Publications by authors named "Sara E Howden"

31 Publications

Generating an iPSC line (with isogenic control) from the PBMCs of an ACTA1 (p.Gly148Asp) nemaline myopathy patient.

Stem Cell Res 2021 Jul 17;54:102429. Epub 2021 Jun 17.

Murdoch Children's Research Institute, Melbourne, Victoria, Australia; Department of Pediatrics, The University of Melbourne, Victoria, Australia. Electronic address:

To produce an in vitro model of nemaline myopathy, we reprogrammed the peripheral blood mononuclear cells (PBMCs) of a patient with a heterozygous p.Gly148Asp mutation in exon 3 of the ACTA1 gene to iPSCs. Using CRISPR/Cas9 gene editing we corrected the mutation to generate an isogenic control line. Both the mutant and control show a normal karyotype, express pluripotency markers and could differentiae into the three cell states that represent embryonic germ layers (endoderm, mesoderm and neuroectoderm) and the dermomyotome (precursor of skeletal muscle). When differentiated these cell lines will be used to explore disease mechanisms and evaluate novel therapeutics.
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http://dx.doi.org/10.1016/j.scr.2021.102429DOI Listing
July 2021

In vivo survival and differentiation of Friedreich ataxia iPSC-derived sensory neurons transplanted in the adult dorsal root ganglia.

Stem Cells Transl Med 2021 Aug 18;10(8):1157-1169. Epub 2021 Mar 18.

Department of Biomedical Engineering, The University of Melbourne, Parkville, Australia.

Friedreich ataxia (FRDA) is an autosomal recessive disease characterized by degeneration of dorsal root ganglia (DRG) sensory neurons, which is due to low levels of the mitochondrial protein Frataxin. To explore cell replacement therapies as a possible approach to treat FRDA, we examined transplantation of sensory neural progenitors derived from human embryonic stem cells (hESC) and FRDA induced pluripotent stem cells (iPSC) into adult rodent DRG regions. Our data showed survival and differentiation of hESC and FRDA iPSC-derived progenitors in the DRG 2 and 8 weeks post-transplantation, respectively. Donor cells expressed neuronal markers, including sensory and glial markers, demonstrating differentiation to these lineages. These results are novel and a highly significant first step in showing the possibility of using stem cells as a cell replacement therapy to treat DRG neurodegeneration in FRDA as well as other peripheral neuropathies.
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http://dx.doi.org/10.1002/sctm.20-0334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8284774PMC
August 2021

Recessive variants impair actin remodeling and cause glomerulopathy in humans and mice.

Sci Adv 2021 Jan 1;7(1). Epub 2021 Jan 1.

Molecular Medicine Program, The Hospital for Sick Children, Toronto, ON, Canada.

Nephrotic syndrome (NS) is a leading cause of chronic kidney disease. We found recessive variants in two families with early-onset NS by exome sequencing. Overexpression of wild-type (WT) , but not cDNA constructs bearing patient variants, increased active CDC42 and promoted filopodia and podosome formation. Pharmacologic inhibition of CDC42 or its effectors, formin proteins, reduced NOS1AP-induced filopodia formation. knockdown reduced podocyte migration rate (PMR), which was rescued by overexpression of WT but not by constructs bearing patient variants. PMR in knockdown podocytes was also rescued by constitutively active or the formin Modeling a patient variant in knock-in human kidney organoids revealed malformed glomeruli with increased apoptosis. mice recapitulated the human phenotype, exhibiting proteinuria, foot process effacement, and glomerulosclerosis. These findings demonstrate that recessive variants impair CDC42/DIAPH-dependent actin remodeling, cause aberrant organoid glomerulogenesis, and lead to a glomerulopathy in humans and mice.
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http://dx.doi.org/10.1126/sciadv.abe1386DOI Listing
January 2021

Plasticity of distal nephron epithelia from human kidney organoids enables the induction of ureteric tip and stalk.

Cell Stem Cell 2021 04 29;28(4):671-684.e6. Epub 2020 Dec 29.

Murdoch Children's Research Institute, Parkville, Melbourne, 3052 VIC, Australia; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, 3052 VIC, Australia; Department of Anatomy and Neuroscience, The University of Melbourne, Melbourne, VIC, Australia. Electronic address:

During development, distinct progenitors contribute to the nephrons versus the ureteric epithelium of the kidney. Indeed, previous human pluripotent stem-cell-derived models of kidney tissue either contain nephrons or pattern specifically to the ureteric epithelium. By re-analyzing the transcriptional distinction between distal nephron and ureteric epithelium in human fetal kidney, we show here that, while existing nephron-containing kidney organoids contain distal nephron epithelium and no ureteric epithelium, this distal nephron segment alone displays significant in vitro plasticity and can adopt a ureteric epithelial tip identity when isolated and cultured in defined conditions. "Induced" ureteric epithelium cultures can be cryopreserved, serially passaged without loss of identity, and transitioned toward a collecting duct fate. Cultures harboring loss-of-function mutations in PKHD1 also recapitulate the cystic phenotype associated with autosomal recessive polycystic kidney disease.
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http://dx.doi.org/10.1016/j.stem.2020.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026527PMC
April 2021

Cellular extrusion bioprinting improves kidney organoid reproducibility and conformation.

Nat Mater 2021 02 23;20(2):260-271. Epub 2020 Nov 23.

Murdoch Children's Research Institute, Melbourne, Victoria, Australia.

Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for renal replacement. Currently, these applications are hampered by organoid variability, nephron immaturity, low throughput and limited scale. Here, we apply extrusion-based three-dimensional cellular bioprinting to deliver rapid and high-throughput generation of kidney organoids with highly reproducible cell number and viability. We demonstrate that manual organoid generation can be replaced by 6- or 96-well organoid bioprinting and evaluate the relative toxicity of aminoglycosides as a proof of concept for drug testing. In addition, three-dimensional bioprinting enables precise manipulation of biophysical properties, including organoid size, cell number and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number. This facilitates the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Hence, automated extrusion-based bioprinting for kidney organoid production delivers improvements in throughput, quality control, scale and structure, facilitating in vitro and in vivo applications of stem cell-derived human kidney tissue.
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http://dx.doi.org/10.1038/s41563-020-00853-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855371PMC
February 2021

Generating Kidney Organoids from Human Pluripotent Stem Cells Using Defined Conditions.

Methods Mol Biol 2020 ;2155:183-192

Murdoch Children's Research Institute, Parkville, VIC, Australia.

The ultimate goal of regenerative medicine is to have access to an unlimited supply of specific cell types on demand, which can be used as effective therapies for a wide range of intractable disorders. With the availability of human pluripotent stem cells (hPSCs) and greatly improved protocols for their directed differentiation into specific cell types, including kidney, this prospect could soon become a reality. We have previously described the generation of kidney organoids from hPSCs. This chapter describes our latest differentiation protocol for generating kidney tissue, which uses a cost-effective and completely defined, xeno-free medium. As with our previous protocol, these complex, multicellular three-dimensional structures are composed of all anticipated kidney cell types including nephrons segmented into the glomerulus, proximal and distal tubule as well as an extensive endothelial network, and renal interstitium. As such, kidney organoids provide useful tools for understanding human development, disease modeling, drug screening/toxicology studies and tissue engineering applications, and may facilitate the development of transplantable hPSC-derived kidney tissue for regenerative medicine purposes in the future.
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http://dx.doi.org/10.1007/978-1-0716-0655-1_15DOI Listing
March 2021

Particle-mediated delivery of frataxin plasmid to a human sensory neuronal model of Friedreich's ataxia.

Biomater Sci 2020 May 9;8(9):2398-2403. Epub 2020 Apr 9.

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, and the Department of Chemical Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia.

Increasing frataxin protein levels through gene therapy is envisaged to improve therapeutic outcomes for patients with Friedreich's ataxia (FRDA). A non-viral strategy that uses submicrometer-sized multilayered particles to deliver frataxin-encoding plasmid DNA affords up to 27 000-fold increase in frataxin gene expression within 2 days in vitro in a stem cell-derived neuronal model of FRDA.
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http://dx.doi.org/10.1039/c9bm01757gDOI Listing
May 2020

A Toolbox to Characterize Human Induced Pluripotent Stem Cell-Derived Kidney Cell Types and Organoids.

J Am Soc Nephrol 2019 10 6;30(10):1811-1823. Epub 2019 Sep 6.

Murdoch Children's Research Institute, Melbourne, Victoria, Australia.

Background: The generation of reporter lines for cell identity, lineage, and physiologic state has provided a powerful tool in advancing the dissection of mouse kidney morphogenesis at a molecular level. Although use of this approach is not an option for studying human development , its application in human induced pluripotent stem cells (iPSCs) is now feasible.

Methods: We used CRISPR/Cas9 gene editing to generate ten fluorescence reporter iPSC lines designed to identify nephron progenitors, podocytes, proximal and distal nephron, and ureteric epithelium. Directed differentiation to kidney organoids was performed according to published protocols. Using immunofluorescence and live confocal microscopy, flow cytometry, and cell sorting techniques, we investigated organoid patterning and reporter expression characteristics.

Results: Each iPSC reporter line formed well patterned kidney organoids. All reporter lines showed congruence of endogenous gene and protein expression, enabling isolation and characterization of kidney cell types of interest. We also demonstrated successful application of reporter lines for time-lapse imaging and mouse transplantation experiments.

Conclusions: We generated, validated, and applied a suite of fluorescence iPSC reporter lines for the study of morphogenesis within human kidney organoids. This fluorescent iPSC reporter toolbox enables the visualization and isolation of key populations in forming kidney organoids, facilitating a range of applications, including cellular isolation, time-lapse imaging, protocol optimization, and lineage-tracing approaches. These tools offer promise for enhancing our understanding of this model system and its correspondence with human kidney morphogenesis.
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http://dx.doi.org/10.1681/ASN.2019030303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779359PMC
October 2019

Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas.

Mol Cell 2019 06 8;74(5):936-950.e5. Epub 2019 Apr 8.

Department of Biological Chemistry, University of Michigan, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA. Electronic address:

CRISPR-Cas systems enable microbial adaptive immunity and provide eukaryotic genome editing tools. These tools employ a single effector enzyme of type II or V CRISPR to generate RNA-guided, precise genome breaks. Here we demonstrate the feasibility of using type I CRISPR-Cas to effectively introduce a spectrum of long-range chromosomal deletions with a single RNA guide in human embryonic stem cells and HAP1 cells. Type I CRISPR systems rely on the multi-subunit ribonucleoprotein (RNP) complex Cascade to identify DNA targets and on the helicase-nuclease enzyme Cas3 to degrade DNA processively. With RNP delivery of T. fusca Cascade and Cas3, we obtained 13%-60% editing efficiency. Long-range PCR-based and high-throughput-sequencing-based lesion analyses reveal that a variety of deletions, ranging from a few hundred base pairs to 100 kilobases, are created upstream of the target site. These results highlight the potential utility of type I CRISPR-Cas for long-range genome manipulations and deletion screens in eukaryotes.
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http://dx.doi.org/10.1016/j.molcel.2019.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6555677PMC
June 2019

Reporter-based fate mapping in human kidney organoids confirms nephron lineage relationships and reveals synchronous nephron formation.

EMBO Rep 2019 04 11;20(4). Epub 2019 Mar 11.

Murdoch Children's Research Institute, Parkville, Vic., Australia

Nephron formation continues throughout kidney morphogenesis in both mice and humans. Lineage tracing studies in mice identified a self-renewing Six2-expressing nephron progenitor population able to give rise to the full complement of nephrons throughout kidney morphogenesis. To investigate the origin of nephrons within human pluripotent stem cell-derived kidney organoids, we performed a similar fate-mapping analysis of the SIX2-expressing lineage in induced pluripotent stem cell (iPSC)-derived kidney organoids to explore the feasibility of investigating lineage relationships in differentiating iPSCs Using CRISPR/Cas9 gene-edited lineage reporter lines, we show that SIX2-expressing cells give rise to nephron epithelial cell types but not to presumptive ureteric epithelium. The use of an inducible (CreERT2) line revealed a declining capacity for SIX2 cells to contribute to nephron formation over time, but retention of nephron-forming capacity if provided an exogenous WNT signal. Hence, while human iPSC-derived kidney tissue appears to maintain lineage relationships previously identified in developing mouse kidney, unlike the developing kidney , kidney organoids lack a nephron progenitor niche capable of both self-renewal and ongoing nephrogenesis.
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http://dx.doi.org/10.15252/embr.201847483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446205PMC
April 2019

Direct reprogramming to human nephron progenitor-like cells using inducible piggyBac transposon expression of SNAI2-EYA1-SIX1.

Kidney Int 2019 05 28;95(5):1153-1166. Epub 2019 Feb 28.

Murdoch Children's Research Institute, Parkville, Melbourne, Australia; Division of Genomics of Development and Disease, Institute for Molecular Biosciences, The University of Queensland, Brisbane, Australia; Department of Pediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Australia. Electronic address:

All nephrons in the mammalian kidney arise from a transient nephron progenitor population that is lost close to the time of birth. The generation of new nephron progenitors and their maintenance in culture are central to the success of kidney regenerative strategies. Using a lentiviral screening approach, we previously generated a human induced nephron progenitor-like state in vitro using a pool of six transcription factors. Here, we sought to develop a more efficient approach for direct reprogramming of human cells that could be applied in vivo. PiggyBac transposons are a non-viral integrating gene delivery system that is suitable for in vivo use and allows for simultaneous delivery of multiple genes. Using an inducible piggyBac transposon system, we optimized a protocol for the direct reprogramming of HK2 cells to induced nephron progenitor-like cells with expression of only 3 transcription factors (SNAI2, EYA1, and SIX1). Culture in conditions supportive of the nephron progenitor state further increased the expression of nephron progenitor genes. The refined protocol was then applied to primary human renal epithelial cells, which integrated into developing nephron structures in vitro and in vivo. Such inducible reprogramming to nephron progenitor-like cells could facilitate direct cellular reprogramming for kidney regeneration.
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http://dx.doi.org/10.1016/j.kint.2018.11.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478531PMC
May 2019

Generating Kidney from Stem Cells.

Annu Rev Physiol 2019 02;81:335-357

Murdoch Children's Research Institute, Parkville, Victoria 3052, Australia; email:

Human kidney tissue can now be generated via the directed differentiation of human pluripotent stem cells. This advance is anticipated to facilitate the modeling of human kidney diseases, provide platforms for nephrotoxicity screening, enable cellular therapy, and potentially generate tissue for renal replacement. All such applications will rely upon the accuracy and reliability of the model and the capacity for stem cell-derived kidney tissue to recapitulate both normal and diseased states. In this review, we discuss the models available, how well they recapitulate the human kidney, and how far we are from application of these cells for use in cellular therapies.
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http://dx.doi.org/10.1146/annurev-physiol-020518-114331DOI Listing
February 2019

Evaluation of variability in human kidney organoids.

Nat Methods 2019 01 20;16(1):79-87. Epub 2018 Dec 20.

Murdoch Children's Research Institute, Melbourne, Victoria, Australia.

The utility of human pluripotent stem cell-derived kidney organoids relies implicitly on the robustness and transferability of the protocol. Here we analyze the sources of transcriptional variation in a specific kidney organoid protocol. Although individual organoids within a differentiation batch showed strong transcriptional correlation, we noted significant variation between experimental batches, particularly in genes associated with temporal maturation. Single-cell profiling revealed shifts in nephron patterning and proportions of component cells. Distinct induced pluripotent stem cell clones showed congruent transcriptional programs, with interexperimental and interclonal variation also strongly associated with nephron patterning. Epithelial cells isolated from organoids aligned with total organoids at the same day of differentiation, again implicating relative maturation as a confounder. This understanding of experimental variation facilitated an optimized analysis of organoid-based disease modeling, thereby increasing the utility of kidney organoids for personalized medicine and functional genomics.
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http://dx.doi.org/10.1038/s41592-018-0253-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6634992PMC
January 2019

Functional Assessment of Patient-Derived Retinal Pigment Epithelial Cells Edited by CRISPR/Cas9.

Int J Mol Sci 2018 Dec 19;19(12). Epub 2018 Dec 19.

Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA.

Retinitis pigmentosa is the most common form of inherited blindness and can be caused by a multitude of different genetic mutations that lead to similar phenotypes. Specifically, mutations in ubiquitously expressed splicing factor proteins are known to cause an autosomal dominant form of the disease, but the retina-specific pathology of these mutations is not well understood. Fibroblasts from a patient with splicing factor retinitis pigmentosa caused by a missense mutation in the splicing factor were used to produce three diseased and three CRISPR/Cas9-corrected induced pluripotent stem cell (iPSC) clones. We differentiated each of these clones into retinal pigment epithelial (RPE) cells via directed differentiation and analyzed the RPE cells in terms of gene and protein expression, apicobasal polarity, and phagocytic ability. We demonstrate that RPE cells can be produced from patient-derived and corrected cells and they exhibit morphology and functionality similar but not identical to wild-type RPE cells in vitro. Functionally, the RPE cells were able to establish apicobasal polarity and phagocytose photoreceptor outer segments at the same capacity as wild-type cells. These data suggest that patient-derived iPSCs, both diseased and corrected, are able to differentiate into RPE cells with a near normal phenotype and without differences in phagocytosis, a result that differs from previous mouse models. These RPE cells can now be studied to establish a disease-in-a-dish system relevant to retinitis pigmentosa.
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http://dx.doi.org/10.3390/ijms19124127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321630PMC
December 2018

Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines.

Development 2019 01 9;146(1). Epub 2019 Jan 9.

Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA

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.
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http://dx.doi.org/10.1242/dev.171686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340149PMC
January 2019

3D organoid-derived human glomeruli for personalised podocyte disease modelling and drug screening.

Nat Commun 2018 12 4;9(1):5167. Epub 2018 Dec 4.

Murdoch Children's Research Institute, Flemington Rd, Melbourne, 3052, VIC, Australia.

The podocytes within the glomeruli of the kidney maintain the filtration barrier by forming interdigitating foot processes with intervening slit diaphragms, disruption in which results in proteinuria. Studies into human podocytopathies to date have employed primary or immortalised podocyte cell lines cultured in 2D. Here we compare 3D human glomeruli sieved from induced pluripotent stem cell-derived kidney organoids with conditionally immortalised human podocyte cell lines, revealing improved podocyte-specific gene expression, maintenance in vitro of polarised protein localisation and an improved glomerular basement membrane matrisome compared to 2D cultures. Organoid-derived glomeruli retain marker expression in culture for 96 h, proving amenable to toxicity screening. In addition, 3D organoid glomeruli from a congenital nephrotic syndrome patient with compound heterozygous NPHS1 mutations reveal reduced protein levels of both NEPHRIN and PODOCIN. Hence, human iPSC-derived organoid glomeruli represent an accessible approach to the in vitro modelling of human podocytopathies and screening for podocyte toxicity.
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http://dx.doi.org/10.1038/s41467-018-07594-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279764PMC
December 2018

Patient-iPSC-Derived Kidney Organoids Show Functional Validation of a Ciliopathic Renal Phenotype and Reveal Underlying Pathogenetic Mechanisms.

Am J Hum Genet 2018 05 26;102(5):816-831. Epub 2018 Apr 26.

Kidney Development, Disease, and Regeneration Group, Murdoch Children's Research Institute, Parkville, VIC 3052, Australia; Department of Paediatrics, University of Melbourne, Parkville, VIC 3052, Australia. Electronic address:

Despite the increasing diagnostic rate of genomic sequencing, the genetic basis of more than 50% of heritable kidney disease remains unresolved. Kidney organoids differentiated from induced pluripotent stem cells (iPSCs) of individuals affected by inherited renal disease represent a potential, but unvalidated, platform for the functional validation of novel gene variants and investigation of underlying pathogenetic mechanisms. In this study, trio whole-exome sequencing of a prospectively identified nephronophthisis (NPHP) proband and her parents identified compound-heterozygous variants in IFT140, a gene previously associated with NPHP-related ciliopathies. IFT140 plays a key role in retrograde intraflagellar transport, but the precise downstream cellular mechanisms responsible for disease presentation remain unknown. A one-step reprogramming and gene-editing protocol was used to derive both uncorrected proband iPSCs and isogenic gene-corrected iPSCs, which were differentiated to kidney organoids. Proband organoid tubules demonstrated shortened, club-shaped primary cilia, whereas gene correction rescued this phenotype. Differential expression analysis of epithelial cells isolated from organoids suggested downregulation of genes associated with apicobasal polarity, cell-cell junctions, and dynein motor assembly in proband epithelial cells. Matrigel cyst cultures confirmed a polarization defect in proband versus gene-corrected renal epithelium. As such, this study represents a "proof of concept" for using proband-derived iPSCs to model renal disease and illustrates dysfunctional cellular pathways beyond the primary cilium in the setting of IFT140 mutations, which are established for other NPHP genotypes.
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http://dx.doi.org/10.1016/j.ajhg.2018.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986969PMC
May 2018

Simultaneous reprogramming and gene editing of human fibroblasts.

Nat Protoc 2018 05 5;13(5):875-898. Epub 2018 Apr 5.

Murdoch Children's Research Institute, The Royal Children's Hospital, Parkville, Victoria, Australia.

The utility of human induced pluripotent stem cells (iPSCs) is enhanced by an ability to precisely modify a chosen locus with minimal impact on the remaining genome. However, the derivation of gene-edited iPSCs typically involves multiple steps requiring lengthy culture periods and several clonal events. Here, we describe a one-step protocol for reliable generation of clonally derived gene-edited iPSC lines from human fibroblasts in the absence of drug selection or FACS enrichment. Using enhanced episomal-based reprogramming and CRISPR/Cas9 systems, gene-edited and passage-matched unmodified iPSC lines are obtained following a single electroporation of human fibroblasts. To minimize unwanted mutations within the target locus, we use a Cas9 variant that is associated with decreased nonhomologous end-joining (NHEJ) activity. This protocol outlines in detail how this streamlined approach can be used for both monoallelic and biallelic introduction of specific base changes or transgene cassettes in a manner that is efficient, rapid (∼6-8 weeks), and cost-effective.
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http://dx.doi.org/10.1038/nprot.2018.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997775PMC
May 2018

Renal Subcapsular Transplantation of PSC-Derived Kidney Organoids Induces Neo-vasculogenesis and Significant Glomerular and Tubular Maturation In Vivo.

Stem Cell Reports 2018 03 1;10(3):751-765. Epub 2018 Mar 1.

Department of Internal Medicine - Nephrology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, the Netherlands; Einthoven Laboratory of Vascular and Regenerative Medicine, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, the Netherlands.

Human pluripotent stem cell (hPSC)-derived kidney organoids may facilitate disease modeling and the generation of tissue for renal replacement. Long-term application, however, will require transferability between hPSC lines and significant improvements in organ maturation. A key question is whether time or a patent vasculature is required for ongoing morphogenesis. Here, we show that hPSC-derived kidney organoids, derived in fully defined medium conditions and in the absence of any exogenous vascular endothelial growth factor, develop host-derived vascularization. In vivo imaging of organoids under the kidney capsule confirms functional glomerular perfusion as well as connection to pre-existing vascular networks in the organoids. Wide-field electron microscopy demonstrates that transplantation results in formation of a glomerular basement membrane, fenestrated endothelial cells, and podocyte foot processes. Furthermore, compared with non-transplanted organoids, polarization and segmental specialization of tubular epithelium are observed. These data demonstrate that functional vascularization is required for progressive morphogenesis of human kidney organoids.
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http://dx.doi.org/10.1016/j.stemcr.2018.01.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5918682PMC
March 2018

GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives.

Stem Cell Reports 2016 09 1;7(3):518-526. Epub 2016 Sep 1.

Murdoch Childrens Research Institute, Flemington Road, Parkville, VIC 3052, Australia; Department of Paediatrics, University of Melbourne, Parkville, VIC 3050, Australia; Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3800, Australia. Electronic address:

The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that β-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives.
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http://dx.doi.org/10.1016/j.stemcr.2016.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5032031PMC
September 2016

A Cas9 Variant for Efficient Generation of Indel-Free Knockin or Gene-Corrected Human Pluripotent Stem Cells.

Stem Cell Reports 2016 09 4;7(3):508-517. Epub 2016 Aug 4.

Murdoch Childrens Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, VIC 3052, Australia; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC 3052, Australia; Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC 3800, Australia.

While Cas9 nucleases permit rapid and efficient generation of gene-edited cell lines, the CRISPR-Cas9 system can introduce undesirable "on-target" mutations within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address this, we fused the Streptococcus pyogenes Cas9 (SpCas9) nuclease to a peptide derived from the human Geminin protein (SpCas9-Gem) to facilitate its degradation during the G1 phase of the cell cycle, when DNA repair by NHEJ predominates. We also use mRNA transfection to facilitate low and transient expression of modified and unmodified versions of Cas9. Although the frequency of homologous recombination was similar for SpCas9-Gem and SpCas9, we observed a marked reduction in the capacity for SpCas9-Gem to induce NHEJ-mediated indels at the target locus. Moreover, in contrast to native SpCas9, we demonstrate that transient SpCas9-Gem expression enables reliable generation of both knockin reporter cell lines and genetically repaired patient-specific induced pluripotent stem cell lines free of unwanted mutations at the targeted locus.
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http://dx.doi.org/10.1016/j.stemcr.2016.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5031952PMC
September 2016

ALPK3-deficient cardiomyocytes generated from patient-derived induced pluripotent stem cells and mutant human embryonic stem cells display abnormal calcium handling and establish that ALPK3 deficiency underlies familial cardiomyopathy.

Eur Heart J 2016 Sep 22;37(33):2586-90. Epub 2016 Apr 22.

Bruce Lefroy Centre for Genetic Health Research, Murdoch Childrens Research Institute, Flemington Road, Parkville 3052, Victoria, Australia Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville 3052, Victoria, Australia

Aims: We identified a novel homozygous truncating mutation in the gene encoding alpha kinase 3 (ALPK3) in a family presenting with paediatric cardiomyopathy. A recent study identified biallelic truncating mutations of ALPK3 in three unrelated families; therefore, there is strong genetic evidence that ALPK3 mutation causes cardiomyopathy. This study aimed to clarify the mutation mechanism and investigate the molecular and cellular pathogenesis underlying ALPK3-mediated cardiomyopathy.

Methods And Results: We performed detailed clinical and genetic analyses of a consanguineous family, identifying a new ALPK3 mutation (c.3792G>A, p.W1264X) which undergoes nonsense-mediated decay in ex vivo and in vivo tissues. Ultra-structural analysis of cardiomyocytes derived from patient-specific and human ESC-derived stem cell lines lacking ALPK3 revealed disordered sarcomeres and intercalated discs. Multi-electrode array analysis and calcium imaging demonstrated an extended field potential duration and abnormal calcium handling in mutant contractile cultures.

Conclusions: This study validates the genetic evidence, suggesting that mutations in ALPK3 can cause familial cardiomyopathy and demonstrates loss of function as the underlying genetic mechanism. We show that ALPK3-deficient cardiomyocytes derived from pluripotent stem cell models recapitulate the ultrastructural and electrophysiological defects observed in vivo. Analysis of differentiated contractile cultures identified abnormal calcium handling as a potential feature of cardiomyocytes lacking ALPK3, providing functional insights into the molecular mechanisms underlying ALPK3-mediated cardiomyopathy.
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http://dx.doi.org/10.1093/eurheartj/ehw160DOI Listing
September 2016

Simultaneous Reprogramming and Gene Correction of Patient Fibroblasts.

Stem Cell Reports 2015 Dec 12;5(6):1109-1118. Epub 2015 Nov 12.

Morgridge Institute for Research, 330 North Orchard Street, Madison, WI 53715, USA; Cell and Regenerative Biology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53707-7365, USA; Department of Molecular, Cellular, and Developmental Biology, University of California Santa Barbara, Santa Barbara, CA 93106, USA.

The derivation of genetically modified induced pluripotent stem (iPS) cells typically involves multiple steps, requiring lengthy cell culture periods, drug selection, and several clonal events. We report the generation of gene-targeted iPS cell lines following a single electroporation of patient-specific fibroblasts using episomal-based reprogramming vectors and the Cas9/CRISPR system. Simultaneous reprogramming and gene targeting was tested and achieved in two independent fibroblast lines with targeting efficiencies of up to 8% of the total iPS cell population. We have successfully targeted the DNMT3B and OCT4 genes with a fluorescent reporter and corrected the disease-causing mutation in both patient fibroblast lines: one derived from an adult with retinitis pigmentosa, the other from an infant with severe combined immunodeficiency. This procedure allows the generation of gene-targeted iPS cell lines with only a single clonal event in as little as 2 weeks and without the need for drug selection, thereby facilitating "seamless" single base-pair changes.
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http://dx.doi.org/10.1016/j.stemcr.2015.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4682122PMC
December 2015

Loss of MITF expression during human embryonic stem cell differentiation disrupts retinal pigment epithelium development and optic vesicle cell proliferation.

Hum Mol Genet 2014 Dec 9;23(23):6332-44. Epub 2014 Jul 9.

Waisman Center, McPherson Eye Research Institute and Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI 53705, USA,

Microphthalmia-associated transcription factor (MITF) is a master regulator of pigmented cell survival and differentiation with direct transcriptional links to cell cycle, apoptosis and pigmentation. In mouse, Mitf is expressed early and uniformly in optic vesicle (OV) cells as they evaginate from the developing neural tube, and null Mitf mutations result in microphthalmia and pigmentation defects. However, homozygous mutations in MITF have not been identified in humans; therefore, little is known about its role in human retinogenesis. We used a human embryonic stem cell (hESC) model that recapitulates numerous aspects of retinal development, including OV specification and formation of retinal pigment epithelium (RPE) and neural retina progenitor cells (NRPCs), to investigate the earliest roles of MITF. During hESC differentiation toward a retinal lineage, a subset of MITF isoforms was expressed in a sequence and tissue distribution similar to that observed in mice. In addition, we found that promoters for the MITF-A, -D and -H isoforms were directly targeted by Visual Systems Homeobox 2 (VSX2), a transcription factor involved in patterning the OV toward a NRPC fate. We then manipulated MITF RNA and protein levels at early developmental stages and observed decreased expression of eye field transcription factors, reduced early OV cell proliferation and disrupted RPE maturation. This work provides a foundation for investigating MITF and other highly complex, multi-purposed transcription factors in a dynamic human developmental model system.
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http://dx.doi.org/10.1093/hmg/ddu351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4222367PMC
December 2014

Gene targeting of human pluripotent stem cells by homologous recombination.

Methods Mol Biol 2014 ;1114:37-55

Department of Cell and Regenerative Biology, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.

The ability of human embryonic stem cells and induced pluripotent stem cells to differentiate into all adult cell types greatly facilitates the study of human development, disease pathogenesis, and the generation of screening systems to identify novel therapeutic agents. Autologous cell therapies based on patient-derived induced pluripotent stem cells also hold great promise for the treatment and correction of many inherited and acquired diseases. The full potential of human pluripotent stem cells can be unleashed by genetically modifying a chosen locus with minimal impact on the remaining genome, which can be achieved by targeting genes by homologous recombination. This chapter will describe a protocol for gene modification of pluripotent stem cells by homologous recombination and several methods for the screening and identification of successfully modified clones.
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http://dx.doi.org/10.1007/978-1-62703-761-7_4DOI Listing
October 2014

Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis.

Proc Natl Acad Sci U S A 2013 Sep 12;110(39):15644-9. Epub 2013 Aug 12.

Morgridge Institute for Research, Madison, WI 53715.

Genome engineering in human pluripotent stem cells (hPSCs) holds great promise for biomedical research and regenerative medicine. Recently, an RNA-guided, DNA-cleaving interference pathway from bacteria [the type II clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) pathway] has been adapted for use in eukaryotic cells, greatly facilitating genome editing. Only two CRISPR-Cas systems (from Streptococcus pyogenes and Streptococcus thermophilus), each with their own distinct targeting requirements and limitations, have been developed for genome editing thus far. Furthermore, limited information exists about homology-directed repair (HDR)-mediated gene targeting using long donor DNA templates in hPSCs with these systems. Here, using a distinct CRISPR-Cas system from Neisseria meningitidis, we demonstrate efficient targeting of an endogenous gene in three hPSC lines using HDR. The Cas9 RNA-guided endonuclease from N. meningitidis (NmCas9) recognizes a 5'-NNNNGATT-3' protospacer adjacent motif (PAM) different from those recognized by Cas9 proteins from S. pyogenes and S. thermophilus (SpCas9 and StCas9, respectively). Similar to SpCas9, NmCas9 is able to use a single-guide RNA (sgRNA) to direct its activity. Because of its distinct protospacer adjacent motif, the N. meningitidis CRISPR-Cas machinery increases the sequence contexts amenable to RNA-directed genome editing.
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http://dx.doi.org/10.1073/pnas.1313587110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3785731PMC
September 2013

Phosphorylation regulates human OCT4.

Proc Natl Acad Sci U S A 2012 May 2;109(19):7162-8. Epub 2012 Apr 2.

Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706-1532, USA.

The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography-MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency.
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http://dx.doi.org/10.1073/pnas.1203874109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3358887PMC
May 2012

Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment.

Stem Cells 2011 Aug;29(8):1206-18

Waisman Center, University of Wisconsin, Madison, Wisconsin, USA.

Differentiation methods for human induced pluripotent stem cells (hiPSCs) typically yield progeny from multiple tissue lineages, limiting their use for drug testing and autologous cell transplantation. In particular, early retina and forebrain derivatives often intermingle in pluripotent stem cell cultures, owing to their shared ancestry and tightly coupled development. Here, we demonstrate that three-dimensional populations of retinal progenitor cells (RPCs) can be isolated from early forebrain populations in both human embryonic stem cell and hiPSC cultures, providing a valuable tool for developmental, functional, and translational studies. Using our established protocol, we identified a transient population of optic vesicle (OV)-like structures that arose during a time period appropriate for normal human retinogenesis. These structures were independently cultured and analyzed to confirm their multipotent RPC status and capacity to produce physiologically responsive retinal cell types, including photoreceptors and retinal pigment epithelium (RPE). We then applied this method to hiPSCs derived from a patient with gyrate atrophy, a retinal degenerative disease affecting the RPE. RPE generated from these hiPSCs exhibited a disease-specific functional defect that could be corrected either by pharmacological means or following targeted gene repair. The production of OV-like populations from human pluripotent stem cells should facilitate the study of human retinal development and disease and advance the use of hiPSCs in personalized medicine.
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http://dx.doi.org/10.1002/stem.674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412675PMC
August 2011

Chemically defined conditions for human iPSC derivation and culture.

Nat Methods 2011 May 10;8(5):424-9. Epub 2011 Apr 10.

Morgridge Institute for Research, Madison, Wisconsin, USA.

We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.
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http://dx.doi.org/10.1038/nmeth.1593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084903PMC
May 2011

Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy.

Proc Natl Acad Sci U S A 2011 Apr 4;108(16):6537-42. Epub 2011 Apr 4.

Morgridge Institute for Research, Madison, WI 53715, USA.

Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a unique approach to gene therapy. Here, we begin to assess whether the mutational load acquired during gene correction of iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected after standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine mutations in protein coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base-pair change, no additional mutations or copy number variation were identified in iPS cells after the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden.
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http://dx.doi.org/10.1073/pnas.1103388108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3080993PMC
April 2011
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