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. 2026 Feb 26;31:101081. doi: 10.1016/j.reth.2026.101081

Anti-inflammatory and immunomodulatory effects of human induced pluripotent stem cells-derived mesenchymal stem/stromal cells and their extracellular vesicles

Yusaku Komoike a,b,1, Daisuke Kamiya b,c,⁎,1, Haruka Sasaki-Ban a,b, Atsushi Matsuda a,b, Hirokazu Matsumoto a,b, Makoto Ikeya b,c,⁎⁎
PMCID: PMC12955577  PMID: 41782806

Abstract

Introduction

Mesenchymal stem/stromal cells (MSCs) are multipotent cells that secrete multiple factors for tissue regeneration. These tissue-derived MSCs can self-renew, but their numbers are limited and decrease with age. The previously established xeno-free MSCs (XF-iMSCs), derived from human induced pluripotent stem cells (hiPSCs) of the neural crest cell (NCC) lineage, can regenerate damaged bone and skeletal muscle. However, the anti-inflammatory and immunomodulatory effects of XF-iMSCs have not been elucidated. Here, we aimed to elucidate the effects of XF-iMSCs and their extracellular vesicles (XF-iEv) on inflammation and immunomodulation.

Methods

XF-iMSCs were generated from hiPSCs using NCCs. Mouse PBMCs and splenocytes were obtained from male C57BL/6 mice. The MSCs were characterized using flow cytometry. Cytokine secretion stimulated by lipopolysaccharides (LPS) or Dynabeads CD3/CD28 was measured by ELISA. The proliferation of CellTrace Violet-labeled effector T cells (Teff) cultured with MSCs or their EVs was analyzed using a suppression assay. EVs were purified from the MSC culture medium using a MagCapture Exosome Isolation Kit. Proteome analysis of the EVs was performed using non-labeled liquid chromatography-tandem mass spectrometry.

Results

XF-iMSCs expressed representative MSC cell surface markers, including CD44, CD73, and CD105, but not CD45 or HLA-DR. XF-iMSCs and human adipocyte-derived MSCs (hAC-MSCs) suppressed LPS-stimulated IL-6 and TNF-α secretion in mouse PBMCs. Suppression of LPS-induced IL-6 and TNF-α secretion by XF-iEv and hAC-MSCs (hAC-Ev) was concentration-dependent. Fifty times-concentrated EVs from both XF-iMSCs and hAC-MSCs strongly suppressed IL-2, IFN-γ, and IL-17 secretion induced by dynabeads CD3/CD28 in mouse splenocytes. The effect of XF-iMSCs against inflammatory and anti-inflammatory cytokine production induced by LPS in hPBMCs was comparable to that of primary human adipocytes, bone marrow, and umbilical cord-derived MSCs. XF-iEv (x50) strongly inhibited TNF-α secretion from LPS-stimulated human PBMCs, although there was no effect on IL-6 secretion. Condition medium (CM) from XF-iMSCs promoted IL-10 secretion but not concentrated XF-iEv (x50). XF-iMSCs suppressed Teff proliferation to a level comparable to that of hAC-MSCs. XF-iEv had 1217 proteins with unique components compared to hAC-Ev.

Conclusions

XF-iMSCs and their EVs exert anti-inflammatory and immunomodulatory effects in human and mouse cell-based assays and have promising therapeutic applications for autoimmune diseases.

Keywords: hiPSCs, MSCs, Extracellular vesicle, anti-inflammation, Immunomodulation

Abbreviations:

EV

extracellular vesicle

iPSCs

human induced pluripotent stem cells (hiPSCs)

MSCs

mesenchymal stem/stromal cells

XF-iMSCs

xeno-free-induced MSCs

AC-MSCs

adipocyte-derived MSCs

BM-MSCs

bone marrow-derived MSCs

UC-MSCs

umbilical cord-derived MSCs

hPBMCs

human peripheral blood mononuclear cells

LPS

lipopolysaccharides

Teff

effector T cells

XF-iEv

EV derived from XF-iMSCs

hAC-Ev

EV derived from AC-MSCs

1. Introduction

Mesenchymal stem/stromal cells (MSCs) are multipotent stem cells found in various body tissues, most commonly the bone marrow, adipocytes, and umbilical cord tissue [1,2]. The International Society for Cellular Therapy (ISCT) committee proposed a definition of human MSCs [3]. According to the criteria, MSCs must be plastic-adherent in culture and show high CD105, CD73, and CD90 expression and low expression of CD45, CD34, CD14, CD11b, CD79alpha, or CD19, and HLA-DR surface molecules. MSCs must differentiate into osteoblasts, adipocytes, and chondroblasts in vitro. Several preclinical and clinical studies have been conducted on cell-based therapies, especially for autoimmune disorders, owing to the unique advantages of MSCs, including their anti-inflammatory and immunomodulatory effects and low immunogenicity [4]. Previously, we established an induction method for xeno-free MSCs (XF-iMSCs) derived from human induced pluripotent stem cells (iPSCs) through the neural crest cell (NCC) lineage to supply stable and high-quality MSCs without animal-derived components, unlike primary MSCs [5]. XF-iMSCs can regenerate damaged bone and skeletal muscle, an ability mediated in part by PXDN and IGF2 production. However, the anti-inflammatory and immunomodulatory effects of XF-iMSCs have not been elucidated.

MSC-derived extracellular vesicles (EVs), recognized as key paracrine mediators, are nano-sized vesicles with sizes ranging from 40 to 200 nm. They facilitate intercellular communication by transporting bioactive molecules originating from parent cells. Emerging research has indicated that EVs from MSCs exhibit notable suppressive effects on various effector cells involved in both innate and adaptive immune responses [6]. Several mechanisms underlying EV secretion have been reported [[7], [8], [9]], and those that can contribute to increased EV secretion.

In this study, we investigated the potential role of XF-iMSCs and their EVs in inflammation and immunomodulation in vitro.

2. Materials and methods

2.1. Mouse primary cell culture

Male C57BL/6 mice (CLEA Japan, Inc.), 8–9 weeks old, were maintained under specific pathogen-free conditions. All animal experiments were in compliance with the standards for humane care and were approved by the Institutional Animal Care and Use Committee (IACUC) of Takeda Pharmaceutical Company, Ltd. (Certification Number: AU-00030589).

Whole blood with 0.1 M EDTA in mice was collected from the inferior vena cava during anesthetization by isoflurane. Blood was diluted by half using extracellular vesicle-depleted (EV-del) 10% FBS/RPMI1640 and slowly laid on lympholyte-mammal (CL5115, Cedarlane), followed by density gradient centrifugation at 1200×g for 20 min. The lymphocyte fraction was obtained and used as primary mouse peripheral blood mononuclear cells (PBMCs) after hemolysis with HLB solution (23905, IBL). Mouse PBMCs were seeded on a 48-well plate at a density of 1 × 106 cells/mL or a 96-well plate at a density of 2 × 106 cells/mL in Ex-del 10% FBS/RPMI1640 medium and stimulated by LPS (10 ng/mL) for 24 h at 37 °C. In contrast, mice were anesthetized with isoflurane, and the spleen was excised after midline laparotomy. The spleen was minced, mixed in HLB solution, and allowed to stand for 5 min for hemolysis, followed by the addition of Ex-del 10% FBS/RPMI1640 medium. The mixture was passed through a 40-μM cell strainer. Splenic lymphocytes were seeded on a 96-well plate at a density of 2 × 106 cells/mL in Ex-del 10% FBS/RPMI1640 medium and stimulated with LPS (10 ng/mL) or dynabeads CD3/CD28 (DB11453, Veritas) for 24 h at 37 °C.

2.2. Human PBMC culture

Primary human PBMCs were purchased from Precision For Medicine (93000-10 M). Human PBMCs were seeded on a 48-well plate at a density of 1 × 106 cells/well in EV-deleted (Ex-del) 10% FBS/RPMI1640 medium and stimulated with 10 ng/mL LPS from Escherichia coli O55:B5 (LPS, L2880-100 MG, Sigma) for 24 h at 37 °C.

2.3. Human MSCs

Human MSCs (adipocyte-derived MSCs, hAC-MSCs; bone marrow-derived MSCs, hBM-MSCs; umbilical cord-derived MSCs, hUC-MSCs) were purchased from PromoCell (Heidelberg, Germany). Xeno-free-induced MSCs (XF-iMSCs) were prepared following a previously reported protocol [5]. Briefly, human iPSCs (1231A3, reprogrammed with episomal vectors, kindly provided by Yamanaka laboratory) were seeded onto iMatrix-511-coated plates or dishes at a density of 3.6 × 103 cells/cm2 in StemFit AK03 N medium and maintained in culture for 4 days. For NCC induction, the cells were cultured in StemFit Basic03 (equivalent to AK03 N without bFGF, Ajinomoto, Tokyo, Japan) with 10 μM SB431542 (FUJIFILM Wako) and 1 μM CHIR99021 (Axon Medchem, Reston, VA, USA) for 10 days. Cells were counted using Countess II FL (Thermo Fisher Scientific). The medium was changed every 2 days from days 0 to 6 and every day from days 7 to 10. For preparing the frozen stock of NCCs, 5 × 105 NCCs were suspended in 500 μl STEM-CELL BANKER GMP grade (Takara, Kusatsu, Japan) and frozen using CoolCell Cell Freezing Containers (Biocision, Kyoto, Japan). Expanded NCCs (passage number 4) were seeded onto fibronectin (Millipore, Bedford, CA, USA)-coated plates at a density of 1 × 104 cells/cm2 in Basic03 supplemented with 10 μM SB431542, 20 ng/mL EGF, and FGF2. The medium was replaced the next day with PRIME-XV MSC Expansion XSFM (FUJIFILM, Irvine Scientific, Tokyo, Japan). Cell morphology started to change approximately 4 days after induction. Cells were passaged every 4 days using Accutase at a density of 1 × 104 cells/cm2. MSCs were cultured in fibronectin-coated culture dishes in PRIME-XV MSC Expansion XSFM. The medium was changed every 3 days. Differentiation protocol for XF-iMSCs into chondrocytes, osteocytes, and adipocytes has been previously described [5].

2.4. Flowcytometry

XF-iMSCs were stained for human MSC-representative cell surface markers using APC-labeled anti-CD44 (BD Pharmingen: 559942), CD45 (BD Pharmingen: 560973), CD73 (BD Pharmingen: 560847), CD105 (eBioscience: 17-1057), and HLA-DR (BD Pharmingen: 340549) mAbs, and analyzed by FACS 14 days after MSC induction. Flow cytometry was performed on the Aria II instrument (BD Biosciences, Franklin Lakes, NJ, USA) following the manufacturer's protocol.

2.5. EV purification

MSC-conditioned medium (CM) was harvested by culturing confluent monolayers of MSCs for 3 days in the culture medium. The MSC-CM for EV purification was collected from passages 9 and 10. EV was purified from the MSC culture medium based on the phosphatidylserine (PS)-affinity method using MagCapture™ Exosome Isolation Kit PS Ver.2 (FujifilmWAKO). The mean particle diameter, mode particle size, and particle concentration of EVs were measured using a NanoSight LM10 (Nanoparticle Tracking Analysis Version 2.3 Build 0033, Fujifilm Wako).

2.6. ELISA

Human or mouse IL-6, TNF-α, IL-10, and IL-2 were measured by ELISA (R&D) following secretion from human PBMCs or mouse PBMCs and splenocytes stimulated by LPS or dynabeads CD3/CD28.

2.7. Suppression assay

MSCs or their EV-mediated suppression of effector T cell proliferation in PBMCs was evaluated following a previously described suppression assay [10]. Briefly, human PBMCs as responder cells were labeled with CellTrace Violet (CTV) and seeded onto 96-well plates at a density of 5 × 105 cells/mL in Treg inspector beads (Miltenyi Biotec). XF-iMSCs or hAC-MSCs were co-cultured with CTV-labeled PBMCs at different ratios (PBMCs: MSCs = 1:0.5, 0.2, 0.1, and 0.05). The cells were harvested after 4 days of co-culture and analyzed for the proliferation of CTV-labeled effector T cells using flow cytometry. Data are presented as % inhibition.

2.8. Cell viability analysis

Human PBMCs (5 × 105 cells/mL) were cultured with conditioned medium (CM) from XF-iMSC, XF-iMSCs- or hAC-MSC-derived EVs in Treg inspector beads for 4 days. The ratio of live cell was calculated based on the FACS labeling of dead cells using Zombie R718 Fixable Viability Kit (Biolegend: 423116). Data are presented as % of live cells in total.

2.9. Proteome analysis

The confluent culture medium was harvested by incubating XF-iMSCs and hAC-MSCs, and their EVs were purified using the MagCapture Exosome Isolation Kit PS ver.2 (FujifilmWAKO). Protein levels in the EVs were measured using a BCA protein assay kit (297-73101, FujifilmWAKO). The purified EV solution was precipitated with acetone, and the dried precipitate was dissolved in a solubilization buffer (8 M urea, 50 mM Tris-HCl, pH 8.0) and subjected to trypsin hydrolysis, followed by desalting. These samples were dissolved in an appropriate solvent (volume ratio of water: acetonitrile: trifluoroacetic acid = 98:2:0.1) and subjected to liquid chromatography-tandem mass spectrometry. Proteome data were processed using Proteome Discoverer version 3.0 (PD 3.0, Thermo Fisher Scientific) and analyzed using Medical ProteoScope. A sequence database search was performed using Mascot software (Matrix Science) through PD 2.2. The search conditions in the Mascot software were as follows: Database: Swiss prot_Human (UniProt 2020 ver.2), amino acid sequences of non-human protein concomitants (The Global Proteome Machine Organization), Enzyme, trypsin; Maximum missed cleavage, 2; Peptide tolerance, ±5 ppm; MS/MS tolerance, ±0.02 Da; Mass, monoisotopic mass; Fixed Modification, Carbamidomethyl (C, +57.021 Da); Variable Modification: Oxidation (M, +15.995).

2.10. Statistical analysis

All data are presented as the mean ± standard deviation (SD). Comparisons between two groups were performed using an unpaired two-tailed Student's t-test or Aspin-Welch's t-test. One-way ANOVA with Dunnett's multiple comparison test was used for comparisons of more than two groups. Statistical significance was set at p < 0.05, 0.01, and 0.001.

3. Results

3.1. Characterization of XF-iMSCs

XF-iMSCs were generated from human iPSCs from the NCC lineage based on a previously established procedure [5]. We found the expression of MSC representative cell surface markers, including CD44, CD73, and CD105, but these were negative for CD45 and HLA-DR (Fig. 1B). XF-iMSCs differentiated into cartilage, bone, and adipose tissues under chondrogenic, osteogenic, and adipogenic induction conditions, respectively (Fig. 1C).

Fig. 1.

Fig. 1

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Characterization of XF-iMSCs.

(A) Representative images of XF-iMSCs colony morphology. Scale bars, 100 μm. (B) Expression of MSC-related surface markers in XF-iMSCs. Gray histograms represent FACS labeling with anti-CD44, CD73, CD105, CD45, and HLA-DR mAb. White histograms represent the corresponding isotype control mAb. (C) Differentiation potentials of XF-iMSC into chondrocyte (left), osteocyte (middle), and adipocyte (right). Scale bars, 100 μm (left and middle), 20 μm (right).

3.2. Effect of XF-iMSCs and their extracellular vesicles in mouse primary cells

We validated the anti-inflammatory and immunomodulatory effects of XF-iMSCs and their extracellular vesicles (EVs) in primary mouse cells. LPS (10 ng/mL) stimulation induced the production of inflammatory cytokines, IL-6 and TNF-α, and anti-inflammatory cytokine, IL-10, in mouse PBMCs. Both XF-iMSCs and hAC-MSCs significantly inhibited IL-6 and TNF-α secretion in LPS-activated mouse PBMCs (Fig. 2). IL-10 secretion was partially inhibited. EVs from XF-iMSCs (XF-iEv) and hAC-MSCs (hAC-Ev) showed inhibitory effects against IL-6 and TNF-α secretion in LPS-activated mouse PBMCs following enrichment of EVs (Fig. 3A). In contrast, XF-iEv (x1 and x2) promoted IL-10 secretion, although highly concentrated in EVs, particularly XF-iEv (x50) and hAC-Ev (x50) inhibited the secretion (Fig. 3A). Regarding the immunomodulatory effect, both XF-iEv and hAC-Ev inhibited IL-2, IFN-γ, and IL-17 in dynabeads CD3/CD28-activated mouse splenocytes (Fig. 3B).

Fig. 2.

Fig. 2

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Effects of XF-iMSCs in mouse peripheral blood mononuclear cells (mPBMCs).

mPBMCs (1 × 106 cells/mL) were stimulated with LPS (10 ng/mL) for 24 h, and levels of IL-6, TNF-α, and IL-10 in culture supernatants were quantified by ELISA. XF-iMSCs or human adipocyte-derived MSCs (hAC-MSCs) were co-cultured at a density of 2 × 105 cells/well with mPBMCs. Data are presented as mean ± SD. ∗∗∗p < 0.001 (Student's t-test), ##p < 0.01; ###p < 0.001 (Dunnett test, vs control group).

Fig. 3.

Fig. 3

Fig. 3

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Effect of XF-iMSCs-secreted EVs in mouse primary cells.

XF-iMSC-secreted EVs (XF-iEv) and human adipocyte-derived MSC-secreted EVs (hAC-Ev) were purified using the MagCapture Exosome Isolation Kit. (A) Mouse peripheral blood mononuclear cells (mPBMCs; 2 × 106 cells/mL) were stimulated with LPS (10 ng/mL) for 24 h. Levels of IL-6, TNF-α, and IL-10 in culture supernatants were quantified by ELISA. (B) Mouse splenocytes (2 × 106 cells/mL) were stimulated with Dynabeads CD3/CD28 for 24 h. Levels of IL-2, IFN-γ, and IL-17 in culture supernatants were quantified by ELISA. hPBMCs were cultured with different concentrations of XF-iEv or hAC-Ev (x1, x2, x10, x50). Data are presented as mean ± SD. +p < 0.05, ++p < 0.01, +++p < 0.001 (Aspin-Welch's t-test), #p < 0.05; ##p < 0.01; ###p < 0.001 (Dunnett test, vs control group).

3.3. Effects of XF-iMSCs and their EVs in primary human PBMCs

Next, we validated the anti-inflammatory effects of XF-iMSCs and their EVs on LPS-induced cytokine secretion by human PBMCs. In the co-culture of XF-iMSCs with human PBMCs, XF-iMSCs relatively inhibited IL-6 and TNF-α secretion induced by 10 ng/ml of LPS, as well as primary human MSCs (hAC-MSCs; bone marrow-derived MSCs, BM-MSCs), except for umbilical cord-derived MSCs (UC-MSCs). All MSCs, including XF-iMSCs, enhanced IL-10 secretion, compared to the control group (Fig. 4). We examined the CM harvested from XF-iMSCs or EVs of XF-iMSCs concentrated fifty times for the PBMCs assay to validate the anti-inflammatory effect of the secretome from XF-iMSCs (Fig. 5). XF-iMSC-CM did not inhibit IL-6 secretion and showed a partial inhibitory effect against TNF-α secretion but increased IL-10 secretion (Fig. 5); XF-iEv (x50) inhibited TNF-α and IL-10 secretion but not IL-6. This suggests that cell-cell contact is critical for the inhibitory mechanism of XF-iMSCs against activated PBMCs. However, their secretory components may contain anti-inflammatory elements.

Fig. 4.

Fig. 4

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Effects of XF-iMSCs in primary human PBMCs.

uman peripheral blood mononuclear cells (hPBMCs, 2 × 106 cells/mL) were stimulated with LPS (10 ng/mL) for 24 h, and levels of IL-6, TNF-α, and IL-10 in culture supernatants were quantified by ELISA. The hPBMCs were cultured with human adipocyte-derived MSCs (hAC-MSCs), bone marrow-derived MSCs (hBM-MSCs), or umbilical cord-derived MSCs at a density of 2 × 105 cells/well. Data are presented as mean ± SD. +++p < 0.001 (Aspin–Welch's t-test). ##p < 0.01; ###p < 0.001 (Dunnett test).

Fig. 5.

Fig. 5

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Effects of XF-iMSCs-derived secretome in primary human PBMCs. Human peripheral blood mononuclear cells (hPBMCs, 1 × 106 cells/mL) were stimulated with LPS (10 ng/mL) for 24 h, and IL-6, TNF-α, and IL-10 in culture supernatants were quantified by ELISA. hPBMCs were cultured in conditioned medium (CM) from XF-iMSCs or fifty times-concentrated iEv. Data are presented as mean ± SD. +++p < 0.001 (Aspin–Welch's t-test). ##p < 0.01; ###p < 0.001 (Dunnett test).

3.4. Immunomodulation by XF-iMSCs and their EVs

Next, we examined whether XF-iMSCs and their EVs exerted immunomodulatory effects on activated T cells. In the suppression assay, the proliferation of CTV-labeled effector T cells (Teff) was suppressed by co-culture with XF-iMSCs in a cell number-dependent manner, showing almost 80% inhibition in the ratio of XF-iMSCs to Teff at 0.5:1 (Fig. 6). The inhibitory effect of hAC-MSCs was similar to that of XF-iMSCs.

Fig. 6.

Fig. 6

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Immunomodulatory effects of XF-iMSCs.

MSC-mediated suppression of effector T cell (Teff) proliferation in CellTrace Violet (CTV)-labeled human PBMCs (hPBMCs) was evaluated by suppression assay. CTV-labeled hPBMCs (5 × 104 cells) were seeded as responder cells on Treg suppression inhibitor beads. XF-iMSCs or hAC-MSCs were co-cultured with CTV-labeled hPBMCs at different ratios (hPBMCs: MSCs = 1:0.5, 0.2, 0.1, and 0.05). The cells were harvested after 4 days of co-culture and analyzed for the proliferation of CTV-labeled effector T cells using flow cytometry. Data are presented as % inhibition. Stim, stimulation; No stim, no stimulation.

3.5. Proteome analysis of XF-iMSC-derived EVs

NanoSight analysis showed that iEv were slightly smaller than hAC-Ev, although the particle and total protein concentrations in iEv were much lower than those in hAC-Ev (Supplementary Table 1A). Proteome analysis of EVs was conducted using non-labeled liquid chromatography-tandem mass spectrometry to identify the potential components responsible for the efficacy of iEv (Supplementary Table 1B). The analysis identified 1217 proteins, including general EV markers (Alix, CD9, CD63, and CD81), in iEv. Among them, some proteins (midkine, immunoglobulin superfamily member 3 (IGSF3), protein shisa-2 homolog (Shisa-2), pleiotrophin, latent-transforming growth factor beta-binding protein 4 (LTBP4)) were more specific to iEv than to hAC-Ev.

4. Discussion

We previously developed an induction protocol for XF-iMSCs from human iPSCs via NCCs, because animal-derived ingredients are not necessary for clinical use [11,12]. We validated the functional ability of XF-iMSCs for skeletal muscle and bone regeneration [5]. Primary MSCs also exert anti-inflammatory and immunomodulatory effects [4]. In this study, XF-iMSCs indeed inhibited the secretion of inflammatory cytokines, IL-6 and TNF-α, from LPS-stimulated both human and mouse PBMCs, comparable to primary human MSCs. XF-iMSCs and primary human MSCs increased the levels of the anti-inflammatory cytokine IL-10. Suppression assays showed that XF-iMSCs strongly inhibited T-cell proliferation. These results demonstrate that XF-iMSCs could be used therapeutically for autoimmune diseases instead of primary MSCs.

In terms of the mechanism of efficacy, MSCs exert an inhibitory effect through cell-cell contact with effector cells or the secretome. In the suppression assay, XF-iMSCs exhibited nearly 80% inhibitory effect against T cell proliferation, with only half the number of T cells, defined prior to the co-culture of cells. In terms of the effect of secretome from cells, the conditioned medium (CM) of XF-iMSCs increased IL-10 secretion in LPS-stimulated PBMCs as well as XF-iMSCs, although the inhibition of IL-6 or TNF-α by XF-iMSC-CM was negligible. Purified and concentrated extracellular vesicles (EVs) from XF-iMSC-CM (XF-iEv) strongly inhibited TNF-α secretion but reduced IL-10 secretion. The difference between the CM and EVs of XF-iMSCs may be related to the process of purification and concentration during the preparation of EVs. On the other hand, neither XF-iEv nor hAC-Ev affected both T cell proliferation and cell survival for 4 days culture, suggesting that the observed reduction in cytokine secretion under LPS or CD3/CD28 stimulation is not attributable to EV-induced cell death, but rather reflects a genuine modulatory effect of XF-iEv and hAC-Ev on inflammatory cytokine production at the cellular level.

EVs secreted by MSCs contain various anti-inflammatory and immunomodulatory components that interact with immune cells [13]. To investigate the content of EVs in this study, we conducted proteome analysis for XF-iEv (Supplementary Table 1). XF-iEv included some distinctive proteins, such as midkine, immunoglobulin superfamily member 3 (IGSF3), protein shisa-2 homolog (Shisa-2), pleiotrophin, latent-transforming growth factor beta-binding protein 4 (LTBP4), compared with those of primary hAC-Ev. Midkines and pleiotrophins are classified under a subfamily of heparin-binding growth factors involved in various activities, such as mitogenicity, cellular survival, angiogenesis, oncogenesis, and stem cell renewal [14]. Their mRNA expression is high in the nervous system during embryonic development [15] and is localized in the radial glial cells of the embryonic brain during the migration and differentiation of neural stem cells [16,17]. Additionally, the induction of neuronal apoptosis by serum deprivation was inhibited by midkine in primary neuronal cultures isolated from mouse cerebral cortices [18]. Midkine are also expressed in neural precursor cells and accelerate their growth [19]. IGSF3 is known as a membrane protein involved in cell adhesion and signal transduction [20]. According to the recent report, IGSF3 was demonstrated the localization of the neural crest and a subset of its derivatives, and the importance of enteric nervous system formation by Igsf3 knockout mouse model [21]. Therefore, EVs from XF-iMSCs are suitable for supporting neural functions and merit further investigation. LTBP4 is a member of the LTBP family and an important regulator of TGF-β signaling. LTBP4 is involved in development, tissue regeneration, and fibrotic diseases [[22], [23], [24]] and highly expressed in the smooth and skeletal muscles [25]. Notably, overexpression of LTBP4 in muscles improves muscular dystrophy by reducing active myostatin protein and increasing skeletal muscle mass [26]. Shisa2, which is localized in the endoplasmic reticulum, is also reported the involvement of myoblast fusion [27]. Previously, we suggested that XF-iMSC transplantation enhances skeletal muscle regeneration [5]. XF-iMSC-secreted EVs, including LTBP4 and Shisa2, may be involved in the repair of muscle injury. In the suppression assay, we did not observe an inhibitory effect of XF-iEv on T cell proliferation although XF-iMSCs alone had a strong inhibitory effect. Considering the source of XF-iMSCs from the NCCs lineage, EVs from XF-iMSCs may be involved in neuronal or skeletal muscle regeneration. On the other hand, the undifferentiated hiPSCs-derived EVs had very few proteins which are related in immunomodulatory and anti-inflammatory proteins as well as neuron or skeletal muscle regeneration. Conversely, unlike XF-iEv and hAC-Ev, the hiPSCs-derived EVs include reprogramming-oriented, epithelial identity, and proliferation-associated proteins such as smoothened homolog, insulin-like growth factor binding protein-2, epithelial cell adhesion molecule, and prominin-1 (CD133) (Supplementary information), which are well known for the suggestive of an elevated risk of malignant transformation, uncontrolled proliferation, and tumorigenicity [[28], [29], [30]]. Notably, the cooperative action of these proteins is likely to further elevate tumorigenic risk, warranting careful consideration in their use.

In summary, we demonstrated that XF-iMSCs and their EVs exert anti-inflammatory and immunomodulatory effects in human and mouse cell-based assays. However, unlike the cells, the data concerning iEv only suggest a partial immunomodulatory capacity. Detailed analysis of the role of XF-iMSCs and iEv in anti-inflammatory effects has also not yet been achieved. Furthermore, the therapeutic efficacy in vivo has not yet been evaluated.

Moving forward, we plan to perform further analysis of iEv in vitro mechanisms. For example, recent reports indicate that MSC-Ev can shift the balance between inflammatory macrophages (M1 macrophages) and anti-inflammatory macrophages (M2 macrophages) towards M2 macrophages [31,32]. We plan to verify whether XF-iEv exhibit a similar effect. Additionally, we intend to evaluate the therapeutic efficacy of XF-iMSCs and iEv in vivo using mouse models of immune-system diseases such as graft-versus-host disease, inflammatory bowel disease and atopic dermatitis.

Grant support

This study was supported by a T-CiRA budget from Takeda Pharmaceutical Company, Limited.

Declaration of competing interest

The other authors declare no competing interests.

Acknowledgments

We sincerely thank the former and current members of the T-CiRA Ikeya project, especially Yayoi Toyooka, Teppei Akaboshi, and Teruyoshi Yamashita, Taiki Nakajima, and Hideyuki Hiyoshi, for their valuable support; Nanako Fujimaki from Orizuru Therapeutics for their technical assistance, and Shin Kaneko, Yoshiaki Kassai, Yasushi Kajii, and Shinya Yamanaka for critically evaluating this study.

Footnotes

Peer review under responsibility of the Japanese Society for Regenerative Medicine.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.reth.2026.101081.

Contributor Information

Daisuke Kamiya, Email: kamiya.daisuke.7s@kyoto-u.jp, d-kamiya@vetanic.com.

Makoto Ikeya, Email: ikeya-g@cira.kyoto-u.ac.jp.

Appendix A. Supplementary data

The following is/are the supplementary data to this article.

Multimedia component 1
mmc1.docx (56.4KB, docx)
Multimedia component 2
mmc2.xlsx (451.1KB, xlsx)

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