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A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney ...

University of Auckland

Corneal diseases such as keratoconus represent a relatively common disorder in the human population. However, treatment is restricted to corneal transplantation, which only occurs in the most advanced cases. Cell based therapies may offer an alternative approach given that the eye is amenable to such treatments and corneal diseases like keratoconus have been associated specifically with the death of corneal keratocytes. The ability to generate corneal keratocytes in vitro may enable a cell-based therapy to treat patients with keratoconus. Human induced pluripotent stem cells (hiPSCs) offer an abundant supply of cells from which any cell in the body can be derived. In the present study, hiPSCs were successfully differentiated into neural crest cells (NCCs), the embryonic precursor to keratocytes, and then cultured on cadaveric corneal tissue to promote keratocyte differentiation. The hiPSC-derived NCCs were found to migrate into the corneal stroma where they acquired a keratocyte-like morphology and an expression profile similar to corneal keratocytes in vivo. These results indicate that hiPSCs can be used to generate corneal keratocytes in vitro and lay the foundation for using these cells in cornea cell-based therapies.

Derivation of Corneal Keratocyte-Like Cells from Human Induced Pluripotent Stem Cells.

Kidney organoids made from pluripotent stem cells have the potential to revolutionize how kidney development, disease, and injury are studied. Current protocols are technically complex, suffer from poor reproducibility, and have high reagent costs that restrict scalability. To overcome some of these issues, we have established a simple, inexpensive, and robust method to grow kidney organoids in bulk from human induced pluripotent stem cells. Our organoids develop tubular structures by day 8 and show optimal tissue morphology at day 14. A comparison with fetal human kidneys suggests that day-14 organoid tissue most closely resembles late capillary loop stage nephrons. We show that deletion of HNF1B, a transcription factor linked to congenital kidney defects, interferes with tubulogenesis, validating our experimental system for studying renal developmental biology. Taken together, our protocol provides a fast, efficient, and cost-effective method for generating large quantities of human fetal kidney tissue, enabling the study of normal and aberrant kidney development.

A Simple Bioreactor-Based Method to Generate Kidney Organoids from Pluripotent Stem Cells.

Mutations in -a gene encoding the cystine transporter cystinosin-cause the rare, autosomal, recessive, lysosomal-storage disease cystinosis. Research has also implicated cystinosin in modulating the mTORC1 pathway, which serves as a core regulator of cellular metabolism, proliferation, survival, and autophagy. In its severest form, cystinosis is characterized by cystine accumulation, renal proximal tubule dysfunction, and kidney failure. Because treatment with the cystine-depleting drug cysteamine only slows disease progression, there is an urgent need for better treatments.

Use of Human Induced Pluripotent Stem Cells and Kidney Organoids To Develop a Cysteamine/mTOR Inhibition Combination Therapy for Cystinosis.

Kidney organoids represent a physiologically advanced model for studying the mechanisms of kidney development and disease. Here, we describe a simple two-step protocol for the differentiation of human pluripotent stem cells into kidney organoids. Our approach involves suspension culture that allows for rapid and cost-effective bulk production of organoids, which is well suited for large-scale assays such as drug screening. The organoids correspond to fetal human kidney tissue and may be of limited use for modeling adult kidney function. For complete details on the use and execution of this protocol, please refer to Przepiorski et al. (2018).

Article	Year
Derivation of Corneal Keratocyte-Like Cells from Human Induced Pluripotent Stem Cells. PloS one\| PubMed ID: 27792791	2016
A Simple Bioreactor-Based Method to Generate Kidney Organoids from Pluripotent Stem Cells. Stem cell reports\| PubMed ID: 30033089	2018
Use of Human Induced Pluripotent Stem Cells and Kidney Organoids To Develop a Cysteamine/mTOR Inhibition Combination Therapy for Cystinosis. Journal of the American Society of Nephrology : JASN\| PubMed ID: 32198276	2020
Protocol for Large-Scale Production of Kidney Organoids from Human Pluripotent Stem Cells. STAR protocols\| PubMed ID: 33377044	2020

Teresa M. Holm

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