Introduction
Fabry disease (OMIM: 301500) is a common X-linked lysosomal storage disorders, which is caused by a deficiency of α-Galactosidase A (GLA) [1]. The defect in GLA activity is caused by genetic mutations in the GLA gene on Xq22.1. Loss of GLA activity caused by a GLA gene mutation leads to progressive accumulation of globotriaosylceramide (Gb3) and its derivatives throughout the body, which leads to various complications, such as angiokeratomas, kidney diseases, cardiovascular diseases, and neuropathy [2]. Until now, several therapies for treating Fabry disease have been applied to the Fabry disease patients. Among the multiple therapeutic strategies developed for the management of Fabry disease, enzyme replacement therapy (ERT) with recombinant human GLA, such as agalsidase alfa (Replagal®) and agalsidase beta (Fabrazyme®), has been the most extensively implemented approach, demonstrating efficacy in reducing Gb3 accumulation and alleviating clinical symptoms [3,4]. However, ERT has notable limitations, such as the requirement for lifelong intravenous infusions, high treatment costs, immune responses against the exogenous enzyme, and insufficient enzyme delivery to critical organs like the heart and kidneys [5]. Additionally, ERT does not completely prevent disease progression, particularly in patients with advanced organ involvement. To overcome these challenges and to gain a deeper understanding of Fabry disease pathophysiology, patient-derived human induced pluripotent stem cells (hiPSCs) have emerged as a valuable model system. hiPSC-derived models recapitulate patientspecific genetic backgrounds and provide a renewable source of disease-relevant cell types, facilitating studies on disease mechanisms and therapeutic development. Furthermore, advances in genome editing technologies, such as CRISPR/Cas9, have enabled not only the correction of pathogenic mutations but also the introduction of disease-causing genetic alterations, allowing for the generation of disease models without direct reliance on patient-derived samples [6]. In a previous report, we have demonstrated that vascular endothelial cells (ECs) differentiated from Fabry disease patient-specific hiPSCs exhibit functional impairments, in part due to enhanced thrombospondin-1 expression [7]. Furthermore, CRISPR/ Cas9-mediated generation of GLA-knockout (GLA-KO) hiPSCs has provided a genetically defined platform for studying Fabry disease without the variability of patient-derived lines [8]. Based on these findings, in the present study, we generated GLA-KO hiPSCs and differentiated them into cardiovascular lineage cells, including vascular ECs and cardiomyocytes (CMs). We then analyzed whether these GLA-KO hiPSC-derived cells could faithfully recapitulate key pathological features of Fabry disease. This isogenic, patient-independent in vitro model provides a new tool to dissect the cellular mechanisms underlying Fabry disease and to explore potential therapeutic interventions targeting cardiovascular complications.
Materials and Methods
1. hiPSC culture and genome editing
The hiPSC line (hFSiPS1) was obtained from the National Stem Cell Bank of Korea and maintained in mTeSR1 medium (STEMCELL Technologies) on Matrigel-coated plates under standard conditions (37℃, 5% CO2) [9]. Cells were passaged every 5–6 days using ReLeSR (STEMCELL Technologies). GLA-KO hiPSCs were generated by transfecting 5 μg of Cas9/ sgRNA plasmid as described previously [8]. Clones harboring a 10-base pair (bp) deletion in the GLA gene were validated by Deep-sequencing and then Sanger sequencing. Deep sequencing-based analysis of genome edited clones was conducted with support from the Center for Genome Engineering at the Institute for Basic Science (IBS) in Daejeon, Republic of Korea.
2. Endothelial differentiation and purification
hiPSCs were differentiated into ECs using a 10-day protocol. Cells were treated with Activin A (120-14P; PeproTech), BMP4 (120-05ET; PeproTech), and CP21R7 (S7954; Selleckchem) (#72112; STEMCELL Technologies) (days 0–3), followed by VEGF-A (100-20; PeproTech), bFGF (100-18B; PeproTech), and forskolin (#72112; STEMCELL Technologies) (days 3–5), and maintained in EGM-2 (C-22111; PromoCell) supplemented with VEGF-A and bFGF (days 5–10). On day 10, CD31 and CD144 expression were assessed by flow cytometry using PE-anti-CD31 (555446; BD Biosciences) and Alexa 647-anti- CD144 (561567; BD Biosciences). CD144+ cells were enriched by CD144 (VE-cadherin) MicroBeads (130-097-857; Miltenyi Biotec) for functional assays, including tube formation on Matrigel (354277; Corning).
3. Cardiomyocyte differentiation
hiPSCs were plated on Matrigel in mTeSR1 with Y-27632 (72308; STEMCELL Technologies), and cardiac differentiation was induced using 12 μM CHIR99021 (SML1046; Sigma- Aldrich) (day 0), followed by 1% B27 minus insulin (A1895601; Gibco) (days 1–2), 5 μM IWP-2 (I0536; Merk) (days 3–4), and full B27 (day 7 onward). Beating CMs were observed by days 8–10 and used after day 14 for analyses.
4. Flow cytometry
Single-cell suspensions were stained with PE-anti-CD31 and Alexa 647-anti-CD144 and analyzed on a BD Aria III cytometer. Data was processed using FlowJo software.
5. α-Galactosidase A enzymatic activity
GLA activity in ECs was measured using an α-Galactosidase Activity Assay Kit (K407-100; BioVision) according to the manufacturer’s instructions.
6. Immunocytochemistry and cell size measurement
Day 30 CMs were fixed in 4% paraformaldehyde, permeabilized, and stained with rabbit anti-cardiac troponin T (cTnT) (ab45932; Abcam) and mouse anti-nuclear factor of activated T cells, cytoplasmic 4 (NFATC4) (sc-271597; Santa Cruz Biotechnology), followed by Alexa Fluor-conjugated secondary antibodies and DAPI. Fluorescence images were acquired using a Nikon Eclipse Ti confocal microscope. Cell size was measured from cTnT-labeled cells using ImageJ.
7. Quantitative reverse transcription polymerase chain reaction
Total RNA was extracted using TRIzol, and cDNA was synthesized using GoScript RT System (A6101; Promega). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed with KAPA SYBR® FAST qPCR Kit (KK4602; Kapa Biosystems) on a StepOnePlus system (Applied Biosystems). Data were normalized to GAPDH and analyzed using the 2–ΔΔCt method. Primer sequences used for qRT-PCR are listed in Table 1.
8. Reactive oxygen species measurement
Mitochondrial and cytoplasmic reactive oxygen species (ROS) were detected using 5 μM MitoSOX Red (M36008; Thermo Fisher Scientific) and CellROX Green (C10444; Thermo Fisher Scientific), respectively. Cells were imaged in real time using the Incucyte® Zoom system (Sartorius), and fluorescence intensity was quantified using Incucyte software.
9. Statistical analysis
All quantitative data are expressed as mean ± standard deviation. Statistical significance between two groups was evaluated using an unpaired two-tailed Student’s t-test. A minimum of three independent biological replicates (n ≥ 3) was included for each condition. All analyses were performed using GraphPad Prism (version 9.0), and p -values less than 0.05 were considered statistically significant.
Results
1. GLA-KO hiPSC-derived endothelial cells retain differentiation potential but lack GLA enzymatic activity
To investigate the impact of GLA deficiency on vascular EC function, we utilized a previously established hiPSC line (hFSiPS1) harboring a 10-bp deletion in exon 3 of the GLA gene, introduced via CRISPR/Cas9-mediated genome editing (Fig. 1A and 1B). Sanger sequencing subsequently confirmed the presence of a hemizygous 10-bp deletion, resulting in a frameshift mutation and disruption of the GLA coding sequence (Fig. 1C). Following genome editing, GLA-KO hiPSCs were differentiated into vascular ECs using a defined stepwise protocol (Fig. 1D). Flow cytometry analysis of the endothelial markers CD31 and CD144 revealed that approximately 45% of differentiated cells in both wild-type (WT) and GLA-KO hiPSCderived populations were CD31/CD144 double-positive (Fig. 1E), indicating that GLA deficiency does not impair endothelial differentiation efficiency. However, enzymatic assays revealed a complete loss of GLA activity in GLA-KO hiPSC-derived ECs, whereas WT hiPSC-derived ECs retained robust enzyme activity (Fig. 1F). Together, these results demonstrate that while GLA deficiency does not affect the endothelial differentiation potential of hiPSCs, it fully abrogates GLA enzymatic function. This system thus provides a genetically defined and functionally validated in vitro model to study Fabry disease-associated vascular pathology.
2. Hypertrophic phenotype in cardiomyocytes derived from GLA-KO hiPSCs
Given that left ventricular hypertrophy is a hallmark clinical feature of Fabry disease, we next differentiated WT and GLA-KO hiPSCs into CMs using a chemically defined monolayer protocol (Fig. 2A). To assess hypertrophic changes, we measured the cell size of day 30 CMs. Immunofluorescence staining for cTnT showed that GLA-KO hiPSC-derived CMs exhibited significantly increased cell size compared to WT counterparts (Fig. 2B and 2C). To further investigate hypertrophic signaling, we examined the localization of NFATC4 ―a transcription factor that translocates into the nucleus in response to hypertrophic stimuli. In GLA-KO hiPSC-derived CMs, NFATC4 was predominantly nuclear, whereas in WT hiPSC-derived CMs, it remained cytoplasmic (Fig. 2D, yellow arrow). These findings indicate that GLA deficiency induces a hypertrophic phenotype in hiPSC-derived CMs, recapitulating features of Fabry cardiomyopathy.
3. GLA-deficient endothelial cells exhibit impaired angiogenic function and elevated oxidative stress
To assess the functional impairment caused by GLA deficiency in ECs, we compared the morphology of WT and GLAKO hiPSC-derived ECs under phase-contrast microscope. Both cell types exhibited a typical cobblestone-like appearance, with no overt morphological differences (Fig. 3A, upper panel). However, when subjected to a Matrigel-based tube formation assay, GLA-KO ECs displayed a markedly impaired ability to form tube-like vascular structures compared to WT ECs, indicating compromised angiogenic function (Fig. 3A, lower panel). To elucidate potential mechanisms underlying this dysfunction, we assessed the expression of inflammatory markers via qRTPCR. GLA-KO ECs exhibited significantly elevated expressions of ICAM1, VCAM1, and SELE (Fig. 3B), suggesting the activation of a pro-inflammatory phenotype. Since inflammation is closely linked to oxidative stress, we evaluated intracellular ROS levels using MitoSOXTM Red and CellROXTM staining. Both mitochondrial and cytoplasmic ROS levels were substantially elevated in GLA-KO ECs relative to WT ECs (Fig. 4A and 4B). Taken together, these findings support a model in which GLA deficiency leads to the lysosomal accumulation of Gb3, triggering excessive production of mitochondrial and cytoplasmic ROS. The resulting oxidative stress amplifies pro-inflammatory signaling cascades, thereby contributing to vascular endothelial dysfunction (Fig. 5).
Discussion
Although clinical manifestations of Fabry disease span various organ systems, including the kidneys, heart, and nervous system, cardiovascular complications are particularly critical and contribute significantly to disease morbidity [10]. In this study, we established a GLA-KO hiPSC model via CRISPR/ Cas9 genome editing tool, and differentiated it into two major cardiovascular lineages to investigate the cellular phenotypes associated with Fabry disease. Our findings demonstrate that GLA-KO hiPSC-derived cardiovascular cells recapitulate key pathological features of the disease, including endothelial dysfunction and CM hypertrophy. Importantly, our data suggest a mechanistic cascade in which Gb3 accumulation promotes ROS production, which in turn amplifies inflammatory signaling and disrupts endothelial homeostasis. These phenotypes are consistent with previous clinical and preclinical findings implicating vascular inflammation and redox imbalance in Fabry pathogenesis [11,12]. Recent literature further supports this link, demonstrating that lysosomal Gb3 accumulation can trigger oxidative stress through NOX2 activation and mitochondrial dysfunction, ultimately impairing autophagy and intracellular trafficking. These interconnected pathways are increasingly recognized as contributors to cardiovascular remodeling and inflammation in Fabry disease [13]. This cellular stress response, including mitochondrial ROS production and autophagic impairment, may in turn contribute to downstream functional consequences such as vascular stiffness, leukocyte adhesion, and eventual microvascular dysfunction―features commonly observed in Fabry patients [14]. This pathophysiological sequence is summarized in our proposed model (Fig. 5) and aligns with accumulating evidence that Fabry disease is not solely a storage disorder, but also a disease of secondary cellular stress responses. In parallel, GLA-KO hiPSC-derived CMs exhibited increased cell size and nuclear translocation of NFATC4, a transcription factor activated during pathological hypertrophy. These results mirror clinical observations of left ventricular hypertrophy in Fabry patients and support the use of patient-independent isogenic models for dissecting Fabryassociated cardiomyopathy mechanisms [15,16]. Given the limitations of current therapies in reversing cardiac pathology, this model offers a valuable translational platform for mechanistic investigation and preclinical drug screening. Nevertheless, this study has several limitations. The 2D in vitro system does not fully recapitulate the complex multicellular and biomechanical environment of human tissues. Moreover, the analysis was limited to two cardiovascular cell types, whereas Fabry disease affects multiple organ systems. Finally, physiological factors such as shear stress and chronic metabolic cues were not included but will be important for future model refinement. Despite these limitations, our isogenic GLA-KO hiPSC-based system provides a robust and versatile platform for modeling Fabry disease. It enables mechanistic dissection of disease-driving processes such as oxidative stress, inflammation, and hypertrophy, and represents a valuable tool for developing and testing new therapeutic strategies, particularly those aimed at targeting the cardiovascular complications of Fabry disease.
