screens and pedigree analyses have identified rare causative mutations that account for less than 5% of cases. Metabolomic surveys of patient populations and GWAS have linked MacTel to decreased circulating levels of serine and elevated levels of neurotoxic 1-deoxysphingolipids (1-dSLs). However, retina-specific, disease-contributing factors have yet to be identified. Here, we used iPSC-differentiated retinal pigmented epithelial (iRPE) cells derived from donors with or without MacTel to screen for novel cell-intrinsic pathological mechanisms. We show that MacTel iRPE cells mimicked the low serine levels observed in serum from patients with MacTel. Through RNA-Seq and gene set enrichment pathway analysis, we determined that MacTel iRPE cells are enriched in cellular stress pathways and dysregulation of central carbon metabolism. Using respirometry and mitochondrial stress testing, we functionally validated that MacTel iRPE cells had a reduction in mitochondrial function that was independent of defects in serine biosynthesis and 1-dSL accumulation. Thus, we identified phenotypes that may constitute alternative disease mechanisms beyond the known serine/sphingolipid pathway.

INVESTIGATING THE ANTI-CATABOLIC PROPERTIES OF NOTOCHORDAL CELLS IN DISC REGENERATION

Low back pain resulting from Interertebral disc (IVD) degeneration is a serious worldwide problem, with poor treatment options available. Notochordal (NC) cells, are a promising therapeutic cell source with anti-catabolic and regenerative effect. However, their behaviour in the harsh degenerate environment is unknown.Porcine NC cells (pNCs), and Human NP cells from degenerate IVDs were cultured in alginate beads to maintain phenotype. Cells were cultured alone or in combination, or co-stimulated with notochordal cell condition media (NCCM), in media to mimic the healthy and degenerate disc environment, together with controls for up to 1 week. Following culture viability, qPCR and proteomic analysis using Digiwest was performed.A small increase in pNC cell death was observed in degenerated media compared to standard and healthy media, with a further decrease seen when cultured with IL-1β. Whilst no significant differences were seen in phenotypic marker expression in pNCs cultured in any media at gene level (ACAN, KRT8, KRT18, FOXA2, COL1A1 and Brachyury). Preliminary Digiwest analysis showed increased protein production for Cytokeratin 18, src and phosphorylated PKC but a decrease in fibronectin in degenerated media compared to standard media. Human NP cells cultured with NCCM, showed a decrease in IL-8 production compared to human NP cells alone when cultured in healthy media. However, gene expression analysis (ACAN, VEGF, MMP3 and IL-1β) demonstrated no significant difference between NP only and NP+NCCM groups.Studying the behaviour of the NCs in in vitro conditions that mimic the in vivo healthy or degenerate niche will help us to better understand their potential for therapeutic approaches. The potential use of NC cell sources for regenerative therapies can then be translated to investigate the potential use of iPSCs differentiated into NC cells as a regenerative cell source.

Getting closer to modeling the gut-brain axis using induced pluripotent stem cells.

The gut microbiome (GM), the gut barrier, and the blood-brain barrier (BBB) are key elements of the gut-brain axis (GBA). The advances in organ-on-a-chip and induced pluripotent stem cell (iPSCs) technology might enable more physiological gut-brain-axis-on-a-chip models. The ability to mimic complex physiological functions of the GBA is needed in basic mechanistic research as well as disease research of psychiatric, neurodevelopmental, functional, and neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. These brain disorders have been associated with GM dysbiosis, which may affect the brain via the GBA. Although animal models have paved the way for the breakthroughs and progression in the understanding of the GBA, the fundamental questions of exactly when, how, and why still remain unanswered. The research of the complex GBA have relied on equally complex animal models, but today's ethical knowledge and responsibilities demand interdisciplinary development of non-animal models to study such systems. In this review we briefly describe the gut barrier and BBB, provide an overview of current cell models, and discuss the use of iPSCs in these GBA elements. We highlight the perspectives of producing GBA chips using iPSCs and the challenges that remain in the field.

Stem Cells In IVF: Hope or Hype?

Perhaps the most obvious link to IVF and stem cells is the development of human Embryonic Stem Cells (hESC) from the inner cell mass of human embryos [4]. Whilst hESC are pluripotent and therefore an excellent candidate for gamete production, very little progress has been made because of the legal, ethical, moral, and religious objections to creating hESC which requires the destruction of a viable human embryo [5]. Induced Pluripotent Stem Cells (iPSC) have a similar history when it comes to IVF. These are somatic cells (e.g., skin cells) which are ‘transformed’ into pluripotent stem cells by the introduction of various genes [6]. These iPSC are pluripotent and autologous, so they have the ability, in theory, to be able to carry out gametogenesis in patients undergoing fertility treatment [7]. The problems with the routine clinical use of iPSC in fertility treatment is the cost but most importantly ongoing safety concerns about iPSC because of the genes needed to be inserted to convert a somatic cell into a stem cell [8]. Mesenchymal stem cells (MSC) can be obtained from adipose tissue, bone marrow, the umbilical cord and even inside teeth [9]. These MSC stem cells can produce bone, connective tissue and adipose tissue and there are some data suggesting that MSC may be useful in premature ovarian failure [10]. The drawbacks of using MSC in the treatment of infertility are the cost and the standardization of MSC to ensure safety and efficacy [11]. All these stem cell types have their problems when considering their use in the treatment of infertility. It may be many years, if ever, before they come to routine clinical practice in the treatment of infertility.

IPSC differentiation into various neuron subtypes

Induced pluripotent stem cells (iPSCs) have been demonstrated to be a valuable tool in modeling diseases, investigating drug mechanisms, and understanding manifestations of disease in specific cell types. In the field of neurology, current studies are investigating the use of iPSCs as a regenerative therapy, which could be extremely beneficial for understanding and treating neurodegenerative diseases. While iPSCs have been used in various studies, a lack of familiarity with all available protocols for neuronal differentiation makes it difficult to identify best practices. Further, because neurological diseases can selectively impact certain types of neurons, it is useful to compile protocols to make different neuronal types. In this article, methods of differentiating iPSCs into various neuron subtypes are described in order to provide a consolidated list of protocols that will aid the analysis of suitable and available methods for specific research protocols. By compiling and summarizing these various methods, future studies can determine which methods are best for their specific research goals, regarding timing, efficiency, and accuracy.

Culturing and Neuronal Differentiation of Human Dental Pulp Stem Cells.

A major issue in studying human neurogenetic disorders, especially rare syndromes affecting the nervous system, is the ability to grow neuronal cultures that accurately represent these disorders for analysis. Although there has been some success in generating induced pluripotent stem cells (iPSC) from both skin and blood, there are still limitations to the collection, production and use of iPSC derived neurons. We have had significant success in collecting and growing human dental pulp stem cells (DPSC) from exfoliated teeth sent directly to our laboratory by the parents of children with a variety of rare neurogenetic syndromes. This protocol outlines our current methods for the growth and expansion of DPSC from exfoliated (baby) teeth. These DPSC can be differentiated into a variety of cell types including osteoblasts, chondrocytes, and mixed neuron and glial cultures. Here we provide our protocol for the differentiation of early passage DPSC cultures into neurons for molecular and cellular studies. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Collection and transportation of exfoliated teeth Basic Protocol 2: Dental pulp extraction Basic Protocol 3: Passage, freezing, and thawing of DPSC cultures Basic Protocol 4: Differentiation of DPSC into mixed neuronal cultures.

Single cell transcriptomic profiling of a neuron-astrocyte assembloid tauopathy model

The use of iPSC derived brain organoid models to study neurodegenerative disease has been hampered by a lack of systems that accurately and expeditiously recapitulate pathogenesis in the context of neuron-glial interactions. Here we report development of a system, termed AstTau, which propagates toxic human tau oligomers in iPSC derived neuron-astrocyte assembloids. The AstTau system develops much of the neuronal and astrocytic pathology observed in tauopathies including misfolded, phosphorylated, oligomeric, and fibrillar tau, strong neurodegeneration, and reactive astrogliosis. Single cell transcriptomic profiling combined with immunochemistry characterizes a model system that can more closely recapitulate late-stage changes in adult neurodegeneration. The transcriptomic studies demonstrate striking changes in neuroinflammatory and heat shock protein (HSP) chaperone systems in the disease process. Treatment with the HSP90 inhibitor PU-H71 is used to address the putative dysfunctional HSP chaperone system and produces a strong reduction of pathology and neurodegeneration, highlighting the potential of AstTau as a rapid and reproducible tool for drug discovery.

IPSC-Derived Airway Epithelial Cells: Progress, Promise, and Challenges.

Induced pluripotent stem cells (iPSCs) generated from somatic cell sources are pluripotent and capable of indefinite expansion in vitro. They provide an unlimited source of cells that can be differentiated into lung progenitor cells for potential clinical use in pulmonary regenerative medicine. This review gives a comprehensive overview of recent progress toward the use of iPSCs to generate proximal and distal airway epithelial cells and mix lung organoids. Furthermore, their potential applications and future challenges for the field are discussed, with a focus on the technological hurdles that must be cleared before stem cell therapeutics can be used for clinical treatment.

Establishment of two iPSC lines from healthy donor with heterozygous mutation in the SLC26A4 gene

The human induced pluripotent stem cell (iPSC) lines, CSUXHEi001-A and CSUXHEi002-A, were generated from peripheral blood mononuclear cells (PBMCs). The donors were couple and each of them has a heterozygous mutation in the SLC26A4 gene. It manifests in their children as Enlarged vestibular aqueduct (EVA). The use of iPSC will allow describing the early stages of hearing loss, which is undoubtedly relevant for identifying key stages of development at which phenotypic manifestations of mutations in the SLC26A4 gene are found.

Tissue-Engineered Models of the Human Brain: State-of-the-Art Analysis and Challenges.

Neurological disorders affect billions of people across the world, making the discovery of effective treatments an important challenge. The evaluation of drug efficacy is further complicated because of the lack of in vitro models able to reproduce the complexity of the human brain structure and functions. Some limitations of 2D preclinical models of the human brain have been overcome by the use of 3D cultures such as cell spheroids, organoids and organs-on-chip. However, one of the most promising approaches for mimicking not only cell structure, but also brain architecture, is currently represented by tissue-engineered brain models. Both conventional (particularly electrospinning and salt leaching) and unconventional (particularly bioprinting) techniques have been exploited, making use of natural polymers or combinations between natural and synthetic polymers. Moreover, the use of induced pluripotent stem cells (iPSCs) has allowed the co-culture of different human brain cells (neurons, astrocytes, oligodendrocytes, microglia), helping towards approaching the central nervous system complexity. In this review article, we explain the importance of in vitro brain modeling, and present the main in vitro brain models developed to date, with a special focus on the most recent advancements in tissue-engineered brain models making use of iPSCs. Finally, we critically discuss achievements, main challenges and future perspectives.

Utility of iPSC-Derived Cells for Disease Modeling, Drug Development, and Cell Therapy.

The advent of induced pluripotent stem cells (iPSCs) has advanced our understanding of the molecular mechanisms of human disease, drug discovery, and regenerative medicine. As such, the use of iPSCs in drug development and validation has shown a sharp increase in the past 15 years. Furthermore, many labs have been successful in reproducing many disease phenotypes, often difficult or impossible to capture, in commonly used cell lines or animal models. However, there still remain limitations such as the variability between iPSC lines as well as their maturity. Here, we aim to discuss the strategies in generating iPSC-derived cardiomyocytes and neurons for use in disease modeling, drug development and their use in cell therapy.