Different responses to DNA damage determine ageing differences between organs

Abstract Organs age differently, causing wide heterogeneity in multimorbidity, but underlying mechanisms are largely elusive. To investigate the basis of organ‐specific ageing, we utilized progeroid repair‐deficient Ercc1Δ /− mouse mutants and systematically compared at the tissue, stem cell and organoid level two organs representing ageing extremes. Ercc1Δ /− intestine shows hardly any accelerated ageing. Nevertheless, we found apoptosis and reduced numbers of intestinal stem cells (ISCs), but cell loss appears compensated by over‐proliferation. ISCs retain their organoid‐forming capacity, but organoids perform poorly in culture, compared with WT. Conversely, liver ages dramatically, even causing early death in Ercc1‐KO mice. Apoptosis, p21, polyploidization and proliferation of various (stem) cells were prominently elevated in Ercc1Δ /− liver and stem cell populations were either largely unaffected (Sox9+), or expanding (Lgr5+), but were functionally exhausted in organoid formation and development in vitro. Paradoxically, while intestine displays less ageing, repair in WT ISCs appears inferior to liver as shown by enhanced sensitivity to various DNA‐damaging agents, and lower lesion removal. Our findings reveal organ‐specific anti‐ageing strategies. Intestine, with short lifespan limiting time for damage accumulation and repair, favours apoptosis of damaged cells relying on ISC plasticity. Liver with low renewal rates depends more on repair pathways specifically protecting the transcribed compartment of the genome to promote sustained functionality and cell preservation. As shown before, the hematopoietic system with intermediate self‐renewal mainly invokes replication‐linked mechanisms, apoptosis and senescence. Hence, organs employ different genome maintenance strategies, explaining heterogeneity in organ ageing and the segmental nature of DNA‐repair‐deficient progerias.


| INTRODUC TI ON
Accumulation of DNA damage is recognized as a principal cause of systemic ageing (Hoeijmakers, 2009;Niedernhofer et al., 2018;Schumacher et al., 2021). DNA lesions interfere with DNA function and activate an intricate DNA damage response (DDR), which triggers repair and decides on cell fate including cell cycle arrest (senescence), mutagenesis, premature differentiation or cell death. Since DNA is at the top of the informational hierarchy, genetic erosion has very diverse, lasting consequences, impairing cell and tissue functioning causing pathology and cancer (Marteijn et al., 2014;Vermeij, Dollé, et al., 2016;Vermeij, Hoeijmakers, & Pothof, 2016). However, how DNA damage shapes ageing and how this relates to organ/tissue-specific ageing trajectories is largely unknown.
Here, we compared intestine and liver, and their stem cells, organoids and damage responses in the Ercc1-deficient mouse model of the XFE human progeroid syndrome (Niedernhofer et al., 2006). ERCC1 is in a complex with XPF involved in damage excision in multiple DNA repair systems: global genome nucleotide excision repair (GG-NER) and transcription-coupled repair (TCR), which remove helix-distorting and transcription-stalling lesions, (deficient in the rare human genetic disorders xeroderma pigmentosum and Cockayne syndrome, respectively (de Laat et al., 1999;Marteijn et al., 2014)), as well as interstrand cross-link repair (defective in Fanconi's anaemia) and single-strand annealing repair of persistent double strand breaks (Ahmad et al., 2008;Gillet & Scharer, 2006;Kuraoka et al., 2000;Niedernhofer et al., 2004). Thus, Ercc1 Δ/− mice carrying one hypomorphic truncation allele and one null allele, harbour defects in four repair systems and combine multiple human genome instability disorders. They have largely normal embryonal development, but show after birth numerous progressive progeroid symptoms (Dolle et al., 2011), strikingly similar to natural ageing, which limit lifespan to 4-6 months (Vermeij et al., 2016b). Using this model, we analysed at tissue, SCs and organoids levels liver and small intestine, which vastly differ in ageing features in order to comprehend why organs and tissues age differently. favours apoptosis of damaged cells relying on ISC plasticity. Liver with low renewal rates depends more on repair pathways specifically protecting the transcribed compartment of the genome to promote sustained functionality and cell preservation.
As shown before, the hematopoietic system with intermediate self-renewal mainly invokes replication-linked mechanisms, apoptosis and senescence. Hence, organs employ different genome maintenance strategies, explaining heterogeneity in organ ageing and the segmental nature of DNA-repair-deficient progerias.

K E Y W O R D S
adult stem cells, DNA damage response, ERCC1, genome maintenance, liver, nucleotide excision repair, organoids, small intestine F I G U R E 1 Aging-related phenotypic features of small intestinal and liver from progeroid Ercc1 Δ/− mice. (a) Intestinal tissue from 15-weekold Ercc1 Δ/− and control mice stained with haematoxylin and eosin. Bars 200 μm. (b,c) Intestinal length (b, n = 2) and perimeter (c, n = 4) from 15-week-old mice of indicated genotypes. (d) Jejunal crypt density of 15-week-old wt and mutant mice. Crypts were counted on paraffinembedded 4μm slices of intestinal tissue. The number of crypts of progeroid Ercc1 Δ/− mice is not significantly reduced in spite of the overall cachexia and decreased organ size, p = 0.7328 (n = 3). (e) Cell density in jejunal crypts of 15-week-old wt and Ercc1 Δ/− mice. Cells were counted on DAPI stained 4μm intestinal tissue slices as in (d), p = 0.5415 (n = 3 mice). (f) Immunofluorescent images of small intestine crypt and villi from sections stained for apoptosis (TUNEL), counterstained with DAPI. Bars 50 μm. (g, h) Apoptosis index in crypts (g) and villi (h), (n = 3 mice). (i) Liver tissue from 15-week-old Ercc1 Δ/− and wt mice assessed for apoptosis (TUNEL). Red arrows: TUNEL + cells; black cut-out depicts a TUNEL + large hepatocyte. Bars 100 μm. (j-m) Apoptosis index in the liver, parenchymal, non-parenchymal (l), and biliary cell (m) population of 15-week-old wt and mutant mice (n = at least 3 mice for WT, n = 5 mice for mutant groups). Quantification of TUNEL + nuclei was performed on DAB-stained liver sections. Data: mean ± SEM. *p < 0.05, **p < 0.01   However, apoptosis-an outcome linked with ageing-related homeostatic deregulation (Muradian & Schachtschabel, 2001) in naturally aged crypts (Martin et al., 1998) appeared significantly elevated with cells dying at the bottom and higher-up in both Ercc1 Δ/− villi and crypts. Hence, apoptosis is not restricted to a specific cellular compartment or stage of differentiation (Figure 1f-h), consistent with a stochastic origin and congruent with accelerated ageing.
Contrary to SI, Ercc1 Δ/− liver is known to suffer from severe ageing pathology (Dolle et al., 2011;Gregg et al., 2012;Vermeij et al., 2016b;Weeda et al., 1997), confirmed for the Lgr5 EGFP Ercc1 Δ/− model used here in Figure S1B-E. TUNEL immunostaining revealed apoptosis to be clearly enhanced in Ercc1 Δ/− liver (Figure 1i-j). Close morphological inspection revealed that nearly all cell types are affected . In fact, the increase of TUNEL + hepatocytes was relatively modest, although, notably, apoptosis included also polykaryons, that is, the equivalent of many diploid hepatocytes. Moreover, hepatocytes expressing cyclin-dependent kinase inhibitor p21 were ~40-fold increased ( Figure S2A-B) and a ~4-fold increase in biliary cells was found ( Figure S2C), consistent with the systemic nature of the repair defect and wide-spread premature ageing features.
p21 expression in mice carrying (liver-specific) DNA repair deficiency is correlated with senescence (Ogrodnik et al., 2017). Recently, Yousefzadeh and coworkers (Yousefzadeh et al., 2020) convincingly demonstrated extensive senescence in 10 organs of Ercc1 Δ/− mice, including liver, starting at the age of 12 weeks, progressively increasing with time. We examined liver tissue of 15-week-old Ercc1 Δ/− mice, but unexpectedly, despite multiple trials and positive controls (see below), did not detect significant loss of LaminB1 and nuclear HMGB1 immunosignals nor significantly increased IL-6 expression that would suggest overt senescence, at this age in our Ercc1 Δ/− mouse line ( Figure S3A-F). Further investigation is warranted to find out whether differences in, for example housing conditions, play a role in the age of onset of senescence in liver, including food, for which Ercc1 Δ/− mutants are extremely sensitive regarding accelerated ageing (Vermeij, Dollé, et al., 2016) and which likely influences antioxidant buffering and DNA damage load (Milanese et al., 2019).
In conclusion, in this and other studies (Dolle et al., 2011;Weeda et al., 1997;Yousefzadeh et al., 2020), Ercc1 Δ/− liver displays numerous accelerated ageing features, in hepatocytes and biliary cholangiocytes, in sharp contrast to SI, although elevated apoptosis in villi and the regenerative and amplifying compartments of crypts suggests altered homeostatic regulation in this organ as well.

| Tissue-specific regenerative responses parallel homeostatic deregulation and pathology in Ercc1 Δ/− small intestine and liver
Previous research provided evidence for limited regenerative proficiency of Ercc1 Δ/− liver following partial hepatectomy , but which cells are implicated and whether SI is affected as well is unknown. Ki67 immunostaining indicated that the proliferative index of Ercc1 Δ/− intestinal crypts did not differ from controls in progeroid Ercc1 Δ/− mice SI displays normal regeneration rates, in spite of increased apoptotic events. However, compared with the very high cell turn-over, cell loss is relatively small and compensatory over-proliferation would likely go undetected. In contrast, in the liver, which normally has low turn-over, homeostasis is severely affected, as for other NER-deficient mouse mutants (Barnhoorn et al., 2014). Hence, DNA repair deficiency triggers cell loss in many cell types in liver, but also enhanced compensatory cell division.

| Distinct responses of Ercc1 Δ/− SC types to unrepaired damage
To further investigate the fate of SCs, we focused on Lgr5 + stem cells responsible for steady-state intestinal homeostasis but also Quantitation of Ki67 + cells per total cells in a field as represented in (c) from 15-week-old mutant and wt mice. More than 5 fields were quantified from n = 7 mice per genotype. (e-g) Proliferative index of various cell populations (identified on morphology and location) in progeroid Lgr5 EGFP Ercc1 Δ/− and wt liver. Note that nearly all cell populations in Ercc1 Δ/− liver show increased proliferation. Over 5 fields were quantified from at least 4 mice per genotype. Data: mean ± SEM, **p < 0.01, ***p < 0.001  damage-induced liver regeneration (Barker et al., 2007;Huch et al., 2013). We crossed Lgr5 EGFP−ires−CreERT2 (Barker et al., 2007) and Ercc1 , 1993;Weeda et al., 1997). We found that a significant fraction of Ercc1 Δ/− Lgr5 EGFP+ cells has enlarged nuclear size. But despite some EGFP + cells in mutant mice looking polyploid ( Figure   S4), overall, nuclei of EGFP + cells seem smaller than the rest of the (GFP − ) population in both genotypes ( Figure S4B). We conclude that, in contrast to intestine, the number of Lgr5 + cells in Ercc1 Δ/− liver is increased. A fraction of LSCs suffers from polyploidy most likely as a consequence of accumulated DNA damage suggesting limited functional potential.

| Functional exhaustion of liver but not intestinal SC populations of progeroid mice
To assess functional consequences of Ercc1 deficiency on SCs, we examined the ability of mutant and WT ISCs to expand into organoids.
After seeding an equivalent number of crypts, organoid-forming capacity of ISCs seems similar for both genotypes ( Figure 4a).
However, mutant differentiated organoids, appeared smaller than WT with lower numbers of organoid-budding crypts ( Ex vivo culture of intestinal and liver SCs from Ercc1 Δ/− mice. (a) Organoids grown from intestinal crypt cell suspensions, derived from 15-weekold Ercc1 Δ/− and wt mice, after 9 days in culture, p = not significant (n = 5 mice per genotype). (b) In vitro cultures of freshly isolated crypts from 15-week-old mice of the indicated genotypes. Images after 9 days in culture. (c) Average organoid size of the indicated genotypes after 9 days in culture (n = 3 independent cultures derived from different mice per group). (d) Average number of crypts budded in organoids of the indicated genotypes after 9 days in culture (at least two cultures for each mouse and 3 mice per genotype). (e) Number of organoids grown after plating bile cell containing liver cell suspensions, derived from 15-week-old Ercc1 Δ/− mice and wt controls (at least two cultures for each mouse and 3 mice per genotype). Organoids were counted at Day 7 of culture. (f) Number of liver organoids grown in secondary cultures, after plating single liver stem cells derived from a primary organoid culture (at least three cultures were measured for each mouse and 3 mice per genotype). Organoids were counted at Day 7 of culture. (g) In vitro cultures of liver organoids grown from bile cell suspensions derived from livers of 15-week-old mice of the indicated genotypes. Images were taken after 7 days in culture. Data: mean ± SEM. *p < 0.05, **p < 0.01 arbitrary units

| Diverse functional outcomes of DNA damage in Ercc1 Δ/− intestinal and liver SCs
Ercc1 Δ/− SI organoids show Ki67 + cell content similar to WT (Figure 5a), indicating that proliferative capacity in vitro is largely unaffected, consistent with the in vivo findings. TUNEL and cleaved Caspase-3 staining revealed that Ercc1 Δ/− SI organoid crypts contain cells undergoing apoptosis (Figure 5b), also recapitulating the mouse tissue phenotype. Interestingly, mutant crypts were found positive for senescence-associated β-galactosidase (SAβ-Gal), indicating the features of senescence (Figure 5c).
In contrast, Ercc1 Δ/− liver organoids display considerably less Ki67 + cells ( Figure 5d) and a significant fraction of SCs was apoptotic or positive for senescence (Figure 5e,f), the latter deviating from our findings in 15-week-old liver ( Figure S3A-F), suggesting that culture conditions are more stressful than in situ and reach the threshold for senescence. LSCs show moderately compromised proliferation (as assessed by EdU incorporation Figure 5d and S6A,B) and γH2AX foci indicative of DNA breaks ( Figure S6C). We found little overlap of γH2AX signal with replicating cells (Figure S6D), consistent with the notion that Ercc1 defects also include repair systems such as TCR (amplified by GG-NER deficiency) linked with transcription, affecting all cell cycle stages.
Flow cytometry for polyploidy showed a trend for more >4n cells in Ercc1 Δ/− organoids, however, increased DNA content did not reach statistical significance ( Figure S6E). Therefore, we measured the perimeter of EdU + nuclei 48 hours after a short pulse, analysing exclusively cells that have undergone replication. Indeed, a significant fraction of Ercc1 Δ/− LSC nuclei seems to be larger and some very large, indicating that they further progress in polyploidization in

| Comparison of functional DNA repair capacity of intestinal and liver stem cells
Since unrepaired DNA damage is the most logical culprit for the SC phenotypes of Ercc1 Δ/− mice, we wished to examine SC responses to different classes of DNA-damaging agents. We chose UV that causes base-pair-disrupting photoproducts mainly repaired by GG-NER; Illudin S, which induces lesions that block transcription and are repaired by TCR (Jaspers et al., 2002); and interstrand cross-links inflicted by cisplatin, which require cross-link repair and mostly affect replication. Ercc1 mutants are deficient in all of these pathways (Marteijn et al., 2014;Niedernhofer et al., 2006). We assessed the ability of cultured repair-proficient ISCs and LSCs to survive and expand into organoids, following increasing doses of the above genotoxins. Surprisingly, although the intestine compared with liver shows less (premature) ageing in WT and when Ercc1 is mutated, ISCs appeared consistently more sensitive to all genotoxins than LSCs (Figure 6a-d). ISCs were previously shown to be more sensitive to γ-irradiation (Barker, 2014). Cytochrome C release following treatment with illudin S and cisplatin illustrates the hypersensitivity of ISCs to apoptotic cell death compared to LSCs (Figure 6e,f).
Apparently, after equal DNA damage ISCs opt for cell death and LSCs prioritize survival. We wondered whether this differential genotoxin sensitivity correlates with differential repair capacity.
Previously, we noted that core NER genes are higher expressed in LSCs than ISCs (Jager et al., 2019). However, expression levels do not always correlate with repair efficiency (Naipal et al., 2015). We examined 6,4-photoproduct resolution after treatment of SCs with UVB, reflecting mainly GG-NER and to a lesser extent TCR activities.
As shown by the remaining 6,4-photoproduct, immunosignals LSCs appear superior in repairing these lesions (Figure 6g). Inefficient repair likely enhances the propensity of ISCs to undergo apoptosis.
Taken together, despite the virtual absence of accelerated ageing features in intestine, ISCs appear inferior in major DNA repair systems when assayed in parallel with LSCs.

| DISCUSS ION
At tissue level, we found no overt features of accelerated ageing in At the organoid level, Ercc1 Δ/− ISCs show ~WT ability to form organoids, which are smaller and contain less crypts, suggesting reduced ability to grow and differentiate. Although proliferative potential is marginally decreased, apoptosis and senescence in these crypts are elevated, in line with time-dependent stochastic DNA damage causing the organoid's limited net growth under culture conditions. In comparison, LSCs appear to be functionally even more compromised in all regards: they form fewer organoids, which are also much smaller and contain fewer proliferative cells, suggesting proliferative exhaustion. Apoptosis, senescence, polyploidy and irregularly shaped nuclei are also increased in liver organoids, accounting for their poor growth. Increased γH2AX foci, mainly in non-replicating cells provides evidence for DNA damage likely related to transcription (rather replication) stress, a genomewide ageing phenomenon, first discovered in Ercc1 Δ/− and Xpg −/− liver and subsequently also in natural ageing (Vermeij, Dollé, et al., 2016), which is associated with functional decline, cell cycle arrest, senescence and cell death. This novel ageing feature occurs primarily in organs with low cell renewal (since DNA replication dilutes damage) and affects expression preferentially of large genes, consistent with accumulating random DNA lesions compromising transcription in a gene-length-dependent manner (Vermeij, Dollé, et al., 2016). The origin of the endogenous DNA damage is unclear, but in our in vitro studies appears largely independent from O 2 levels (see Figure S5b), fitting our observation (Milanese et al., 2019) that TCR-defects trigger a potent anti-oxidant defence (Garinis et al., 2009;Niedernhofer et al., 2006;van der Pluijm et al., 2007).
Directly comparing the DDR and repair properties of WT ISCs and LSCs, we show that ISCs are more sensitive to diverse genotoxins attesting different repair systems. Individual ISC types have distinct damage sensitivities (Shivdasani, 2014). Quiescent Lgr5 + cells appear more resistant, while fast-dividing ISCs have a low apoptotic threshold, as shown for ionizing radiation (Barker, 2014;Barriga et al., 2017). Consistent with low expression of NER genes (Jager et al., 2019), we find ISCs to be less efficient in repair of UVlesions and more sensitive to UV, illudin S and cisplatint damages eliminated by GG-NER, TCR and cross-link repair, respectively, supporting the idea that the entire repair machinery is functionally inferior in ISCs.
As liver exhibits much more ageing features than intestine, one might expect genome maintenance in ISCs to be superior. However, in a direct comparison, repair in LSCs appears superior. This explains why Ercc1 (and all combined TCR/GG-NER) mutants display much less ageing in intestine than liver (and, as evident from human and mouse TCR/GG-NER mutants, also e.g., neurons and kidney). damage to accumulate, rendering repair less critical, and hence, prefer they opt for apoptosis. The high proliferation rate of ISCs and the reserve capacity probably can easily compensate for the loss of a relatively small fraction of cells.
The above strategy may be less suitable when cell turnover is slower with more time for lesions to accrue, increasing dependence on repair. This matches with the severe functional and numerical SC exhaustion in Ercc1 mutant mice for the hematopoietic system (Cho et al., 2013;Prasher et al., 2005;Rossi et al., 2007), in which SCs have an estimated average turn-over of ~2 months. Presumably, exhaustion of HSCs in the Ercc1 mutant is largely caused by its replicationassociated cross-link repair defect, as in Fanconi's anaemia (FA), in line with the notion that defects in the ERCC1 partner XPF can cause FA (Bogliolo et al., 2013;Kuraoka et al., 2000).
Presumably, the investments in high intestinal tissue turn-over cannot be afforded by many other organs and cell turn-over is incompatible with the primary function of, for example most post-mitotic neurons. Moreover, the intestine resides in a very hostile environment, including the microbiome, with all metabolites passing through its epithelium rendering the organ with the highest exposure to exogenous compounds, explaining its 3-5 day cell turn-over. Although liver, as main detoxification organ, is also exposed to numerous toxics, we assume that its superior repair and high damage tolerance enable hepatocytes to live longer. Since cells in liver replicate only occasionally,

F I G U R E 7
Tentative model for organ-specific anti-ageing strategies. Remaining cellular lifespan largely determines which DNA damage response strategy is preferred by organs/tissues to counteract ageing. For intestine, cell death is preferred, as cells have to function only for 3-5 days and cell loss can be compensated by increased (stem) cell proliferation. Obviously, this is very energy-demanding (daily a human body produces 200 gram intestinal epithelium) and unaffordable for many organs. Other tissues with continuous but slower cell renewal such as the hematopoietic system (average cell turn-over ~2 months) rely mostly on replication-related repair (such as NHEJ/HR and XLR) and apoptosis (Hoeijmakers, 2001). Skin, as an organ with high UV exposure and also intermediate cell renewal combines GG-NER and TCR with apoptosis and premature differentiation of damaged stem cells (Kim et al., 2020). Finally, tissues with slow (e.g., liver, on average ~1 year) or no cell turn-over (e.g., the central nervous system, life time) depend on constitutive (cell cycle independent) DNA repair systems, most notably TCR to permit long-term unperturbed use of the transcribed compartment of the genome, needed for sustained proper cellular functioning. Global genome repair systems (base and nucleotide excision repair) are probably important for all organs and tissues for preventing mutagenesis and permit survival. TLS allows replication bypass of lesions to rescue stalled replication and cellular proliferative capacity, however, at the expense of elevated mutagenesis and cancer risk. Cellular (replicative) senescence opposes cell death in most organs. This model explains the segmental nature of repair-deficient progeroid syndromes, in which inherited deficiencies in different repair systems are associated with a different subset of organs and tissues displaying accelerated ageing.

1
Not including mismatch repair, which is a replication error correction system, important for preventing mutations and cancer, particularly in highly proliferative tissues such as intestine. Blood they must rely on replication-independent DNA repair pathways.

| Immunohistochemistry and immunocytochemistry, microspcopy, FACS and flow cytometric analysis
Immunohistochemistry and immunocytochemistry of intestinal and liver tissues were done according to established procedures and detailed in supplementary materials, including the specific reagents (e.g., antibodies) used. Stainings of paraffin-embedded SI and liver tissue sections and liver organoids for senescence markers were performed as described (Baar et al., 2017). Procedures for microscopy, FACS and flow cytometric analysis and equipment are specified in Supplementary materials.

| In vitro small intestinal organoid culture
Intestinal organoid formation from crypts was performed as described (Sato et al., 2009

| In vitro liver organoid culture
Growth of organoid cultures from liver bile duct enriched cell suspensions was performed as described (Broutier et al., 2016;Huch et al., 2013Huch et al., , 2015. A total of 100,000 cells was mixed with 50 μl of Matrigel and plated on 24-well plates. Culture medium was refreshed every two days, and organoids were passaged every 8 to 10 days. Growth of liver organoids from single cells was performed with fluorescence-activated cell sorting of cell suspensions derived from TrypLE-digested primary cultures.

| EdU incorporation assay
In intestinal and liver organoid culture medium, EdU was added for 2 h at a final concentration of 10 μM, cultures were washed with PBS, replenished and at the appropriate time points, fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Edu immunofluorescence was performed using Click-iT ® EdU Alexa Fluor ® Imaging Kit (Invitrogen), according to manufacturers' instructions.

| Genotoxic sensitivity assay
Organoids were dissociated into single cell suspension using TrypLE reagent and passed through a 40 μm cell strainer. Following cell counting, 10,000 single ISCs and 500 LSCs were resuspended in 20 μl Matrigel and plated in a 48-well plate. Cisplatin and illudin S were diluted in HBSS at appropriate concentrations and added to each well. Two hours after treatment medium was removed and replaced with appropriate stem cell culture medium supplemented with 10μM Rock inhibitor Y27632 (Sigma). For ISC cultures, Gsk3 inhibitor CHIR-9921 (Axon MedChem) was added to the culture medium at a final concentration of 10 μM.
For UV sensitivity assessment, suspensions of single stem cells were plated in 12-well plates at different densities and centrifuged at 600 relative centrifugal force for 20 min. at 32°C, medium was carefully removed, and the cells at the well bottom were exposed to the indicated doses of UV (254nm, TUV Lamp Philips). Cells were collected in culture medium, centrifuged at 1500 rpm, resuspended in Matrigel and plated in triplicates in 48-well plates. Organoids were counted 5 days after plating.

| Assessment of DNA lesion resolution in SCs
Organoids were grown in 8-well tissue culture slides, exposed to UVB (200J/m 2 , Philips 40W/12RS UVB lamp) and at selected time points fixed (2% formalin, 20 min. at RT), washed with PBS and 0.1 M glycine, permeabilized with 0,5% Triton-X-100 in PBS for 20 min. RT and treated with 2N HCL for 30 min to denature DNA. After extensive washes with PBS and incubation with blocking solution (1% BSA in PBS), 150μl of anti-6,4-PP antibody (COSMO-BIO, Cat#CAC-NM-DND-002) mix (1:500 in blocking solution) was added to each well, and the slides were transferred to a 37 0 C incubator for 3 h.
Organoids were washed 3 times with PBST (PBS and 0.05% Triton-X-100) and incubated with secondary antibody (Alexa Fluor 555) diluted 1:100 in PBS for 2 h at 37°C. Nuclei were counterstained with Hoechst for 30min. Subsequently, Prolong Diamond mounting medium (Thermo Fisher) was added and slides were incubated overnight in a freezer. Images were captured with a confocal microscope.
Fluorescence measurements and analysis were performed using image J software.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.