Journal: International Journal of Molecular Sciences

Article Title: Definition of Synovial Mesenchymal Stem Cells for Meniscus Regeneration by the Mechanism of Action and General Amp1200 Gene Expression

doi: 10.3390/ijms251910510

Figure Lengend Snippet: Cell clustering by Amp1200. Lots of SyMSCs (4 donors: S1, S2, S3 and S4), ADSCs (3 donors: A1, A2 and A3), BMSCs (3 donors: B1, B2 and B3), RECs (3 donors: R1, R2 and R3), FBs, iMSCs, WPs, PAECs, T cells, and CD14+ monocytes were clustered by the expression level of genes obtained by Amp1200. The black lines at the top and left showed the results of the clustering. A similar clustering analysis using all 25,193 human genes is shown in .

Article Snippet: Human SyMSCs (Articular Engineering, Northbrook, IL, USA, CDD-H-2910-N), human ADSCs (Lonza, Basel, Switzerland, PT-5006), human BMSCs (Lonza, Basel, Switzerland, PT-2501), human RECs (PuREC, Izumo, Japan, 387-16591), and iMSCs (FUJIFILM Cellular Dynamics, Madison, WI, USA, iCell MSC R1098) were all cultured in MEMα (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: Activin-A enhances mTOR signaling to promote aberrant chondrogenesis in fibrodysplasia ossificans progressiva

doi: 10.1172/JCI93521

Figure Lengend Snippet: (A and B) Knockdown experiments of mTORC complex components assessed by GAG/DNA (A) and Alcian blue staining (B) in a 2DCI assay of FOP-iMSCs. Scale bar: 200 μm. (C and D) Phosphorylation of SMAD1/5/8 (C) and SMAD2/3 (D) in FOP-iMSCs. Serum-starved FOP-iMSCs were pretreated with 10 nM rapamycin (Rapa), 1 μM DMH1, or 1 μM SB-431542 for 1 hour, and after a 1-hour stimulation with Activin-A, the cells were harvested. (E) p-S6 was enhanced in FOP-iMSCs compared with resFOP-iMSCs In 2DCI assays, p-S6 was enhanced in Activin-A–stimulated FOP-iMSCs compared with levels detected in resFOP-iMSCs. The strength of resFOP at each time point was set at 1. (F) Enhanced p-S6 was inhibited by mTOR (Rapa), PI3K (LY), or AKT (Ipa) inhibitors 24 hours after treatment with Activin-A and inhibitors in FOP-iMSCs. Treatment with 10 μM DMH1, 10 μM BIRB 796 (BIRB), 10 μM LY294002 (LY), 1 μM ipatasertib (Ipa), or 10 nM rapamycin was used. Results represent the mean ± SEM. n = 3 (A and C–F). *P < 0.05, **P < 0.01, and ***P < 0.001, by Dunnett’s multiple comparisons t test compared with the DMSO-treated control stimulated with Activin-A (C, D, and F); with the siRNA-transfected negative control, with or without Activin-A stimulation (A); and by Student’s t test compared with resFOP-iMSCs stimulated with Activin-A (E). Data are representative of 2 independent experiments (A, B, E, and F).

Article Snippet: FOP- (right leg) and resFOP-iMSCs (left leg) (4 × 10 6 cells, respectively) were transplanted into the gastrocnemius muscle of 6- to 8-week-old NOD/ShiJic-scid Jcl (NOD/SCID) mice (CLEA Japan) with C3H-DoxOn-hINHBA (5 × 10 5 cells), which can continuously expose Activin-A to the transplanted iMSCs in vivo ( 38 ).

Techniques: Staining, Transfection, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: Activin-A enhances mTOR signaling to promote aberrant chondrogenesis in fibrodysplasia ossificans progressiva

doi: 10.1172/JCI93521

Figure Lengend Snippet: (A) Venn diagram of genes highly upregulated in FOP-iMSCs compared with resFOP-iMSCs 24 hours after chondrogenesis induction stimulated with Activin-A, BMP-7, or TGF-β3. (B) Genes highly upregulated in FOP-iMSCs 24 hours after chondrogenesis induction stimulated with Activin-A, but not with BMP-7 or TGF-β3. (C) Expression of ENPP2 during the chondrogenic induction of FOP-iMSCs and resFOP-iMSCs. The expression level of resFOP (0 h) was set at 1. (D and E) ENPP2 inhibitors (HA, HA130; PF, PF-8380) (D) and ENPP2 knockdown (E) reduced p-S6 levels. (D) The cells were harvested 24 hours after treatment with Activin-A and ENPP2 inhibitors, and (E) siRNA–transfected cells were harvested 48 hours after treatment with Activin-A. Rapa, 100 nM rapamycin; HA, 10 μM HA130; PF, 10 μM PF-8380. (F) Serum-starved FOP-iMSCs were treated with 100 μM LPA, and 30 minutes after stimulation, the cells were harvested. Results represent the mean ± SEM. n = 1 (A and B) and n = 3 (C–F). *P < 0.05, **P < 0.01, and ***P < 0.001, by Student’s t test compared with LPA (–) control (F), by Dunnett’s multiple comparisons t test compared with FOP-iMSCs stimulated with Activin-A (D), or with negative control siRNA-transfected FOP-iMSCs stimulated with Activin-A (E). Data are representative of 2 independent experiments (D and F).

Article Snippet: FOP- (right leg) and resFOP-iMSCs (left leg) (4 × 10 6 cells, respectively) were transplanted into the gastrocnemius muscle of 6- to 8-week-old NOD/ShiJic-scid Jcl (NOD/SCID) mice (CLEA Japan) with C3H-DoxOn-hINHBA (5 × 10 5 cells), which can continuously expose Activin-A to the transplanted iMSCs in vivo ( 38 ).

Techniques: Expressing, Transfection, Negative Control

Journal: iScience

Article Title: Embryonic lineage-specific iPSC-derived mesenchymal stem/stromal cells exhibit different morphologies and intrinsic functions

doi: 10.1016/j.isci.2025.114482

Figure Lengend Snippet: Osteogenic differentiation potential varies among MSCs derived from different developmental origins (A) Bright-field microscopy images reveal that SM-iMSCs undergo early calcification detectable by alizarin red staining on day 7, with Limb-iMSCs showing calcification by day 14, followed by cNCC-iMSCs, while tNCC-MSCs show appearance of high-density cell nodule (red arrow) and LPM-iMSCs are only in the early stage of mineralization. (B) Quantification of calcification by alizarin red staining at days 7 and 14 shows the highest calcification levels in SM-iMSCs, followed by Limb-iMSCs. (Data are presented as mean ± SD, n = 3, independent experiments, one-way ANOVA followed by Tukey’s test, full results of all pairwise comparisons are provided in ). (C) Alizarin red-staining areas of SM-iMSCs were highest among the iMSCs after 14 days in osteogenic culture. (Data are presented as mean ± SD, n = 3, independent experiments). (D) Comparative qPCR analysis at day 7 shows that all iMSC types, including BM-MSCs, significantly upregulate RUNX2 gene, a key regulator of osteogenesis, with SM-iMSCs displaying the highest level of RUNX2 expression. (Data are presented as mean ± SD, n = 3, independent experiments). (E) Enriched pathways in SM-iMSCs related to ossification ( n = 2, independent experiments). (F) Identified ossification pathway in GO terms analysis validated by comparing extracted genes with traditional databases such as GeneCards, UniProt, AmiGo 2, and NCBI. (G) qPCR result validates the RNA-seq analysis as osteogenesis-related genes such as RUNX2, MMP13 , and COL1A1 are upregulated in SM-iMSCs. (Data are presented as mean ± SD, n = 2, independent experiments). (H) Osteogenic marker BMPR1B upregulated in all iMSCs except for LPM-iMSCs, consistent with the functional experiment. (Data are presented as mean ± SD, n = 2, independent experiments). p value notification: ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Article Snippet: Chondrogenic spheroids (day 21) from cNCC-iMSCs and SM-iMSCs were transplanted subcutaneously into the horizontal incisions on the backs of 8-week-old mice (male, NSG line, The Jackson Laboratory).

Techniques: Derivative Assay, Microscopy, Staining, Expressing, RNA Sequencing, Marker, Functional Assay

Journal: iScience

Article Title: Embryonic lineage-specific iPSC-derived mesenchymal stem/stromal cells exhibit different morphologies and intrinsic functions

doi: 10.1016/j.isci.2025.114482

Figure Lengend Snippet: iMSCs display different adipogenic and chondrogenic differentiation potential (A) Phase-contrast images and oil red O staining showed that cells with accumulated triglyceride droplets appeared in all samples and were highest in number in the Limb-iMSC samples (scale bars, 50 μm, representative data from 3 independent experiments). (B) Quantitative analysis of oil red O staining showed that Limb-iMSC samples exhibited the highest results among all samples. (Data are presented as mean ± SD, n = 3, independent experiments). (C) qPCR at D28 showed PPARG upregulation in all samples, with ADIPOQ and CEBPB significantly higher in Limb-iMSCs. (Data are presented as mean ± SD, n = 3, independent experiments). (D) Alcian blue staining demonstrated that all iMSC types have chondrogenic potential, as evidenced by the deposition of glycosaminoglycans in the extracellular matrix after 7 days of culture in chondrogenic differentiation medium (scale bars, 200 μm, representative data from 3 independent experiments). (E) Quantitative analysis of extracted GAGs showed that the SM-iMSC samples had the highest GAG content among all groups; “μg” denotes the total mass of sulfated glycosaminoglycans (sGAGs) quantified per iMSC pellet. (Data are presented as mean ± SD, n = 3, independent experiments, one-way ANOVA followed by Tukey’s test, full results of all pairwise comparisons are provided in , p value notification: ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). (F) qPCR results indicated that the chondrogenic marker SOX9 was upregulated in all iMSC samples compared to negative control fibroblasts, confirming successful chondrogenic induction, with COL2A1 expression significantly higher in the cNCC-iMSC samples. (n = 2, independent experiments). (G) Specific hypertrophic marker COL10A1 significantly upregulated in SM-iMSC samples. (n = 2, independent experiments). (H) Further GO term analysis reveals pathways related to chondrogenesis in cNCC-iMSCs compared to LPM-iMSCs. (n = 2, independent experiments). (I) Ossification and hypertrophy-related pathways enriched in SM-iMSCs (n = 2, independent experiments).

Article Snippet: Chondrogenic spheroids (day 21) from cNCC-iMSCs and SM-iMSCs were transplanted subcutaneously into the horizontal incisions on the backs of 8-week-old mice (male, NSG line, The Jackson Laboratory).

Techniques: Staining, Marker, Negative Control, Expressing

Journal: iScience

Article Title: Embryonic lineage-specific iPSC-derived mesenchymal stem/stromal cells exhibit different morphologies and intrinsic functions

doi: 10.1016/j.isci.2025.114482

Figure Lengend Snippet: All iMSCs can be differentiated into chondrogenic spheroids in 3D culture (A) After 21 days of chondrogenic differentiation, all iMSC spheroids were stained with Alcian blue, indicating glycosaminoglycan production (scale bars, 50 μm, representative data from 3 independent experiments) with chondrocytes resting in well-structured lacunae (red arrows) and column-like structure in hypertrophic region in SM-iMSCs’ chondrogenic spheroid (black arrow). (B) Hypertrophic markers were strongly expressed in SM-iMSC chondrogenic spheroids and other iMSC samples, except for cNCC-iMSC chondrogenic spheroids (scale bar, 200 μm, representative data from 3 independent experiments). (C) Illustration explains the mechanism of von Kossa testing for dystrophic hypertrophy in chondrogenic pellet. (D) Images show that spheroids from cNCC-iMSCs were stable in the in vivo setting with chondrocytes resting in well-structured lacunae (red arrows), while spheroids from SM-iMSCs showed calcification by von Kossa test (scale bars, 50 μm).

Article Snippet: Chondrogenic spheroids (day 21) from cNCC-iMSCs and SM-iMSCs were transplanted subcutaneously into the horizontal incisions on the backs of 8-week-old mice (male, NSG line, The Jackson Laboratory).

Techniques: Staining, In Vivo

Journal: iScience

Article Title: Embryonic lineage-specific iPSC-derived mesenchymal stem/stromal cells exhibit different morphologies and intrinsic functions

doi: 10.1016/j.isci.2025.114482

Figure Lengend Snippet: Transcriptomic profiling reveals distinct clusters among iMSCs derived from a single parental iPSC line (A) The PCA plot illustrates that the intermediate cell populations cluster according to their embryonic origins. The iMSCs form three distinct clusters: one cluster comprises cNCC-iMSCs and tNCC-iMSCs, another includes SM-iMSCs and Limb-iMSCs, and the third cluster consists of LPM-iMSCs. (B) A heatmap of global gene expression further supports these findings, displaying clustering patterns consistent with the PCA results. (C) Focusing on the top 100 most variably expressed genes during differentiation from iPSCs to expanded intermediates (PN4) to iMSCs, the heatmap aligns with the global expression patterns, highlighting significant changes in gene expression associated with the transition to iMSCs. (D) GO term analysis reveals enriched pathways within each cluster, which correspond to their known biological properties and functions. (E) qPCR results demonstrate that the expression levels of COL1A1 and COL1A2 , markers for mesenchymal stem cells, are significantly upregulated in the iMSC samples compared to the parental iPSCs. (Data are presented as mean ± SD, n = 2, independent experiments).

Article Snippet: Chondrogenic spheroids (day 21) from cNCC-iMSCs and SM-iMSCs were transplanted subcutaneously into the horizontal incisions on the backs of 8-week-old mice (male, NSG line, The Jackson Laboratory).

Techniques: Derivative Assay, Gene Expression, Expressing

Journal: iScience

Article Title: Embryonic lineage-specific iPSC-derived mesenchymal stem/stromal cells exhibit different morphologies and intrinsic functions

doi: 10.1016/j.isci.2025.114482

Figure Lengend Snippet: Transcriptomic profile comparison between iMSCs and tMSCs (A) PCA plot showing distinct clustering of iMSCs and tMSCs, and further analysis reveals close cluster of cNCC-iMSCs and DPSCs. (B) Correlation plot of global gene expression indicates a high correlation score between iMSCs and tMSCs, highlighting their transcriptomic similarities. (C) GO term analysis identifies enriched pathways in tMSCs, including macroautophagy and lipid metabolism. (D) Enriched pathways in iMSCs include those related to proliferation (e.g., cell growth and stem cell population maintenance) and differentiation (e.g., osteogenesis and chondrogenesis), suggesting a higher potential of iMSCs for osteoblast and chondrocyte differentiation. (E) Functional analysis reveals that SM-iMSCs show the earliest positive response to alizarin red staining (scale bars, 200 μm).

Article Snippet: Chondrogenic spheroids (day 21) from cNCC-iMSCs and SM-iMSCs were transplanted subcutaneously into the horizontal incisions on the backs of 8-week-old mice (male, NSG line, The Jackson Laboratory).

Techniques: Comparison, Gene Expression, Functional Assay, Staining