遗传疾病类器官建模：囊性纤维化与多囊肾病

以下为英文全文（English full text）

Abstract

Genetic diseases affecting epithelial organs have been particularly amenable to organoid modeling due to the accessibility of tissue and the ability to derive organoids from patient-specific induced pluripotent stem cells (iPSCs) or adult stem cells. This article focuses on two exemplar diseases—cystic fibrosis (CF) and polycystic kidney disease (PKD)—that have emerged as flagship applications for genetic disease organoid modeling. We provide detailed technical protocols for deriving and characterizing CF and PKD organoids, discuss disease-specific phenotypic assays, and review the integration of CRISPR gene editing for mechanistic studies and therapeutic development.

1. Introduction

The modeling of genetic diseases has historically been limited by the availability of patient-specific tissues and the lack of human-relevant in vitro systems. Animal models of genetic diseases often fail to recapitulate the human phenotype due to species-specific differences in gene function, physiology, and disease modifiers. Organoid technology has overcome these limitations by enabling the generation of patient-specific tissue models from small biopsy samples or by differentiating patient iPSCs into disease-relevant organ types [1].

Cystic fibrosis and polycystic kidney disease represent two distinct classes of genetic disorders: CF is a monogenic disease caused by mutations in a single gene (CFTR) and affects multiple organs (lung, intestine, pancreas, liver); PKD is a ciliopathy that affects primarily the kidney and can be caused by mutations in multiple genes (PKD1, PKD2, PKHD1, DZIP1L). Both diseases have been successfully modeled using organoids, and these models have been integrated into clinical decision-making and drug development pipelines [2].

2. Cystic Fibrosis Organoid Models

2.1 Disease Background and Rationale for Organoid Modeling

Cystic fibrosis is caused by mutations in the CFTR gene, which encodes a chloride and bicarbonate channel expressed in epithelial cells. Loss of CFTR function leads to defective ion transport, mucus accumulation, chronic inflammation, and progressive organ damage. The CFTR modulator drugs ivacaftor, lumacaftor, tezacaftor, and elexacaftor have transformed treatment for patients with responsive mutations, but approximately 10% of CF patients carry mutations that do not respond to current therapies.

Intestinal organoids have emerged as the most clinically validated CF organoid model. The rationale is straightforward: CFTR is highly expressed in intestinal epithelial cells, and the forskolin-induced swelling (FIS) assay provides a direct, quantitative readout of CFTR function. Forskolin elevates intracellular cAMP, which activates CFTR; in wild-type organoids, CFTR-mediated chloride secretion drives fluid influx and organoid swelling, while in CF organoids lacking functional CFTR, this swelling is absent or markedly reduced [3].

2.2 CF Intestinal Organoid Establishment

CF intestinal organoids are established from rectal suction biopsies (1–2 mm²), duodenal biopsies, or surgical specimens. Rectal biopsies are particularly advantageous because they can be obtained safely and repeatedly, enabling longitudinal monitoring of drug responses.

The tissue is placed in ice-cold Advanced DMEM/F12 with 10 mM HEPES and 1% P/S. The specimen is washed three times in ice-cold PBS and minced into 1 mm fragments. The tissue is digested in 5 mL of collagenase/dispase solution (Advanced DMEM/F12 with 2 mg/mL collagenase II, 1 mg/mL dispase, and 10 μM Y-27632) at 37°C on a rocker for 30–60 minutes. The digestion is monitored microscopically; crypt-like structures should be visible [4].

The digested mixture is centrifuged at 300 × g for 5 minutes, washed twice in wash medium, and resuspended in cold basement membrane matrix (Matrigel or GBiowit Intestinal Organoid Matrix). The suspension is plated as 40–50 μL droplets in 24-well plates and polymerized at 37°C for 20 minutes.

The complete CF intestinal organoid medium (CF-IOGM) contains:

• Advanced DMEM/F12 (base)
• 1× B-27 supplement (without vitamin A)
• 1× N-2 supplement
• 1.25 mM N-acetylcysteine
• 10 mM nicotinamide
• 50 ng/mL human recombinant EGF
• 100 ng/mL recombinant Noggin
• 1 μg/mL recombinant R-spondin 1
• 50% Wnt3a-conditioned medium (or 100 ng/mL recombinant Wnt3a)
• 10 nM [Leu15]-gastrin
• 500 nM A83-01
• 10 μM SB202190
• 10 μM Y-27632 (first 3 days)
• 100 μg/mL Primocin

2.3 Forskolin-Induced Swelling (FIS) Assay

The FIS assay is the cornerstone functional test for CF intestinal organoids. The protocol is as follows:

1. Organoids are harvested from Matrigel using cold Cell Recovery Solution on ice for 30 minutes.
2. The organoids are washed in PBS and resuspended in 10 μL of basal medium per well in a 96-well plate.
3. Baseline images are acquired using brightfield microscopy.
4. Organoids are incubated in medium containing 1 μM forskolin and 100 μM IBMX (phosphodiesterase inhibitor) for 60–120 minutes at 37°C.
5. Post-treatment images are acquired at the same magnification and focal plane.
6. Organoid surface area is quantified using ImageJ or automated image analysis software.
7. The swelling index is calculated as: (Area_post - Area_pre) / Area_pre × 100%.

Wild-type organoids typically show a swelling index of 50–150%, while CF organoids with severe mutations (e.g., F508del homozygous) show a swelling index of <10%. CFTR modulators are tested by pre-incubating organoids with the drug for 24 hours before performing the FIS assay. Ivacaftor (a potentiator) restores swelling to F508del/F508del organoids to approximately 20–40% of wild-type levels, while the triple combination (elexacaftor + tezacaftor + ivacaftor) achieves near-normal swelling in responsive genotypes [5].

2.4 Clinical Translation and Drug Screening

The FIS assay using rectal organoids has been developed into a clinical diagnostic test (the "organoid assay") that predicts patient responses to CFTR modulators. In prospective studies, the organoid assay predicted clinical response with 100% positive predictive value and 90% negative predictive value for ivacaftor responsiveness. This is particularly valuable for patients with rare mutations where clinical trial data are lacking [6].

High-throughput screening of CF organoids has been used to identify novel CFTR modulators. Libraries of thousands of compounds can be tested for their ability to restore forskolin-induced swelling in F508del organoids. This approach has identified novel corrector and potentiator scaffolds that are being advanced toward clinical development [7].

3. Polycystic Kidney Disease Organoid Models

3.1 Disease Background

Polycystic kidney disease is the most common inherited cause of kidney failure. Autosomal dominant PKD (ADPKD) is caused by mutations in PKD1 or PKD2 and affects approximately 1 in 1,000 individuals. Autosomal recessive PKD (ARPKD) is caused by mutations in PKHD1 and presents in infancy or childhood with enlarged, cystic kidneys. The pathogenesis involves defective function of polycystin proteins (PC1, PC2) in primary cilia, leading to increased cAMP signaling, cell proliferation, and fluid secretion into developing cysts.

Kidney organoids derived from human pluripotent stem cells (hPSCs) provide a powerful model for studying PKD pathogenesis. Unlike animal models, human kidney organoids form nephron-like structures with segment-specific identities (podocytes, proximal tubules, distal tubules, collecting duct cells) and develop cysts when carrying PKD mutations [8].

3.2 PKD Kidney Organoid Differentiation Protocol

Kidney organoids are generated from hPSCs using a directed differentiation protocol. The key steps are:

1. hPSCs are maintained in feeder-free conditions in mTeSR1 medium.
2. Induction of primitive streak (Day 0–2): CHIR99021 (GSK3β inhibitor, 8 μM) in APEL medium with 1% penicillin-streptomycin.
3. Induction of intermediate mesoderm (Day 2–4): FGF9 (20 ng/mL) + heparin (1 μg/mL) in APEL medium.
4. Induction of ureteric bud and metanephric mesenchyme (Day 4–7): CHIR99021 (3 μM) + FGF9 (20 ng/mL) in APEL medium.
5. Self-organization into kidney organoids (Day 7–14): Organoids are transferred to low-attachment plates in APEL medium with FGF9 (20 ng/mL) and heparin.
6. Maturation (Day 14–28): Organoids are maintained in kidney organoid maturation medium (Advanced DMEM/F12, 1× B-27, 1× N-2, 10 ng/mL EGF, 20 ng/mL FGF9) [9].

For PKD modeling, the hPSCs carry disease-causing mutations either naturally (patient-derived iPSCs) or introduced via CRISPR-Cas9. CRISPR-derived PKD1−/− and PKD2−/− hESC lines have been extensively characterized and shown to develop cystic structures in kidney organoids [10].

3.3 Cystogenic Phenotype Assessment

PKD organoids develop cysts through distinct mechanisms depending on the culture conditions:

• Static suspension culture: PKD1−/− and PKD2−/− organoids develop cysts spontaneously in 6–15% of organoids by differentiation day 20–28. Forskolin (10 μM) treatment enhances cyst formation by elevating cAMP levels, with 60–80% of mutant organoids developing cysts within 48–72 hours of treatment. Wild-type organoids do not develop cysts under these conditions [11].
• Flow-induced cystogenesis: When PKD organoids are cultured in microfluidic chips with continuous perfusion, flow-induced mechanical stress activates mechanosensors (RAC1, c-FOS) and triggers cyst formation in PKHD1−/− organoids. This flow-induced cystogenesis is restricted to CDH1+ distal tubules and is abrogated by pharmacological inhibition of RAC1 or integrin signaling. This model is particularly relevant for ARPKD, as it recapitulates the collecting duct localization of cysts [12].

Cyst quantification parameters include:

• Cyst incidence: percentage of organoids with visible cysts
• Cyst size: maximum diameter of cystic structures
• Cyst growth rate: change in cyst area over time
• Tubule segment contribution: identification of proximal (LTL+) vs. distal (CDH1+) tubule-derived cysts

3.4 Drug Screening in PKD Organoids

PKD organoids have been used to screen for cystogenic modifiers and therapeutic candidates:

• Tolvaptan (vasopressin V2 receptor antagonist): The only FDA-approved drug for ADPKD. Tolvaptan reduces cAMP signaling and inhibits cyst expansion in PKD organoids. Vasopressin stimulation promotes cyst formation in PKD1−/− organoids, and tolvaptan abrogates this effect [13].
• Octreotide (somatostatin analog): Reduces cAMP levels and inhibits cyst growth in PKD organoids.
• Metformin: Activates AMPK and inhibits cystogenesis in PKD2−/− organoids.
• CFTR inhibitors: Inhibit cyst fluid secretion and reduce cyst growth in PKD organoids.

High-throughput screening platforms using 384-well plates and automated imaging have been developed to evaluate thousands of compounds for cystogenic inhibition in PKD organoids. These screens have identified novel targets including mTOR, Src, and MEK/ERK pathway components [14].

4. CRISPR Gene Editing in Genetic Disease Organoids

4.1 Disease Modeling via CRISPR Knockout

CRISPR-Cas9 enables the introduction of specific disease-causing mutations into wild-type hPSCs, followed by organoid differentiation to generate isogenic disease models. For PKD, CRISPR-derived biallelic PKD1 and PKD2 frameshift mutations in the H9 hESC line produce kidney organoids that develop cystic structures, while isogenic wild-type controls remain non-cystic. The use of base editing to introduce precise nonsense mutations (e.g., PKD1 R2430X, Q3838X) has achieved even higher cyst formation rates (75–90% of organoids) [15].

For CF, CRISPR can introduce specific CFTR mutations (F508del, G551D, W1282X) into wild-type hPSCs or iPSCs. The resulting intestinal organoids show the expected CFTR functional defects and can be used to test mutation-specific responses to modulators.

4.2 Gene Correction and Therapeutic Proof-of-Concept

CRISPR-mediated correction of disease-causing mutations in patient-derived iPSCs provides definitive proof that the mutation is responsible for the observed phenotype. In PKD, correction of PKD1 mutations in patient iPSCs restored normal organoid morphology and prevented cyst formation. In CF, correction of CFTR mutations restored forskolin-induced swelling to normal levels [16].

These gene-corrected isogenic pairs are powerful tools for:

• Validating genotype-phenotype relationships
• Screening for mutation-agnostic therapies
• Developing gene therapy approaches (e.g., CRISPR-based gene correction in vivo)
• Investigating modifier genes that influence disease severity

4.3 Prime Editing and Base Editing Advances

Prime editing and base editing technologies enable the introduction of precise point mutations without double-strand DNA breaks, reducing the risk of off-target effects and unwanted chromosomal rearrangements. These approaches are particularly valuable for modeling diseases caused by single-nucleotide variants and for therapeutic gene correction in clinical applications [17].

Conclusion

Genetic disease organoid modeling has transformed the study of cystic fibrosis and polycystic kidney disease. CF intestinal organoids have already entered clinical practice as diagnostic tools, while PKD kidney organoids are advancing toward therapeutic screening and drug development applications. The integration of CRISPR gene editing, organ-on-chip technology, and high-throughput screening is expanding the capabilities of these models, bringing us closer to personalized therapies for genetic diseases.

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