Equine induced pluripotent stem cells are responsive to inflammatory cytokines before and after differentiation into musculoskeletal cell types

Persistent inflammation is associated with the poor regeneration of musculoskeletal tissues. Embryonic stem cells (ESCs) have an attenuated response to inflammatory cytokines, but there are mixed reports on the response of induced pluripotent stem cells (iPSCs) to inflammation. Horses provide a relevant large animal model for studying musculoskeletal tissue diseases and the testing of novel therapies. The aim of this study was to determine if equine iPSCs are responsive to the inflammatory cytokines IL-1β, TNFα and IFN-γ in their undifferentiated state, or following differentiation into tendon and cartilage-like cells. We demonstrated that in undifferentiated iPSCs, the cytokines induce NF-κB P65 and STAT1 nuclear translocation which leads to cell death, decreased OCT4 expression and increased expression of inflammatory genes. Following differentiation towards cartilage-like cells exposure to the cytokines resulted in STAT1 nuclear translocation, changes in cartilage gene expression and increased expression of matrix metalloproteinases (MMPs) and inflammatory genes. Exposure of iPSC-derived tendon-like cells to the cytokines resulted nuclear translocation of NF-κB P65 and STAT1, altered tendon gene expression, increased MMP expression and increased expression of inflammatory genes. Equine iPSCs are therefore capable of responding to inflammatory stimulation and this may have relevance for their future clinical application. Supplementary Information The online version contains supplementary material available at 10.1007/s11626-023-00800-3.


Introduction
Musculoskeletal injuries to tissues such as tendon and cartilage are a major cause of morbidity in humans (Badley et al. 1994;Zhang and Jordan 2010;Sheth et al. 2017).Horses also suffer from naturally occurring injuries to these tissues and provide a relevant, large animal model for their study (Ribitsch et al. 2020).In horses, tendon injuries and cartilage damage occur frequently (Williams et al. 2001;Goodrich and Nixon 2006;Avella et al. 2009;Russell et al. 2017) and are a leading cause of retirement (Lam et al. 2007;Shrestha et al. 2021).This is due to the poor natural regeneration of these tissues, formation of scar tissue and propensity to reinjure (Dyson 2004;Ribitsch et al. 2021).Injuries to these tissues lead to increased inflammatory cytokine production (Hosaka et al. 2002;KAMM et al. 2010;Bertuglia et al. 2016;Morita et al. 2017;Dakin et al. 2018b).Although some inflammation may help to initiate tissue repair, evidence suggests that the persistence of inflammation may contribute to poor tissue regeneration and subsequent fibrotic healing (Dakin et al. 2013(Dakin et al. , 2014(Dakin et al. , 2018a;;van der Kraan 2019).
We have previously demonstrated that the inflammatory cytokine Interleukin 1 beta (IL-1β) has negative effects on tendon cell gene expression and functional ability to contract a three-dimensional (3D) collagen gel (McClellan et al. 2019a).In contrast, we found that horse embryonic stem cell (ESC) derived tendon cells do not respond to IL-1β (McClellan et al. 2019a).This is because they have very low expression of the IL-1 receptor 1 (IL1R1) and high expression of both the decoy receptor IL-1 receptor 2 (IL1R2) and IL-1 receptor antagonist protein (IL1RA) (McClellan et al. 2019a).
Human and mouse ESCs have also been shown to have an attenuated response to inflammatory cytokines (Allen et al. 2000;Guo et al. 2015;D'Angelo et al. 2017;Chen et al. 2020), even after differentiation to a range of cell types including osteoblasts (Sidney et al. 2014), fibroblasts (Chen et al. 2020), smooth muscle cells (Zampetaki et al. 2007) and endothelial cells (Zampetaki et al. 2007;Rajan et al. 2008;Glaser et al. 2011).The relevance of this for the clinical translation of cell products to aid tissue repair and regeneration is not clear.On the one hand, it may be of benefit if the transplanted cells do not respond to, or perpetuate local inflammation.On the other hand, mouse ESCs and ESC-derived endothelial cells fail to respond to the bacterial endotoxin lipopolysaccharide (LPS) (Zampetaki et al. 2006) and undifferentiated human ESCs fail to activate an anti-viral response to double stranded RNA (dsRNA) (Chen et al. 2010).If the cells have low innate immunity and cannot detect or respond to infection, this may be problematic following transplantation (Guo et al. 2015).
Induced pluripotent stem cells (iPSCs) are derived by the reprogramming of adult cells back into a pluripotent state.As the adult cells have already developed innate immunity and cytokine responsiveness, it is of interest to determine if this is "reset" during reprogramming.Mouse iPSCs have a poor antiviral response to Baculovirus infection (Chen et al. 2012) and introduction of double stranded RNA into the cytoplasm (Chen et al. 2010).While undifferentiated human iPSCs do not respond to Tumour Necrosis factor alpha (TNFα) and Interferon gamma (IFN-γ) (Chen et al. 2020).However, human iPSCs which have been differentiated into pancreatic beta cells (Demine et al. 2020), cardiomyocytes (Saraf et al. 2021), astrocytes (Hyvärinen et al. 2019), neurons (Hagman et al. 2019) or chondrocytes (Lee et al. 2017) all show some responsiveness to inflammatory cytokines including IL-1β, TNFα and IFNγ.Although in some cases, the response appears reduced compared to primary adult cells (Lee et al. 2017).
We have previously demonstrated that equine iPSCs can differentiate into tendon-like cells in vitro (Bavin et al. 2015) and the successful differentiation of equine iPSCs to chondrocytes has been reported by others (Quattrocelli et al. 2016).Recently, we have shown that in adult tissue derived tendon cells, a combination of inflammatory cytokines (IL-1β, TNFα and IFN-γ) has an additive effect in inducing more damaging consequences for tendon cell gene expression and function (Smith et al. 2023).Here we aim to determine if equine iPSCs are responsive to these inflammatory cytokines either in their undifferentiated state or following differentiation towards tendon and chondrocyte-like cells.

Materials and Methods
An overview of the experimental approach used in this study is shown in Fig. 1.
iPSC basic media was replaced every 48 h until iPSC colonies began to appear and reached a large enough size to manually pick selected colonies.These colonies were used to establish clonal iPSC lines.iPSCs were mechanically passaged in the presence of 2 μM Thiazovivin (Miltenyi Biotec, Bisley, UK).iPSCs were used at passage 8-15 for all the conditions in this study.

Equine iPSC differentiation
For embryoid body formation, the iPSCs were passaged onto low attachment plates (Corning Life Sciences, Corning, NY) in basic media without feeder cells, LIF or bFGF for up to 14 d.Equine iPSCs were allowed to spontaneously differentiate into derivatives of all three germ layers by culturing in the absence of feeder cells, in basic media (without LIF and bFGF) for 10 d, as previously described (Bavin et al. 2015).Equine iPSCs were directed to differentiate into tenocyte-like cells by culturing in the absence of feeder cells in basic media (without LIF and bFGF) supplemented with 20 ng/ml TGF-β3 (Peprotech) for 14 d as previously described (Bavin et al. 2015).Equine iPSCs were directed to differentiate into cartilage-like cells by culturing in the absence of feeder cells, in basic media (without LIF and bFGF) supplemented with 10 ng/ml TGF-β1 (Peprotech) and 100 nM dexamethasone (Sigma-Aldrich) for 21 d.In each case, media was replenished every 3-4 days.
Nuclear fluorescent intensity (NFI) was quantified by measuring the mean grey scale of the nucleus using ImageJ software.NFI data is presented as fold change in intensity of the inflammatory cytokine treated cells, compared with untreated controls.
RNA extraction, cDNA synthesis and quantitative PCR iPSCs were cultured under undifferentiated or differentiated conditions prior to stimulation with inflammatory cytokines.Stimulation was performed with TNFα (10 ng/mL), IL-1β (1 nM) and IFN-γ (100 ng/mL) for 72 h (Smith et al. 2023).Unstimulated cells served as controls.Following stimulation, RNA was collected using Tri-reagent (Sigma-Aldrich) and extracted using the RNeasy mini kit (Qiagen, UK) following the manufacturer's instructions.Purified RNA was treated with the DNA-free™ DNA removal kit (Invitrogen, Thermo Fisher) according to the manufacturer's instructions to remove genomic DNA contamination.cDNA was synthesised from 1 µg of treated RNA using a sensiFAST cDNA synthesis kit (Bioline, UK). 2 µl of cDNA (corresponding to 20 ng) was used for each quantitative PCR (qPCR) reaction.Primer sequences can be found in Table 2. qPCR was performed in duplicate using SYBR Green containing supermix (Bioline) on a Bio-rad C1000 Touch Thermal Cycler (Bio-rad, UK).PCR cycle parameters were as follows: 95°C (10 min), followed by 45 cycles of 95°C (15 s), 60°C (15 s) and 72°C (15 s).Following this, a melt curve was produced with readings taken every 1°C from 65°C to 95°C.Relative gene expression levels were normalised with the housekeeping gene 18s rRNA using the 2 −ΔΔCt method (Livak and Schmittgen 2001).qPCR data is presented as a fold change in gene expression of the inflammatory cytokine treated cells, compared with untreated controls.
Alcian blue staining and leaching Undifferentiated equine iPSCs and iPSC-derived cartilage-like cells were fixed in 3% PFA for 20 min prior to washing and staining in Alcian blue stain solution (1% w/v in 3% acetic acid solution, pH 2.5, Sigma, B8438) following the manufacturer's instructions.Briefly, cells were stained with Alcian blue for 1 h at  RT.After removing the solution the cells were washed three times with water.Finally, samples were left to dry for up to 1 h at RT. Leaching was performed using 6M Guanidine-HCl (Sigma-Aldrich) for 1 h in the dark at RT. Measurements were made on a microplate absorbance reader (Mplex_Infinite M200; Tecan, Switzerland) at 650 nm.

Statistical analysis
All the assays were performed on three biological replicates for each cell type.Statistical analysis was performed using Excel (Microsoft™) and SPSS (version 28.0.0.0.; IBM, UK).All data sets were tested to ensure Gaussian distribution using the Shapiro Wilks normality test and equal variance using Levene's test for equality of variances.For comparisons of two groups Student's t-tests were used.Data with more than two groups was analysed using one-way ANOVA with a Tukey's post-hoc test.When data was not normally distributed, the data was log transformed or a non-parametric Kruskal Wallis test was performed followed by Dunn's pairwise comparisons.In all cases a p value of < 0.05 was considered statistically significant.

Inflammatory cytokines have negative effects on undifferentiated equine iPSCs
The iPSCs lines used in this study were characterized by their expression of pluripotency markers, capacity to generate embryonic bodies and undergo spontaneous differentiation into derivatives of endoderm, ectoderm and mesoderm when cultured in the absence of feeder cells, LIF and bFGF.(Supplementary Fig. 1).An overview of the experimental procedures is shown in Fig. 1.Undifferentiated equine iPSCs show predominantly cytoplasmic staining of NF-κB P65 under non-stimulated conditions.In response to a combination of IL-1β, TNFα and IFN-γ, NF-κB P65 is translocated to the nucleus within 1 h and quantification of the nuclear staining is significantly increased.Similarly, in unstimulated equine iPSCs there is a low level of both nuclear and cytoplasmic STAT1.However, after stimulation with IL-1β, TNFα and IFN-γ there is significant accumulation of nuclear STAT1.In contrast, JNK1/2/3 shows a similar nuclear and cytoplasmic localisation in both stimulated and non-stimulated cells and p38 MAPK shows nuclear and cytoplasmic staining which decreases following stimulation with cytokines (Fig. 2A and B).
Although no overt change in cell morphology was observed following 1 h of cytokine stimulation, after 72 h of cytokine stimulation we observed considerable cell death within equine iPSC colonies, despite the feeder cell layer being unaffected (Fig. 2C).This coincided with a significant decrease in the pluripotency gene OCT4 and a very large, significant increase in the inflammatory associated gene IL-6 (over 8000-fold).There was also an induction of IL-1β and a large (over 300-fold), but not significant increase in TNFα (due to high levels of variability in the degree of change between the replicates).Other genes associated with inflammation such as STAT3, TGF-β1 and COX2 showed no, or small and not significant, changes in expression in response to 72 h of cytokine stimulation (Fig. 2D).
Inflammatory cytokines induce translocation of NF-κB P65 and STAT1 and result in increased expression of inflammatory genes in spontaneously differentiated equine iPSCs We next allowed the iPSCs to spontaneously differentiate for 10 days into a mixed cell population by the removal of the feeder cells, LIF and bFGF.These conditions are sufficient to allow cells derived from all three germ layers to be produced ( (Bavin et al. 2015) and Supplementary Fig. 1).Similar to the undifferentiated iPSCs, exposure to all three cytokines together produced an increase in nuclear NF-κB P65 (Fig. 3A).However, due to the variability between the replicates and across the coverslips (likely due to the heterogeneous differentiation) the quantification of nuclear staining showed no significant differences between conditions (Fig. 3B).STAT1 showed a predominantly nuclear localisation and was not affected by the cytokine exposure.JNK1/2/3 showed a strong nuclear and weaker cytoplasmic staining in the control conditions lacking cytokines, and a strong, more defined nuclear staining following exposure to all three cytokines.However, the intensity of nuclear staining was not quantitatively increased (Fig. 3B).p38 MAPK shows a fairly low level of nuclear and cytoplasmic staining under both stimulated and non-stimulated conditions (Fig. 3A and B).
Following 72 h of exposure to all three cytokines, we measured the expression of inflammation-associated genes.We saw very large increases in IL6 and TNFα (over 2000fold), but the variability between replicates meant that these changes were not statistically significant.The cytokine treatment also led to an induction of IL-1β and a small, but significant increase in STAT3 expression.TGF-β1 and COX2 showed no changes in expression (Fig. 3C).

Inflammatory cytokines activate STAT1 signalling in cartilage-like cells derived from equine iPSCs and lead to significant changes in gene expression
Undifferentiated equine iPSCs show little Alcian blue staining.However, following 21 days of culture in the presence of TGF-β1 (and absence of feeder cells, LIF and bFGF) all iPSC colony outgrowths stained positively for Alcian blue indicating glycosaminoglycan deposition (Fig. 4A).Stain leaching and quantification demonstrated a significant increase in Alcian blue levels following differentiation (Fig. 4B).After 21 days of differentiation, the cartilage-like cells were exposed to the inflammatory cytokines.After 1 h of exposure, we observed a significant increase in nuclear STAT1, but no change in the nuclear protein levels of NF-κB P65, JNK1/2/3 or p38 MAPK (Fig. 4C and D).
After 72 h exposure to all three cytokines the expression of the cartilage associated gene SOX9 showed a small, but significant increase.Whereas ACAN and COMP showed decreases in expression, which was significant for COMP.CNMD expression was unaffected.MMP1 and MMP3 showed robust, but not significant increases in gene expression, and MMP8 was induced following cytokine exposure.In contrast, MMP2, 9 and 13 showed no or smaller changes in expression.IL6 and TNFα showed large increases in expression but the differences were not significant and IL-1β expression was only detected following cytokine exposure.STAT3 expression showed a small, but significant increase, and TGF-β1 and COX2 showed no changes in expression (Fig. 4E).

Inflammatory cytokines induce translocation of NF-κB P65 and alter gene expression in iPSC-derived tendon-like cells
We have previously demonstrated that equine iPSCs upregulate tendon associated gene and protein expression following 14 days of culture in the presence of TGF-β3 (and absence of feeder cells, LIF and bFGF) (Bavin et al. 2015).Here, we demonstrate that this population of cells are capable of responding to inflammatory cytokines in a similar manner to that which we recently described for primary tenocytes isolated from the superficial digital flexor tendon of horses (Smith et al. 2023).In response to a 1 h exposure to all three cytokines, the iPSC-tendon-like cells exhibit a significant increase in NF-κB P65 nuclear staining (Fig. 5A and B).There is also a trend for a small increase in nuclear NF-κB p65 staining following IL-1β and TNFα exposure alone.In contrast, STAT1 undergoes a trend for increased nuclear staining following exposure to IFN-γ alone or all three cytokines together.JNK1/2/3 shows strong nuclear staining under control conditions which is not further altered by cytokine exposure, and p38 shows very weak staining under all conditions (Fig. 5A and B).This pattern is very similar to that which we observed in primary tendon cells (Smith et al. 2023).72 h following exposure to all three cytokines, we observed a significant downregulation of tendon associated genes SCX, COL1A1 and TNMD and a trend for an increase in TNC.COMP and THBS4 expression levels were not changed (Fig. 5C).MMP expression was altered with a robust (but not significant) increase in MMP1 and significant increases in MMP3 and MMP13.Inflammatory gene expression was also altered with over a 200-fold increase in IL6 and large increases in IL-1β and TNFα.But due to the variability in the scale of the fold change between biological replicates, these increases were not significant.Other genes associated with inflammation such as STAT3, TGF-β1 and COX2 showed no, or small and not significant changes in expression in response to 72 h of cytokine stimulation.

Discussion
We have previously demonstrated that, like human and mouse ESCs and their derivatives (D'Angelo et al. 2017(D'Angelo et al. , 2018)), equine ESC-derived tendon cells do not respond to the inflammatory cytokine IL-1β (McClellan et al. 2019a).Conflicting reports on the responsiveness of iPSCs and their differentiated progeny to inflammatory cytokines have been published (Lee et al. 2017;Hagman et al. 2019;Hyvärinen et al. 2019;Chen et al. 2020;Demine et al. 2020;Saraf et al. 2021) and to date, there are no reports examining this in equine iPSCs.
Here we demonstrate that undifferentiated equine iPSCs do respond to a combination of IL-1β, TNFα and IFN-γ.After 1 h they demonstrate increased nuclear staining of both NF-κB P65 and STAT1 and after 72 h they have a decreased expression of the pluripotency factor OCT4 and increased expression of inflammatory associated genes including IL-1β and TNFα, which suggests a positive feedback loop.IL6, which is a known target gene of NF-κB (Liu et al. 2017), was also upregulated in response to inflammatory stimulation.STAT3 expression is associated with inflammation and fibrosis, and is upregulated in response to IL6 signalling (Kasembeli et al. 2018).Nevertheless, we did not observe any changes in STAT3 expression within the time frame studied.Future work should confirm if the increased IL6 gene expression correlates with an increase in IL6 protein levels.Cytokine exposure also resulted in observable cell death after 72 h which did not occur in any of the differentiated cell types we studied.However, we did not perform any cell viability assays to quantify this.A previous report on undifferentiated human iPSCs found that these cells exhibited no response to TNFα alone or TNFα plus IFN-γ (Chen et al. 2020).We did not look at the effect of these specific conditions on the undifferentiated equine iPSC cells.
The spontaneous differentiation of the equine iPSCs resulted in a mixed population of cells, representing derivatives of all three germ layers.Similar results to the undifferentiated iPSCs were observed, with an increase in NF-κB P65 nuclear localisation, increased IL6 and TNFα expression and induction of IL-1β.However, none of these increases were significant, likely due to the very heterogenous population of cells produced.In contrast, we did observe a small but significant increase in STAT3.
We then directed the iPSCs towards cartilage or tendonlike cells using TGF-β1/dexamethasone or TGF-β3 respectively.Following cartilage differentiation, we observed positive Alcian blue staining for glycosaminoglycans, and although we had expression of cartilage-associated genes, we did not quantify the heterogeneity of the resulting population.TGF-β1/dexamethasone has previously been shown to drive the chondrogenic differentiation of human iPSCs (Saitta et al. 2014), but a two stage protocol via a mesenchymal intermediate may increase the efficiency of differentiation (Diederichs et al. 2019) and this could be optimised in future work.Nevertheless, we were able to demonstrate that the iPSC-derived cartilage-like cells were able to respond to inflammatory cytokines with a significant increase in STAT1 nuclear localisation, and changes in cartilage-associated, MMP and inflammatory gene expression.We observed an increase in the early cartilage transcription factor SOX9 and a decrease in the more mature cartilage markers ACAN and COMP.This may suggest that the efficiency of the differentiation is reduced in the presence of inflammatory cytokines, but we did not directly test this here.We also observed increases in MMP, IL6, IL-1β, TNFα and STAT3 gene expression.Human iPSC-derived chondrocytes have also been shown to increase IL6, MMP3 and SOX9 expression in response to IL-1β (Lee et al. 2017).However, in this study, the authors demonstrated that the scale of the response was lower in iPSC-derived chondrocytes and juvenile chondrocytes than it was in adult chondrocytes.A limitation of our study is that we did not carry out the same experiments with primary equine chondrocytes to determine if the iPSC-cartilage like cells responded to inflammation to a similar degree to primary cells.
We have previously demonstrated equine iPSCs cultured for 14 days in the presence of TGF-β3 (and absence of feeder cells, LIF and bFGF) results in an increase in tendonassociated gene and protein expression (Bavin et al. 2015).Here, we have confirmed that tendon-associated genes are expressed following our differentiation protocol, but we did not measure protein expression.However, it is unlikely that we have a homogenous population of cells as we have previously shown they fail to contract a 3-D collagen gel; a measure of cell mediated matrix re-organisation (Bavin et al. .The stepwise differentiation of human iPSCs to tendon cells has been reported previously (Nakajima and Ikeya 2021), but these approaches have not yet been applied to equine cells and accurate tendon cell markers are lacking (Li et al. 2021).Despite the probable heterogeneity within our population, here we show that equine iPSC tendon-like cells exhibit a similar response to that which we have recently observed in primary equine tendon cells (Smith et al. 2023).For example, both primary tenocytes and iPSC tendon-like cells have significantly increased nuclear NF-κB P65 staining after exposure to all three cytokines.In the primary tenocytes, this was also seen to occur in response to IL-1β or TNFα alone, but the increase in the iPSC tendon-like cells was not significant in these conditions (possibly due to the heterogenous nature of the differentiation).In primary tenocytes, nuclear STAT1 is significantly increased in response to IFN-γ exposure (Smith et al. 2023), and a similar trend is observed in the iPSC tendon-like cells, although the increase is not significant.In both the primary tenocytes and iPSC tendon-like cells no increase in JNK1/2/3 or p38 MAPK is observed in response to any of the inflammatory cytokines.However, a limitation of our study is that we only utilised immunofluorescence to determine nuclear localisation and in future studies other techniques, such as western blot, could be used to validate the results.
Similarly, in both the primary tenocytes and the iPSC tendon-like cells we observed significant decreases in tendonassociated genes such as SCX and COL1A1 along with an increase in TNC and MMP1, 3, 8 and 13.However, the scale of the changes in the MMP genes is much larger in the primary tenocytes (over 100-fold for MMP3 and 8 (Smith et al. 2023)).Again, this may reflect the heterogeneity of the iPSC tendon-like cell population.In this study we did not measure the expression of the tendon-associated proteins and further work is required to determine if the observed changes in gene expression are also reflected at the protein level.Here, we also examined the expression of inflammatory genes and found a similar pattern as for the undifferentiated and other differentiated iPSCs, with a large increase in IL6, IL-1β and TNFa and a smaller increase in STAT3.COX2 is another target gene of NF-κB (Nakao et al. 2000) and has previously been shown to be increased in response to IL-1β and TNFα exposure in mouse, rat and human tenocytes (Oreff et al. 2021).Interestingly, this increase was not observed in either sheep or horse tenocytes (Oreff et al. 2021) and in our study we saw no increase in COX2 expression in response to cytokine stimulation in any of the equine iPSC derived cell types investigated.TGFβ can drive fibrosis and is overexpressed during a tendon injury (Morita et al. 2016).IL-1β exposure of human tenocytes from diseased tissues leads to an upregulation in TGF-β1 expression, but this does not occur in tenocytes isolated from healthy tissues (Morita et al. 2019).Similarly, we did not observe any changes in TGF-β1 expression in our iPSC-derived tendon-like cells, or any of the other cell types studied.
Together, this work demonstrates that equine iPSCs both pre and post differentiation into multiple lineages are able to respond to the inflammatory cytokines IL-1β, TNFα and IFN-γ.This supports work using iPSCs from other species that have demonstrated cytokine responsiveness following lineage differentiation (Lee et al. 2017;Hagman et al. 2019;Hyvärinen et al. 2019;Demine et al. 2020;Saraf et al. 2021) The equine iPSCs used in this study have been characterised for their expression of pluripotency markers and ability to differentiate into derivatives of endoderm, mesoderm and ectoderm in vitro (Bavin et al. 2015;Baird et al. 2018Baird et al. , 2019)).We have previously demonstrated that this method of reprogramming generates karyotypically normal iPSCs (Bavin et al. 2015), however the specific clonal iPSC lines used in this study did not undergo karyotyping.Furthermore, the ability of the iPSCs to form teratomas in vivo has not been tested

◂
and therefore it is possible that complete reprogramming to a pluripotent state equivalent to the ESCs has not been achieved.However, further work is also required to determine if ESCderived cells develop responsiveness with longer differentiation protocols, or after in vivo transplantation is also required to determine what factors are necessary for the establishment of cytokine responsiveness during development.
Human adult tenocytes have been shown to establish an inflammation memory giving them an increased responsiveness to future stimuli (Dakin et al. 2017(Dakin et al. , 2018b)).Our work on equine tenocytes (Smith et al. 2023), ESCs (McClellan et al. 2019a) and iPSCs in this study has investigated only the short-term effects of a single inflammatory cytokine stimulation.Future work to determine the longer term effects of both short-term, long term and repeated exposure to cytokines is required in order to better mimic the response of cells to acute and chronic inflammation.

Conclusions
In conclusion, we have demonstrated that equine iPSCs and their differentiated progeny are negatively affected by shortterm exposure to inflammatory cytokines which may have relevance for their clinical application.

MMP3
Matrix Metallopeptidase 3 (Stromelysin-1) TGG ACC TGG AAA AGT TTT GG GAC CAA GTT CAT GAG CAG CA MMP8 Matrix Metallopeptidase 8 (Neutrophil collagenase) TTT GAT GGA CCC AAT GGA AT TTC ATG GGC AGC AAC AAT AA MMP9 Matrix Metallopeptidase 9 GAG ATC GGG AAT CAT CTC CA CCA AGA GTC GCC AGT ACC TC OCT4 Octamer-binding transcription factor 4 GAT ATA CCC AGG CCG ATG TG GTC GTT TGG CTG AAC ACC TT SCX Basic helix-loop-helix transcription factor scleraxis CCC AAA CAG ATC TGC ACC TT ATC CGC CTC TAA CTC CGA AT SOX9 Transcription factor SOX-9 GCT CTG GAG ACT TCT GAA CGA GTA ATC CGG GTG GTC CTT CT STAT3 Signal transducer and activator of transcription 3 AGT GAC CAG GCA GAA GAT GC CTG TTG TCG CCT CTT CCA GT TGF-β1 Transforming Growth Factor-β1 CGC GTG CTA ATG GTG GAA AA GAG CTC CGA CGT GTT GAA GA THBS4 Thrombospondin-4 GGG AAA TGG GGT TAC CTG TT CGG GTA GCA GGG ATG ATA TT TNC Tenascin AAC CCG TCC AAA GAG ACC TT GCG TGG GAT GGA AGT ATC AT TNFα Tumor necrosis factor alpha AAA GGA CAT CAT GAG CAC TGA AAG GGG CCC CCT GCC TTCT TNMD Tenomodulin GTC CCT CAA GTG AAG GTG GA CCT CGA CGG CAG TAA ATA CAA

Figure 2 .
Figure 2. Response of undifferentiated equine iPSCs to inflammatory cytokine stimulation.(A) Immunofluorescence staining of NF-κB, STAT1, JNK1/2/3 and p38 MAPK in equine iPSCs after 1 h of exposure to IL-1β, TNFα and IFN-γ (all 3) stimulation compared to the control (no stimulation).DAPI staining of the nucleus is shown in blue.Images are representative of three biological replicates.Scale bar = 50 µm.(B) Quantification of the relative nuclear fluorescent intensity of NF-κB, STAT1, JNK1/2/3 and p38 MAPK after 1 h of cytokine stimulation (all 3) shown as a fold change compared to the

Figure 3 .
Figure 3. Response of spontaneously differentiated iPSCs to inflammatory cytokine stimulation.(A) Immunofluorescence staining of NF-κB P65, STAT1, JNK1/2/3 and p38 MAPK in equine iPSCs that have been allowed to spontaneously differentiate for 10 days and then exposed to IL-1β, TNFα and IFN-γ (all 3) for 1 h compared to the control (no stimulation).DAPI staining of the nucleus is shown in blue.Images are representative of three biological replicates.Scale bar = 50 µm.(B) Quantification of the relative nuclear fluorescent

Figure 4 .
Figure 4. Response of cartilage-like cells derived from iPSCs to cytokine stimulation.(A) Alcian blue staining of undifferentiated iPSCs (top row) and cartilage-like cells derived from iPSCs (bottom row).Scale bars = 1 mm (left column) and 200 µm (right column).(B) Quantification of Alcian blue absorbance at 650 nm following stain leaching in undifferentiated iPSCs and iPSC-derived cartilagelike cells.Error bars represent the S.E.M from three biological replicates.*p < 0.05.(C) Immunofluorescence staining of NF-κB P65, STAT1, JNK1/2/3 or p38 MAPK in cartilage-like cells derived from iPSCs after 1 h of cytokine stimulation (all 3) compared to the control (no stimulation).DAPI staining of the nucleus is shown in blue.Images are representative of three biological replicates.Scale bar = 50 µm.D Quantification of the relative nuclear fluorescent intensity of NF-κB P65, STAT1, JNK1/2/3 or p38 MAPK after 1 h of cytokine stimulation (all 3) compared to the control (no stimulation).Error bars represent the S.E.M of three measurements from each of three biological replicates.*p < 0.05.E Fold change in cartilage-associated, MMP and inflammatory gene expression in cartilagelike cells following stimulation with all 3 cytokines (IFN-γ, TNFα and IL-1β) compared to the no cytokine control and shown on a log2 scale.Induced = expression only detected following exposure to the cytokines and not in the control conditions.Error bars represent the S.E.M of three biological replicates, *p < 0.05.◂ . However, it contrasts with our work on equine ESCs.While we have not yet examined the response of equine ESC-derived tendon cells to TNFα, we have previously shown that equine ESC-derived tendon cells do not respond to the inflammatory cytokine IL-1β (McClellan et al. 2019a).However, they are capable of responding to IFN-γ as an upregulation of MHC I was observed (McClellan et al. 2019b).We found that the lack of response to IL-β was due to a reduced IL1R1 expression, an increased expression of the decoy IL1R2 receptor and increased IL1 receptor antagonist protein gene expression (McClellan et al. 2019a).It is not clear if this reflects a lack of IL-1β receptor expression in the inner cell mass cells of horse blastocysts from which the ESCs are derived.However, in bovine embryos it has been shown that IL1R1 is present on trophectoderm cells, but not in inner cell mass cells (Correia-Álvarez et al. 2015).It would be of interest to compare cytokine receptor expression in horse ESCs and iPSCs before and after differentiation to understand the differences in the responsiveness of the cells to inflammatory stimulation.

Figure 5 .
Figure 5. Response of tendon-like cells derived from equine iPSCs to cytokine stimulation.(A) Immunofluorescence staining of NF-κB P65, STAT1, JNK1/2/3 or p38 MAPK in tendon-like cells derived from iPSCs after 1 h of cytokine stimulation (IL-1β, TNFα and/or IFN-γ stimulation) compared to the control (no stimulation).DAPI staining of the nucleus is shown in blue.Images are representative of three biological replicates.Scale bar = 50 µm.(B) Quantification of the relative nuclear fluorescent intensity of NF-κB P65, STAT1, JNK1/2/3 or p38 MAPK after 1 h of cytokine stimulation (IL-1β, TNFα and/or IFN-γ) compared to the control (no stimulation).Error bars represent the S.E.M of three measurements from each of three biological replicates.*p < 0.05.(C) Fold change in tendon-associated, MMP and inflammatory gene expression in tendon-like cells following stimulation with all 3 cytokines (IFN-γ, TNFα and IL-1β) compared to the no cytokine control for 72 h and shown on a log2 scale.N.D = not detected.Error bars represent the S.E.M of three biological replicates, *p < 0.05.

Table 2 .
Primers used in qPCR