Modeling of lung phenotype of Hermansky–Pudlak syndrome type I using patient-specific iPSCs

Background Somatic cells differentiated from patient-specific human induced pluripotent stem cells (iPSCs) could be a useful tool in human cell-based disease research. Hermansky–Pudlak syndrome (HPS) is an autosomal recessive genetic disorder characterized by oculocutaneous albinism and a platelet dysfunction. HPS patients often suffer from lethal HPS associated interstitial pneumonia (HPSIP). Lung transplantation has been the only treatment for HPSIP. Lysosome-related organelles are impaired in HPS, thereby disrupting alveolar type 2 (AT2) cells with lamellar bodies. HPSIP lungs are characterized by enlarged lamellar bodies. Despite species differences between human and mouse in HPSIP, most studies have been conducted in mice since culturing human AT2 cells is difficult. Methods We generated patient-specific iPSCs from patient-derived fibroblasts with the most common bi-allelic variant, c.1472_1487dup16, in HPS1 for modeling severe phenotypes of HPSIP. We then corrected the variant of patient-specific iPSCs using CRISPR-based microhomology-mediated end joining to obtain isogenic controls. The iPSCs were then differentiated into lung epithelial cells using two different lung organoid models, lung bud organoids (LBOs) and alveolar organoids (AOs), and explored the phenotypes contributing to the pathogenesis of HPSIP using transcriptomic and proteomic analyses. Results The LBOs derived from patient-specific iPSCs successfully recapitulated the abnormalities in morphology and size. Proteomic analysis of AOs involving iPSC-derived AT2 cells and primary lung fibroblasts revealed mitochondrial dysfunction in HPS1 patient-specific alveolar epithelial cells. Further, giant lamellar bodies were recapitulated in patient-specific AT2 cells. Conclusions The HPS1 patient-specific iPSCs and their gene-corrected counterparts generated in this study could be a new research tool for understanding the pathogenesis of HPSIP caused by HPS1 deficiency in humans. Supplementary Information The online version contains supplementary material available at 10.1186/s12931-021-01877-8.


PCR analysis of the 16 bp duplication in HPS1 patient-specific genomic DNA
Genomic DNA was extracted from the cloned cells using PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Touch down PCR was performed using KOD -Plus-NEO (TOYOBO) with a forward primer, 5'-GGTCCCTTCTGCTGTAATGC-3', and a reverse primer, 5'-GCTGCGTGAAGGAAGTACG-3', for the exon 15 of HPS1 gene (amplicon size: healthy donor, 240 bp; HPS1 patient, 256 bp) [6,7] with the thermal cycling condition as follows: 94°C for 2 min; 98 °C for 10 sec, and 74 °C for 20 sec in 5 cycles; 98 °C for 10 sec and 72 °C for 20 sec in 5 cycles; 98 °C for 10 sec and 70 °C for 20 sec in 5 cycles; 98 °C for 10 sec and 68 °C for 20 sec in 30 cycles; 68 °C for 5 min. Each PCR product was loaded on 5% polyacrylamide Mini Protien TBE precast gel (Bio-Rad) and electrophoresed at 100 V for 1 h in TBE buffer (Bio-Rad). The gels were stained with Midori Green Advance (NIPPON genetics) and analyzed using ChemiDoc Touch MP (Bio-Rad). Out of 92 clones, 3 had a single amplicon of approximately the same size as the healthy-donor-derived control iPSCs and were selected for downstream analysis. These amplicons were treated with ExoSAP-IT Express (Thermo Fisher Scientific) and sequenced to confirm the gene correction.
Permeabilized specimens were immersed in the blocking buffer of PBS containing 5% normal donkey serum (EMD-Millipore) and 1% BSA (Sigma-Aldrich) for 30 min. They were immunostained with the 5 primary antibodies overnight at 4 °C and secondary antibodies for 1 h. The staining conditions for each antibody are listed in Table S1 and S2. Blocking buffer supplemented with 0.1% Hoechst 33342 (Dojindo) was used for dilution of secondary antibodies. For organoids, each section was mounted in ProLong Gold Antifade Mountant (Thermo Fisher Scientific) and imaged using BZ-X710 microscope or TCS SP8 confocal microscope (Leica Microsystems).

Flow cytometry
PBS containing 1% BSA and 10 μM Y-27632 was used for cell suspension, washing and antibody dilution.
FACSAriaIII was used for all FACS analysis in the present study. Cells were dissociated using Accutase (Innovative Cell Technologies) for undifferentiated iPSCs in 2D culture or 0.1% Trypsin/EDTA (Thermo Fisher Scientific) for organoids, as described previously [8]. For intracellular staining, they were fixed with 4% paraformaldehyde/PBS for 15 min and permeabilized with ice-cold methanol at −30 °C for at least 30 min. The cells were immunostained with primary and secondary antibodies at 4 °C for 20 min.
The staining conditions for each antibody are listed in Table S1 and S2. For live cell staining, dead cells were removed using Propidium iodide (Dojindo) or SYTOX Blue Dead Cell Stain.

Western blotting
HPS1-overexpressing A549 cell extracts were prepared as positive controls for the detection of full-length HPS1. A549 cells were obtained from ATCC and cultured as described previously [9]. N-terminally HAtagged HPS1 cDNA cloned into pCAG-Neo (Fujifilm Wako) was transfected into A549 cells using the Lipofectamine LTX Reagent (Thermo Fisher Scientific), according to the manufacture's protocol.
Transfected A549 cells and undifferentiated iPSCs were lysed with the Pierce IP Lysis Buffer (Thermo Fisher Scientific) supplemented with cOmplete TM Mini Protease Inhibitor Cocktail (Merck). Each lysate was centrifuged at 15,000 × g for 15 min and supernatant was collected. Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), and protein concentration and volume were matched among samples (5 mg/mL, 1 mL for iPSCs). For detection of HPS4 and β-Actin, about 5% of each lysate was separated, added 1/4 th volume of 4X Laemmli sample buffer (supplemented with 2-mercaptoethanol; Bio-Rad) relative to lysate volume, and boiled for 5 min. Two microgram of mouse anti-human HPS1 antibody (Santa Cruz Biotechnology #sc-101435) was added into each of the remaining cell lysates and incubated overnight with rotation at 4 °C. Protein G Mag Sepharose Xtra (Cytiva) magnetic beads were washed, resuspended in 50 μL/sample of Pierce IP Lysis Buffer, added to each cell lysate and incubated with rotation for 1 h. After three cycles of magnetic capture with the sepharose beads and resuspension in PBS containing 0.1% BSA and 2 mM EDTA, the supernatant was completely removed from each sample using a magnet. Laemmli sample buffer (4X) supplemented with 2-mercaptoethanol was diluted to 2X with Pierce IP Lysis Buffer. Then, 30 μL of buffer was added to each sample with sepharose beads and boiled for 5 min. Supernatants were collected using a magnet and were used to detect HPS1. Each sample was loaded on polyacrylamide (7.5% for HPS1, 4-20% for HPS4 and β-Actin) Mini Protien TGX precast gels (Bio-Rad), electrophoresed in Tris Glycine SDS buffer (Bio-Rad) at 130 V for 90 min, and transferred to methanol-activated Immobilon-P polyvinylidene difluoride (PVDF) membrane (Merck, 0.2 μm) in Tris Glycine buffer (Bio-Rad) supplemented with 20% methanol at 100 V for 90 min at 4 °C. The membranes were immersed in TBS-T (Takara Bio) containing 3% BSA for HPS1 and β-Actin, or 1:2 diluted Blocking One (Nacalai Tesque) with TBS-T for HPS4, and incubated for 1 h.
Next, the membranes were immunostained with primary antibodies overnight at 4 °C. After three washings with TBS-T, they were immunostained with HRP-conjugated secondary antibodies for 1 h. The staining conditions for each antibody are listed in Table S1 and S2. Can Get Signal Solution 2 (Toyobo) was used for dilution of secondary antibodies. Pierce ELC plus Western Blotting Substrate (Thermo Fisher Scientific) was used for detection of chemiluminescence, according to the manufacturer's protocol. Images were obtained using ChemiDoc Touch MP.

qRT-PCR
Total RNA was extracted with PureLink RNA Mini Kit (Thermo Fisher Scientific), and cDNA was synthesized with ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo), according to the manufacturer's protocol. The primers used in this study are listed in Tables S3 and S4. TaqPath qPCR Master Mix, CG (Thermo Fisher Scientific), or Power SYBR Green Master Mix (Thermo Fisher Scientific) was used for qPCR in the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) or ABI7300 Real-Time PCR System (Thermo Fisher Scientific). Each gene expression was normalized using the eukaryotic 18S rRNA, and was presented as a relative value to the human adult lung 5 donor pool (BioChain #R1234152-P, lot A811037).

Generation of CPM-isolated lung bud organoids
CPM-isolated lung bud organoids (C-LBOs) were cultured in the LBO medium: Iscove's modified Dulbecco's medium (IMDM; Fujifilm Wako) and Ham's F-12 (Fujifilm Wako) were mixed in 3:1 ratio and supplemented with 0.5% N-2 supplement (Thermo Fisher Scientific), 1% B-27 supplement, 50 U/mL penicillin-streptomycin, 0.05 mg/mL L-ascorbic acid, 1% GlutaMax, 0.4 mM monothioglycerol, 3 μM CHIR99021, 10 ng/mL BMP4, 10 ng/mL FGF10, and 50 nM all-trans retinoic acid. Isolated 5 × 10 3 CPM + lung epithelial progenitor cells were suspended in the LBO medium supplemented with 10 μM Y-27632, seeded into Prime Surface 96U plate (Sumitomo Bakelite), and centrifuged at 100 × g for 2 min on Day 9 0. They were incubated for 7 days to form a spheroid in each well with 50% medium replaced every other day. On Day 7, a spheroid collected from each well was mixed with 2-3 spheroids in a tube, and they were allowed to settle spontaneously and the supernatant was removed. Next, 50 μL of ice-cold growth factor reduced Matrigel (Corning) was placed in a 24-well cell culture insert (Corning) and allowed to solidify replaced with DCIK medium every 2-3 days. NaPi2B + or GFP-SPC + cells were isolated every 14 days and continuously cultured in AOs to maintain and allow the maturation of AT2 cells, as described previously [1,8].

Measurement of organoid sizes
Live cell images of C-LBOs and AOs were obtained using the BZ-X710 microscope. Images were combined, and the diameter of each organoid was measured using BZ-X Analyzer (Keyence). All 3 C-LBOs in a well were measured in 4 independent experiments, whereas 20 randomly selected AOs in a well were measured in 6 independent experiments.

RNA-seq
Total RNAs of FACS-sorted NaPi2B high or SPC-GFP + cells from AOs were extracted and DNA was removed using the RNeasy micro kit (Qiagen), according to the manufacturer's protocol. The RNA integrity number calculated by 2100 BioAnalyer (Agilent Technologies) for each sample was confirmed to be > 8.0. Sequence libraries for NaPi2B high cells were synthesized using the TruSeq Stranded mRNA (illumina). For SPC-GFP + cells, 10 ng of total RNA was reverse-transcribed, amplified with 7 cycles using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech Laboratories), and converted to sequence libraries using Nextera XT DNA Library Preparation Kit (Illumina). The library was sequenced using 100 bp paired-end method by NovaSeq 6000 (Illumina).

Bioinformatic analysis of RNA-seq data
Publicly available fastq files were downloaded from Sequence Read Archive: GSE163575, lung epithelial cell progenitors; GSE121999, LBOs; GSE131768, adult AT2 cells; GSE94555, AT2 cells from idiopathic pulmonary fibrosis (IPF). Sequenced reads were trimmed using fastp [11] and aligned to the GRCh38 using STAR 2.7.3a [12]. RSEM was used to calculate transcripts per million (TPM) values [13]. Low expression genes, with total TPM values of the corresponding gene expression in the data set under 6, were excluded for listing differentially expressed genes (DEGs) and conducting principal component analysis (PCA). R package DESeq2 [14] was used to identify DEGs, and the volcano plot was visualized using R package "ggplot2". Gene ontology (GO) enrichment analysis was performed using the PANTHER online software [15]. PCA of log2 (TPM + 0.01) was performed using the R function "prcomp", and the results were visualized using the R package "ggbiplot". A hierarchical clustering for log2 (TPM + 0.01) of lung epithelial cell markers was performed using "heatmap.2" in the R package "gplots".

Proteomic analysis
AO cells were carefully dissociated with 0.1% Trypsin-EDTA, as described previously [8]. The cells were immunostained with mouse anti-human EpCAM antibody (Santa Cruz Biotechnology #sc-66020) and and quantified using the Scaffold DIA software program (Proteome Software). Next, 3098 proteins with false discovery rate < 0.01 were used for downstream analysis. GO enrichment analysis was performed using the PANTHER online software for upregulated proteins with log2 (fold change) > 0.5, or downregulated proteins with log2 (fold change) < −0.5. GSEAPreranked test was run using the genes ordered by log2 (fold change) [17].

Evaluation of intracellular reactive oxygen species (ROS) levels
Two-dimensional cultured A549 cells were stained with highly sensitive DCFH-DA Dye (Dojindo) and 200 nM MitoTracker Deep Red FM (Thermo Fisher Scientific), as per the manufacture's protocol. The stained cells were washed twice with HBSS (+) and analyzed using BZ-X710 microscope or FACSAriaIII.

Transmission electron microscope
Lamellar bodies were examined using transmission electron microscope (Hitachi; H-7650) as described previously [9]. Briefly, AOs were incubated in a fixative solution, en bloc stained, and embedded in Epon 812 (Nacalai Tesque). Uranyl acetate and lead citrate were used to stain the ultrathin sections.   Data are presented as mean ± SEM (n = 6 from 3 independent experiments). One-way ANOVA with Tukey's multiple comparisons test: **P < 0.01, ***P < 0.001.   We used Kaminski/Rosas data set (www.ipfcellatlas.com).  left, normal; center, sparsely expanded lamellar structure but not spherical; right, excessively fused lamellar bodies.     Table S5 (separate file). RNA-seq processed data of NaPi2B high cells isolated from alveolar organoids. Table S6 (separate file). RNA-seq processed data of EpCAM + cells isolated from lung bud organoids, reanalysis of the publicly available data (GSE121999).