scholarly journals Multicellular modeling of ciliopathy by combining iPS cells and microfluidic airway-on-a-chip technology

2021 ◽  
Vol 13 (601) ◽  
pp. eabb1298
Author(s):  
Naoyuki Sone ◽  
Satoshi Konishi ◽  
Koichi Igura ◽  
Koji Tamai ◽  
Satoshi Ikeo ◽  
...  
Keyword(s):  
Planar Cell Polarity ◽  
Mucociliary Clearance ◽  
Fluid Shear Stress ◽  
Disease Modeling ◽  
Cell Sheet ◽  
Recurrent Pneumonia ◽  
Chip Technology ◽  
Human Ipscs ◽  
Airway Reconstruction

Mucociliary clearance is an essential lung function that facilitates the removal of inhaled pathogens and foreign matter unidirectionally from the airway tract and is innately achieved by coordinated ciliary beating of multiciliated cells. Should ciliary function become disturbed, mucus can accumulate in the airway causing subsequent obstruction and potentially recurrent pneumonia. However, it has been difficult to recapitulate unidirectional mucociliary flow using human-derived induced pluripotent stem cells (iPSCs) in vitro and the mechanism governing the flow has not yet been elucidated, hampering the proper humanized airway disease modeling. Here, we combine human iPSCs and airway-on-a-chip technology, to demonstrate the effectiveness of fluid shear stress (FSS) for regulating the global axis of multicellular planar cell polarity (PCP), as well as inducing ciliogenesis, thereby contributing to quantifiable unidirectional mucociliary flow. Furthermore, we applied the findings to disease modeling of primary ciliary dyskinesia (PCD), a genetic disease characterized by impaired mucociliary clearance. The application of an airway cell sheet derived from patient-derived iPSCs and their gene-edited counterparts, as well as genetic knockout iPSCs of PCD causative genes, made it possible to recapitulate the abnormal ciliary functions in organized PCP using the airway-on-a-chip. These findings suggest that the disease model of PCD developed here is a potential platform for making diagnoses and identifying therapeutic targets and that airway reconstruction therapy using mechanical stress to regulate PCP might have therapeutic value.

scholarly journals Human iPSC-Based Modeling of Central Nerve System Disorders for Drug Discovery

2021 ◽  
Vol 22 (3) ◽  
pp. 1203
Author(s):  
Lu Qian ◽  
Julia TCW
Keyword(s):  
Drug Discovery ◽  
Disease Modeling ◽  
Three Dimensional ◽  
Structural Complexity ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Central Nerve System ◽  
Human Ipscs ◽  
Nerve System

A high-throughput drug screen identifies potentially promising therapeutics for clinical trials. However, limitations that persist in current disease modeling with limited physiological relevancy of human patients skew drug responses, hamper translation of clinical efficacy, and contribute to high clinical attritions. The emergence of induced pluripotent stem cell (iPSC) technology revolutionizes the paradigm of drug discovery. In particular, iPSC-based three-dimensional (3D) tissue engineering that appears as a promising vehicle of in vitro disease modeling provides more sophisticated tissue architectures and micro-environmental cues than a traditional two-dimensional (2D) culture. Here we discuss 3D based organoids/spheroids that construct the advanced modeling with evolved structural complexity, which propels drug discovery by exhibiting more human specific and diverse pathologies that are not perceived in 2D or animal models. We will then focus on various central nerve system (CNS) disease modeling using human iPSCs, leading to uncovering disease pathogenesis that guides the development of therapeutic strategies. Finally, we will address new opportunities of iPSC-assisted drug discovery with multi-disciplinary approaches from bioengineering to Omics technology. Despite technological challenges, iPSC-derived cytoarchitectures through interactions of diverse cell types mimic patients’ CNS and serve as a platform for therapeutic development and personalized precision medicine.

scholarly journals Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells

2020 ◽  
Author(s):  
Finn J. Hawkins ◽  
Shingo Suzuki ◽  
Mary Lou Beermann ◽  
Cristina Barillà ◽  
Ruobing Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Basal Cell ◽  
Pluripotent Stem Cells ◽  
Airway Epithelium ◽  
Disease Modeling ◽  
Airway Epithelial ◽  
Human Ipscs ◽  
Lung Airways

SummaryThe derivation of self-renewing tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would shorten the time needed to engineer mature cell types in vitro and would have broad reaching implications for the field of regenerative medicine. Here we report the directed differentiation of human iPSCs into putative airway basal cells (“iBCs”), a population resembling the epithelial stem cell of lung airways. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato) we track and purify these cells over time, as they first emerge from iPSC-derived foregut endoderm as developmentally immature NKX2-1GFP+ lung progenitors which then augment a TP63 program during subsequent proximal airway epithelial patterning. These cells clonally proliferate, initially as NKX2-1GFP+/TP63tdTomato+ immature airway progenitors that lack expression of the adult basal cell surface marker NGFR. However, in response to primary basal cell medium, NKX2-1GFP+/ TP63tdTomato+ cells upregulate NGFR and display the molecular and functional phenotype of airway basal stem cells, including the capacity to clonally self-renew or undergo multilineage ciliated and secretory epithelial differentiation in air-liquid interface cultures. iBCs and their differentiated progeny recapitulate several fundamental physiologic features of normal primary airway epithelial cells and model perturbations that characterize acquired and genetic airway diseases. In an asthma model of mucus metaplasia, the inflammatory cytokine IL-13 induced an increase in MUC5AC+ cells similar to primary cells. CFTR-dependent chloride flux in airway epithelium generated from cystic fibrosis iBCs or their syngeneic CFTR-corrected controls exhibited a pattern consistent with the flux measured in primary diseased and normal human airway epithelium, respectively. Finally, multiciliated cells generated from an individual with primary ciliary dyskinesia recapitulated the ciliary beat and ultrastructural defects observed in the donor. Thus, we demonstrate the successful de novo generation of a tissue-resident stem cell-like population in vitro from iPSCs, an approach which should facilitate disease modeling and future regenerative therapies for a variety of diseases affecting the lung airways.

scholarly journals Prospective Isolation of Chondroprogenitors from Human iPSCs Based on Cell Surface Markers Identified using a CRISPR-Cas9-Generated Reporter

2019 ◽  
Author(s):  
Amanda Dicks ◽  
Chia-Lung Wu ◽  
Nancy Steward ◽  
Shaunak S. Adkar ◽  
Charles A. Gersbach ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Disease Modeling ◽  
Cartilage Tissue Engineering ◽  
Culture Conditions ◽  
Cartilage Tissue ◽  
Human Ipscs ◽  
Chondroprogenitor Cells ◽  
Cell Source ◽  
Induced Pluripotent

SUMMARYArticular cartilage shows little or no capacity for intrinsic repair, generating a critical need for regenerative therapies for joint injuries and diseases such as osteoarthritis. Human induced pluripotent stem cells (hiPSCs) offer a promising cell source for cartilage tissue engineering andin vitrohuman disease modeling; however, heterogeneity and off-target differentiation remain a challenge. We used a CRISPR-Cas9-editedCOL2A1-GFPknock-in reporter hiPSC line, coupled with a surface marker screen, to identify a novel chondroprogenitor population expressing CD146, CD166, and PDGFRβ, but not CD45. Under chondrogenic culture conditions, these triple positive chondroprogenitor cells demonstrated decreased heterogeneity as measured by single cell RNA sequencing, as well as more robust and homogenous matrix production with significantly higher chondrogenic gene expression. Overall, this study has identified a unique hiPSC-derived subpopulation of chondroprogenitors that are CD146+/CD166+/PDGFRβ+/CD45-and exhibit high chondrogenic potential, providing a purified cell source for cartilage tissue engineering or disease modeling studies.

IN VITRO METHODS FOR THE STUDY OF THE ADENOHYPOPHYSIAL FUNCTIONS TO SECRETE GONADOTROPHIN

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S27-S40 ◽  
Author(s):  
T. Kobayashi ◽  
T. Kigawa ◽  
M. Mizuno ◽  
T. Watanabe
Keyword(s):  
Cell Culture ◽  
Organ Culture ◽  
Cultured Cells ◽  
Cell Sheet ◽  
Short Term ◽  
Hypothalamic Extract ◽  
Numerous Cell ◽  
Single Cell Culture ◽  
Almost All

ABSTRACT There are several in vitro methods to analyse the function of the adenohypophysis or the mechanisms of its regulation. The present paper deals with single cell culture, organ culture and short term incubation techniques by which the morphology and gonadotrophin-secreting function of the adenohypophysis were studied. In trypsin-dispersed cell culture, the adenohypophysial cells showed extensive propagation to form numerous cell colonies and finally develop into a confluent monolayer cell sheet covering completely the surface of culture vessels. Almost all of the cultured cells, however, became chromophobic, at least at the end of the first week of cultivation, when gonadotrophin was detectable neither in the culture medium nor in the cells themselves. After the addition of the hypothalamic extract, gonadotrophin became detectable again, and basophilic or PAS-positive granules also reappeared within the cells, suggesting that the gonadotrophs were stimulated by the extract to produce gonadotrophin. In organ culture and short term incubation, the incorporation of [3H] leucine into the adenohypophysial cells in relation to the addition of hypothalamic extract was examined. It was obvious that the ability to incorporate [3H] leucine into the gonadotrophs in vitro was highly dependent upon the presence of the hypothalamic extract.

scholarly journals Restoration of Osteogenesis by CRISPR/Cas9 Genome Editing of the Mutated COL1A1 Gene in Osteogenesis Imperfecta

2021 ◽  
Vol 10 (14) ◽  
pp. 3141
Author(s):  
Hyerin Jung ◽  
Yeri Alice Rim ◽  
Narae Park ◽  
Yoojun Nam ◽  
Ji Hyeon Ju
Keyword(s):  
Osteogenesis Imperfecta ◽  
Type I Collagen ◽  
Disease Modeling ◽  
Bone Fragility ◽  
Osteogenic Potential ◽  
Type I ◽  
Gene Correction ◽  
Col1a1 Gene ◽  
Collagen Formation

Osteogenesis imperfecta (OI) is a genetic disease characterized by bone fragility and repeated fractures. The bone fragility associated with OI is caused by a defect in collagen formation due to mutation of COL1A1 or COL1A2. Current strategies for treating OI are not curative. In this study, we generated induced pluripotent stem cells (iPSCs) from OI patient-derived blood cells harboring a mutation in the COL1A1 gene. Osteoblast (OB) differentiated from OI-iPSCs showed abnormally decreased levels of type I collagen and osteogenic differentiation ability. Gene correction of the COL1A1 gene using CRISPR/Cas9 recovered the decreased type I collagen expression in OBs differentiated from OI-iPSCs. The osteogenic potential of OI-iPSCs was also recovered by the gene correction. This study suggests a new possibility of treatment and in vitro disease modeling using patient-derived iPSCs and gene editing with CRISPR/Cas9.

scholarly journals Rapid target validation in a Cas9-inducible hiPSC derived kidney model

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yasaman Shamshirgaran ◽  
Anna Jonebring ◽  
Anna Svensson ◽  
Isabelle Leefa ◽  
Mohammad Bohlooly-Y ◽  
...  
Keyword(s):  
High Efficiency ◽  
Disease Modeling ◽  
Genetic Manipulation ◽  
Disease Model ◽  
Genetically Engineered ◽  
Model Systems ◽  
Target Validation ◽  
Direct Differentiation ◽  
Kidney Organoids

AbstractRecent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.

scholarly journals Rapid Fabrication of Microfluidic Devices for Biological Mimicking: A Survey of Materials and Biocompatibility

Micromachines ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 346
Author(s):  
Hui Ling Ma ◽  
Ana Carolina Urbaczek ◽  
Fayene Zeferino Ribeiro de Souza ◽  
Paulo Augusto Gomes Garrido Carneiro Leão ◽  
Janice Rodrigues Perussi ◽  
...  
Keyword(s):  
Shear Stress ◽  
Cell Viability ◽  
Cellular Response ◽  
Fluid Shear Stress ◽  
Umbilical Vein ◽  
Microfluidic Devices ◽  
In Vitro Assays ◽  
Stress Effect

Microfluidics is an essential technique used in the development of in vitro models for mimicking complex biological systems. The microchip with microfluidic flows offers the precise control of the microenvironment where the cells can grow and structure inside channels to resemble in vivo conditions allowing a proper cellular response investigation. Hence, this study aimed to develop low-cost, simple microchips to simulate the shear stress effect on the human umbilical vein endothelial cells (HUVEC). Differentially from other biological microfluidic devices described in the literature, we used readily available tools like heat-lamination, toner printer, laser cutter and biocompatible double-sided adhesive tapes to bind different layers of materials together, forming a designed composite with a microchannel. In addition, we screened alternative substrates, including polyester-toner, polyester-vinyl, glass, Permanox® and polystyrene to compose the microchips for optimizing cell adhesion, then enabling these microdevices when coupled to a syringe pump, the cells can withstand the fluid shear stress range from 1 to 4 dyne cm2. The cell viability was monitored by acridine orange/ethidium bromide (AO/EB) staining to detect live and dead cells. As a result, our fabrication processes were cost-effective and straightforward. The materials investigated in the assembling of the microchips exhibited good cell viability and biocompatibility, providing a dynamic microenvironment for cell proliferation. Therefore, we suggest that these microchips could be available everywhere, allowing in vitro assays for daily laboratory experiments and further developing the organ-on-a-chip concept.

scholarly journals Silencing of Vangl2 attenuates the inflammation promoted by Wnt5a via MAPK and NF-κB pathway in chondrocytes

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Ke Zhang ◽  
Zhuoying Li ◽  
Yunyang Lu ◽  
Linyi Xiang ◽  
Jiadong Sun ◽  
...  
Keyword(s):  
Western Blotting ◽  
Planar Cell Polarity ◽  
Type Ii Collagen ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Inflammatory Microenvironment ◽  
Functional Roles ◽  
Protein Levels ◽  
Oa Chondrocytes

Abstract Background The Wnt planar cell polarity (PCP) pathway is implicated in osteoarthritis (OA) both in animals and in humans. Van Gogh-like 2 (Vangl2) is a key PCP protein that is required for the orientation and alignment of chondrocytes in the growth plate. However, its functional roles in OA still remain undefined. Here, we explored the effects of Vangl2 on OA chondrocyte in vitro and further elucidated the molecular mechanism of silencing Vangl2 in Wnt5a-overexpressing OA chondrocytes. Methods Chondrocytes were treated with IL-1β (10 ng/mL) to simulate the inflammatory microenvironment of OA. The expression levels of Vangl2, Wnt5a, MMPs, and related proinflammatory cytokines were measured by RT-qPCR. Small interfering RNA (siRNA) of Vangl2 and the plasmid targeting Wnt5a were constructed and transfected into ATDC5 cells. Then, the functional roles of silencing Vangl2 in the OA chondrocytes were investigated by Western blotting, RT-qPCR, and immunocytochemistry (ICC). Transfected OA chondrocytes were subjected to Western blotting to analyze the relationship between Vangl2 and related signaling pathways. Results IL-1β induced the production of Vangl2, Wnt5a, and MMPs in a time-dependent manner and the significantly increased expression of Vangl2. Vangl2 silencing effectively suppressed the expression of MMP3, MMP9, MMP13, and IL-6 at both gene and protein levels and upregulated the expression of type II collagen and aggrecan. Moreover, knockdown of Vangl2 inhibited the phosphorylation of MAPK signaling molecules (P38, ERK, and JNK) and P65 in Wnt5a-overexpressing OA chondrocytes. Conclusions For the first time, we demonstrate that Vangl2 is involved in the OA process. Vangl2 silencing can notably alleviate OA progression in vitro by inhibiting the expression of MMPs and increasing the formation of the cartilage matrix and can inhibit the proinflammatory effects of Wnt5a via MAPK and NF-κB pathway. This study provides new insight into the mechanism of cartilage inflammation.

scholarly journals The CAD risk locus 9p21 increases the risk of vascular calcification in an iPSC-derived VSMC model

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anja Trillhaase ◽  
Beatrice Schmidt ◽  
Marlon Märtens ◽  
Undine Haferkamp ◽  
Jeanette Erdmann ◽  
...  
Keyword(s):  
Coronary Artery ◽  
Vascular Calcification ◽  
Alizarin Red ◽  
Vitro Assay ◽  
Human Ipscs ◽  
Increased Risk ◽  
Risk Locus ◽  
9P21 Locus ◽  
Cad Risk

Abstract Background Coronary artery disease (CAD) is the leading cause of death worldwide. Chromosome locus 9p21 was the first to be associated with increased risk of CAD and coronary artery calcification (CAC). Vascular calcification increases the risk for CAD. Vascular smooth muscle cells (VSMCs) are one of the major cell types involved in the development of vascular calcification. Methods So far, mainly animal models or primary SMCs have been used to model human vascular calcification. In this study, a human in vitro assay using iPSC-derived VSMCs was developed to examine vascular calcification. Human iPSCs were derived from a healthy non-risk (NR) and risk (R) donor carrying SNPs in the 9p21 locus. Additionally, 9p21 locus knockouts of each donor iPSC line (NR and R) were used. Following differentiation, the iPSC-derived VSMCs were characterized based on cell type, proliferation, and migration rate, along with calcium phosphate (CaP) deposits. CaP deposits were confirmed using Calcein and Alizarin Red S staining and then quantified. Results The data demonstrated significantly more proliferation, migration, and CaP deposition in VSMCs derived from the R and both KO iPSC lines than in those derived from the NR line. Molecular analyses confirmed upregulation of calcification markers. These results are consistent with recent data demonstrating increased calcification when the 9p21 murine ortholog is knocked-out. Conclusion Therefore, in conclusion, genetic variation or deletion of the CAD risk locus leads to an increased risk of vascular calcification. This in vitro human iPSC model of calcification could be used to develop new drug screening strategies to combat CAC.

scholarly journals Kindlin-2 mediates mechanotransduction in bone by regulating expression of Sclerostin in osteocytes

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Lei Qin ◽  
Xuekun Fu ◽  
Jing Ma ◽  
Manxia Lin ◽  
Peijun Zhang ◽  
...  
Keyword(s):  
Bone Loss ◽  
Mechanical Stimulation ◽  
Fluid Shear Stress ◽  
Weight Bearing ◽  
Underlying Mechanism ◽  
Long Bones ◽  
Cell Orientation ◽  
Cytoskeleton Organization

AbstractOsteocytes act as mechanosensors in bone; however, the underlying mechanism remains poorly understood. Here we report that deleting Kindlin-2 in osteocytes causes severe osteopenia and mechanical property defects in weight-bearing long bones, but not in non-weight-bearing calvariae. Kindlin-2 loss in osteocytes impairs skeletal responses to mechanical stimulation in long bones. Control and cKO mice display similar bone loss induced by unloading. However, unlike control mice, cKO mice fail to restore lost bone after reloading. Osteocyte Kindlin-2 deletion impairs focal adhesion (FA) formation, cytoskeleton organization and cell orientation in vitro and in bone. Fluid shear stress dose-dependently increases Kindlin-2 expression and decreases that of Sclerostin by downregulating Smad2/3 in osteocytes; this latter response is abolished by Kindlin-2 ablation. Kindlin-2-deficient osteocytes express abundant Sclerostin, contributing to bone loss in cKO mice. Collectively, we demonstrate an indispensable novel role of Kindlin-2 in maintaining skeletal responses to mechanical stimulation by inhibiting Sclerostin expression during osteocyte mechanotransduction.

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