EMBO/SourceData huggingface dataset:
Figure 6miR-130a represses VGLL4 and PD-L1 protein levels. A549 cells were transfected with control and miR-130a mimic and subjected to immunoblot analysisInhibition of miR-130a by its sponge enhances IFNγ-inducible PD-L1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected to immunoblotInhibition of miR-130a by its sponge enhances IFNγ-inducible PD-L1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected t qRT-PCR analysisDecreased YAP target gene expression by inhibition of miR-130a using its sponge in A549 cellsYAP5SA expression attenuates IFNγ-inducible PD-L1 expression. A549 cells were transduced with control, YAP-5SA or YAP-S94A lentivirus and treated with IFNγ for the indicated times and subjected to immunoblot analysisqRT-PCR analysis of cell lysates from control, YAP-5SA or YAP-S94A expressing A549 cells treated with IFNγ for the indicated timesControl or YAP-overexpressing LLC cells were transplanted into nude mice (upper panel) or C57BL/6 (lower panel) mice, and tumor-growth kinetics was measured at the indicated times. Tumor volumes at the end time points were analyzed and shown at the right panelmiR-130a regulates IRF1 3′UTR sensor activity. A549 cells were transfected with WT or mutant IRF1 3′UTR sensor plasmids together with miR-130a mimic. Sensor activities were determined by dual luciferase assayYAP5SA induces miR-130a expression and represses IRF1 expression. A549 cells were transduced with control or YAP-5SA lentivirus and subjected to qRT-PCR analysisInhibition of miR-130a by its sponge enhances IFNγ-inducible IRF1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected to qRT-PCR analysisSuppression of IFNγ-inducible PD-L1 expression by YAP5SA is compromised in miR-130a KO A549 cells. PD-L1 expression levels were analyzed by qRT-PCR in IFNγ-treated control or miR-130a KO A549 cells pre-transduced with lenti-YAP5SA virus
Figure 7Representative immunohistochemical staining for VGLL4 and IRF2BP2 in human non-small cell lung cancer tissues. IHC score is indicated. Scale bar: 50 μmStaining of VGLL4 and IRF2BP2 on human non-small cell lung cancer tissue arrays containing 71 samples was quantified as described in the Materials and Methods section. Correlation of VGLL4 and IRF2BP2 expressions was significant (Pearson correlation test; R = 0.6341, P < 0.001) Correlation analysis of VGLL4 and PD-L1 expression on human non-small cell lung cancer tissue arrays containing 71 samplesKaplan-Meier survival analysis of patients with high (n=74) or low (n=130) VGLL4 mRNA levels from GSE31210, a lung cancer data set. Survival curves are calculated according to the Kaplan-Meier method
Figure 1B, C. Detection of CST at stalled and active forks with SIRF assay in U2OS (B) and HCT116 (C) cells. To detect CST at stalled forks, cells were pulse-labeled with EdU for 8 min, followed by 4 mM HU treatment for 3 h. To detect CST at active forks, SIRF was performed without HU treatment. Representative SIRF images of CTC1, STN1 and PCNA on normal or stalled forks are shown. Scale bars: 10 µm. Relative SIRF fluorescence intensity was calculated by normalizing to untreated samples and then to their respective EdU-SIRF signals. Mean value in each sample is labeled in the graph. N: the number of cells analyzed in each sample.
Figure 2Western blot showing STN1 knockdown. shLUC (siRNA targeting luciferase) was used as the control.CST deficiency causes ssDNA accumulation upon fork stalling. HeLa cells with STN1 knockdown were incubated with 10 µM BrdU for 48 h, followed by 2 mM HU treatment for 3 h, and subsequently stained with BrdU antibody under a non-denaturing condition to detect ssDNA. N, the number of cells analyzed in each condition. Relative BrdU fluorescence intensity was quantitated by Image J.Scheme of DNA fiber analysis and representative fiber images.DNA fiber analysis of U2OS cells with STN1 knockdown and its RNAi-resistant Flag-STN1 WT re-expression with and without mirin treatment.DNA fiber analysis of BJ cells with STN1 knockdown by two siRNA sequence with and without mirin treatment.DNA fiber analysis of HCT116 cells with STN1 knockdown with and without mirin treatment.DNA fiber analysis of U2OS cells showing that SMARCAL1-mediated fork reversal is needed for nascent-stand degradation observed in CST-deficient cells.DNA fiber analysis of U2OS cells showing that ZRANB3-mediated fork reversal is needed for nascent-strand degradation observed in CST-deficient cells.
Figure 3SIRF detection of MRE11 at stalled forks in STN1-deficient U2OS cells. Representative SIRF images of MRE11 at normal or stalled replication forks are shown. Scale bars: 10 µm.ssDNA accumulation analysis in STN1-deficient U2OS cells with and without mirin treatment. Representative images of native BrdU staining are shown.
Figure 4Coomassie blue stained SDS-PAGE gel (15%) of purified human MRE11.(i) Scheme shows the nuclease activity of MRE11 in degrading 5' Cy3-labeled substrates (25 nt + 60 nt with phosphorothioate bonds on both ends). (ii) The 5' Cy3-labeled substrates (80 nM) were incubated with the indicated concentration of MRE11 with or without the Mn2+ cofactor at 37°C for 20 min. Reactions were stopped by SDS and proteinase K and resolved on 27% denatured polyacrylamide gel. (iii) The results are graphed, and error bars represent the standard deviation (± SD) calculated from at least three independent experiments.Coomassie blue stained SDS-PAGE gel (15%) of purified human CST complex (CTC1 wild- type and ∆700N).DNA binding activity of wild-type CST complex and Δ700N mutant complex. The 5' Cy3- labeled substrates (80 nM) were incubated with the indicated concentrations of CST. Samples were analyzed by 0.8 % agarose gel.MRE11 degradation analysis. 5' Cy3-labeled substrates (80 nM) were pre-incubated with indicated concentrations of CST wild-type (i) or ∆700N mutant variant (ii) at 37°C for 5 min. Reactions were completed by adding MRE11 (200 nM) for an additional 20 min incubation and then stopped by SDS and proteinase K. Samples were resolved in 27% denatured polyacrylamide gel. The results are graphed and error bars represent the standard deviation (± SD) calculated from at least three independent experiments.
Figure 5Deletion of the N-terminal 700 aa of CTC1 abolishes CTC1 localization at telomeres. U2OS stably expressing vector (V), WT Myc-CTC1 and Myc-∆700N were treated with or without HU and co-stained with Myc (red) and TRF2 (green) antibodies. Boxed areas are amplified in inserts to indicate CTC1/TRF2 colocalization (yellow).Scale bars: 10 μm. Quantification of percent of cells with >3 CTC1/TRF2 colocalization foci were from three independent experiments. In each experiment, >150 cells from each sample were analyzed.   ∆700N does not affect CST complex formation. HEK293T cells were co-transfected with Myc- CTC1 or Myc-∆700N, His6-STN1 and HA-TEN1. Co-IP was performed with Myc antibody to pulldown His6- STN1 and HA-TEN1. Full-length Myc-CTC1 was prone to degradation during immunoprecipitation.      ∆700N retains RAD51 interaction. HEK293T cells were co-transfected with Myc-CTC1 or Myc-∆700N, His6- STN1, HA-TEN1 and Flag-RAD51 or vector control (V), and treated with HU (2 mM, 16 h). Co-IP was performed with Flag antibody to pulldown Myc-CTC1.   DNA fiber analysis of CTC1 depleted U2OS cells with RNAi-resistant WT or ∆700N re-expression. Mean values in each sample are listed at the top of the graph. N, the number of cells analyzed in each condition, ***
Figure 6Micronuclei (MN) formation in BRCA2- and STN1-deficient cells. Co-depletion was achieved by transfecting siBRCA2 in U2OS cells expressing shSTN1. Representative DAPI staining images are shown. Arrows point to MN. Scale bars: 20 µm. Average percentages of nuclei containing MN from three independent experiments are presented. In each experiment, >300 nuclei were analyzed per sample.Anaphase bridges (arrows) in BRCA2- and STN1-deficient U2OS cells. Scale bar: 10 µm. Average percentages of anaphase bridges from three independent experiments are presented.   γH2AX induced by BRCA2 knockdown and STN1 knockdown in U2OS cells. Nuclei containing ≥ 5 foci were considered as positive γ-H2AX staining. Results were from three independent knockdown experiments. In each experiment, >80 cells were analyzed per sample.   Co-depletion of STN1 and BRCA2 significantly impairs DNA replication. Scale bar: 50 µm. Results were from three independent knockdown experiments. In each experiment, >180 nuclei were analyzed per sample.Co-depletion of STN1 and BRCA2 increases chromosome instabilities. U2OS cells with siBRCA2 and/or shSTN1 knockdown were treated with HU (2 mM, 3 h). Representative metaphase images show aberrant chromosomes (red arrows). Boxed areas are amplified and shown at the bottom of images. Scale bars: 20 µm. Two independent knockdown and chromosome spread experiments were performed. N, the number of metaphase spreads analyzed in each sample.
Figure 7SIRF analysis of RAD51 binding to forks. Representative RAD51 SIRF images at normal or stalled forks are shown. Scale bars: 10 µm. Two independent treatments were performed and scatter plot of RAD51-bound replication sites from one experiment is shown. Mean values in each sample are listed at the top of the graph. N, number of cells analyzed in each sample in one experiment.RAD51 foci formation in U2OS CTC1 knockdown cells was rescued by RNAi-resistant WT but not by ∆700N. Cells were treated with HU (2 mM, 16 h) and stained with anti-RAD51. Representative images are shown. Scale bars: 10 µm. Scatter plot of RAD51 foci number with and without HU treatment is shown. Mean values in each condition are listed at the top of the graph.DNA fiber analysis of nascent-strand DNA degradation in U2OS cells with STN1 and RAD51 co-depletion. Three independent treatments were performed and results from one biological replicate are shown. N, the number of DNA fibers analyzed in each sample in one experiment. Western blots showing protein knockdown in DNA fiber assays are included.
Figure 1F Whole-mount visualization of mitochondria in injured hypoglossal motor axon at 5 days after injury. G Immunostaining of injured hypoglossal nerves for GFP and cytochrome c (Cytc) at 5 days after injury. H Number of mitochondria in injured axons of Atf3:BAC2 Tg and Rpt3 CKO mice at 5 days after injury (n= 5 mice per group). I Total number of GFP-positive injured motor neurons in Atf3:BAC2 Tg and Rpt3 CKO mice at 5 days after injury (n= 4 mice per group). J Number of GFP-positive mitochondria in soma at 5 days after injury. Data are shown as the mean ± s.e.m. (n= 3 mice). Data information: Data are shown as the mean ± s.e.m., ** P = 0.0005 in (H), no significant difference (P = 0.8915) in (I) and * P = 0.0068 in (J), determined by unpaired t-test. Dashed lines reveal hypoglossal nucleus in axon in (F and G). Scale bars, 5 μm in (F) and 2.5 μm in (G).
Figure 2C Thionine-stained motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice at 7, 14 and 17 days after hypoglossal nerve axotomy. Arrows indicate the loss of motor neurons. D The graph showing the percentage of surviving motor neurons on the injured side compared with that on the control side. Data are shown as the mean ± s.e.m. (** P = 0.0002, *** P < 0.0001, determined by unpaired t-test, n = 5 mice per group at each time point). Data information: Dashed lines outline hypoglossal nucleus in XII, hypoglossal nucleus; cc, central canal. Scale bars, 150 μm in C)
Figure 3C Hypoglossal motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice before and at 5 days (5d) and 28 days (28d) after injury, immunostained by AnkG and GFP. Arrows indicate the AIS stained by AnkG. Scale bars, 20 μm. D Quantification of the number of the AIS in control (Cont) and injured (Inj) hypoglossal motor neurons of Atf3:BAC2 and Rpt3 CKO mice. Data are expressed as mean ± s.e.m. * P = 0.043, ** P = 0.0162, *** P = 0.008, **** P < 0.0001, determined by one-way ANOVA followed by Tukey post hoc analysis, n.d., not detected, n = 5 mice for each group. E Quantification of the AIS length in control (Cont) and injured (Inj) hypoglossal motor neurons of Atf3:BAC2 and Rpt3 CKO mice. Data are expressed as mean ± s.e.m. * P = 0.0087, ** P = 0.0024, *** P = 0.0007, determined by one-way ANOVA followed by Tukey post hoc analysis, n.d., not detected, n = 5 mice for each group.
Figure 4B Ubiquitination assay of AnkG protein. The lysates of COS-7 cells expressing the indicated truncated AnkG tagged with GST and HA-Ubiquitin were pulled down with glutathione Sepharose beads, followed by western blotting with anti-HA and anti-GST antibodies.C Western blot of hypoglossal nuclei (left) and nerves (right) at 5 days after injury with anti-AnkG, RPT3 and GAPDH antibodies. AnkG products of 190/ 270/ 480 kDa are indicated by arrowheads. The positions of the degraded products by calpain are shown by asterisks at ~95 kDa and 72 kDa.
Figure 5C The localization of AnkG and GFP-labeled mitochondria in the AIS of uninjured MN of Thy1-Mito Tg mice and injured motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice. D Graphs show the GFP and AnkG fluorescent intensity scans corresponding to (C) over a 20 μm line running from the soma to the axon. E Graph represents the percentage (%) of GFP intensity in the AIS, compared with that in the AIS of Thy1-Mito mouse (n = 5 mice per group). Data information: For (E, data are shown as the mean ± s.e.m., ** P = 0.0003, *** P < 0.0001 in (E determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. In closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. Scale bars, 5 μm in C,F Whole-mount observation of GFP- or CFP-labeled mitochondria (mtGFP or mtCFP) in the proximal region of peripheral hypoglossal (XII) nerve as shown in (A). Dashed lines outline the axon. G Graph shows mitochondrial number in hypoglossal (XII) axon (n = 5 mice). Data information: For G, data are shown as the mean ± s.e.m., ** P = 0.0003, *** P < 0.0001 in G) determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 5 μm inI Altered localization of the GFP-labeled mitochondria in the AIS of injured motor neurons of AnkG shRNA AAV-infected Rpt3 CKO mice at 5 days after injury. J Fluorescent intensity scans of GFP and AnkG corresponding to (I). K Graph represents the percentage (%) of GFP intensity in the AIS, compared with that in the AIS of Atf3:BAC2 Tg mouse treated with AnkG shRNA (n = 5 mice per group). Data information: For data are shown as the mean ± s.e.m. , ** P = 0.0064 in (K) determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. In I), closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. Scale bars, 5 μm in I)L Thionine-stained hypoglossal motor neurons of Rpt3 CKO mice at 10 days after injury. Rpt3 CKO mice are infected by AnkG shRNA or scramble shRNA AAVs. Dashed lines outline hypoglossal nucleus. XII, hypoglossal nucleus; cc, central canal. M The percentage of surviving motor neurons on the injured side compared with that on the control side in Rpt3 CKO mice after infection of AnkG shRNA or scramble shRNA AAVs (n= 4 mice per group). Data information: For M), data are shown as the mean ± s.e.m. , * P = 0.0405, ** P = 0.003 in (M), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 100 μm in (L).
Figure 6B Whole-mount visualization of GFP- or CFP-labeled mitochondria (mtGFP or mtCFP) in sciatic nerve. Dashed lines indicate the boundary of axon. Scale bar, 5 μm. C Representative kymographs showing the behavior of mtCFP in uninjured sciatic nerves of Thy1-Mito Tg and mtGFP in injured sciatic nerves of Atf3;BAC2 and Rpt3 CKO mice.D-F The number of mitochondria (D), percentage of motile mitochondria (E) and stationary mitochondrial site (F) in axons. Data are shown as the mean ± s.e.m. *** P = 0.0001, **** P < 0.0001 in (D), ** P = 0.0006, *** P = 0.0001, **** P < 0.0001 in (E), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected, n = 5 mice for each group.
Figure 7C Immunostaining of spinal cord in Atf3:BAC Tg and Atf3;SOD1 mice at P70. The boxed regions are magnified in lower panels. Arrows indicate GFP- and ATF3- positive motor neurons (MNs). Dashed lines outline the edge of the gray matter in spinal cord. D The percentage (%) of GFP-positive MNs (black bar) in ChAT-positive MNs of Atf3:BAC and Atf3;SOD1 mice at P70 (n = 5 mice per each group). E The percentage (%) of ATF3, MMP-9 or osteopontin (OPN)-positive MNs (black bar) in GFP-positive MNs of Atf3;SOD1 mice at P65-70 (n = 5-7 mice per group). Data information: For (D, E, data are shown as the mean ± s.e.m., Scale bars , 50 μm in (C; upper panel), 20 μm in (C; lower)F Immunostaining of AnkG in ChAT-positive spinal MN of Atf3:BAC and SOD1 and in GFP-positive spinal MN of Atf3;SOD1 mouse at P70. G Quantification of the AIS length (n = 5 mice per group). Data information: For G, data are shown as the mean ± s.e.m., no significant difference in (G) determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 10 μm in (FH The localization of AnkG and GFP-labeled mitochondria in uninjured spinal MN of Thy1-Mito (without ATF3 induction), traumatically sciatic nerve-injured MN of Atf3:BAC (with ATF3 induction) and pathologically damaged MN of Atf3;SOD1 mice (with ATF3 induction). Closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. I Fluorescent intensity scan of GFP and AnkG in corresponding to (H). J The percentage (%) of GFP intensity in the AIS, compared with that in the AIS of uninjured MNs in Thy1-Mito mice (n = 5 mice per group). Data information: For data are shown as the mean ± s.e.m. , ** P = 0.0008, *** P = 0.0007 in (J), ** determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 5 μm in (H).K Whole-mount observation of mtGFP or mtCFP in sciatic nerve. Dashed lines outline the axon. L The number of mtGFP or mtCFP in sciatic nerve (n = 5 mice) Data information: For L), data are shown as the mean ± s.e.m., , ** P = 0.0036, **** P <0.0001 in (L), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 10 μm in K)
Figure 1B. Whole exome sequencing was carried out and analysis revealed a missense p.L165F variant (c.493 C>T) in the ALX1 homeodomain, heterozygous in the parents (ALX1165L/165F), wildtype in the unaffected sibling (ALX1165L/165L), and homozygous in both affected subjects (ALX1165F/165F).E. Immunofluorescence staining for pluripotent markers SSEA4, OCT4, SOX2, and TRA-1-60 and alkaline phosphatase staining of iPSC clones. One representative iPSC clone is shown for each genotype. Scale bar: 400 µm.F. Expression of pluripotent (OCT4, NANOG), endoderm (Endo., AFP, GATA4, FOXA2), ectoderm (Ecto., NESTIN, GFAP, SOX1), and mesoderm (Meso., BRACH. (BRACHYURY), RUNX1, CD34) gene markers for ALX1165L/165L (green), ALX1165L/165F(red), and ALX1165F/165F (blue) iPSC relative to undifferentiated cells (UND). Data are represented as pooled mean ± SEM of three experiments on three clones from each genotype. Significance: p=0.0167 for OCT4, p=0.0005 for NANOG, p= 0.000004 for AFP, p=0.0082 for GATA4, p=0.0137 for FOXA2, p=0.00002 for NESTIN, p=0.0167 for GFAP, p=0.0014 for SOX1, p=0.0117 for BRACHYURY, p=0.0008 for RUNX1 and p=0.0068 for CD34 when comparing undifferentiated and differentiated ALX1165L/165L iPSC. p=0.0013 for OCT4, p=0.0011 for NANOG, p=0.0000003 for AFP, p=0.0003 for GATA4, p=0.0063 for FOXA2, p=0.0001 for NESTIN, p=0.027 for GFAP, p =0.000002 for SOX1, p=0.000009 for BRACHYURY, p=3e-9 for RUNX1 and p=0.000006 for CD34 when comparing undifferentiated and differentiated ALX1165F/165L iPSC. p=0.0201 for OCT4, p=0.006 for NANOG, p=1x10-12 for AFP, p=5x10-13 for GATA4, p=0.0031 for FOXA2, p=0.0292 for NESTIN, p=0.00001 for GFAP, p=6x10-7 for SOX1, p=0.0204 for BRACHYURY, p=0.0009 for RUNX1 and p=0.000003 for CD34 when comparing undifferentiated and differentiated ALX1165F/165F iPSC. Data from each clone were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant.
Figure 2B. Images of iPSC and iPSC-derived NCC at Days 0, 14, and passage 4 following differentiation. Scale bars: 400 µm (Day 0), 200µm (Day 14, passage 4).
Figure 3Gene expression analysis across NCC differentiation of unaffected control ALX1165L/165L (green), heterozygous ALX1165L/165F (magenta) and homozygous ALX1165F/165F iPSC: ALX1, neural plate border specifier genes ZIC1, PAX7, PAX3, MSX1, MSX2, DLX5; neural crest specifier genes FOXD3, P75, TFAP2A, SNAI2, TWIST1; and lineage specifier gene HAND2. The RT-qPCR relative expression values were normalized to RPLP0 and GAPDH expression. Data is represented as pooled mean ± SEM of three experiments on three clones from each genotype. Exact p-values are provided in Table EV1. Data from each clone were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant.
Figure 4A. Homozygous ALX1165F/165F NCC (blue) showed an increase in sensitivity to apoptosis when compared to control ALX1165L/165L NCC (black). The data on the left represents the mean percentage of Annexin V positive cells, indicative of apoptosis, as determined by FACS analysis, with the data on the right being an example of one such experiment. Apoptosis was induced by immersion in a 55°C water bath for ten minutes. Representative experiment for each condition is shown. Data is represented as pooled mean ± SEM of three independent experiments. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant. . *: Significantly different from the basal apoptosis rate: p=3x10-12 between control NCC basal apoptosis and induced apoptosis, and between ALX1165F/165F NCC basal apoptosis and induced apoptosis. **: Significantly different from control NCC (p=0.0004). B. Expression levels of cyclins CCNA2 (blue) and CCND1 (orange) in NCC at passages 2 and 3 of ALX1165L/165L and ALX1165F/165F NCC. The RT-qPCR relative expression values were normalized to RPLP0 and GAPDH expression. Data is represented as pooled mean ± SEM of three experiments on three clones from each genotype. *: Significantly different from control NCC at passage 2 (p=0.001 between control and ALX1165F/165F NCC at passage 2 for CCNA2, p=0.0052 for CCND1). **: Significantly different from control NCC at passage 3 (p=0.0494 between control and ALX1165F/165F NCC for CCNA2, p=0.0008 for CCND1).C. Fluorescence Activated Cell Sorting (FACS) experiments showed that control ALX1165L/165L NCC (green) exhibited increased expression of mesenchymal markers CD90, CD105, and CD73 with culture time (passages 1 through 4), whereas homozygous ALX1165F/165F NCC (blue) showed a consistent expression of the markers expressed at passage 1 throughout. Further, control ALX1165L/165L NCC showed a downregulation of CD57 expression with culture time, while ALX1165F/165F NCC maintained the same level of CD57 across passages. Data are presented as the mean percentage of positive-stained cells across passage numbers. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant. *: Significantly different from control NCC. For CD90, p=0.0013 at passage 3 and p=0.0207 at passage 4. For CD105, p=0.0016 at passage 2, p=0.00004 at passage 3 and p=0.0021 at passage 4. For CD73, p=0.0060 at passage 2, p=0.00004 at passage 3 and p=0.0114 at passage 4. For CD57, p=0.0026 at passage 2, p=0.000003 at passage 3 and p=0.000007 at passage 4.