The application of iPSC-derived kidney organoids and genome editing in kidney disease modeling

Induced pluripotent stem cells (iPSCs) are somatic cells that have been transcriptionally reprogrammed to an embryonic stem cell (ESC)-like state. iPSCs are a renewable source of diverse somatic cell types and tissues matching the original patient, including nephron-like kidney organoids. iPSCs have been derived representing several kidney disorders, such as ADPKD, ARPKD, Alport syndrome, and lupus nephritis, with the goals of generating replacement tissue and ‘disease in a dish’ laboratory models. Cellular defects in iPSCs and derived kidney organoids provide functional, personalized biomarkers, which can be correlated with genetic and clinical information. In proof of principle, disease-specific phenotypes have been described in iPSCs and ESCs with mutations linked to polycystic kidney disease or focal segmental glomerulosclerosis. In addition, these cells can be used to model nephrotoxic chemical injury. Recent advances in directed differentiation and CRISPR genome editing enable more specific iPSC models and present new possibilities for diagnostics, disease modeling, therapeutic screens, and tissue regeneration using human cells. This review outlines growth opportunities and design strategies for this rapidly expanding and evolving field.

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

Scientific Reports ◽

10.1038/s41598-021-95986-5 ◽

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.

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Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs

Scientific Reports ◽

10.1038/s41598-021-89312-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Pan P. Li ◽

Russell L. Margolis

Keyword(s):

Genome Editing ◽

Disease Modeling ◽

Neurodegenerative Disorder ◽

Cag Repeat ◽

Spinocerebellar Ataxia Type ◽

End Joining ◽

Human Ipscs ◽

Non Homologous End Joining ◽

Transient Overexpression ◽

Donor Template

AbstractCas9 nucleases permit rapid and efficient generation of gene-edited cell lines. However, in typical protocols, mutations are intentionally introduced into the donor template to avoid the cleavage of donor template or re-cleavage of the successfully edited allele, compromising the fidelity of the isogenic lines generated. In addition, the double-stranded breaks (DSBs) used for editing can introduce undesirable “on-target” indels within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address these problems, we present an optimized protocol for precise genome editing in human iPSCs that employs (1) single guided Cas9 nickase to generate single-stranded breaks (SSBs), (2) transient overexpression of BCL-XL to enhance survival post electroporation, and (3) the PiggyBac transposon system for seamless removal of dual selection markers. We have used this method to modify the length of the CAG repeat contained in exon 7 of PPP2R2B. When longer than 43 triplets, this repeat causes the neurodegenerative disorder spinocerebellar ataxia type 12 (SCA12); our goal was to seamlessly introduce the SCA12 mutation into a human control iPSC line. With our protocol, ~ 15% of iPSC clones selected had the desired gene editing without “on target” indels or off-target changes, and without the deliberate introduction of mutations via the donor template. This method will allow for the precise and efficient editing of human iPSCs for disease modeling and other purposes.

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TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery

Acta Naturae ◽

10.32607/20758251-2014-6-3-19-40 ◽

2014 ◽

Vol 6 (3) ◽

pp. 19-40 ◽

Cited By ~ 94

Author(s):

A. A. Nemudryi ◽

K. R. Valetdinova ◽

S. P. Medvedev ◽

S. M. Zakian

Keyword(s):

Genome Editing ◽

Dna Sequences ◽

Genome Engineering ◽

Disease Modeling ◽

Regulation Of Gene Expression ◽

Hereditary Disease ◽

Transcription Activator ◽

Wide Range ◽

High Standards ◽

Phenotype Formation

Precise studies of plant, animal and human genomes enable remarkable opportunities of obtained data application in biotechnology and medicine. However, knowing nucleotide sequences isnt enough for understanding of particular genomic elements functional relationship and their role in phenotype formation and disease pathogenesis. In post-genomic era methods allowing genomic DNA sequences manipulation, visualization and regulation of gene expression are rapidly evolving. Though, there are few methods, that meet high standards of efficiency, safety and accessibility for a wide range of researchers. In 2011 and 2013 novel methods of genome editing appeared - this are TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems. Although TALEN and CRISPR/Cas9 appeared recently, these systems have proved to be effective and reliable tools for genome engineering. Here we generally review application of these systems for genome editing in conventional model objects of current biology, functional genome screening, cell-based human hereditary disease modeling, epigenome studies and visualization of cellular processes. Additionally, we review general strategies for designing TALEN and CRISPR/Cas9 and analyzing their activity. We also discuss some obstacles researcher can face using these genome editing tools.

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Stem Cells Applications in Therapeutics and Site-Specific Genome Editing Through CRISPR Cas9 System

Journal of Stem Cell Research and Transplantation ◽

10.26420/jstemcellrestransplant.2021.1033 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Javed M ◽

◽

Khan A ◽

Mukheed M ◽

◽

...

Keyword(s):

Stem Cells ◽

Genome Editing ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Cell Types ◽

Multipotent Stem Cells ◽

Site Specific ◽

Regenerative Medicines ◽

Induced Pluripotent ◽

Adult Cells

Stem cells ae immature cells that have ability to differentiate into all specific and mature cells in body. The two main characteristics of stem cells are selfrenewable and ability to differentiate into all mature, functional and adult cells types. There are the two major classes a) pluripotent stem cells which have potential to differentiate in all adult cell and b) multipotent stem cells which have capacity to differentiate into many adult cells but not in all cell types. Due to the self-renewable ability stem cells are use in therapeutics, tissue regeneration, disease modeling and regenerative medicines and to treat cardiovascular diseases, neural disorders such as Parkinson’s disease and most importantly to treat carcinomas. The human induced pluripotent stem cells provide a great platform to study and treatment of human diseases because these are able to differentiate into many functional and specialized adult cells of body. The genome editing tools such as CRISPR Cas9 system and TALENs are used to generate multiple DNA variants in hPSCs by inducing site specific mutations, frame shift mutation and deletion. In present days CRISPR Cas9 is more efficient and frequent method for genome editing which is derived from bacterial cell.

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Genome editing in large animals: current status and future prospects

National Science Review ◽

10.1093/nsr/nwz013 ◽

2019 ◽

Vol 6 (3) ◽

pp. 402-420 ◽

Cited By ~ 7

Author(s):

Jianguo Zhao ◽

Liangxue Lai ◽

Weizhi Ji ◽

Qi Zhou

Keyword(s):

Regenerative Medicine ◽

Genome Editing ◽

Biomedical Research ◽

Human Disease ◽

Gene Editing ◽

Disease Modeling ◽

Current Status ◽

Future Prospects ◽

Recent Advances ◽

Large Animals

AbstractLarge animals (non-human primates, livestock and dogs) are playing important roles in biomedical research, and large livestock animals serve as important sources of meat and milk. The recently developed programmable DNA nucleases have revolutionized the generation of gene-modified large animals that are used for biological and biomedical research. In this review, we briefly introduce the recent advances in nuclease-meditated gene editing tools, and we outline these editing tools’ applications in human disease modeling, regenerative medicine and agriculture. Additionally, we provide perspectives regarding the challenges and prospects of the new genome editing technology.

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Stem cell-based treatment of kidney diseases

Experimental Biology and Medicine ◽

10.1177/1535370220915901 ◽

2020 ◽

Vol 245 (10) ◽

pp. 902-910

Author(s):

Binbin Pan ◽

Guoping Fan

Keyword(s):

Chronic Kidney Disease ◽

Stem Cells ◽

Acute Kidney Injury ◽

Stem Cell ◽

Kidney Disease ◽

Cell Therapy ◽

Kidney Diseases ◽

Kidney Injury ◽

Great Promise ◽

Kidney Organoids

Kidney dysfunction, including chronic kidney disease and acute kidney injury, is a globally prevalent health problem. However, treatment regimens are still lacking, especially for conditions involving kidney fibrosis. Stem cells hold great promise in the treatment of chronic kidney disease and acute kidney injury, but success has been hampered by insufficient incorporation of the stem cells in the injured kidney. Thus, new approaches for the restoration of kidney function after acute or chronic injury have been explored. Recently, kidney organoids have emerged as a useful tool in the treatment of kidney diseases. In this review, we discuss the mechanisms and approaches of cell therapy in acute kidney injury and chronic kidney disease, including diabetic kidney disease and lupus nephritis. We also summarize the potential applications of kidney organoids in the treatment of kidney diseases. Impact statement Stem cells hold great promise in regenerative medicine. Pluripotent stem cells have been differentiated into kidney organoids to understand human kidney development and to dissect renal disease mechanisms. Meanwhile, recent studies have explored the treatment of kidney diseases using a variety of cells, including mesenchymal stem cells and renal derivatives. This mini-review discusses the diverse mechanisms underlying current renal disease treatment via stem cell therapy. We postulate that clinical applications of stem cell therapy for kidney diseases can be readily achieved in the near future.

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