KIT D816V Mast Cells Derived from Induced Pluripotent Stem Cells Recapitulate Systemic Mastocytosis Transcriptional Profile

Mast cells (MCs) represent a population of hematopoietic cells with a key role in innate and adaptive immunity and are well known for their detrimental role in allergic responses. Yet, MCs occur in low abundance, which hampers their detailed molecular analysis. Here, we capitalized on the potential of induced pluripotent stem (iPS) cells to give rise to all cells in the body and established a novel and robust protocol for human iPS cell differentiation toward MCs. Relying on a panel of systemic mastocytosis (SM) patient-specific iPS cell lines carrying the KIT D816V mutation, we generated functional MCs that recapitulate SM disease features: increased number of MCs, abnormal maturation kinetics and activated phenotype, CD25 and CD30 surface expression and a transcriptional signature characterized by upregulated expression of innate and inflammatory response genes. Therefore, human iPS cell-derived MCs are a reliable, inexhaustible, and close-to-human tool for disease modeling and pharmacological screening to explore novel MC therapeutics.


Introduction
Mast cells (MCs) are rare but key cells of the hematopoietic system with a pivotal role in innate and adaptive immune responses [1,2]. They act as sentinels of the immune system and are frequently located in tissues, which are directly exposed to environmental challenges, such as the gastro-intestinal, respiratory, urogenital tracts, and skin [3]. Following activation, MCs modulate immune responses by secreting a vast number of mediators, including cytokines, chemokines, proteases among others, or by direct cell-cell contact [4]. As a result, MCs efficiently respond to infection or injury by recruiting and modulating the activity of effector cells. However, MCs are still a rather poorly studied cell of the hematopoietic system and the origin of MC precursors during embryogenesis and in the adult are currently being explored [5,6]. Recently, independent studies showed that peripheral blood circulating CD34 + KIT + FcER1 + cells are MC precursors that can be differentiated in vitro into mature functional MCs [7][8][9].
In line with the pleiotropic functions of MCs, their abnormal proliferation, infiltration, and accumulation in organs, such as the spleen, liver, skin, or bone marrow (BM), drives a heterogenous pathology, referred to as systemic mastocytosis (SM) [10,11]. The burden of . KIT D816V and control iPS cells generated a similar number of hematopoietic cells (Welch's t-test, p = 0.4512, KIT D816V, n = 7; control, n = 9). The CD34 + population on day 14 is shown on the right. KIT D816V and control iPS cells generated a similar number of CD34 + HSPC (Welch's t-test, p = 0.27, KIT D816V, n = 7; control, n = 9). For the graphs in (C), box and whiskers plots show minimum and maximum values, and "+" indicates the mean value. ns indicates not significant.

KIT D816V iPS Cell-Derived MCs Recapitulate Features of SM MCs
In the final step of our protocol, MC maturation was performed by culture for additional 60-70 days in the presence of hyper-IL-6 and SCF (Table 2). After this step, a high purity (≥95% CD45 + KIT high ) MC population was obtained. Acidic toluidine blue staining of KIT D816V and control MCs revealed cells displaying metachromatic granules in the cytoplasm and dense non-lobulated nuclei. MCs were also tryptase positive ( Figure 2D). KIT D816V and control MCs showed similar granularity and size as demonstrated by FSC and SSC analysis ( Figure 2E). Flow cytometry analysis further revealed higher surface expression of CD25 and CD30 in KIT D816V MCs in comparison to control MCs ( Figure 2F). These results are in agreement with clinical data where aberrant expression of either of these markers is used as a further criterion in the SM disease diagnosis [10]. CD2 expres- The total number of hematopoietic cells obtained per 96-well plate after 14 days of differentiation in the spin-EB step is shown (left graph). KIT D816V and control iPS cells generated a similar number of hematopoietic cells (Welch's t-test, p = 0.4512, KIT D816V, n = 7; control, n = 9). The CD34 + population on day 14 is shown on the right. KIT D816V and control iPS cells generated a similar number of CD34 + HSPC (Welch's t-test, p = 0.27, KIT D816V, n = 7; control, n = 9). For the graphs in (C), box and whiskers plots show minimum and maximum values, and "+" indicates the mean value. ns indicates not significant.
In the first step, embryoid body (EB) formation is initiated by the spin-EB method, where forced aggregation of single-cell iPS cell suspension is achieved by centrifugation in a 96-well format. Mesoderm commitment followed by hematopoietic differentiation is induced by the sequential application of a defined combination of cytokines ( Figure 1A and Table 2). Hematopoietic cells start to be released from EB on day 8-10. On day 14, for each 96-well plate, an average of 2.786 ± 2 × 10 6 hematopoietic cells (mean ± SD, n = 7) were obtained for KIT D816V iPS cells, while an average of 3.83 ± 3.3 × 10 6 hematopoietic cells (mean ± SD, n = 9) were obtained for control iPS cells ( Figure 1C, left graph).
The second step promotes the hematopoietic expansion of CD34 + HSPC for 10-14 days in a myeloid cell supporting medium (supplemented with IL-3, hyper-IL-6 [38], Flt3L, and SCF, Table 2). At the end of this step, MC progenitors were obtained, quantified, and isolated by FACS sorting for CD45 + KIT high cells (Figure 2A). A homogenous population of MC progenitors with multi-lobulated nuclei and with low cytoplasmic granularity was observed by acidic toluidine blue staining (Figure 2A, right panel). Recapitulating our previous data [33] and in line with the aberrant expansion of KIT D816V MCs in SM patients, we observed a trend toward an increased number of mutated CD45 + KIT high MC progenitors in comparison to control cells ( Figure 2B). KIT D816V MC progenitors also displayed enlarged cell size, shown by FSC analysis in comparison to control cells, while no difference in cell granularity was noted ( Figure 2C). The enlarged cell size observed for KIT D816V MCs may indicate a more advanced maturation status, as MCs have been shown to increase in size upon maturation [39]. Highlighting the potential of iPS cells to generate substantial amounts of rare cell types, such as MCs, starting with a 96-well plate seeded with 5 × 10 5 iPS cells, we routinely obtained 3.6 ± 2 × 10 6 KIT D816V MC progenitors (n = 8) and 1.9 ± 1.8 × 10 6 control MC progenitors (n = 10; Figure 2B, right graph).

KIT D816V iPS Cell-Derived MCs Recapitulate Features of SM MCs
In the final step of our protocol, MC maturation was performed by culture for additional 60-70 days in the presence of hyper-IL-6 and SCF (Table 2). After this step, a high purity (≥95% CD45 + KIT high ) MC population was obtained. Acidic toluidine blue staining of KIT D816V and control MCs revealed cells displaying metachromatic granules in the cytoplasm and dense non-lobulated nuclei. MCs were also tryptase positive ( Figure 2D). KIT D816V and control MCs showed similar granularity and size as demonstrated by FSC and SSC analysis ( Figure 2E). Flow cytometry analysis further revealed higher surface expression of CD25 and CD30 in KIT D816V MCs in comparison to control MCs ( Figure 2F). These results are in agreement with clinical data where aberrant expression of either of these markers is used as a further criterion in the SM disease diagnosis [10]. CD2 expression was not detected on the surface of KIT D816V or control MC. CD33, a pan-myeloid marker also used as a criterion in SM disease diagnosis and a potential therapeutic cell surface target, was highly expressed in both, KIT D816V and control MCs ( Figure 2F) [11,40].
Interleukin 4 (IL-4) is a cytokine produced by lymphocytes, eosinophils, basophils, and MCs. IL-4 acts on MCs, affecting their proliferation and the profile of the mediators they secrete, and upregulates FcER1α surface expression while downregulating the KIT surface expression [41][42][43]. In this context, we investigated the IL-4 effect on FcER1α and KIT surface expression in mature iPS cell-derived MCs. We observed that KIT D816V MCs have higher FcER1α surface expression in comparison to control cells, indicating a more mature or activated status. Upon IL-4 treatment, FcER1α surface expression was increased in both KIT D816V and control MCs with a more pronounced effect on the latter ( Figure 2G, left panel). In agreement with previously reported data, KIT surface expression was downregulated in both, KIT D816V and control MC, upon IL-4 treatment ( Figure 2G, right panel). We further investigated if iPS cell-derived MCs were able to degranulate upon FcER1α activation by IgE. In line with the observed surface expression of FcER1α, both KIT D816V and control MCs degranulated upon IgE stimulation, as shown by β-hexosaminidase activity in the culture supernatant ( Figure 2H and Supplemental Figure S1). marker also used as a criterion in SM disease diagnosis and a potential therapeutic cell surface target, was highly expressed in both, KIT D816V and control MCs ( Figure 2F) [11,40].   Figure S2). In line with reported ultrastructural data for human MCs, iPS cell-derived MCs displayed thin cytoplasmic projections, modest Golgi-associated endoplasmic reticulum, and cytoplasmic membranedelimited secretory granules with distinct electron densities and granularity. Of note, "scroll-like" granules, typical of MCs, were also observed [44,45]. In KIT D816V MCs, we also observed a pronounced enlargement of secretory granule compartments with reduced granular content (Supplemental Figure S2). These structures are indicative of MC activation that may lead to secretory granule membrane fusion and piecemeal degranulation, a process in which MCs release their granule contents but retain the granule membranes within the cytoplasm [44].
Altogether, our data show that the protocol for iPS cell differentiation toward MCs described here generates a large amount of functional MCs and very well recapitulates features associated with KIT D816V neoplastic MCs in SM.

KIT D816V iPS Cell-Derived MCs Recapitulate the Gene Expression Profile of SM MCs
To gain further information on the functionality and the impact of the KIT D816V mutation on iPS cell-derived MCs, we performed global gene expression analysis on mature KIT D816V and control MCs by RNA-Seq. Confirming their MC identity, 11 out of 14 MC signature genes described by Motakis and colleagues were highly expressed in iPS cell-derived MCs ( Figure 4A and Supplemental Figure S3A) [46].
Both KIT D816V and control MCs showed high expression of tryptase genes (TPSAB1, TPSB2, and TPSD1) and enzymes associated with the biosynthesis of MC mediators (HDC, HPGDS, LTC4S). In line with our flow cytometry data ( Figure 2G) and previous work, two subunits of the FcER1 (FCER1A, MS4A2) showed higher expression in KIT D816V MCs than control ( Figure 4A and Supplemental Figure S3A). In addition, we observed higher expression of Mas-related G protein-coupled receptor X2 (MRGPRX2) in KIT D816V MCs (Supplemental Figure S3B). MRGPRX2 has a pivotal role in IgE cross-linking-independent MC activation [47]. We further observed high expression of gene coding for granule membrane-associated proteins such as VAMP8 and VAMP3, in KIT D816V MCs (Supplemental Figure S3C). Expression of lysosomal associated membrane proteins LAMP3 (CD63), LAMP1 (CD107a), and lipid raft resident transmembrane adaptor molecule LAT2, all involved in MC degranulation, was also higher in KIT D816V cells compared to control (Supplemental Figure S3D).
Gene set enrichment analysis (GSEA) revealed an upregulation in KIT D816V MCs of pathways related to IFN-α, -β, and -γ signaling, innate and adaptive immune responses (including viral response), inflammation, cytokine signaling, and MC activation ( Figure 4B,C, Supplemental Figure S4). This observation is in line with the work of Teodosio and colleagues, who described a similar gene expression profile in MCs isolated from the BM of KIT D816V SM patients [48]. In this context, we observed a strong enrichment of genes found deregulated in primary SM MCs in the KIT D816V MC transcriptome ( Figure 5A). We further validated selected key genes by RT-qPCR and confirmed in iPS cell-derived KIT D816V MC trend toward the upregulation of interferon signaling/inflammatory response pathways (IFIT2, STAT1, and CCL23, Figure 5B). Next, we investigated the expression of genes involved in pathways that were also found to be deregulated in primary SM MCs. We observed significant upregulation in LAT2 (MC degranulation), APOL1 (lipid metabolism), CHF and SERPING1 (complement regulation), and CASP1 (apoptosis) in KIT D816V iPS cell-derived MCs ( Figure 5C). In conclusion, transcriptome analysis confirmed the identity of iPS cell-derived MCs and, additionally, revealed a more activated phenotype of KIT D816V MC. Moreover, KIT D816V MCs derived from iPS cells recapitulate the gene expression profile of primary KIT D816V SM MCs, characterized by the upregulation of pathways involved in innate and adaptive immune responses and inflammation.

KIT D816V iPS Cell-Derived MCs Recapitulate the Gene Expression Profile of SM MCs
To gain further information on the functionality and the impact of the KIT D816V mutation on iPS cell-derived MCs, we performed global gene expression analysis on ma-  Both KIT D816V and control MCs showed high expression of tryptase genes (TPSAB1, TPSB2, and TPSD1) and enzymes associated with the biosynthesis of MC mediators (HDC, HPGDS, LTC4S). In line with our flow cytometry data ( Figure 2G) and previous work, two subunits of the FcER1 (FCER1Α, MS4A2) showed higher expression in KIT D816V MCs than control ( Figure 4A and Supplemental Figure S3A). In addition, we ob-

Discussion
Here, we presented a novel and robust protocol for the differentiation of patient-specific iPS cells toward the MC lineage in a feeder-free culture system. By using previously established and characterized SM patient-derived KIT D816V and control iPS cell lines, we built further on our efforts to exploit patient-specific iPS cells as a model for SM [33]. The panel of iPS cell lines used in this work allowed us to confirm the robustness of our protocol, as MCs were obtained from nine different iPS cell lines generated from three patients ( Table 1). In addition, the only xenobiotic supplement in our protocol is bovine serum albumin (BSA), used in the first two days and replaced afterward by human serum

Discussion
Here, we presented a novel and robust protocol for the differentiation of patientspecific iPS cells toward the MC lineage in a feeder-free culture system. By using previously established and characterized SM patient-derived KIT D816V and control iPS cell lines, we built further on our efforts to exploit patient-specific iPS cells as a model for SM [33]. The panel of iPS cell lines used in this work allowed us to confirm the robustness of our protocol, as MCs were obtained from nine different iPS cell lines generated from three patients (Table 1). In addition, the only xenobiotic supplement in our protocol is bovine serum albumin (BSA), used in the first two days and replaced afterward by human serum albumin (HSA). The defined composition of our basal medium, especially at the myeloid expansion and MC maturation stage, allows the precise assessment of factors influencing MC development and function. Another highlight of our protocol is the use of low-cost basal medium (Table 2), in contrast with commercially available media and kits commonly used in iPS cell differentiation protocols (e.g., StemDiff, StemPro34, and StemSpan) [49,50]. With this basal medium and optimized cytokine cocktails, MCs are efficiently produced at the end of three steps: first, we rely on feeder-free iPS cell culture and mesodermhematopoietic commitment through the formation of 3D EB structures in the presence of BMP-4, VEGF, and SCF; second, myeloid cell expansion is performed in the second step, driven by SCF, IL-3, Flt3L, and hyper-IL-6; and third, MC maturation is achieved in the presence of SCF and hyper-IL-6.
In our protocol, CD34 + MACS isolation of HSPC allows the synchronization of differentiation kinetics, a key step when evaluating the impact of molecular lesions, culture conditions, or drugs on MC development and function. Similarly, CD45 + KIT high FACS sorting enables the maturation step to occur in a homogenous MC population, without the potential interference of other hematopoietic cell types and the cytokines they might secrete. However, omitting CD34 + MACS selection does not compromise MC differentiation, but it is recommended to deplete for tissue culture plastic-adherent macrophages during the myeloid/MC maturation step.
The KIT D816V iPS cell-derived MCs obtained via our protocol recapitulate phenotypic features of SM MCs, such as abnormal expansion and aberrant surface expression of CD25 and CD30. Furthermore, mutant MCs showed higher FcER1 surface expression in comparison to control cells. The gene coding for MRGPRX2, a pivotal receptor in non-IgEmediated MC activation and responsible for the connection between MC-mediated immune regulation and neurologic stimuli, showed higher expression in KIT D816V MCs than in control MCs [47]. TEM analysis of iPS cell-derived MCs revealed MC typical cytoplasmic secretory granules and indicated a more activated degranulating status of KIT D816V MCs. The observation of large membrane delimited structures with low granule content, more abundant in KIT D816V MCs, suggests the occurrence of piecemeal degranulation, a process of MC degranulation upon activation commonly observed under inflammatory conditions [3]. Altogether, our data indicate a more activated status of KIT D816V MC, in agreement with SM clinical features [51].
Teodosio and colleagues reported a common gene expression profile for MCs isolated from the BM of KIT D816V patients with different SM entities [48]. Global gene expression analysis of KIT D816V and control iPS cell-derived MCs recapitulated this transcriptional profile in mutated cells. GSEA revealed an upregulation of pathways related to innate and adaptive immune response, anti-viral response, cytokine signaling, IFN signaling, inflammatory response, and MC activation in KIT D816V MCs. In the BM of SM patients, MCs are in close and constant interaction with BM niche components (e.g., stroma cells, vasculature, and other hematopoietic cells) and soluble mediators that are mostly lacking in our in vitro differentiation system. Therefore, the fact that in vitro generated KIT D816V iPS cell-derived MCs recapitulate the key transcriptional features of KIT D816V SM patientderived MCs strongly suggests that this signature is driven by the KIT D816V mutation and the constitutive activation of KIT signaling pathways. KIT signaling is not only essential for MC development and survival but has also been shown to impact MC function and modulate cell migration and adhesion [52]. Moreover, in synergy with FcER1 engagement, KIT signaling enhances MC degranulation and production of cytokines, such as IL-6 [53,54]. Hence, the constitutive activation of oncogenic KIT D816V likely leads to the deregulated MC maturation and activation features we observed in our KIT D816V iPS cell-derived MCs, such as high FcER1A and MRGPRX2 expression and upregulated cytokine signaling and MC activation pathways.
MCs originate from BM but mature in their final tissue of residence, which has a direct impact on the mature MC phenotype and function [3]. Therefore, we envision that better models to study MC biology, disease, and therapeutic targeting need to be developed. An attractive approach consists of 3D coculture systems of iPS cell-derived immature MCs with fibroblasts, endothelial, and mesenchymal cells in 3D matrixes. These niche-mimicking constructs should provide a model closer to the in vivo situation, such as MC niches in the connective tissue or the BM niche hosting SM MCs. In this context, MC maturation may be achieved by niche-secreted factors, such as SCF and IL-6. In addition, pathological scenarios can be recapitulated by using iPS cell-derived MCs harboring disease-relevant mutations (e.g., KIT D861V, JAK2 V617F) or by adding effector molecules, such as pro-inflammatory cytokines. In summary, our protocol reported here paves the way for implementing patientspecific iPS cell-derived MCs as reliable tools to investigate MC biology, pathomechanisms, and drug response, overcoming the cell number and availability limitations faced when using primary MC.

The iPS Cell Lines and iPS Cell Culture
The iPS cell lines used in this study were generated and characterized in our previous work and are listed in Table 1 [32,33]. Patient 1 and 2 iPS cell lines were generated from peripheral blood mononuclear cells, and patient 3 iPS cell lines were generated from BM mononuclear cells. The iPS cells were cultured with StemMACS iPS-Brew XF (Miltenyi Biotec, Bergisch Gladbach, Germany) on 6-well plates coated with Matrigel (Corning, USA) following the manufacturer's recommendations. Culture passaging was performed with Accutase (PAN-Biotech GmbH, Aidenbach, Germany) or 0.2 mM EDTA PBS solution (both from Gibco, USA).

The iPS Cell Differentiation toward MCs
The iPS cells were cultured as described above up to a confluency of 70-80%. On day 0 of the first step ( Figure 1A), cells were treated with Accutase (PAN-Biotech GmbH, Aidenbach, Germany) for 4 min at 37 • C, followed by a wash step with KO-DMEM (Gibco, USA). A total of 5 × 10 5 iPS cells were seeded per well in a 96-well U-bottom suspension culture plate (Greiner, Frickenhausen, Germany) in d0 Spin EB medium (please refer to Table 2 for medium composition) followed by centrifugation at 360× g for 7 min. Cells were incubated at 37 • C and 5% CO 2 throughout the entire differentiation protocol. On day 2, day 2 Spin EB medium was added to the wells. From day 3 onwards, 50 µL of the medium was removed from the wells, and 50 µL of fresh medium was added ( Table 2). On day 14, spin EB and hematopoietic stem/progenitor cells (HSPC) derived thereof were harvested, passed through a 40 µm cell strainer (Greiner), and subjected to CD34 + magnetic-activated cell sorting (MACS) as described below. In the second step, CD34 + HSPC (≤1 × 10 6 cells/mL) were cultured for 10-14 days in a hematopoietic progenitor medium ( Table 2) for myeloid cell expansion. Partial medium change was performed every 3 days. In the final step, CD45 + KIT high immature MCs were FACS sorted as described below and further cultured in MCs' maturation medium ( Table 2) for 60-70 days (≤1 × 10 6 cells/mL). Partial medium change was performed every 3-4 days. At the end of this step, MCs were referred to as mature MCs.

Magnetic Activated Cell Sorting (MACS)
The iPS cell-derived hematopoietic cells were harvested on day 14 of Spin EB differentiation and passed through a 40 µm cell strainer (Corning). After centrifugation at 350× g for 5 min, cells were resuspended in MACS/FACS buffer (5% FCS, 2 mM EDTA in PBS, all from Gibco, USA). Cells were subjected to MACS using the human CD34 MicroBead Kit and LS MACS Columns (both from Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer's instructions.

Fluorescence Activated Cell Sorting (FACS)
FACS analysis was performed with a BD FACS Canto II (BD Bioscience, USA). The antibodies used for FACS are listed in Table 3. Briefly, 1-2 × 10 5 cells pre-washed in MACS/FACS buffer were incubated with the diluted antibody solution (dilutions were performed in MACS/FACS buffer and the antibodies used, and respective dilutions are listed in Table 3) and incubated for 30 min at 4 • C, protected from light. Cells were washed once and resuspended in 500 µL MACS/FACS and subjected to FACS analysis. FACS sorting was performed with a FACS Aria II 3L (BD Bioscience, USA). Sorted cells were further cultured in an MCs' maturation medium for 60-70 days. Data analysis was performed with FlowJo™ Software (BD Life Sciences, USA) and GraphPad Prism.
For tryptase staining, cytospin preparations with 1-2 × 10 5 mature MCs were performed as described above and fixed with acetone. Immunohistochemical staining against MC tryptase was performed with Flex Kit DAKO/Agilent (DAKO, Carpinteria, CA, USA). Samples were incubated with an anti-human tryptase antibody (Table 3) for 30 min, followed by the application of a staining enhancer. Next, secondary antibody staining was performed, followed by visualization with horseradish peroxidase and DAB (DAKO). Counterstaining was performed with hematoxylin (DAKO).

IL-4 Stimulation of MC
Mature iPS cell-derived MCs (1-2 × 10 5 cells/mL) were treated for 4 days with 10 ng/mL IL-4 (Peprotech, UK) in MCs' maturation medium supplemented with SCF and hyper IL-6 ( Table 2). After the incubation period, MCs were subjected to flow cytometry analysis as described above.

MC Degranulation Assay
MC degranulation assay was performed as described by Kuehn and colleagues [55]. Briefly, mature MCs were starved for 4 h in MCs' maturation medium (Table 2) without SCF and hyper IL-6 supplementation, followed by stimulation with 100 ng/mL biotinylated IgE (DIA HE1B) for 16 h at 37 • C with 5% CO 2 . Cells were washed 3× with HEPES buffer, and 3 × 10 5 cells were seeded in 90 µL of HEPES buffer per well in a 96-well plate. Cells were then incubated with 0.1 µg/mL DNP-HSA (Dinitrophenyl-human serum albumin conjugate, Sigma-Aldrich, USA) for 30 min at 37 • C. Secreted hexosaminidase activity was measured by incubating culture supernatant with PNAG (p-nitrophenyl N-acetyl-β-Dglucosamine, Sigma-Aldrich) solution (3.5 mg/mL) for 90 min at 37 • C. Enzymatic activity was measured by absorbance at 405 nm with a reference filter at 620 nm.

RT-qPCR
RNA isolation of iPS cell-derived MCs was performed with NucleoSpin RNA Kit (Macherey-Nagel, Düren, Germany), and cDNA synthesis was performed with MultiScribe reverse transcriptase (High-Capacity cDNA Reverse Transcriptase Kit, Thermo Fisher Scientific, USA). For RT-qPCR, the FAST SYBR Green master mix (Thermo Fisher Scientific) was used, and runs were performed using a StepOnePlus Real-Time cycler. Primers (Eurofins Genomics, Ebersberg, Germany) are listed in Table 4. Data analysis and heatmap plots were conducted with GraphPad Prism and MeV-Multiple Experiment Viewer (http: //mev.tm4.org/, accessed on 12 December 2022), respectively.

Transmission Electron Microscopy
The iPS cell-derived MCs were washed once with PBS and fixed with 3% glutaraldehyde for at least 2 h at RT. Next, cells were embedded in 5% low melting agarose (Merck, Darmstadt, Germany), followed by washing in 0.1 M Soerensen's phosphate buffer (Merck) and post-fixed in 25 mM sucrose buffer (Merck) containing 1% OsO4 (Roth, Karlsruhe, Germany). Samples were dehydrated by performing an ascending ethanol series repeating the last step three times (30,50,70, 90, and 100% ethanol; 10 min each step). Dehydrated samples were incubated in propylene oxide (Serva, Heidelberg Germany) for 30 min, in a mixture of EPON resin (Serva) and propylene oxide (1:1) for 1 h, and in pure EPON for 1 h. EPON polymerization was conducted at 90 • C for 2 h. Ultrathin sections of 70-100 nm were performed with a Reichert Ultracut S ultramicrotome (Leica, Wetzlar, Germany) equipped with a diamond knife (Leica) and picked up on copper-rhodium grids (Plano, Wetzlar, Germany). Contrast enhancement was performed by staining with 0.5% uranyl acetate and 1% lead citrate (both EMS, Munich, Germany). Samples were viewed at an acceleration voltage of 60 kV using a Zeiss Leo 906 (Carl Zeiss, Jena, Germany) transmission electron microscope. Image processing and analysis were performed using ImageJ [56].