Derivation and Correction of Proband iPSCs

Gene-corrected and uncorrected iPSC lines were generated from proband dermal fibroblasts according to a one-step reprogramming and gene-editing protocol as previously described.^{27, 31} Fibroblasts were harvested with TrypLE (Thermo Fisher) 2 days after passaging and resuspended in Buffer R (Thermo Fisher) at a final concentration of 1 × 10⁷ cells/mL. 100 μL of the cell suspension was added to a tube containing reprogramming plasmids (pEP4E02SET2K, pEP4E02SEN2L, pEP4E02SEM2K, and pSimple-miR302/367), mRNA encoding SpCas9-Gem, a plasmid encoding a single guide RNA that overlaps the proband-specific variant (c.634G>A [p.Gly212Arg]), and a repair template plasmid carrying 942 bp (chr16: 1591716–1592658, UCSC Genome Browser hg38) corresponding to sequence within IFT140. The repair template included a 3 bp synonymous change to act as a Cas9-blocking mutation and to facilitate the identification of gene-corrected iPSC clones by allele-specific PCR. Electroporation was performed in a 100 μL tip with the following conditions: two pulses at 1,400 V for 20 ms. After electroporation, cells were plated on a 6-well Matrigel-coated (BD Biosciences) plate and maintained in fibroblast media until 4 days after transfection and then switched to Essential 7 medium (Essential 8 [E8] medium without TGFβ; Thermo Fisher) supplemented with 100 μM sodium butyrate and changed every other day as described previously.³² Sodium butyrate was removed from the media after the appearance of the first iPSC colonies at around day 10. Gene-corrected iPSCs were identified by allele-specific PCR and confirmed by Sanger sequencing. One gene-corrected clone and one uncorrected iPSC clone were selected and expanded for further analysis. Karyotype was confirmed by Infinium CoreExome-24 DNA microarray (Illumina), and cells tested negative for mycoplasma contamination. Pluripotency of iPSC lines was confirmed by flow cytometry after staining with antibodies to pluripotency markers (EPCAM, CD9, and SSEA4).

Cell Culture and Directed Differentiation to Kidney Organoids

Undifferentiated iPSCs were maintained in feeder-free conditions on human embryonic stem cell (hESC)-qualified Matrigel in E8 media. 1 day before differentiation, iPSCs were dissociated from 60%–70% confluence with TrypLE (Thermo Fisher) and plated at 3.5 × 10⁴ cells/cm² in a 6-well plate on hESC-qualified Matrigel (BD Biosciences) in 2.5 mL E8 media supplemented with Revitacell (Thermo Fisher). The following morning, cells were treated with 8 μM CHIR99021 in APEL2 basal media (STEMCELL Technologies) supplemented with 3.5% Protein Free Hybridoma Media 2 (PFHM2) and Antibiotic-Antimycotic (Life Technologies) for 4 days, followed by FGF9 (200 ng/mL) and heparin (1 μg/mL) for 3 days, during which media were changed every 2 days. At day 7, cells were dissociated with 0.05% trypsin, and 0.25 × 10⁶ cells were spun three times at 300 × g in 1.5 mL tubes. With a large-bore P200 pipette tip, pellets were transferred to a Transwell 0.4 μm pore polyester membrane (CLS3450 Corning). Pellets were cultured over APEL2, 3.5% PFHM2, and Antibiotic-Antimycotic supplemented with 5 μM CHIR 99021 for 1 hr and then over FGF9 (200 ng/mL) and heparin (1 μg/mL) for 5 days. Transwell culture continued without added growth factors for up to 24 days, during which media where changed three times a week.

Immunofluorescence

Organoids were fixed at the desired time point with 2% paraformaldehyde for 20 mins on ice and washed three times with PBS. Organoids were incubated with 10% donkey serum and 0.3% Triton X and PBS overnight at 4°C. Primary antibodies were incubated for 48 hr in the blocking buffer at 4°C. After six washes with 0.3% Triton X and PBS, secondary antibodies were incubated overnight at 4°C. Where applicable, DAPI staining was applied at 1:1,000 for 3 hr. After four washes with PBS, organoids underwent immunofluorescent imaging with a Zeiss LSM 780 confocal microscope. For analysis of ciliary length, three images were captured from two separate experiments. 100 cilia per image were measured in ZEN (v.2.3, Zeiss), and statistical analyses were performed in GraphPad Prism with an unpaired t test and Welch’s correction for unequal variance.

Spheroid culture assays were fixed and stained according to a previously published protocol.³³ Matrigel was dissolved, and spheroids were fixed with 4% paraformaldehyde at room temperature for 30 min. Spheroids were washed with DPBS with 1 mM CaCl₂ and 1 mM MgCl₂ and blocked with a gelatin-based permeabilization buffer (DPBS, 1 mM CaCl₂, 1 mM MgCl₂, 7 mg/mL gelatin, and 0.5% Triton X) for 1 hr before overnight incubation with primary antibodies in permeabilization buffer at 4°C. Spheroids were washed four times with permeabilization buffer and incubated with secondary antibodies overnight. After incubation with FITC-conjugated phalloidin for 1 hr and DAPI for 10 min, spheroids were washed with DPBS with CaCl₂ and MgCl₂ three times and mounted with Prolong Diamond Antifade Mountant.

Primary antibodies used included anti-NPHS1 (Bioscientific, AF4263, 1:300), biotinylated Lotus tetragonolobus lectin (LTL; Vector Laboratories, B-1325, 1:300), anti-CDH1 (BD Biosciences, 610181, 1:300), anti-CDH1 (Cell Signaling Technologies, 3195S, 1:300), anti-GATA3 (R&D Systems, AF2605, 1:300), anti-acetylated tubulin (Sigma Aldrich, T6793, 1:500), anti-IFT140 (Proteintech, 17460-1-AP, 1:100), anti-WDR19 (Proteintech, 13647-1-AP, 1:100), anti-IFT88 (Proteintech, 13967-1-AP, 1:100), anti-EPCAM-AF488 (Biolegend, 324210, 1:300), anti-ZO1 (Life Technologies, 40-2300, 1:200), anti-beta catenin (Sigma Aldrich, C2206, 1:200), and Phalloidin-FITC (Sigma-Aldrich, P5282, 1:1,000).

Secondary antibodies included Streptavidin Alexa Fluor 405 conjugate (Life Technologies, S32351, 1:400), Alexa Fluor 488 Donkey anti-mouse IgG (H+L) (Invitrogen, A21202, 1:400), Alexa Fluor 568 Donkey anti-rabbit (H+L) (Invitrogen, A10042, 1:400), Alexa Fluor 647 Donkey anti-sheep (H+L) (Invitrogen, A21448, 1:400), Alexa Fluor 647 Donkey anti-goat IgG (H+L) (Invitrogen, A21447, 1:400), Alexa Fluor 647 Donkey anti-mouse IgG (H+L) (Invitrogen, A31571, 1:400), and Alexa Fluor 568 Donkey anti-mouse IgG (H+L) (Invitrogen, A10037, 1:400).

Ciliary Morphology Assessment

Raw confocal images of cilia were median filtered (radius = 2) and segmented with a Fiji script³⁴ for implementation of the watershed, region-labeling, and volume-filtering (minimum = 200 voxels) functions from the MorpholibJ library.³⁵ A custom Python script was used for processing labeled images to isolate and maximum-project labeled regions representing a single cilium and then randomly select a subset of cilia from the combined dataset for scoring. The program generates blinded and unblinded scoring tables referencing a randomized, multi-image TIFF of individual cilia sampled from both genotypes. Image sets were spiked with automatically generated images (solid grayscale circles) so the scorer could reference their position within a 1,000 image set while scoring. TIFFs were manually scored and referenced back to their genotype with the unblinded table. Proportions were compared with chi-square contingency tests in GraphPad Prism.

RNA Sequencing

RNA from the epithelial fraction of whole kidney organoids was extracted with the PureLink RNA Mini Kit (Invitrogen), including treatment with the PureLink DNase Set (Invitrogen), and sequencing libraries were prepared according to Illumina’s TruSeq Stranded Total RNA protocol with RiboZero rRNA depletion. In total, six samples (three from each iPSC clone) were sequenced with an Illumina NextSeq 500 sequencer at the Translational Genomics Unit of Murdoch Children’s Research Institute. The data were submitted to the Gene Expression Omnibus (GEO) (accession number GEO: GSE107230). The raw reads were quality and adaptor trimmed with Trimmomatic v.0.35³⁶ and mapped to the human genome (hg38) with STAR v.2.5.2a³⁷ in the two-pass mapping mode. Reads were summarized over genes with featureCounts³⁸ and GENCODE v.20 annotation. Genes that had expression levels of at least one count per million in at least three samples were kept for further analysis. The data were TMM normalized³⁹ and voom transformed.⁴⁰ DGE was initially identified with robust paired moderated t tests in the limma R Bioconductor package⁴¹ with a false-discovery rate cutoff of less than 5%. This identified 1,097 downregulated and 1,244 upregulated genes in the corrected iPSC versus uncorrected iPSC lines. Testing for enrichment of the Broad Institute’s Hallmark gene sets (see Web Resources) was performed with CAMERA.⁴² To further refine the list of differentially expressed genes, we removed 570 genes from the DGE analysis that we had previously identified as being highly variable from experiment to experiment and therefore likely false positives (Table S3) and restricted our focus to significantly up- and downregulated DGE (adjusted p < 0.01, n = 954). Gene-set enrichment analysis of these 308 genes was performed with the ToppGene Suite.⁴³ Analysis of downregulated DGE (log fold change < −0.7, n = 308) was performed with an adjusted p value cutoff < 0.05. Histograms and heatmaps were generated with GraphPad Prism (v.7.01). Protein interactomes were generated with STRING v.10.5,⁴⁴ and nodes were recolored from GraphPad Prism heatmaps in Adobe Illustrator (v.2015.2.1).

cDNA was generated from RNA samples with the GoScript Reverse Transcription Kit (Promega), and qPCR validation of selected differentially expressed genes was performed with the SensiFAST SYBR Lo-ROX Kit (BIOLINE) and QuantStudio5 Real-Time PCR System (Thermo Fisher). Oligonucleotide primers are listed in Table S7.

Magnetic-Activated Cell Sorting (MACS) and Cyst Culture

12 whole organoids were dissociated in 2.0 mL TrypLE Select (Thermo Fisher) for 12 min and inactivated by dilution with chilled MACS buffer solution (PBS, 2 mM EDTA, and 0.5% bovine serum albumin). The cell solution was passed sequentially through a 70, 40, and 30 μm sieve. Cells were counted, centrifuged at 300 × g for 5 min, and resuspended in 300 μL of MACS buffer and 100 μL of EPCAM⁺ (CD326) Microbeads (Miltenyi Biotech) for 30 min. Cells were rinsed with MACS buffer and centrifuged twice before resuspension in 500 μL of MACS buffer and passage through an MS MACS column according to the manufacturer’s protocol (Miltenyi Biotec). The EPCAM⁺ fraction was eluted gently from the column in chilled Renal Epithelial Cell Growth Medium 2 (RECGM2, Promocell). 1.0 × 10⁵ cells in 100 μL of RECGM2 were mixed with 100 μL Matrigel GFR (BD Biosciences) and plated immediately into an 8-well chamber slide. The cell-Matrigel suspension was incubated at 37°C for 30 min before the addition of 200 μL RECGM2 supplemented with 1× Revitacell overnight. RECGM2 without Revitacell was refreshed daily until spheroids were visible by bright-field microscopy. After fixation and immunofluorescent staining (as above), spheroids were imaged with a Zeiss LSM 780 confocal microscope. 100 consecutive spheroids were assessed per condition, and proportions were compared statistically in GraphPad Prism (v.7.01) with Fisher’s exact test. Cilia per nucleus were manually counted in ten epithelial cysts imaged at high resolution in Fiji with a Cell Counter plugin. Mean cilia per nucleus expressed as a percentage for each clone were compared in GraphPad Prism with an unpaired t test and Welch’s correction for unequal variance.

Simultaneous Reprogramming and Gene Correction of Proband Fibroblasts

A previously described one-step protocol that combines both an episomal-based reprogramming strategy and CRISPR/Cas9-mediated gene editing²⁷ was used to generate clonal uncorrected and gene-corrected iPSC lines within a single experiment (Figure 2A). The Cas9 repair template included correction of the c.634G>A variant as well as an additional synonymous 3 bp change to facilitate the identification of corrected clones and prevent Cas9-mediated cleavage after successful homology-directed repair (Figure 2A). Gene correction was identified in 1 of 44 screened iPSC clones (2.3%). One uncorrected proband (henceforth referred to as PR) and the isogenic, gene-corrected clone (GC) were carried forward to further experiments. Pluripotency was confirmed by culture morphology and staining for common pluripotency markers and subsequent flow cytometry (Figures S1A and S1B).

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Figure 2

Gene Correction, Transcript Analysis, and Differentiation to Kidney Organoid

(A) The gene-correction template for the c.634G>A variant contains the corrected 1 bp substitution (blue) and a synonymous downstream 3 bp substitution (green) for ease of identification by Sanger sequencing. Successfully reprogrammed clones were screened for unsuccessful (red nuclei, PR) or successful (green nuclei, GC) gene correction.

(B) DNA sequencing demonstrates the c.634G>A variant in the PR clone (red arrowhead) and successful correction of this variant in the GC clone (green arrowhead), as well as the synonymous 3 bp change in this allele (gray shading).

(C) Sanger-sequenced RNA (copy DNA) transcripts suggest that the c.634G>A allele is not detectable as a stable transcript, indicated by a single chromatogram peak at both the c.634G>A locus (red arrowhead) and the c.2176C>G variant (blue arrowhead) in the PR clone. The double chromatogram peak at the synonymous 3 bp substitution (gray) and the c. 2176C>G variant locus (not gene-corrected; blue) in the GC clone indicates that gene correction has rescued this transcripts.

(D) qPCR identifies 50% relative mRNA expression in PR iPSCs compared with control cells (data represent the mean + 95% CI; p = 0.0001).

(E) Capillary western blot reveals 43% relative IFT140 expression in PR differentiated renal epithelial cells compared with GC cells (data represent the mean + 95% CI; p < 0.001).

(F) Bright-field images of kidney organoids on day 25 of culture (day 18 of aggregate culture). Scale bars, 1 mm.

(G) Immunofluorescence images of whole organoids demonstrate balanced expression of glomerular precursors (NPHS1, white), proximal tubule, (LTL, blue), distal tubule (CDH1, green), and collecting duct (CDH1, green; GATA3, red). Scale bars, 1 mm (low-magnification images) and 50 μm (high-magnification images).

Gene Correction of iPSCs Rescues Unstable mRNA Transcript

The proband-specific variants were confirmed after Sanger sequencing of PCR amplicons spanning exon 6 or exon 18 of the IFT140 locus (Figure 2C, red arrowhead), whereas loss of the c.634G>A variant and gain of the synonymous 3 bp change were confirmed in gene-corrected iPSCs.

Total RNA was extracted, and RT-PCR was performed to amplify IFT140 transcripts from GC and PR iPSCs, which were then sequenced. This analysis revealed that only the c.2176C>G allele was detectable at the transcriptional level in PR iPSCs (Figure 2C), indicating that the c.634G>A variant results in defective splicing of exon 6 and that the incorrectly spliced transcripts are potentially targeted for degradation. Hence, only one IFT140 allele is likely to be transcribed and translated. In contrast, transcription from both alleles was detected in GC iPSCs, as evidenced by the presence of the synonymous 3 bp change adjacent to the corrected variant (Figure 2C). This was also supported by the finding of 53% IFT140 mRNA expression and 33% quantitative protein in the PR clone relative to the GC clone (Figures 2D and 2E).

Kidney Organoid Tubules Demonstrate Ciliary Morphology Consistent with Defective Retrograde IFT

Multicellular kidney organoids were differentiated from both PR and GC iPSC lines (Figure 2F) through adaptation of a protocol previously published by Takasato et al.⁴⁹ to one that used fully defined serum- and feeder-free culture conditions (Figure S1C). Organoids produced from both lines were validated as reaching a renal endpoint by the contiguous immunofluorescent localization of nephrin (NPHS1, staining glomerular precursors), LTL (staining proximal tubule), cadherin 1 (CDH1, staining distal tubule), and co-immunofluorescence for CDH1 and GATA3 (collecting duct) (Figure 2G).

Previous IFT140-null mouse models demonstrated shortened primary cilia with swollen ciliary tips in developing limb buds.²² It should be noted that dual IFT140 knockout resulted in embryonic lethality.²² Primary cilia stained with acetylated tubulin and ARL13B were clearly identified in the tubular epithelium of iPSC-derived kidney organoids by immunofluorescence (Figures 3A and 3D). Automated image processing was used to generate a random series of images of individual cilia from CDH1⁺ regions of kidney organoids (n = 900 cilia) for blinded scoring (Figure 3A). A clubbed morphology similar to that seen in the IFT140-null mouse was evident in the majority of PR cilia (59%; 95% confidence interval [CI] = 54%–63%), whereas a similar majority of GC cilia (60%; 95% CI = 56%–65%) demonstrated wild-type morphology (Figure 3B). PR cilia were significantly shorter than GC cilia, and the difference between PR and GC mean ciliary length increased with time in aggregate culture (Figure 3C). Ciliary IFT140 localization within club-shaped cilia in GATA3⁺ regions of organoid tubules demonstrated increased IFT140 staining of the swollen ciliary axoneme. Other IFT-A and IFT-B proteins demonstrated a similar intensification of fluorescence in the ciliary tip, consistent with defective retrograde IFT (Figure 3D).

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Figure 3

Proband Organoid Tubules Demonstrate Abnormal Cilia, which Are Rescued by Gene Correction

(A) Cilia of CDH1⁺ tubular epithelial cells demonstrate classical morphology associated with defective retrograde IFT: short with swollen ciliary tips (left and middle panels; scale bars, 2 μm). Individual cilia were isolated from these images (right panels; scale bars, 500 nm) and shuffled for blinded scoring of morphology.

(B) Quantitative output of blinded cilia-morphology analysis demonstrates predominantly clubbed cilia in PR organoids (59%) and predominant wild-type morphology in gene-corrected cilia (61%) (n = 900; p < 0.0001, Chi-square test).

(C) PR cilia were shorter than GC cilia at every culture time point, such that an increasing difference was noted with time in culture (n = 600 cilia per condition; p < 0.0001, Welch’s t test; error bars represent the mean + 95% CI).

(D) IFT components IFT140, WDR19, and IFT88 were all found to accumulate in the PR ciliary tip. Gene-corrected organoids displayed a wild-type distribution (scale bar, 1 μm).

Transcriptional Profiling Identifies Dysfunctional Cellular Processes Resulting from c.634G>A

The isolated transcriptional effects of the c.634G>A variant were investigated by comparison of the bulk RNA sequencing (RNA-seq) profiles (accession number GEO: GSE107230) of the epithelial (EPCAM⁺) cell fractions isolated from iPSC-derived kidney organoids by MACS (Figure 4A). According to co-immunofluorescence analysis, this EPCAM⁺ fraction included proximal tubule (LTL⁺) and collecting duct structures (GATA3⁺) as well as an epithelial structure resembling Bowman’s capsule surrounding visceral epithelial cells (Figures S2A and S2B). Validation of EPCAM⁺ purification by MACS was confirmed by flow cytometry (Figure S2C). Replicates clustered appropriately on a principle-component analysis with clear separation between PR and GC transcription profiles upon unbiased clustering (Figures 4B and 4C).

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Figure 4

RNA-Seq of Epithelial Sub-fractions of Organoids Demonstrates Dysfunctional Cellular Processes Resulting from IFT140 Variants

(A) Diagrammatic representation of epithelial MACS from dissociated organoids. Dissociated organoids were incubated with magnetic beads conjugated with EPCAM antibody and passed through a magnetic field, trapping epithelial cells that could be gently eluted for downstream application after removal of the magnetic field.

(B) Principal-component analysis of primary RNA-seq data demonstrates clustering of PR and GC replicates.

(C) Heatmap demonstrates consistency of gene expression across replicates.

(D) Top GO terms output from ToppGene gene-list enrichment of set 1 (combined up- and downregulated genes with significant differential expression, adjusted p < 0.01; top graph) and set 2 (genes with log fold change > 0.7 favoring GC and significant differential expression < 0.05; bottom graph). Bars represent the −log₁₀(p value), and the adjacent fraction represents the number of genes within the dataset (numerator) and the number of genes within the GO gene list (denominator). Similar GO terms with a near-identical DGE representation and GO term were filtered out to avoid repetition. Colors are as follows: blue, GO molecular function; purple, GO biological process; and orange, GO cellular component.

For the purposes of gene enrichment analysis, the primary DGE dataset (Table S2) was filtered for genes that have previously exhibited high transcriptional variability between repeated differentiation experiments of a human wild-type iPSC cell line (CRL1502, clone C32).⁵⁰ This transcriptional “noise” is batch associated and likely to result from slight variations in relative maturation between individual organoids. Any of the top 1,000 most variable genes within the CRL1502 dataset were excluded from further analysis (n = 570; Table S3).

Gene enrichment analysis of the remaining genes with significant DGE (adjusted p value < 0.01, n = 954; Table S4) returned gene ontology (GO) terms illustrating disturbances in cell adhesion and cytoskeletal interaction pathways (Figure 4D). This analysis returned one single human phenotype term, “vitreoretinal degeneration” (HP:0000655), consistent with the proband phenotype (Figure S3A) and inclusive of CRB1 (OMIM: 604210), a critical component of the highly conserved Crumbs polarity complex, which is strongly associated with retinitis pigmentosa (OMIM: 600105) and Leber’s congenital amaurosis (OMIM: 613835).⁵¹ Cell polarity complexes are highly conserved, critical regulators of epithelial polarity and modulate asymmetrical cytoskeletal organization within the cell and cell-junction integrity in order to preserve apicobasal expression of transmembrane receptors and transporters.^{52, 53} Differential expression of Crumbs complex genes within the primary RNA-seq dataset suggested downregulation in PR renal epithelia compared with GC epithelia (Figure S3B). Transcriptional differences within the associated PAR polarity complex were less consistent between replicates with the exception of CDC42 (OMIM: 116952), which was downregulated in PR (Figure S3C). Of interest, animal knockout models of Cdc42^−/− develop a NPHP-like renal histology and demonstrate loss of photoreceptor cilia.⁵⁴ CDC42 was also a central node within a STRING protein interaction network of DGE highlighted within the “adherens Junction” term (GO:0005912; Figures S4A and S4B). EGFR was also a significantly downregulated central interacting gene product identified by the STRING protein interactome. EGF signaling is strongly associated with the proliferative epithelial phenotype seen in ADPKD cysts, and inhibition of this signaling pathway reduces cyst progression in animal models (reviewed in Harskamp et al.⁵⁵). The observation of CDK1 (int:CDK1) and CDK2 (int:CKD2) in the top five interacting gene products is of interest given the increasing evidence of benefit in both animal and cellular nephronophthisis models treated with CDK inhibitors.^{56, 57, 58, 59}

IFT-related ciliopathy is often associated with increases in canonical Wnt, Shh, and Notch signaling pathways, which are usually amplified by treatment with a pathway agonist.^{24, 26, 60} Although these pathways did not feature prominently in ToppGene enrichment analysis, all three were upregulated in Hallmark gene-list enrichment analyses of the primary dataset before the exclusion of highly variable genes (Figures S4D–S4F). In a murine Ift140 HoxB7-Cre knockout model, transcriptional changes in these signaling pathways were not observed until postnatal days 15–20.⁶¹ The present study, however, demonstrates pathway changes consistent with the existing literature within a model that at a transcriptional level most closely resembles first-trimester human fetal kidney.⁶

Gene enrichment analysis of downregulated DGE reflected disturbances to apical and basolateral plasma membranes, the apical junction complex, and transporter activities associated with these cellular membranes (Figure 4D). Additional downregulated GO outputs of interest included “negative regulation of epithelial cell proliferation” (GO:0050680) and “axonemal dynein complex assembly” (GO:0070286) (Figures S4C and S4D). RNA-seq of selected differentially expressed genes from these GO terms was validated by qPCR (Figure S5).

We thank Pei Xuan Er and Irene Ghobrial for assistance with iPSC derivation and maintenance, Alison Graham for iPSC pluripotency flow cytometry, and Andrew Lonsdale for assistance with bioinformatic graphical outputs. This study was funded by the National Health and Medical Research Council of Australia (NHMRC) (GNT1098654), Kidney Health Australia, the Royal Brisbane and Women's Hospital Foundation, the Royal Children’s Hospital Foundation, and the Murdoch Children’s Research Institute. The Murdoch Childrens Research Institute is supported by the Victorian Government’s Operational Infrastructure Support Program. T.A.F. is a recipient of the NHMRC Postgraduate Scholarship (GNT1114409) and RACP Jacquot Award. M.H.L. is an NHMRC Senior Principal Research Fellow and has consulted for and received research funding from Organovo Inc.