Effect of M694V mutation in the MEFV gene, associated with Familial Mediterranean fever, on the morphology of iPSC-derived macrophages

1. Introduction

Familial Mediterranean fever (FMF) is a systemic autoinflammatory disorder characterized by recurrent episodes of fever and polyserositis (e.g. peritonitis, pleuritis, synovitis) symptoms. The FMF carrier frequencies are high in several eastern Mediterranean populations, ranging from 37–39% in Armenians and Iraqi Jews, to 20% in Turks, North African and Ashkenazi Jews, and Arabs which leads to a significant economic burden [1,2]. The disease is mostly caused by recessively inherited mutations in MEFV, which encodes pyrin protein playing an important role in inflammatory processes [3]. There are two “mutation hot-spots” located in the 2nd (E148Q) and 10th (M694V, M694I, M680I и V726A) exons. These mutations account for over 90% of all FMF cases [4]. Mutated pyrin causes an exaggerated inflammatory response by uncontrolled interleukin-1 (IL-1) secretion [5]. Besides the advances in molecular genetics of FMF, the molecular mechanisms underlying the disease are not fully understood. These questions have been studied using a battery of experimental and in silico methods. Thus, molecular dynamic simulations gain insight into the role of mutations on pyrin structure, function, and interactions [6,7]. Another study of polymorphonuclear neutrophils of FMF patients suggests increased sensitivity of mutated pyrin inflammasome towards cytoskeletal modifications in the absence of pathogens [8]. A recent study using different cell types (synovial fibroblasts, monocytes, macrophages) showed that inflammation-related functional assays have an anti-inflammatory effect of miR-197-3p [9]. Various cell-line-based models have been developed for a more comprehensive understanding of the etiology and pathogenesis of FMF [10]. Also, gene editing with CRISPR/Cas9 has been used to understand the effect of the MEFV E583A mutation on IL-1β secretion [11]. However, immortalized cell lines have their limitations in mimicking the disease of interest since they do not account for patient genetic variability [12], may accumulate mutations and lack genetic and cellular diversity, and mostly represent cancer-derived cells [13]. On the other side, patient primary cells have limited potential for cultivation and maintenance, posing limitations for experiments.

Induced pluripotent stem cells (iPSCs) are considered a unique tool for studying molecular genetic mechanisms of this disease, disease modeling, and screening of potential drugs [14,15,16]. The main advantage of iPSCs is the almost unlimited ability of cultivation and differentiation serving as a proper source of pluripotent stem cells and any type of cells in the living organism. IPSCs were successfully used for studying autoinflammatory [17,18], neurodegenerative [17,18,19,20,21,22,23,24], and other diseases. There are few attempts to create iPSCs for FMF patients, for example, a Turkish patient with a homozygous missense mutation (p.Met694Val) in the MEFV gene [25].

In this paper, we generated iPSCs from an Armenian FMF patient carrying a homozygous c.2080A>G (M694V) mutation in the MEFV gene. Molecular-genetic characterization proved their stemness characteristics. We further differentiated these cells into macrophage-like cells. The morphology analysis showed considerable differences between derived macrophages with mutated MEFV gene compared with macrophages derived from iPSCs with wild-type MEFV [26].

2. Results

2.1. Generation and Characteristics of iPSCs Associated with the MEFV Gene Mutation

A 20-year-old patient was admitted to the Rheumatology Department of Mikayelyan University Hospital with symptoms relevant to mixed thoracoabdominal form of FMF, pain in joints, arthritis, erysipeloid erythema, and fever. Genetic analysis of the patient revealed pathogenic homozygous missense mutation c.2080A>G (p.M694V, rs61752717) in exon 10 of the MEFV gene. We isolated peripheral blood mononuclear cells (PBMCs) in a Ficoll gradient and reprogrammed them using episomal vectors OCT4, KLF4, L-MYC, SOX2, LIN28, and Trp53 [27]. As a result, 10 independent cell lines were obtained, one of which was characterized in detail. All resulting cell lines have a large nuclear-cytoplasmic ratio, grow in densely packed iPSC-like single-layer colonies (Figure 1A), and express the early stem cell marker endogenous alkaline phosphatase (Figure 1B). Cultivation of the resulting cells was carried out on a substrate of mitotically inactivated mouse embryonic fibroblasts (MEF).

One cell line was selected for detailed characterization and was registered in the Human Pluripotent Stem Cell Registry (hPSCreg, https://hpscreg.eu, accessed on 14 March 2024) under the name RAUi002-A. We carried out qualitative (immunofluorescence) and quantitative (RT-qPCR) analyzes of this line for markers of pluripotent cells. Both analyzes demonstrate the presence in cells obtained from a patient with FMF, associated with the genetic variant c.2080A>G (M694V) in the MEFV gene, expression of the transcription factors OCT4, SOX2, and NANOG (Figure 1C,D), as well as immunofluorescence showing the expression of surface marker SSEA-4 (Figure 1C). Cytogenetic analysis (G-banding) of the obtained cells showed the presence of a normal karyotype (46,XY) (Figure 1E).

One of the main tests of pluripotency is the ability of cells to give rise to all three germ layers (ectoderm, mesoderm, and endoderm). We performed spontaneous differentiation in embryoid bodies (Figure 1F) and used immunofluorescence analysis of differentiated cells to show the expression of mesoderm markers (α-smooth muscle actin (αSMA) and the surface marker CD29), ectoderm (tubulin β 3 (TUBB3/TUJ1) and mature neural cell markers methionine aminopeptidase 2 (MAP2)), and endoderm (alpha-fetoprotein (AFP) and cytokeratin 18 (CK18)) (Figure 1G). Thus, we have demonstrated that the resulting cells are pluripotent, and we will refer to them as iPSCs.

To confirm the presence of a pathogenic mutation in the resulting iPSC line, Sanger sequencing of DNA isolated from the patient's PBMCs and from the RAUi002-A iPSC line was performed and compared to DNA from a conditionally healthy patient. We found a substitution at position 2080 A to G in exon 10 of the MEFV gene in both samples compared to control DNA (Figure 1H, location of substitution indicated by arrow). In addition, to confirm the origin of the iPSCs derived from the patient's PBMCs, STR analysis was performed on the DNA of the PBMC sample and the RAUi002-A line. 25 loci from both samples were analyzed and their identity was shown (data available on request from the authors). This suggests that iPSCs data obtained from an FMF patient can serve as a tool to study the contribution of the p.M694V mutation in the MEFV gene to the pathogenesis of FMF disease.

RAUi002-A iPSCs were also analyzed for the presence/absence of residual episomes and culture contamination with mycoplasma. Both PCR analyses showed their complete absence (Figure 1I).

Summary characteristics of the RAUi002-A iPSC line are shown in Table 1.

2.2. Generation and Characteristic of Macrophages from RAUi002-A iPSCs

The resulting iPSC line was specifically differentiated into macrophages to obtain a relevant cell type for further studies on the pathogenesis of FMF. The previously obtained iPSC line K7-4Lf/ICGi022-A was used as a control cell line in the experiment [26]. Differentiation of iPSCs into macrophages was achieved by adding cytokines such as interleukin-3 (IL-3) and macrophage colony-stimulating factor (M-CSF) to the differentiating embryoid bodies (Figure 2A,B) to differentiation along the myeloid pathway and form a homogeneous population of monocytes. As a result, from day 14 of differentiation and over 3 weeks, monocytes were produced in the culture medium that adhered to the plastic and terminally differentiated into macrophage-like cells in the presence of M-CSF. Immunofluorescence for markers specific for mature macrophages, CD14 and CD45, confirmed that the resulting cells were macrophages (Figure 2). IPSC-derived macrophages from a healthy donor were found to have a classic cloaked, spreading morphology (Figure 2C), whereas macrophages with the pathogenic p.M694V mutation in the MEFV gene had an elongated morphology with many rounded cells (Figure 2D, middle photo, white arrows).

3. Discussion

In this study, we used the technology of reprogramming PBMCs into a pluripotent state to obtain patient-specific iPSCs from a patient with FMF associated with the pathogenic mutation p.M694V in the MEFV gene. The resulting cell line meets all the requirements of pluripotent cells, has a stem cell-like morphology, a normal karyotype, and is capable of producing derivatives of three germ layers. These cells demonstrated their ability to differentiate into macrophages which are one of the key cells involved in the disease pathogenesis [28].

Research related to the establishment of patient-derived iPSCs is expected to be a promising avenue for elucidating the pathogenesis of the diseases, disease therapy, and for drug discovery [29]. They became attractive tools for studying neurodegeneration [21,22,23,24,30], cardiac dysfunction [31,32,33], and genetic disorders, such as Duchenne’s muscular dystrophy [34]. Recently these approaches have been actively used for modeling immune-related diseases, such as systemic lupus erythematosus, systemic sclerosis, rheumatoid arthritis, and autoinflammatory syndromes (for review see in [35]). It has been shown that various cell types differentiated from patient-derived iPSCs can be further used for research into the pathogenesis of these diseases.

To our knowledge, a few attempts to generate iPSCs from FMF patients have been made [14,25]. Fidan et al. (2015) reported a cell line derived from fibroblasts of an FMF patient carrying homozygous p.Met694Val mutation in the MEFV gene [25]. In our study, we successfully reprogrammed PBMCs of FMF patient with homozygous MEFV gene mutation (M694V). This method is less invasive for the patients. Moreover, we performed the differentiation of stem cells into macrophages and analyzed morphological differences between healthy and diseased macrophages. The morphology of macrophage-like cells derived from control iPSCs was significantly different from that of cells derived from iPSCs with a mutation in the MEFV gene. Control macrophage-like cells had a flattened morphology, whereas patient-derived cells had an elongated morphology with a large number of rounded, dying cells. We saw that under the same culture conditions, macrophages with a mutation in the MEFV gene are less viable, most likely due to a pathogenic mutation. These results are well aligned with the previous observations about the structural and functional features of FMF patients’ primary immune cells. Thus, studies indicated characteristics of aged/activated cells (small cell size and granularity, up-regulated CXCR4) for polymorphic neutrophils from the patients in acute flares, while in remission mixed morphology (normal cell size and granularity, up-regulated CD11b, CD49d, CXCR4, and CD62L) has been described [8].

One of the advantages of iPSC-derived macrophages is the preservation of the initial phenotype of the cells. In previous research, it has been shown that iPSC-derived cells at different stages of differentiation demonstrate a complete switch of iPSCs to cells expressing a monocyte, macrophage, or dendritic cell-specific gene profile. Moreover, iPSC-derived LPS-induced macrophages induce the expression of classic macrophage pro-inflammatory response markers [36]. Moreover, the ability to grow an unlimited number of iPSCs and differentiate them into various cells opens multiple avenues for studying FMF pathogenesis, performing drug candidate screening, and developing gene-based therapies. Using patient-specific iPSCs from FMF patients and the CRISPR/Cas9 genome editing system, it will be possible to generate modified isogenic iPSC lines with the corrected mutation in the future. Such cell platforms will be valuable in understanding the effects of the mutations on pyrin inflammasome dysfunction in FMF.

4. Materials and Methods

4.1. Ethics Statement

The study was approved by the Ethics Committee of the Institute of Molecular Biology NAS RA (IRB 00004079, Protocol N3 from 23.08.2021). A patient provided informed consent about the use of the blood sample for planned analysis. ICGi022-A iPSC cell line obtained from a healthy donor [26] was used as a control in the experiments of macrophage differentiation and analysis of the morphological features of mutant and wild-type MEFV-carrying cells.

4.2. Detection of MEFV Mutation

Mutations in the MEFV gene in the FMF patient was determined by commercially available qPCR assay for 26 most common mutations (FMF Multiplex real-time CPR kit, SNP Biotechnology RnD Ltd, Turkey).

4.3. Reprogramming of PBMCs into iPSCs

PBMCs of a patient with FMF were isolated as described here [22]. iPSCs were obtained by overexpression of reprogramming factors OCT4, KLF4, L-MYC, SOX2, LIN28, and Trp53 using a set of episomal vectors (ID Addgene #41855–58, #41813–14) as previously described [22].

IPSCs were propagated onto feeder layer of mitotically inactivated mouse embryonic fibroblasts (MEF) in iPSC-medium: 82% KnockOut DMEM medium, 15% KoSR, 2 mM Gluta-MAX, 100 U/ml penicillin-streptomycin, 0.1 mM MEM NEAA (all Thermo Fisher Scientific, Waltham, MA, USA), 0.1 mM β-Mercaptoethanol (Sigma-Aldrich, Darmstadt, Germany), 10 ng/ml basic FGF (SCI Store, Moscow, Russia).

IPSCs were passaged using TrypLE Express (Thermo Fisher Scientific, Waltham, MA, USA), splitting 1:10 in the iPSC medium with the addition of 2 µM Thiazovivin (Sigma-Aldrich, Darmstadt, Germany) for the first 24 hours.

4.4. In Vitro Spontaneous Differentiation of RAUi002-A into Three Germ Layers

Differentiation capacity of the iPSCs was estimated by spontaneous differentiation in embryoid bodies as described earlier [37].

4.5. Immunofluorescent Staining of RAUi002-A iPSC Line

For immunofluorescence staining, cells growing on Chambered Coverglass 8-well plates (Thermo Fisher Scientific, Waltham, MA, USA) were fixed with 4% PFA (Sigma-Aldrich, Darmstadt, Germany), permeabilized with 0.5% Triton-X (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 30 minutes, and incubated in blocking buffer containing 1% BSA (Sigma-Aldrich, Darmstadt, Germany) in PBS at room temperature. Primary antibodies were diluted in a blocking buffer according to Table 2. Cell preparations were incubated with primary antibodies overnight at +4°C. Preparations were washed with PBS twice for 15 min, and secondary antibodies were added for 1.5 hours at room temperature. After incubation, cell preparations were washed twice with PBS and stained with DAPI. Manufacturers, catalog numbers and dilutions of all used antibodies are listed in Table 2. The preparations were analyzed using Nikon Eclipse Ti-E microscope and NIS Elements software.

4.6. qPCR Analysis of Expression of Pluripotency Markers in RAUi002-A iPSC Line

For RNA isolation, 2*10⁶ cells were lysed in 1 ml TRIzol reagent (Ambion by Life technologies, Carlsbad CA, USA), and processed according to the manufacturer's protocols. The cDNA was synthesized by reverse transcription of 1 μg RNA using M-MuLV reverse transcriptase (Biolabmix, Novosibirsk, Russia).

Quantitative PCR (qPCR) was performed on a LightCycler 480 II system (Roche, Basel, Switzerland) using BioMaster HS-qPCR SYBR Blue 2 × (Biolabmix, Novosibirsk, Russia) with the following program: 95 °C 5 min; 40 cycles: 95 °C 10 s, 60 °C 1 min. The primers used are listed in Table 2. qPCR reactions for each sample were run in triplicate. CT values of the samples for NANOG, OCT4, and SOX2 expression were normalized to beta-2-microglobulin (B2M), and the results were processed using the ΔΔCT method.

4.7. Karyotyping of RAUi002-A iPSC Line

Karyotype analysis was performed as described earlier [22]. For chromosome banding, samples were stained with DAPI (4,6-diamino-2-phenylindole) solution (200 ng/mL, in 2xSSC) for 5 minutes, rinsed in 2xSSC buffer and water. Air-dried slides were covered with 7-10 μL antifade (Vector, United States) under a coverslip. Analysis of preparations was performed using an Axioplan 2 microscope (Zeiss, Germany) equipped with a CV-M300 CCD camera (JAI Corp., Japan) at the Center for Collective Use of Microscopic Analysis of Biological Objects at the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences. ISIS 5 software (MetaSystems Group, Inc., United States) was used for metaphase processing and chromosome folding.

4.8. Genotyping of RAUi002-A iPSC Line

Sanger sequencing was used to confirm the mutation in the MEFV gene in the RAUi002-A iPSC line. To confirm the absence of MEFV mutations, Sanger sequencing was performed also for the line K7-4Lf/ICGi022-A used as a control sample. The list of primers used is shown in Table 2. Genome DNA was isolated using Quick-DNA Miniprep Kit (Zymo Research, Irvine, CA, USA). PCR reactions were run on a T100 thermal cycler (Bio-Rad) using BioMaster HS-Taq PCR-Color (2×) (Biolabmix, Novosibirsk, Russia) with the program: 95 ◦C, 3 min; further 35 cycles: 95 ◦C, 30 s; 60 ◦С, 30 s; 72 ◦C, 30 s; and 72 ◦C, 5 min. For Sanger sequencing, we used BigDye Terminator V. 3.1. Cycle Sequencing Kit (Applied Biosystems, Austin, TX, USA). Sequencing reactions were analyzed on an ABI 3130XL genetic analyzer at the Genomics Center of the SB RAS (http://www.niboch.nsc.ru/doku.php/corefacility, accessed on 14 March 2024).

STR profiling was performed using AmpFlSTR Identifiler (Applied Biosystems) and Investigator HDplex (QIAGEN) by Genoanalytica company (https://www.genoanalytica.ru, accessed on 14 March 2024).

4.9. Detection of Mycoplasma and Reprogramming Vectors in RAUi002-A iPSC Line

The presence of episomal reprogramming vectors and mycoplasma contamination was assessed by PCR (95°C, 5 min; 35 cycles: 95°C, 15 s, 62°C, 15 s, 72°C, 20 s) using primers listed in Table 2 [27,38].

4.10. Differentiation of RAUi002-A iPSC Line into Macrophages

Differentiation of iPSCs into macrophages was performed according to a previously published protocol [39] with modifications. IPSCs were placed on a Petri dish (D60 mm) coated with mitotically inactivated MEFs. Dense iPSC colonies were detached with 0.15% collagenase type IV (Thermo Fisher Scientific, Waltham, MA, USA), washed with medium, and transferred to a Petri dish (D60 mm) coated with 1% agarose (Sigma-Aldrich, Darmstadt, Germany) in iPSC medium without the addition of bFGF. On day 4 of culture, the formed embryoid bodies were transferred to 3 wells of a 6-well plate coated with 0.1% gelatin (Sigma-Aldrich, Darmstadt, Germany) for spreading and differentiation into monocyte-like cells in RPMI medium supplemented with 10% fetal bovine serum, 2 mM GlutaMax, 100 U/ml penicillin-streptomycin, 0. 1 mM MEM NEAA, 1 mM sodium pyruvate (all Thermo Fisher Scientific, Waltham, MA, USA), 0.1 mM 2-mercaptoethanol (2-mce, Sigma-Aldrich, Darmstadt, Germany), 25 ng/ml IL-3 and 100 ng/ml M-CSF (both SCI Store, Moscow, Russia). From day 14-19 of culture, the cell suspension was collected from embryoid bodies containing monocyte-like cells, centrifuged at 300 g for 5 min, and seeded onto Chambered Coverglass 8-well plates pretreated with 0.1% gelatin for immunofluorescence staining.