Induced Pluripotent Stem Cells (iPSCs) Derived 3D Human Lung Organoids from Different Ethnicities to Understand the SARS-CoV2 Severity/Infectivity Percentage.
Induced Pluripotent Stem Cells (iPSCs) Derived 3D Human Lung Organoids from Different Ethnicities to Understand the SARS-CoV2 Severity/Infectivity Percentage.
Project description:Recent generation of patient-specific induced pluripotent stem cells (PS-iPSCs) provides significant advantages for cell- and gene-based therapy. Establishment of iPSC-based therapy for skin diseases requires efficient methodology for differentiating iPSCs into both keratinocytes and fibroblasts, the major cellular components of the skin, as well as the reconstruction of skin structures using these iPSC-derived skin components. We previously reported generation of keratinocytes from human iPSCs for use in the treatment of recessive dystrophic epidermolysis bullosa (RDEB) caused by mutations in the COL7A1 gene. Here, we developed a protocol for differentiating iPSCs into dermal fibroblasts, which also produce type VII collagen and therefore also have the potential to treat RDEB. Moreover, we generated in vitro 3D skin equivalents composed exclusively human iPSC-derived keratinocytes and fibroblasts for disease models and regenerative therapies for skin diseases, first demonstrating that iPSCs can provide the basis for modeling a human organ derived entirely from two different types of iPSC-derived cells.
Project description:Three-dimensional (3D) brain organoids derived from human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), appear to recapitulate the brain's 3D cytoarchitectural arrangement and provide new opportunities to explore disease pathogenesis in the human brain. Human iPSC (hiPSC) reprogramming methods, combined with 3D brain organoid tools, may allow patient-derived organoids to serve as a preclinical platform to bridge the translational gap between animal models and human clinical trials. Studies using patient-derived brain organoids have already revealed novel insights into molecular and genetic mechanisms of certain complex human neurological disorders such as microcephaly, autism, and Alzheimer's disease. Furthermore, the combination of hiPSC technology and small-molecule high-throughput screening (HTS) facilitates the development of novel pharmacotherapeutic strategies, while transcriptome sequencing enables the transcriptional profiling of patient-derived brain organoids. Finally, the addition of CRISPR/Cas9 genome editing provides incredible potential for personalized cell replacement therapy with genetically corrected hiPSCs. This review describes the history and current state of 3D brain organoid differentiation strategies, a survey of applications of organoids towards studies of neurodevelopmental and neurodegenerative disorders, and the challenges associated with their use as in vitro models of neurological disorders.
Project description:Numerous protocols have been described for producing neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. Although nearly all such methods yield at least partial segments of retinal structure with a mature appearance, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoids when viewed across multiple differentiations and hPSC lines. In an effort to understand better the current capabilities and limitations of these cultures, we generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time by light microscopy, immunocytochemistry, metabolic imaging and electron microscopy. We also employed optical coherence tomography and 3D imaging techniques to assess and compare whole or broad regions of organoids to avoid selection bias. Results from this study led to the development of a practical staging system to reduce inconsistencies in retinal organoid cultures and increase rigor when utilizing them in developmental studies, disease modeling and transplantation.
Project description:The pathogenesis of idiopathic pulmonary fibrosis (IPF), an intractable interstitial lung disease, is unclear. Recessive mutations in some genes implicated in Hermansky-Pudlak syndrome (HPS) cause HPS-associated interstitial pneumonia (HPSIP), a clinical entity that is similar to IPF. We previously reported that HPS1-/- embryonic stem cell-derived 3D lung organoids showed fibrotic changes. Here, we show that the introduction of all HPS mutations associated with HPSIP promotes fibrotic changes in lung organoids, while the deletion of HPS8, which is not associated with HPSIP, does not. Genome-wide expression analysis revealed the upregulation of interleukin-11 (IL-11) in epithelial cells from HPS mutant fibrotic organoids. IL-11 was detected predominantly in type 2 alveolar epithelial cells in end-stage IPF, but was expressed more broadly in HPSIP. Finally, IL-11 induced fibrosis in WT organoids, while its deletion prevented fibrosis in HPS4-/- organoids, suggesting IL-11 as a therapeutic target. hPSC-derived 3D lung organoids are, therefore, a valuable resource to model fibrotic lung disease.
Project description:COVID-19 has become difficult to contain in our interconnected world. In this article, we discuss some approaches that could reduce the consequences of COVID-19. We elaborate upon the utility of camelid single-domain antibodies (sdAbs), also referred to as nanobodies, which are naturally poised to neutralize viruses without enhancing its infectivity. Smaller sized sdAbs can be easily selected using microbes or the subcellular organelle display methods and can neutralize SARS-CoV2 infectivity. We also discuss issues related to their production using scalable platforms. The favorable outcome of the infection is evident in patients when the inflammatory response is adequately curtailed. Therefore, we discuss approaches to mitigate hyperinflammatory reactions initiated by SARS-CoV2 but orchestrated by immune mediators.
Project description:Human induced pluripotent stem cells (iPSCs) are ideal for developing personalized medicine. However, the spontaneous differentiation of human iPSCs under conventional 2D and 3D cultures results in significant heterogeneity and compromised quality. Therefore, a method for effectively isolating and expanding high-quality human iPSCs is critically needed. Here, a biomimetic microencapsulation approach for isolating and culturing high-quality human iPSCs is reported. This is inspired by the natural proliferation and development of blastomeres into early blastocyst where the early embryonic stem cells-containing core is enclosed in a semipermeable hydrogel shell known as the zona pellucida (Zona). Blastomere cluster-like human iPSC clusters are encapsulated in a miniaturized (≈10 nanoliter) hyaluronic acid (HA)-rich core of microcapsules with a semipermeable Zona-like hydrogel shell and subsequently cultured to form pluripotent human iPSC spheroids with significantly improved quality. This is indicated by their high expression of pluripotency markers and highly efficient 3D cardiac differentiation. In particular, HA is found to be crucial for isolating the high-quality human iPSCs with the biomimetic core-shell microencapsulation culture. Interestingly, the isolated human iPSCs can maintain high pluripotency even after being cultured again in 2D. These discoveries and the bioinspired culture method may be valuable to facilitate the human iPSC-based personalized medicine.
Project description:Human induced pluripotent stem cells (hiPSCs) promise a great number of future applications to investigate retinal development, pathophysiology and cell therapies for retinal degenerative diseases. Specific approaches to genetically modulate hiPSC would be valuable for all of these applications. Vectors based on adeno-associated virus (AAV) have shown the ability for gene delivery to retinal organoids derived from hiPSCs. Thus far, little work has been carried out to investigate mechanisms of AAV-mediated gene delivery and the potential advantages of engineered AAVs to genetically modify retinal organoids. In this study, we compared the early transduction efficiency of several recombinant and engineered AAVs in hiPSC-derived RPE cells and retinal organoids in relation to the availability of their cell-surface receptors and as a function of time. The genetic variant AAV2-7m8 had a superior transduction efficiency when applied at day 44 of differentiation on retinal organoids and provided long-lasting expressions for at least 4 weeks after infection without compromising cell viability. All of the capsids we tested transduced the hiPSC-RPE cells, with the AAV2-7m8 variant being the most efficient. Transduction efficiency was correlated with the presence of primary cell-surface receptors on the hiPS-derived organoids. Our study explores some of the mechanisms of cell attachment of AAVs and reports long-term gene expression resulting from gene delivery in retinal organoids.
Project description:The current coronavirus disease (COVID-19) outbreak caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV2) has emerged as a threat to global social and economic systems. Disparity in the infection of SARS-CoV2 among host population and species is an established fact without any clear explanation. To initiate infection, viral S-protein binds to the Angiotensin-Converting Enzyme 2 (ACE2) receptor of the host cell. Our analysis of retrieved amino acid sequences deposited in data bases shows that S-proteins and ACE2 are rich in cysteine (Cys) residues, many of which are conserved in various SARS-related coronaviruses and participate in intra-molecular disulfide bonds. High-resolution protein structures of S-proteins and ACE2 receptors highlighted the probability that two of these disulfide bonds are potentially redox-active, facilitating the primal interaction between the receptor and the spike protein. Presence of redox-active disulfides in the interacting parts of S-protein, ACE2, and a ferredoxin-like fold domain in ACE2, strongly indicate the role of redox in COVID-19 pathogenesis and severity. Resistant animals lack a redox-active disulfide (Cys133-Cys141) in ACE2 sequences, further strengthening the redox hypothesis for infectivity. ACE2 is a known regulator of oxidative stress. Augmentation of cellular oxidation with aging and illness is the most likely explanation of increased vulnerability of the elderly and persons with underlying health conditions to COVID-19.
Project description:Autism spectrum disorder (ASD) represents a group of neurodevelopmental diseases characterized by persistent deficits in social communication, interaction, and repetitive patterns of behaviors, interests, and activities. The etiopathogenesis is multifactorial with complex interactions between genetic and environmental factors. The clinical heterogeneity and complex etiology of this pediatric disorder have limited the development of pharmacological therapies. The major limit to ASD research remains a lack of relevant human disease models which can faithfully recapitulate key features of the human pathology and represent its genetic heterogeneity. Recent advances in induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells of patients into all types of patient-specific neural cells, have provided a promising cellular tool for disease modeling and development of novel drug treatments. The iPSCs technology allowed not only a better investigation of the disease etiopathogenesis but also opened up the potential for personalized therapies and offered new opportunities for drug discovery, pharmacological screening, and toxicity assessment. Moreover, iPSCs can be differentiated and organized into three-dimensional (3D) organoids, providing a model which mimics the complexity of the brain's architecture and more accurately recapitulates tissue- and organ-level disease pathophysiology. The aims of this review were to describe the current state of the art of the use of human patient-derived iPSCs and brain organoids in modeling ASD and developing novel therapeutic strategies and to discuss the opportunities and major challenges in this rapidly moving field.
Project description:Inflammatory bowel disease (IBD) patients are generally at an increased risk for viral diseases. Viral infections of tissue affected by chronic IBD are insufficiently understood, especially in the context of flare-ups; while some studies have confidently shown aggravation of symptoms in large cohorts, others found no association. Although not considered at an increased risk of Coronavirus Disease 2019 (COVID-19), IBD patients suffer more frequent gastrointestinal symptoms during COVID-19 and a worse outcome has been associated with certain IBD medications and disease activity. For this reason, we generated intestinal epithelial organoids from ileum with Crohn’s disease (CD) and colon with ulcerative colitis (UC) together with healthy controls. Each group included five to six patients. Using RNA sequencing we showed that IBD organoids were not more susceptible to infection. The highest viral load two days after infection was in healthy ileum and is correlated to the expression of SARS-CoV-2 entry receptor ACE2 and proteases CTSL/B. Pathway analyses determined an apparently weaker defense reaction in CD organoids to SARS-CoV-2 which could simply be due to the lower viral load compared to the healthy group. In contrast to CD, there was an activation of viral defense in infected UC colon, especially interferon signaling, resembling highly infected healthy ileum. This was not seen in healthy colon and was unexpected since UC and healthy colon did not differ in viral load. In uninfected IBD organoids compared to healthy, there was an already stronger basal expression of genes implicated in viral defense, for example, of viral sensors OAS1 and OASL in CD and interferon regulatory transcription factor IRF7 in UC. Some were further influenced by SARS-CoV-2 infection with notable differences between CD and UC. To conclude, infection with SARS-CoV-2 did not lead to higher viral numbers in IBD organoids providing reassurance for patients in remission. Still, we observed a more pronounced epithelial defense reaction to the virus in UC colon, prompting further investigations on whether this could be the reason for stronger gastrointestinal symptoms of COVID-19 seen in IBD patients or if it could lead to a consequent IBD flare.