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Edited Human Kidney Organoids Reveal Mechanisms of Disease in Podocyte Development

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Edited Human Kidney Organoids Reveal Mechanisms of Disease in Podocyte Development EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS Gene-Edited Human Kidney Organoids Reveal Mechanisms of Disease in Podocyte Development a,b,c,d* e,f* g* h YONG KYUN KIM, IDO REFAELI, CRAIG R. BROOKS, PEIFENG JING, a,b,c,d e,f a,b,c,d h RAMILA E. GULIEVA, MICHAEL R. HUGHES, NELLY M. CRUZ, YANNAN LIU, a,b,c,d c,i c,d,j,k a,d ANGELA J. CHURCHILL, YULIANG WANG, HONGXIA FU, JEFFREY W. PIPPIN, a Division of Nephrology, h a,d l e,f b LIH Y. L IN, STUART J. SHANKLAND, A. WAYNE VOGL, KELLY M. MCNAGNY, Kidney Research Institute, a,b,c,d cInstitute for Stem Cell and BENJAMIN S. FREEDMAN Regenerative Medicine, • • • • dDepartment of Medicine, Key Words. Pluripotent stem cells Kidney Adhesion receptors Differentiation jDivision of Hematology, Gene targeting • Developmental biology • Focal segmental glomerulosclerosis • Department of Medicine, and Nephrogenesis • Cell adhesion • Foot processes • Slit diaphragm • Podocalyxin • kDepartment of Nephrin • Podocin • Genome editing • Biophysics Bioengineering, University of Washington School of Medicine, Seattle, ABSTRACT Washington, USA; eThe Biomedical Research Centre, A critical event during kidney organogenesis is the differentiation of podocytes, specialized epi- fDepartment of Medical thelial cells that filter blood plasma to form urine. Podocytes derived from human pluripotent Genetics, and lDepartment of stem cells (hPSC-podocytes) have recently been generated in nephron-like kidney organoids, but Cellular and Physiological the developmental stage of these cells and their capacity to reveal disease mechanisms remains Sciences, University of British unclear. Here, we show that hPSC-podocytes phenocopy mammalian podocytes at the capillary Columbia, Vancouver, British loop stage (CLS), recapitulating key features of ultrastructure, gene expression, and mutant phe- Columbia, Canada; gDivision notype. hPSC-podocytes in vitro progressively establish junction-rich basal membranes (neph- 1 1 1 1 of Nephrology, Vanderbilt rin podocin ZO-1 ) and microvillus-rich apical membranes (podocalyxin ), similar to CLS University School of podocytes in vivo. Ultrastructural, biophysical, and transcriptomic analysis of podocalyxin-knock- Medicine, Nashville, out hPSCs and derived podocytes, generated using CRISPR/Cas9, reveals defects in the assembly Tennessee, USA; of microvilli and lateral spaces between developing podocytes, resulting in failed junctional migra- hDepartment of Electrical tion. These defects are phenocopied in CLS glomeruli of podocalyxin-deficient mice, which cannot Engineering and iPaul G. produce urine, thereby demonstrating that podocalyxin has a conserved and essential role in Allen School of Computer mammalian podocyte maturation. Defining the maturity of hPSC-podocytes and their capacity to Science and Engineering, reveal and recapitulate pathophysiological mechanisms establishes a powerful framework for University of Washington, studying human kidney disease and regeneration. STEM CELLS 2017;35:2366–2378 Seattle, Washington, USA *Contributed equally. SIGNIFICANCE STATEMENT Correspondence: Kelly M. Human pluripotent stem cells (hPSCs) have recently been differentiated into complex kidney orga- McNagny, Ph.D., The Biomedical noids. Generation of podocytes—the filtering cells of the kidney—in these organoids is of great Research Centre, University of British Columbia, Vancouver, interest, because podocytes are highly specialized cells that regenerate poorly in vivo and are British Columbia, Canada V6T 1Z3. challenging to culture using conventional methods. However, hPSC-podocytes differ from cultured Telephone: 1-604-822-7824; Fax: podocytes in appearance and gene expression, and it is important to determine the developmen- 1-604-822-7815; e-mail: kelly@ tal stage of hPSC-podocytes and their ability to recapitulate disease phenotypes. This study brc.ubc.ca; or Benjamin S. Freed- man, Ph.D., Division of Nephrol- addressed these questions by comparing hPSC-podocytes to developing podocytes in human and ogy, Department of Medicine, mouse kidneys. hPSC-podocytes are shown mimic developing podocytes in vivo at the capillary University of Washington School loop stage of glomerular maturation, both structurally and in disease phenotype, providing new of Medicine, 850 Republican St, insight into the genetic mechanisms of podocyte specialization. This establishes a powerful new Box 358056, Seattle, Washington tool for genetic studies of human podocyte development and disease. 98109, USA. Telephone: 1-206- 685-4653; Fax: 1-206-685-1357; e-mail: [email protected] transplant is required to sustain life. These INTRODUCTION Received February 16, 2017; treatments are of limited availability and effi- accepted for publication Humans are born with a fixed number of kid- cacy, prompting interest in new therapeutic September 4, 2017; first strategies based on the expansion of nephron published online in STEM CELLS ney subunits, called nephrons. Progressive and EXPRESS September 14, 2017. irreversible reduction in nephron number progenitor cell populations that arise during causes end-stage renal disease, affecting two kidney development [1–5], with the ultimate http://dx.doi.org/ million people worldwide, in which kidney goal of generating new kidney tissues for 10.1002/stem.2707 function fails, and either dialysis or kidney transplantation [6–9]. STEM CELLS 2017;35:2366–2378 www.StemCells.com VC AlphaMed Press 2017 Kim, Refaeli, Brooks et al. 2367 Human pluripotent stem cells, or hPSCs, are both self- male), and NPHS1-GFP 201B7 iPSCs (Kumamoto University; renewing and pluripotent, providing a renewable source of diverse female). Passages used were between 30 and 60. Kidney orga- cells and tissues for laboratory studies and regeneration [10, 11]. noid differentiation was performed as described previously hPSCs include both embryonic stem cells (ESCs) derived from [5]. hPSCs were plated at a density of 45,000 cells per well in embryos and induced pluripotent stem cells (iPSCs) reprog- mTeSR1 (STEMCELL Technologies, Vancouver, Canada, http:// rammed from adult cells. Recently, multiple groups have published www.stemcell.com) 1 10 mM Y27632 (LC Laboratories, protocols describing the generation of kidney tissues from hPSCs Woburn, MA, http://www.lclabs.com) on glass plates (LabTek) [3–5]. In these protocols, hPSCs differentiate stepwise, first into coated with 3% GelTrex (ThermoFisher Scientific) (day 23), primitive streak mesendoderm, subsequently into nephron pro- which was changed to 1.5% GelTrex in mTeSR1 (day 22), genitor cells expressing sine oculis-related 2 (SIX2), and finally into mTeSR1 (day 21), RPMI (ThermoFisher Scientific, Waltham, organoids containing segments of distal tubules, proximal tubules, MA, http://www.thermofisher.com) 1 12 mM CHIR99021 (Toc- and podocytes [3–5, 12–14]. This provides a new resource for ris, Bristol, UK, http://www.tocris.com) (day 0), RPMI 1 B27 modeling human kidney disease and regeneration. supplement (ThermoFisher Scientific) (day 1.5), and fed every The generation of hPSC-derived podocytes (hPSC-podo- 2–3 days to promote kidney organoid differentiation. Organo- cytes) in these cultures is particularly interesting because pri- ids were fixed on day 18, unless otherwise noted. To fix, an mary podocytes are highly specialized, terminally differentiated equal volume of phosphate-buffered saline (PBS) (Thermo- epithelial cells that have been challenging to study in vitro or Fisher Scientific) 1 8% paraformaldehyde (Electron Microscopy regenerate in vivo [15–17]. Mature podocytes have elaborate Sciences, Hatfield, PA, http://www.emsdiasum.com) was basal membrane extensions (foot processes), which are linked added to the media for 15 minutes, and the sample was sub- together by specialized junctions (slit diaphragms), and interdig- sequently washed three times with PBS. Kidneys (days 60– itate around glomerular capillaries to form a sieve-like filter for 120) were obtained from the Laboratory of Developmental the blood [18–20]. Failure to properly form or maintain these Biology (University of Washington) with informed consent and structures results in defective urine production, which can be approval of the institutional review board. To generate cryo- fatal [21–23]. hPSC-podocytes express several markers associ- sections, halved kidneys were fixed in PBS 1 4% paraformalde- ated with podocytes, such as WT1, podocalyxin, synaptopodin, hyde for 1 hour, incubated overnight in 30% sucrose (Sigma, and nephrin, suggesting that these cells may be useful for dis- St. Louis, MO, http://www.sigmaaldrich.com) in water, ease modeling experiments and possibly cell therapy [4, 5, 24]. mounted in Tissue-Tek (Sakura, Torrance, CA, http://www. To establish the validity of this new system and advance the sakuraus.com), and flash frozen with liquid nitrogen. For par- field, it is important to determine the developmental stage of affin sections, tissues were fixed overnight with methacarn: hPSC-podocytes and their ability to phenocopy genetic disease. 60% absolute methanol, 30% chloroform, 10% glacial acetic Microarray datasets of purified hPSC-podocytes show significant acid (Sigma), and subsequently paraffin-embedded. Paraffin overlap with published mouse and human datasets, but the top tissue sections were deparaffinized with three 2-minute genes do not cluster clearly with kidney tissues [24]. The washes in xylene, followed by 100%, 85%, and 70% ethanol, rounded, tightly clustered appearance of hPSC-podocytes also and heated in citrate buffer pH 6.0 (Sigma) in a pressure differs markedly from that of cultured
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