The Generation of an Induced Pluripotent Stem Cell Line from a Patient with Phenylketonuria

Abstract

Phenylketonuria (PKU), an autosomal recessive genetic disorder, has been documented to exhibit over 950 distinct mutations. This condition primarily affects the metabolism of phenylalanine, which is affected by a deficiency in the hepatic enzyme phenylalanine hydroxylase. The optimal treatment for PKU disease remains to be determined, necessitating further research. The severity of the disease and the most effective treatment method vary depending on the specific mutation, which necessitates the development of personalized treatment strategies. In this study, we successfully established induced pluripotent stem cell (iPSC) lines from the blood of a PKU patient with the R243Q mutation via Sendai virus-based reprogramming (R243Q-iPSCs). The established R243Q-iPSCs exhibited characteristics of pluripotency, as confirmed through quantitative reverse transcription polymerase chain reaction, western blot, immunocytochemistry, and karyotype analysis. Furthermore, these iPSCs not only successfully differentiated into hepatocytes but also exhibited a complete PKU disease phenotype. These results provide a valuable foundation for PKU disease research, including physiological studies of PKU, gene therapy, drug screening, and the development of platforms for novel cell therapy approaches.

Introduction

The prevalence of phenylketonuria (PKU) exhibits variation across different geographical regions. However, on average, the condition manifests in approximately 1 in 10,000 newborns worldwide (1). The most notable aspect of this disease is its genetic variability, with studies conducted thus far identifying over 950 mutations in the phenylalanine hydroxylase (PAH) gene (2). These mutations influence the observed phenotype, contingent upon an individual’s genetic composition, such as blood phenylalanine (Phe) concentrations, residual PAH enzymatic activity, and responsiveness to treatment. Consequently, interindividual differences in both clinical severity and treatment outcomes are observed (2-4). Among East Asian populations, p.Arg243Gln (R243Q) mutation has been identified as the most prevalent (4, 5). The homozygous R243Q genotype has been identified as the most prevalent genotype in these populations (genotype frequency=5.6%), and this mutation has been demonstrated to be strongly associated with the classical PKU phenotype (4, 6). Therefore, R243Q may be a clinically relevant and representative target for the development of therapeutic strategies and in vitro disease modeling in East Asian populations.

In addition, advancements in science and technology are prompting a shift in the PKU treatment approach toward personalized strategies. The generation of induced pluripotent stem cells (iPSCs) derived from PKU patients with genetic mutations, p.[F39L];[c.1066-11G>A] and p.[F299C];[R408W], has been successfully achieved (7). In addition, there is an active research focus on in vivo-based studies, including experiments to correct a murine PKU model using adeno-associated virus-based CRISPR/Cas9 editing (8) and the evaluation of embryo-based gene editing using a humanized PKU mouse model (9). However, it is essential to note that the degree of genetic similarity between mice and humans is approximately 85%, indicating that there are significant differences in their genetic composition. Consequently, the direct comparison of safety and efficacy results from mouse models to humans remains inherently limited. Besides, this can lead to concerns in light of social and ethical demands to reduce animal experimentation (10). To ensure the safety and reliability of clinical studies, it is necessary to develop stem cell platforms based on genotypes with a high mutation frequency.

In this study, we screened the Korean PKU mutation information and successfully produced iPSCs using peripheral blood mononuclear cells (PBMCs) from patients with the R243Q mutation, which was identified as the most common mutation. Furthermore, the iPSCs produced in this manner successfully achieved stable hepatic differentiation, thereby creating an in vitro platform for PKU disease treatment. This platform is expected to serve as a valuable preclinical model for various applications such as organoid-based disease modeling, therapeutic testing, genotype-specific metabolic analysis, and evaluation of gene-editing strategies.

Materials and Methods

Peripheral blood mononuclear cells isolation and culture

Peripheral blood was obtained with consent from a 15-year-old male PKU patient (R243Q mutation) (Soonchunhyang University Hospital, SCHUH 2023-12-006-001). PBMCs from a healthy donor (WT) were purchased from Koma Biotech (Cat. PBMNC015C, LOT. #2308170175). PBMCs were isolated using RosetteSep^TM Human Progenitor Cell Basic Pre-Enrichment Kit (STEMCELL Technologies) and density gradient centrifugation with Lymphoprep^TM in SepMate^TM-15 tubes (STEMCELL Technologies). Isolated cells were cultured for 5 days in StemSpan^TM SFEM II with Erythroid Expansion Supplement (STEMCELL Technologies).

Reprogramming and culture of iPSCs, and embryoid body generation

PBMCs (3×10⁵) were reprogrammed into iPSCs using the CytoTune^TM-iPS 2.0 Sendai Kit (Invitrogen). After 1 day, cells were transferred to Matrigel-coated plates (Corning) and cultured in TeSR^TM-E7^TM/ReproTeSR^TM (STEMCELL Technologies) for 3 days. iPSCs picked after colony formation were maintained in mTeSR^TM1 (STEMCELL Technologies) supplemented with 1% penicillin-streptomycin (Gibco) and 1 μM Y-27632 (Tocris) for 24 hours. These cells were passaged at least 20 times before use. For embryoid body (EB) generation, cells were dissociated with TrypLE (Gibco) and seeded at 3×10⁶ cells/well into Aggrewell^TM plates (STEMCELL Technologies), cultured in mTeSR^TM1 with 10 μM Y-27632 for 1 day, and transferred to 60 mm ultra-low-attachment dishes (Corning).

Hepatic differentiation

The iPSCs were seeded at a density of 2.5×10⁵ cells/cm² 1 day before differentiation into definitive endoderm. To differentiate the definitive endoderm, the medium was changed to definitive endoderm medium for 4 days (Supplementary Table S1). The media were changed daily. On day 4, the media were changed to hepatoblast media and remained so for 5 days (Supplementary Table S1). Media changes were performed every other day. The hepatocyte media were treated for 5 days (Supplementary Table S1). Media changes were performed every other day.

Reverse transcription polymerase chain reaction

The absence of the Sendai reprogramming vector was confirmed by PCR after passage 20. Total RNA was isolated using TRIzol (Invitrogen), following the manufacturer’s protocol. Then, cDNA was synthesized using an AccuPower RT Premix Kit (Bioneer). Reverse transcription PCR was performed using a T100 thermal cycler (Bio-Rad). Each of the synthesized 1 μg cDNA templates and primers (SeV, c-Myc, and Klf4) was used with AmfiSure^TM ONE PCR Master Mix (GenDEPOT) for PCR with a T100^TM thermal cycler. The primer sequences are listed in Supplementary Table S2. The cycling conditions were as follows: denaturation at 95℃ for 30 seconds, annealing at 55℃ for 30 seconds, and elongation at 72℃ for 30 seconds, repeated 30 times. Analyze the PCR products using 2% agarose gel electrophoresis.

RNA extraction, quantitative reverse transcription polymerase chain reaction, and western blot

Total RNA was extracted using TRIzol, and cDNA synthesis was performed with a kit on a T100 Thermal Cycler (Bio-Rad). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was conducted using SYBR Green (Bio-Rad) on a CFX Duet (Bio-Rad), with mRNA expression normalized to GAPDH. Primer sequences are listed in Supplementary Table S2. For western blotting, proteins were extracted with RIPA buffer (Biosesang) containing protease and phosphatase inhibitors (GenDEPOT) and quantified using a BCA assay (ThermoFisher Scientific). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis separated samples were transferred to PVDF membranes (Bio-Rad), and blocked with 5% Skim milk (Bio-Rad) for 1 hour at RT. Membranes were incubated with 1st antibodies overnight at 4℃, followed by HRP-conjugated 2nd antibodies for 1 hour at RT (Supplementary Table S3). Detection was performed using ECL substrate (ThermoFisher Scientific) with an Amersham Imager 680 (General Electric).

Short tandem repeat analysis

Short tandem repeat (STR) profiling was performed to verify the cell lines. Genomic DNA was extracted from primary R243Q-PBMCs and R243Q-iPSCs. The STR loci were amplified using the AmpFlSTR^Ⓡ Identifiler^Ⓡ Plus PCR Amplification Kit (Applied Biosystems), following the manufacturer’s protocol. A SimpliAmp^TM PCR Cycler was used to conduct PCR, and the amplified fragments were analyzed by capillary electrophoresis using a 3730XL DNA Analyzer. Alleles were assigned using GeneMapper^Ⓡ Software v5.0, and identity was confirmed by comparing 15 autosomal STR markers and the amelogenin locus.

Immunocytochemistry

Cells were fixed with 4% PFA (Biosesang) for 10 minutes at RT, blocked with 10% donkey serum (Abcam), 1% BSA (ThermoFisher Scientific), and 0.3% Triton X-100 (Sigma-Aldrich) for 1 hour, and incubated with 1st antibodies overnight at 4℃ (Supplementary Table S3). Second antibodies were applied for 2 hours at RT (Supplementary Table S3). Nuclei were counterstained with Hoechst 33342 (ThermoFisher Scientific). Alkaline phosphate staining was done using the Vector^Ⓡ Red Substrate Kit (Vector Labs). Images were acquired with a Nikon Ts2R fluorescence microscope.

Karyotype analysis

A karyotype analysis was conducted at Gendix Incorporated. Cells were treated with 500 μL of colcemid (10 μg/mL, Gibco) and incubated at 37℃ with 5% CO₂ for 1 hour to arrest metaphase. Cells were centrifuged, resuspended in 0.075 M potassium chloride, incubated for 25 minutes at 37℃, and fixed with Carnoy’s fixative. Chromosomes were stained with Giemsa (Sigma-Aldrich) to generate GTG-banded karyotypes and analyzed using ChIPS-Karyo software (GenDix). The karyotype was 46, XY (n=20 metaphases).

Sanger sequencing

The PAH gene R243Q mutation (c.728G>A) was confirmed by Sanger sequencing (ABI 3730xl).

Measurement of phenylalanine hydroxylase levels

PAH was quantified using an ELISA kit (Human Phenylalanine Hydroxylase ELISA Kit, Abbexa, abx152625) according to the manufacturer’s instructions. The sample used was conditioned medium collected from cell cultures. Total protein concentrations in the samples were measured separately using a BCA assay kit (ThermoFisher Scientific), to normalize PAH levels if required, and the sample was measured at an optical density of 450 nm.

Mycoplasma test

Mycoplasma was tested with the MycoStrip^Ⓡ Kit (InvivoGen) (Supplementary Fig. S3).

Results

Reprogramming of PBMCs from a PKU patient into iPSCs

Given the variability in both disease severity and treatment response that is associated with PKU, it is imperative to identify the most prevalent mutation types in Korean patients, thereby facilitating the development of a customized treatment approach. To this end, a genetic analysis was conducted on 204 patients with PKU who visited Soonchunhyang University Hospital. The results of this analysis indicated that 14.7% of the patients had the p.Arg243Gln (R243Q) mutation, followed by IVS4-1G>A and p.Tyr204Cys (Table 1). Therefore, the objective of this study was to generate iPSCs from the peripheral blood of patients with the R243Q mutation and utilize them for subsequent research. To address the need for personalized therapies tailored to specific mutation types, iPSC lines carrying PKU-associated mutations have been established. The underlying process is depicted schematically in Fig. 1A. PBMC samples were obtained from the blood of a PKU patient. Following the isolation of hematopoietic progenitor cells from the patient’s blood, the cells were cultivated in StemSpan^TM SFEM II medium supplemented with Erythroid Expansion Supplement (Fig. 1B). The medium was changed once every two days over a period of 7 days. A population of erythroid progenitor cells with a higher purity was obtained on Day 7 compared to Day 0 (Fig. 1B). On the 7 day, the cells were infected with CytoTune^TM-iPS 2.0 Sendai virus, and the following day, the cells were transferred to a medium coated with Matrigel, after which the attachment of the cells was observed. Subsequently, the medium was substituted with TeSR^TM-E7^TM/ReproTeSR^TM, and the formation of iPSC colonies was observed from Day 13. By Day 19, colonies with distinct morphologies were confirmed (Fig. 1B). Finally, on the 25th day, colonies were manually isolated and subsequently cultured in mTeSR^TM1. On the 28th day, the process of reprogramming iPSCs was successfully completed. The obtained iPSCs were stabilized through subculture. After eliminating the potential confounding effects of residual reprogramming factors, an electrophoresis test was conducted on the Sendai virus (Fig. 1C). RT-PCR was used to amplify each sample, targeting SeV, c-Myc, and Klf4. The positive control confirmed the presence of SeV-derived transgenes. In contrast, the R243Q iPSCs showed no visible band in the electrophoresis result. These results indicated that the generated R243Q-iPSCs were free of exogenous SeV components and suitable for downstream analyses. Moreover, to confirm that the generated iPSCs originated from the same source as the non-reprogrammed R243Q patient-derived primary PBMCs, we performed STR analysis (Table 2, Supplementary Fig. S1, S2). The results demonstrated the presence of identical profiles across all genetic loci. Consequently, the iPSCs that were generated did not undergo genetic mutations during the reprogramming process, thereby indicating the successful generation of R243Q patient-derived iPSCs.

Table 1.

Relative frequencies of PAH mutations in 204 independent Korean PKU patients

Genotype	n (%)	Genotype	n (%)
R243Q	30 (14.7)	S70[del]	4 (2.0)
IVS4-1G>A	27 (13.2)	V399=	3 (1.5)
Y204C	21 (10.3)	IVS10-3C>T	3 (1.5)
Y356*	14 (6.9)	A447P	2 (1.0)
c.442-1G>A	12 (5.9)	N207D	2 (1.0)
A259T	9 (4.4)	R111*	2 (1.0)
R413P	8 (3.9)	c.728G>A	2 (1.0)
R176*	6 (2.9)	c.441-1G>A	2 (1.0)
Y325*	6 (2.9)	exon 5-6 del	2 (1.0)
A345T	5 (2.5)	IVS10-14C>G	2 (1.0)
P281L	5 (2.5)	IVS10-3C>G	2 (1.0)
T278I	5 (2.5)	A202T	1 (0.5)
V388M	5 (2.5)	A408G	1 (0.5)
D84Y	1 (0.5)	R241C	1 (0.5)
G239S	1 (0.5)	R261Q	1 (0.5)
G332Q	1 (0.5)	R261X	1 (0.5)
I306L	1 (0.5)	T266R	1 (0.5)
IVS7-5G>A	1 (0.5)	T278L	1 (0.5)
L115T	1 (0.5)	T325*	1 (0.5)
L255S	1 (0.5)	V423A	1 (0.5)
L48S	1 (0.5)	W187X	1 (0.5)
L95del	1 (0.5)	c.775G>A	1 (0.5)
P69S	1 (0.5)	c.970-3C>G	1 (0.5)
Q419R	1 (0.5)	c.115T＋79	1 (0.5)
R158Q	1 (0.5)	Total	204

Number of patients (%): (%=allele frequency/total subject chromosome).

PAH: phenylalanine hydroxylase, PKU: phenylketonuria.

Fig. 1.

Generation of iPSC from PBMC derived from a PKU patient. (A) The workflow for iPSC generation from a PKU patient’s PBMC. (B) Samples were collected from a 15-year-old Asian male with consent. Cell morphologies were obtained using a bright-field microscope throughout the workflow. Scale bar=100 μm. (C) Detection of residual Sendai virus vectors in iPSCs by RT-PCR (primer: SeV, c-Myc, and Klf4). The positive control RNA was obtained at day 10 during iPSC generation. The R243Q-iPSC sample was obtained at passage 20. iPSC: induced pluripotent stem cell, PBMC: peripheral blood mononuclear cell, PKU: phenylketonuria, RT-PCR: reverse transcription polymerase chain reaction.

Table 2.

STR profile comparison between R243Q patient-derived PBMCs and corresponding iPSC line

Comparison	Sample			Sample		Number of shared alleles (reference & sample)
Marker	R243Q normal PBMC			R243Q-iPSC
	Allele			Allele
AMEL	X	Y		X	Y	2
CSF1PO	10	11		10	11	2
D13S317	8	10		8	10	2
D16S539	10	12		10	12	2
D18S51	15	16		15	16	2
D19S433	13	-		13	-	1
D21S11	32	32.2		32	32.2	2
D2S1338	22	24		22	24	2
D3S1358	14	15		14	15	2
D5S818	12	13		12	13	2
D7S820	9	12		9	12	2
D8S1179	12	13		12	13	2
FGA	21	-		21	-	1
TH01	6	7		6	7	2
TPOX	8	11		8	11	2
vWA	17	-		17	-	1
Allele No.	Total # of reference allele (A)			Total # of sample allele (B)		Total # of shared allele (A)
	29			29		29
% Match	Match algorithm=$\frac{C\times 2}{A+B}$					100%

STR: short tandem repeat, PBMCs: peripheral blood mononuclear cells, iPSC: induced pluripotent stem cell, -: not available.

Evaluation of pluripotency and quality of generated iPSCs

Normal PBMCs, obtained from a patient with PKU, were reprogrammed using a Sendai virus (Fig. 1, 2A, 2B). We established two iPSC cell lines. One is from a PKU patient with the R243Q mutation (R243Q), and the other is from PBMCs of a healthy donor (WT). Both WT and R243Q iPSC lines were validated and exhibited typical morphological features, including the formation of compact, multicellular colonies with well-defined, smooth borders (Fig. 2B). Additionally, they demonstrated the ability to form EBs, which is typical feature of iPSC (Fig. 2C), and exhibited positive Alkaline phosphate activity (Fig. 2D). In addition, all iPSC lines exhibited elevated mRNA expression levels of the pluripotency markers OCT4, SOX2, and NANOG compared to non-reprogrammed PBMCs by qRT-PCR analysis (Fig. 2E). In analyzing the protein expression levels of OCT4 and SOX2 in the iPSCs (Fig. 2F), it was confirmed that the expression levels of OCT3/4 and SOX2 increased in both the WT and R243Q to levels that were similar to those of iPSCs derived from previously produced fibroblasts (11). All iPSCs exhibited positive staining for the pluripotency markers OCT4, SOX2, NANOG, SSEA4, TRA-1-60, and TRA-1-81 (Fig. 2G). A subsequent analysis of the generated iPSC revealed positive staining for OTX2 (ectoderm marker), brachyury (mesoderm), and SOX17 (endoderm) (Fig. 2H). Furthermore, both WT-iPSC and R243Q iPSC exhibited a normal karyotype (46, XY) (Fig. 2I). Finally, it was confirmed that the iPSCs utilized for hepatic differentiation were free of mycoplasma contamination (Supplementary Fig. S3), thereby indicating the suitability of the used cell lines for experimental use. In sum, the generated iPSCs have pluripotency and the capacity for three germ layer differentiation, without genetic mutations.

Fig. 2.

Characterization of the generated iPSC cell lines. (A) Normal PBMCs were generated using blood from a patient with PKU. The WT and R243Q iPSCs exhibited (B) compact, round colonies with defined edges and (C) successfully generated EB in suspension culture, which is a morphological characteristic of iPSCs. (D) WT and R243Q iPSCs showed strong positive Alkaline phosphate staining. (E) The generated iPSC cell lines exhibited significantly higher expression levels of OCT4, SOX2, and NANOG compared to normal PBMCs. Additionally, these cell lines demonstrated a profile consistent with established iPSCs, as determined by qRT-PCR (n≥3). (F) OCT4 and SOX2 protein expression was detected in established iPSCs and in newly generated WT and R243Q iPSC lines. (G) WT and R243Q iPSC were expressed pluripotency marker (green: OCT4, SOX2, NANOG, red: SSEA4, TRA-1-60, TRA-1-81, blue: Hoechst33258). (H) Both lines were positive for the specific markers OTX2 (ectoderm), brachyury (mesoderm), and SOX17 (endoderm). (I) Karyotype analysis confirmed a normal 46, XY chromosomal complement in both WT and R243Q iPSC lines. Scale bar=100 μm. iPSC: induced pluripotent stem cell, PBMC: peripheral blood mononuclear cell, PKU: phenylketonuria, EB: embryoid body, qRT-PCR: quantitative reverse transcription polymerase chain reaction. *p<0.05, **p<0.01, ***p<0.001.

Hepatic differentiation of iPSCs and functional characterization

Hepatocyte differentiation was conducted in 3 steps and depicted in Fig. 3A. In the definitive endoderm, cells become flattened and dispersed (Fig. 3B), and in the hepatoblast stage, the cells adopt a morphology of closely packed epithelial-like clusters. In the hepatocyte stage, cells transformed, becoming enlarged polygonal cells with well-defined borders that resemble human hepatocytes. A1AT, ALB, and AFP are well-known for hepatic markers (12). WT and R243Q hepatocytes strongly expressed these markers compared to each hepatoblast (Fig. 3C). Furthermore, WT and R243Q hepatocytes demonstrated robust cytoplasmic expression of AFP and ALB (Fig. 3D). Expression of HNF4α, a protein known to be expressed in the nucleus of hepatocytes, was also observed in the present experiments (Fig. 3D) (13). Taken together, WT and R243Q iPSCs can be fully differentiated into hepatocytes and exhibit characteristic hepatic features at the mRNA and protein levels.

Fig. 3.

Establishment of a hepatic differentiation protocol and characterization of iPSC-derived hepatocytes. (A) It is the 3-step process for generating hepatocyte-derived iPSCs from WT and R243Q. (B) Both lines can be differentiated into definitive endoderm, hepatoblasts, and hepatocytes within 13 days. iPSC-derived cells exhibited polygonal morphology with a high cytoplasm-to-nucleus ratio, characteristic of hepatocyte cells. (C) Fully differentiated hepatocytes derived from WT and R243Q were significantly more highly expressed hepatocyte markers (A1AT, ALB, and AFP) than hepatoblasts. (D) WT and R243Q iPSC-derived cells expressed hepatocyte markers: HNF4α and ALB (green), AFP (red), and nuclei were counterstained with Hoechst 33258 (blue). Right: quantify the relative expression of HNF4α, ALB, and AFP in a single cell. Scale bar=100 μm. iPSC: induced pluripotent stem cell. ***p<0.001.

iPSC-derived hepatocyte preserved genetic mutation and PKU phenotype

To verify the suitability of the differentiated R243Q hepatocytes as a reliable model of the PKU phenotype, additional validation experiments were conducted. Initially, the WT hepatocyte sequence at codon 243 of the PAH gene is CGA, which encodes arginine (Arg) (Fig. 4A). In contrast, in the R243Q hepatocytes, this codon underwent a substitution of CAA, resulting in a glutamine (Gln) replacement. Hepatic differentiation does not appear to affect the R243Q mutation when confirmed using Sanger sequencing (Fig. 4A). Besides, differentiated R243Q hepatocytes verified whether the R243Q mutation led to a reduction in PAH protein. ELISA was conducted to measure PAH level (Fig. 4B). An equal amount of total protein, as determined through BCA assay, was utilized for subsequent ELISA analysis. The PAH protein expression levels in WT hepatocytes were found to be 13.01±0.034 ng/mL, while in R243Q hepatocytes, these levels were 4.68±0.20 ng/mL. It represents a 2.78-fold reduction, which is approximately 36% of the levels observed in WT hepatocytes.

Fig. 4.

Retention of the R243Q mutation and reduced PAH metabolism in iPSC-derived hepatocytes. (A) Sanger sequencing of hepatocytes derived from R243Q iPSCs confirmed the retention of the c.728G>A (R243Q) mutation, with a codon change from CGA (arginine) to CAA (glutamine). (B) ELISA analysis revealed that R243Q-derived hepatocytes had significantly impaired PAH activity compared to WT hepatocytes. iPSC: induced pluripotent stem cell, PAH: phenylalanine hydroxylase. ***p<0.001.

Discussion

PKU is an example of a congenital disorder of amino acid metabolism that is caused by a genetic deficiency of PAH. This liver-derived enzyme that plays a pivotal role in the conversion of Phe to tyrosine within hepatocytes (14). Given that PKU stems from a deficiency of the liver’s main expressed enzyme (PAH), the development of a system that can recapitulate disease-specific functions at the hepatocyte level and test direct gene correction or cell replacement strategies is imperative (15). Therefore, in this study, iPSCs were successfully induced from PBMCs of a PKU patient with the R243Q mutation. In addition, we confirmed that these cells can be differentiated into hepatocytes, and the PKU-specific phenotypes are also maintained in vitro. This finding verified that the iPSC-derived hepatocytes functioned as a suitable model, capable of recapitulating the genotype-based disease phenotype. These results suggest that they have the potential to serve as an in vitro disease model platform for the development and validation of personalized treatment strategies.

In a previous genetic study of 33 unrelated Korean patients with PKU, a total of 63 mutant PAH alleles were identified, among which p.Arg243Gln (R243Q) accounted for 8 alleles, indicating its status as a recurrent mutation in this population (16). Similarly, in a separate analysis of 79 Korean patients with PKU or hyperphenylalaninemia, 39 distinct PAH mutations were identified, including 10 novel variants (nine missense mutations and one splice-site mutation). Among them, R243Q, IVS4−1G>A, and E6−96A>G were the most frequently observed, together comprising 32% of all mutant alleles (17). Furthermore, R243Q, IVS4−1G>A, and E6−96A>G have been documented as some of the most prevalent mutations in Asian populations (17). In our study, R243Q also emerged as the most frequent mutation observed in the Korean PKU genotype database. This consistency with prior reports underscores the clinical relevance and high prevalence of the R243Q mutation in East Asian populations. Accordingly, the iPSC-derived hepatocyte model established in this study, based on the homozygous R243Q genotype, provides a clinically representative and genetically meaningful in vitro platform for modeling PKU and evaluating genotype-specific therapeutic strategies.

The therapeutic efficacy of PKU is associated with the PAH genotype, which influences the outcome of BH4 supplementation, enzyme substitution, and gene therapy (17-21). For instance, classic PKU, including R243Q, exhibits limited response to standard treatments and may necessitate more aggressive interventions. Conversely, mild mutations are indicative of residual enzyme activity and may be sufficient for dietary modifications or cofactor therapy. These results emphasize the necessity of novel research approaches targeted towards specific diseases and patient mutations to evaluate genotype-based treatment strategies. The homozygous R243Q iPSC-derived hepatocyte model established in this study is a representative example of a mutation-specific in vitro platform that meets this need.

Especially, the expression level of PAH protein in R243Q hepatocytes was approximately 36% of the wild-type level, as measured by ELISA. This result is consistent with previously reported average PAH protein expression in classical PKU variants, which was 35.6% relative to wild-type controls (22). These results support the physiological relevance of our in vitro model. Although Himmelreich’s studies have reported near-absent PAH levels (<5%) for homozygous R243Q mutants, such variation may arise from differences in detection methodology (western blot, ELISA). Moreover, our model utilizes iPSC-derived hepatocytes, while in prior studies, COS-7 monkey kidney cells were used. Taken together, these findings support the validity of our platform for modeling severe PKU phenotypes and for evaluating genotype-specific therapeutic responses.

In summary, the present study established an in vitro disease model that reflects the classical PKU phenotype by differentiating PBMC-derived iPSCs from a PKU patient with the homozygous R243Q genotype into hepatocyte. This model provides a mutation-specific cellular environment that can reflect genotype-specific therapeutic responsiveness and has high potential as a preclinical testing platform for drug screening and gene therapy efficacy evaluation. Specifically, the implementation of a 3D culture system through the production of liver organoids establishes a physiologically sophisticated environment that has the potential to supersede existing animal models and enhance the precision of assessments of organ function and drug response. Subsequent studies employing technologies such as co-culture with nonparenchymal liver cells will further strengthen the clinical translation potential of this model. Consequently, this model can function as a valuable framework for the development and validation of gene editing-based precision therapeutic strategies for PKU.

Supplementary Materials

Supplementary data including three tables and three figures can be found with this article online at https://doi.org/10.15283/ijsc25073.