SALL3 mediates the loss of neuroectodermal differentiation potential in human embryonic stem cells with chromosome 18q loss

SALL3 mediates the loss of neuroectodermal differentiation potential in human embryonic stem cells with chromosome 18q loss | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SALL3 mediates the loss of neuroectodermal differentiation potential in human embryonic stem cells with chromosome 18q loss Claudia Spits, Yingnan Lei, Diana Al Delbany, Nuša Krivec, Marius Regin, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3100381/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Human pluripotent stem cell (hPSC) cultures are prone to genetic drift, as cells that have acquired specific genetic abnormalities experience a selective advantage in vitro. These abnormalities are highly recurrent in hPSC lines worldwide, but currently their functional consequences in differentiating cells are scarcely described. An accurate assessment of the risk associated with these genetic variants in both research and clinical settings is therefore lacking. In this work, we established that one of these recurrent abnormalities, the loss of chromosome 18q, impairs neuroectoderm commitment and affects the cardiac progenitor differentiation of hESCs. We show that downregulation of SALL3 , a gene located in the common 18q loss region, is responsible for failed neuroectodermal differentiation. Knockdown of SALL3 in control lines impaired differentiation in a manner similar to the loss of 18q, while transgenic overexpression of SALL3 in hESCs with 18q loss rescued the differentiation capacity of the cells. Finally, we show by gene expression analysis that loss of 18q and downregulation of SALL3 leads to changes in the expression of genes involved in pathways regulating pluripotency and differentiation, including the WNT, NOTCH, JAK-STAT, TGF-beta and NF-kB pathways, suggesting that these cells are in an altered state of pluripotency. Biological sciences/Stem cells/Pluripotent stem cells/Embryonic stem cells Biological sciences/Stem cells/Stem-cell differentiation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), are self-renewing cells that can give rise to any cell type originating from any of the three embryonic germ layers 1 , 2 . This makes hPSCs an attractive resource for in vitro disease modelling, developmental biology research, drug discovery, and cell transplantation therapy. A substantial number of clinical trials are underway using hPSC-derived cell products, including for the treatment of age-related macular degeneration, spinal cord injury and type 1 diabetes 3 – 5 . An important hurdle for both safe clinical translation and the reliable use of hPSCs as in vitro research models is the occurrence of cell culture drift due to the acquisition of genetic abnormalities 5 – 8 . A subset of these genetic aberrations are highly recurrent and are found in hPSC lines worldwide. These recurrent changes vary in size from single-nucleotide point mutations to large chromosome structural variants, the most common being gains of chromosomes 1q, 12p, 17, 20, and X and losses of 10p, 18q and 22p, as well as mutations in TP53 6,7,9–12 . Other genetic changes include epigenetic variations, including erosion of X-chromosome inactivation 13 , 14 , mutations in the mitochondrial genome 15 , 16 and an array of other point mutations and structural variants spread throughout the genome 10 , 11 These recurrent genetic changes arise from the pool of common variations in hPSC cultures via different cell competition mechanisms. hPSCs are prone to replication stress, leading to DNA damage, which in turn is a source of de novo genetic variation 17 – 21 . For instance, up to 20% of cells in an hESC culture carry de novo structural variants, but only a minority of them have the potential to confer a selective advantage to the cells 20 , 22 , 23 , leading to the mutant cells rapidly outcompeting their genetically balanced counterparts 24 – 27 . The exact traits that the different chromosomal abnormalities confer on undifferentiated cells, as well as the specific driver genes of these traits, are only well established for the gain of 20q11.21 28 . This abnormality confers a decreased sensitivity to apoptosis-inducing events due to increased expression of the gene BCL2L1 , located in the minimal gained region 6 , 26 , 27 . For gains of 12p, it is considered that NANOG drives at least part of the growth advantage of the cells 29 , and cells with a complex karyotype carrying all the most common abnormalities (gains of 1, 12, 17 and 20q) can outcompete the other cells by corralling and mechanical compression 24 . An important concern about these genetic variants is whether and how they alter the differentiation capacity of hPSCs and potentially prime differentiated cells for malignant transformation 8 , 30 – 32 . The gain of 1q is common in cancers, particularly lung adenocarcinoma, breast invasive carcinoma and liver hepatocellular carcinoma 33 , and appears to confer a growth advantage to cells during differentiation from hESCs to neural precursors 34 . Moreover, variants in 1q21.1 can alter neurodevelopmental trajectories upon hiPSC differentiation, with the deletion of 1q21.1 accelerating neuronal production and its duplication delaying the transition from neural progenitor cell to neuron 35 . Gains in chromosome 12 are frequently found in testicular germ cell tumors 36 . hPSCs with trisomy 12 display a reduced tendency toward spontaneous differentiation 29 , 37 , and gain of 12p13.31 results in an overall reduction in trilineage differentiation capacity and foci of residual pluripotent cells during hepatic differentiation 38 , 39 . The highly recurrent gain of 20q11.21 impairs the neuroectodermal lineage commitment of hPSCs 40 , 41 , and hESCs with 20q11.1q11.2 amplification have a reduced propensity to differentiate down the hematopoietic lineage, maintain more immature phenotypes along the neural differentiation trajectory and generate teratomas with foci of undifferentiated cells 42 . The gain of chromosome 17 is common in neuroblastomas, testicular germ cell tumors and breast cancers 36 . hPSC lines with a gain of chromosome 17 show altered differentiation patterns in embryoid bodies 43 , and the amplification of WNT3A and WNT9 on chromosome 17q21.31 alters neuronal differentiation 44 . Deletions of chromosome 18q are one of the rarer recurrent structural chromosomal abnormalities in hPSCs. This deletion was first reported as a single event by Maitra et al. in 2005 45 , and our group later found 18q deletions in three different hESC lines at relatively early passages, always as part of a derivative chromosome 18 28 . A large study by Amps et al . in 2011 revealed 5 instances of this deletion 46 , A large study by Amps et al . in 2011 revealed 5 instances of this deletion, and WiCell has reported that 4% of the 7300 hPSC cultures evaluated over nearly eight years carried an 18q deletion (WiCell Cytogenetics Lab, https://www.wicell.org/media.acux/29102c0e-e88e-426b-ab7d-bac4c2a9ec6a ). Chromosome 18q loss is common in cancers, especially gastrointestinal tract cancers 33 , and is linked to several disorders, including congenital malformations, developmental delays, and intellectual disability 47 . However, the impact of 18q loss on the functional properties of hPSCs is unknown. Therefore, the aim of this work was to examine the functional effects of 18q deletions during hPSC differentiation into the three embryonic germ layers and to determine the molecular mechanisms involved. RESULTS The minimal common region of 18q loss spans 14 genes expressed in undifferentiated hESCs and includes SALL3 We initially identified 18q losses in the hESC lines VUB04 and VUB26, in the form of a derivative chromosome 18. VUB04 presented a deletion at 18q21.2qter and a duplication at 5q14.2qter, and VUB26 showed the minimal 18q loss region (18q23qter) and a duplication at 7q33qter (Fig 1A. Sup. Fig. 1 and Sup. Table 1) 48 . These two hESC lines were not used in the present work because they further genetically drifted and acquired gain of 1q and 20q11.21, but their analysis helped to narrow down the common 18q deletion region. In this study, we used two other hESC lines bearing derivative chromosomes 18 involving a loss of 18q losses (hESC del18q ), VUB14 del18q and VUB13 del18q , as well as three chromosomally balanced lines (hESC WT ), VUB14 WT , and VUB04 WT and VUB03 WT , which served as controls for VUB13 del18q since VUB13 WT was lost (details on the karyotypes of the lines and their characterization are shown in Sup. Fig. 1 and Sup. Table 1). We used shallow genome sequencing to confirm the karyotypes before starting the experiments and all lines were routinely inspected with qPCR assays targeting recurrent chromosomal abnormalities (1q, 12p, 20q11.21 and 17q) to confirm their genomic stability for the duration of the different experiments. The 18q losses exhibited a common loss region from bp 75,773,285 to 80,373,285, spanning 37 loci. Bulk RNA sequencing of undifferentiated hESCs indicated that 14 genes within this region are expressed in undifferentiated hESCs, with counts per million greater than one in at least two samples (Fig. 1A). Of these coding genes, ADNP2 , SALL3 and TXNL4A had the highest expression and showed decreased transcript levels in mutant cells. ADNP2 and TXNL4A have no known function in hPSC. ADNP2 is predicted to be a transcription factor, and its silencing increases oxidative stress-mediated cell death 49 . TXNL4A is a component of the U5 small ribonucleoprotein particle, which is involved in pre-mRNA splicing and is associated with Burn-McKeown syndrome 50 . SALL3 was more promising as candidate driver gene as it has previously been reported to regulate the differentiation propensity of hiPSC lines 51 . Kuroda et al . showed that hiPSC lines expressing high levels of SALL3 differentiated preferentially into ectoderm, while hiPSC lines expressing lower levels of SALL3 tended to differentiate into mesoderm and endoderm 51 . It has also been shown that SALL3 interacts with the Mediator complex in neural stem cells 52 and is related to the development of the nervous system 53 . Considering these previous findings, we hypothesized that decreased expression of SALL3 as a result of a loss of one copy of the gene could alter the differentiation capacity of hESCs del18q . hESCs with 18q loss show impaired neuroectoderm differentiation As a first step, we investigated the effect of 18q deletion on hESC ectoderm lineage commitment. hESC WT and hESC del18q were subjected to neuroectoderm differentiation for 8 days using LDN193189 (LDN), SB431542 (SB) and retinoic acid (RA) 54 (Fig. 1B). We measured the mRNA levels of different neuroectoderm markers to evaluate neuroectoderm differentiation efficiency and the expression of the undifferentiated state markers NANOG and POUF51 (Fig. 2A and Sup Fig. 2A). VUB13 del18q and VUB14 del18q had significantly lower mRNA levels of PAX6 , NES and SOX1 , which were decreased by 3-fold, 15-fold, and 25-fold, respectively, compared to the levels in hESC WT (p≤0.0001 unpaired t-test), indicating a decreased neuroectodermal differentiation efficiency in hESC del18q . POU5F1 and NANOG mRNA expression levels were almost undetectable for all differentiated cells (Sup Fig. 2A). We also evaluated the differentiation of hESC WT and hESC del18q cells by immunostaining (Fig. 2B-C). We observed a lower percentage of PAX6 positive cells in differentiated hESC del18q than in differentiated hESC WT cells (45% vs 70%, p = 0.0114, unpaired t-test, Fig. 2C), which was consistent with the decrease in the levels of PAX6 mRNA. Taken together, these results show that hESCs del18q differentiation into neuroectoderm is impaired, and rather than to remain undifferentiated state, they miss-specify. hESC del18q readily differentiates into mesendoderm derivatives but shows abnormal cardiomyocyte progenitor differentiation We further investigated the impact of 18q deletion on the mesendoderm differentiation capacity of hESCs by differentiating hESC WT and hESC del18q into, in this case cardiac progenitors and hepatoblasts. Thus, we induced the differentiation of hESC WT and hESC del18q into mesoderm using a 5-day cardiac progenitor induction protocol as described previously 55 (Fig. 1B). We evaluated the mRNA levels of the cardiac progenitor markers GATA4 , ISL1 , NKX2-5 and PDGFRA (Fig. 3A). hESC del18q showed 2-fold lower levels of GATA4 mRNA (p = 0.0012 unpaired t- test) (Fig. 3A). hESC del18q lines expressed ISL1 at 15-fold higher levels, on average (p=0.0016, unpaired t-test), than hESC WT lines. We found no significant difference in the expression levels of NKX2-5 or PDGFRA between the control and mutant cardiac progenitor groups (p=0.11 and p=0.42, unpaired t-test, Fig. 3A). We also evaluated the proportion of differentiated cardiac progenitor and undifferentiated cells by immunostaining for GATA4 and POU5F1. The percentage of GATA4-positive cells was not statistically significantly different in hESC del18q than in hESC WT (p=0.2942, unpaired t-test, Fig. 3B-C), mainly due to the low differentiation efficiency of VUB03 WT , while the cells were all POU5F1 negative. Taken together, and bearing in mind the temporal expression of these markers during cardiac differentiation 56,57 , the results suggest that hESC del18q may experience differentiation delays or arrest, reaching an ISL1 high GATA4 low stage at day 5 but remaining less mature than their hESC WT counterparts. Next, we differentiated hESC WT and hESC del18q into hepatoblasts by applying the modified differentiation protocol for 8 days as described previously 58 (Fig. 1B). We measured the expression levels of the hepatoblast markers HNF4A , AFP , ALB and FOXA2 (Fig. 4A). We found no significant difference in the mRNA expression levels of HNF4A , ALB , and FOXA2 between hESC WT and hESC del18q (p>0.05, unpaired t-test), while AFP had a lower expression in hESC del18q (Fig. 4A, p=0.0095, unpaired t-test). We further evaluated hepatoblast differentiation by immunostaining for HNF4A and found that the percentages of HNF4A - positive cells in hESC del18q cells were similar to those in the wild type (Fig. 4B-C, p=0.2514, unpaired t-test). For both mesodermal and endodermal lineage commitment, all hESC WT and hESC del18q lines displayed low mRNA and protein levels of undifferentiated state markers (Sup Fig. 2B-C), indicating a loss of pluripotency for all cell lines during differentiation. Overall, our results indicate that hESC WT and hESC del18q differentiate into definitive mesoderm and endoderm and that there may be a delay or impairment in the progression of hESC del18q toward the cardiac progenitor stage. The differences in gene expression observed during hepatoblast differentiation are likely due to between-line variation in differentiation propensity rather than to the 18q deletion itself. Downregulation of SALL3 impairs neuroectoderm differentiation but does not affect differentiation into mesoderm and endoderm We next examined SALL3 mRNA expression levels in undifferentiated cells and found that SALL3 expression was significantly lower in hESC del18q than in hESC WT (Sup Fig. 3A), supporting the notion that SALL3 could be a key gene in the altered differentiation capacity of hESCs del18q . W e first generated SALL3 knockdown (KD) lines from t hree hESC WT lines (VUB02 WT , VUB03 WT and VUB04 WT ) by transducing a lentiviral vector containing shRNA targeting the SALL3 transcript (hESC WT_ SALL3 KD ) or a nontargeting shRNA as a control (hESC WT-NT ). We confirmed the knockdown efficiency of the generated hESC WT SALL3 KD lines by measuring the SALL3 mRNA expression levels and found that they were reduced by 30%, 40% and 20% in VUB02 WT_ SALL3 KD , VUB03 WT_ SALL3 KD and VUB04 WT_ SALL3 KD respectively, compared to the controls (Sup Fig. 3B). Next, we generated hESC del18q with stable overexpression of SALL3 (hESC del18q_ SALL3 OE ) by transducing VUB13 del18q and VUB14 del18q with the SALL3 lentiviral vector, and we verified the overexpression by measuring SALL3 mRNA levels, which were significantly increased in VUB13 del18q_ SALL3 OE (3-fold) and VUB14 del18q_ SALL3 OE (5-fold) compared to controls (Sup Fig. 3C). To investigate the role of SALL3 in regulating hESC differentiation propensity, we first induced neuroectodermal differentiation in hESC WT_ SALL3 KD , hESC del18q_ SALL3 OE , and the corresponding control cells. All three hESC WT_ SALL3 KD lines had lower levels of all NE markers than nontarget controls (Fig. 5A and Sup Fig. 4A-B). PAX6 protein levels were also reduced in hESC WT_ SALL3 KD (Fig. 5B-C), with only 20% of the cells expressing PAX6, compared to 80% of PAX6 positive cells in the control group (Fig. 5B-C). Our results show that SALL3 suppression in hESC WT recapitulates the impaired NE differentiation seen in hESC del18q lines. In contrast, hESC del18q_ SALL3 OE cells efficiently differentiated into neuroectoderm cells, accompanied by a significant increase in the mRNA levels of PAX6 , SOX1 , and NES (Fig. 5A and Sup Fig. 4A-B); moreover, hESC del18q­ SALL3 OE cultures included more PAX6 positive cells than hESC del18q cultures (60% vs 20%, respectively) (Fig. 5B-C). These results indicate that exongenous SALL3 expression can rescue the impairment of ectoderm differentiation caused by 18q loss. Next, we induced the differentiation of the different hESC lines into cardiac progenitors. The three hESC WT_ SALL3 KD lines differentiated inconsistently toward mesoderm fates. VUB04 WT_ SALL3 KD showed lower levels of GATA4 (Fig. 6A, p=0.0003, unpaired t-test), whereas compared to the controls, VUB03 WT_ SALL3 KD and VUB02 WT_ SALL3 KD showed no differences in GATA4 expression (Fig. 6A). Additionally, the percentage of GATA4 + cells detected by immunostaining in hESC WT_ SALL3 KD followed the same pattern, consistent with the mRNA levels of each line (Fig. 6B-C). The hESC del18q_ SALL3 OE cells exhibited variable marker profiles, with increases in GATA4 expression at both the mRNA (Fig. 6A, p=0.004, unpaired t-test) and protein levels for VUB13 del18q_ SALL3 OE but no difference in GATA4 mRNA levels (Fig. 6A, p=0.3622, unpaired t-test) and a slight decrease in GATA4 protein levels in VUB14 del18q_ SALL3 OE (Fig. 6B-C). Similarly, the mRNA expression levels of other markers, NKX2-5 , ISL1 , and PDGFRA , showed no consistent trend in the hESC WT_ SALL3 KD groups and exhibited no consistent differences in hESCs del18q and hESC del18q SALL3 OE (Sup Fig. 5A-C). When hESCs were differentiated into hepatoblast, SALL3 downregulation in hESC WT resulted in increased HNF4A , ALB , and FOXA2 mRNA expression (Fig. 7A and Sup Fig. 4B-C). The percentage of HNF4A positive cells was higher in VUB03 WT SALL3 KD cells but not in VUB04 WT_ SALL3 KD and VUB02 WT_S ALL3 KD cells compared to controls (Fig. 7B-C). The mRNA expression of another marker, AFP , also did not show consistent changes (Sup Fig. 4A). Upon overexpression of SALL3 , the differentiation profiles into hepatoblast did not show the expected mirroring effect. The mRNA expression of all the hepatoblast markers HNF4A , ALB , AFP and FOXA2 increased in VUB14 del18q_ SALL3 OE cells, consistent with the changes observed in the HNF4A protein level, but this same effect was not observed in VUB13 del18q_ SALL3 OE cells (Fig. 7 and Sup Fig. 4). Overall, these results suggest that the effect of SALL3 on mesoderm and endoderm differentiation may be line-specific and is not the reason for the delayed progression seen during cardiac differentiation in hESCs 18q . Downregulation of SALL3 and loss of 18q result in the deregulation of genes in pathways associated with pluripotency and differentiation To gain deeper insight into the effect of SALL3 downregulation on the global transcriptomic profile of cells with 18q loss, we carried out bulk RNA sequencing of hESC WT-NT (N=6), hESC WT_ SALL3 KD (N=6), hESC WT (N=6), VUB13 del18q (N=4) and VUB13 del18q_ SALL3 OE (N=5) cells. To estimate the similarity among the samples, we generated a distance clustering heatmap with global row scaling (Fig. 8A) and performed principal component analysis (Fig. 8B). The heatmap shows that while VUB13 del18q and hESC WT_ SALL3 KD cluster together, they cluster apart from the WT cell lines, as well as from VUB13 del18q_ SALL3 OE . The samples in this last group clustered more closely to the WT cell lines than to their unmodified VUB13 del18q (Fig. 8A). This pattern was also reflected in the first dimension of the PCA (Fig. 8B), where hESC WT , hESC WT-NT and VUB13 del18q_ SALL3 OE clustered more closely with each other than with VUB13 del18q and hESC WT_ SALL3 KD (Sup Fig. 6A). These results suggest that the downregulation of SALL3 in WT cells is sufficient to alter the transcriptome such that its profile is closer to that of an hESC line with a loss of 18q, and SALL3 overexpression in hESC del18q can restore the transcriptome to a near-WT state. Taken together, these findings support the our hypothesis that the differences between hESC WT and hESC del18q are mostly driven by the downregulation of SALL3 due to the loss of one copy of this gene. For this reason, we pooled the hESCs WT-NT with the untreated hESCs WT for further analysis. Next, we carried out differential gene expression analysis to gain further insight into which genes and pathways form the basis of the differences between hESC WT and hESC del18q and which of these are driven by SALL3 . Figure 8B-D shows volcano plots of the differentially expressed genes in the different groups. We considered this differential expression to be significant at a |log 2 fold-change| > 1.0 and false discovery rate (FDR)< 0.05. VUB13 del18q shows 891 and 448 genes that are significantly upregulated and downregulated in hESC del18q , respectively, compared to the WT cells (Fig 8C). Downregulation of SALL3 in WT cells led to the differential expression of 745 genes, of which 399 were upregulated and 346 were downregulated (Fig 8D). Overexpression of SALL3 in VUB13 del18q resulted in the upregulation of 121 genes and the downregulation of 605 genes (Fig 8E). To elucidate which of the transcriptional differences between hESC WT and hESC del18q are mediated by SALL3 , we investigated the overlaps in up- and downregulated genes across conditions. First, we found that 231 and 93 genes were commonly up- and downregulated, respectively, between VUB13 del18q and hESC WT_ SALL3 KD , representing 26% of the differentially expressed genes in hESCs with an 18q deletion (Fig 8F and G). Next, we compared these subsets of genes to the genes with altered gene expression in hESC del18q upon overexpression of SALL3 . We compared the genes that were upregulated by SALL3 knockdown and by loss of 18q to those downregulated by overexpression of SALL3 in hESC 18q , and vice versa (Fig 8F and G). Out of the 231 upregulated genes shared by the VUB13 del18q and hESC WT_ SALL3 KD groups, 173 genes showed increased expression in VUB13 del18q_ SALL3 OE (Fig 8F). Additionally, 18 of the 93 downregulated genes shared by VUB13 del18q and hESC WT_ SALL3 KD displayed increased expression in VUB13 del18q SALL3 OE (Fig 8G). This further refined the gene set to a core of 191 genes that are the most strongly regulated by SALL3 in hESCs, both by the loss of a copy of the gene itself and by the modulation of its expression (Supplementary Table 2). The other differences in gene expression are likely associated with the loss of other genes in 18q or with the overexpression of genes in the duplicated regions of chromosomes 5 and 7, which are part of the derivative chromosome 18 in hESC del18q . Finally, to identify potential molecular targets and elucidate the underlying functional mechanisms that contribute to the observed impairment of differentiation capacity in hESC del18q , we analyzed the differential gene expression of the three groups using Gene Set Enrichment Analysis (GSEA) and the MSigDB database. Specifically, we focused on the Kyoto Encyclopedia of Genes and Genomes (KEGG) and WikiPathways databases within the C2 library and the pathways of the H library. We filtered the significant pathways based on a normalized enrichment score |NES| > 1, a p-value < 0.05, and the proportion of leading-edge genes accounting for over 30% of the entire gene set involved in the pathway. Figures 8H and I show Venn diagrams of the overlap between significantly enriched pathways in the C2 and H libraries, respectively, for each of the three groups. The full list can be found in the Supplementary Table 3. In total, we found 13 pathways that overlapped among the three groups, all 13 of which were positively enriched in both VUB13 del18q and hESC WT_ SALL3 KD and negatively enriched in VUB13 del18q_ SALL3 OE (Fig 8J shows the cell-type relevant pathways; the whole set can be found in the Supplementary Table 4). Because of the critical role that these pathways play in pluripotency maintenance and differentiation, we analyzed the expression of pluripotency-associated genes, and we found that NANOG, POU5F1, LIN28, SOX2, PODXL, SUSD2, MYC, FOXD3 and DPPA3 are overexpressed in VUB13 del18q , with all but DPPA3 and POU5F1 also being overexpressed upon SALL3 KD and downregulated by transgenic SALL3 overexpression in VUB13 del18q (Sup Fig. 7A). Discussion In this study, we examined the repercussions of the loss of chromosome 18q on the differentiation capacity of hESCs. For this, we used an early-stage differentiation approach to generate lineage-specific cell types representing the three germ layers: neuroectoderm, hepatoblast and cardiac progenitors. Our in vitro lineage commitment studies indicated that the deletion of 18q in hESCs impaired neuroectodermal differentiation and delayed cardiac progenitor differentiation, while no consistent differences were observed in the commitment toward hepatoblasts. To study the mechanistic basis for these changes in differentiation, we looked at the genes located in the minimal region of loss. We found that decreased SALL3 expression due to the loss of one copy of the gene was sufficient to result in the observed decreased neuroectoderm differentiation but not to modulate cardiac and hepatoblast differentiation. In this sense, our results are only partially aligned with those obtained by Kuroda et al. 51 . These authors found that downregulating SALL3 resulted not only in decreased neuroectoderm differentiation, similar to our results, but also in increased cardiac progenitor differentiation. While we can only speculate about the reasons for these differences, it is likely that the genetic background of the cell lines plays an important role 59 . Also, given that SALL3 is a modulator of DNMT3A activity 60 , the preexisting epigenetic marks in each of the lines, particularly histone modifications, may influence the recruitment of DNMT3A to methylate the DNA 61 , 62 . Furthermore, it is possible that other genes located in 18q, or in the gain regions of chromosomes 5 and 7, cause cell-line specific effects. In line with this reasoning, the gene expression analysis revealed that only part of the divergence between hESCs with 18q loss and their chromosomally normal isogenic counterparts was related to the differential expression of SALL3 . Interestingly, the core set of deregulated genes were associated with pathways involved in maintenance of and exit from the undifferentiated pluripotent state, and key regulators of pluripotency were both upregulated in hESCs 18q and regulated by SALL3 expression. For instance, TGF-beta signaling is key to the maintenance of the primed pluripotent state 63 . TGF-beta and BMP4 signaling are also core genes in regulating the balance between neuroectoderm differentiation and mesendoderm in both humans and mice 64 . Notch activation mediates TGF-β signaling during hESC and mesenchymal stem cell differentiation into smooth muscle cells 65 , and its inhibition supports naïve state consolidation in rodent models 63 . The cytokine tumor necrosis factor alpha has been shown to negatively regulate the differentiation of various cell types, including cardiomyocytes 66 , embryoid bodies 67 and osteoblasts 68 , 69 . Additionally, nuclear factor-κB inhibition has been found to mediate naïve pluripotency in mice 70 . Overall, these results suggest that downregulation of SALL3 due to the loss of 18q alters undifferentiated-state maintenance in hESCs by affecting pluripotency-associated pathways, and these changes have profound effects on the differentiation capacity of the cells. With hPSCs steadily moving into clinical trials 71 and being broadly used as a cell source for in vitro modelling of, for instance, developmental processes and diseases, determining the impact of recurrent genetic abnormalities is critical 72 – 74 . Work from our group and others is beginning to generate a detailed picture showing how these genetic abnormalities affect differentiation in a cell lineage-specific manner. For instance, 20q11.21 gain impairs neuroectoderm commitment without affecting mesendoderm induction 75 , 76 , and recently, it has been shown that cells with an isochromosome 20q are not able to survive RPE differentiation and display overall disruptions in the ability to correctly differentiate 77 . In this work, we show that 18q loss specifically impairs neuroectoderm commitment and appears to delay cardiac differentiation. Taken together, these results highlight the importance of the genetic screening of hPSC cultures to ensure that these abnormalities do not pass unnoticed. In a research setting, chromosomal abnormalities could lead to confounding effects that decrease the reliability and reproducibility of the work, and in a clinical setting, they could lead at best to decreased therapeutic efficacy and at worst to tumorigenesis 72–7478,79 . Losses of chromosome 18q are recurrently found in cancers 33 and have been shown to be early events in midgut carcinoids 80 , 81 . Loss of heterozygosity on chromosome 18q is associated with significantly decreased survival in head and neck squamous cell carcinoma patients, and the methylation status of the SALL3 promoter correlates with shortened disease-free survival 82 – 84 . Furthermore, three cancer suppressor genes have been identified in 18q, PIGN, MEX3C and ZNF516, which play roles in replication stress and in cervical neoplasia 85 – 87 . In this context, several studies have found that even when chromosomally abnormal hPSCs are capable of differentiating, they display altered gene expression patterns suggestive of malignant transformation 72 , 88 – 91 . Furthermore, the abnormalities seen in hPSCs could be regarded as a first hit in cancerous transformation. Cells that are already genetically abnormal upon transplantation are not cancerous yet but may have a higher chance of undergoing oncogenic transformation as they could require fewer additional genetic hits to initiate the process 74 , 78 , 79 . With this in mind, centers involved in clinical work subject their hPSC cultures and hPSC-based products to genetic screening prior to their use in patients. This typically involves the use of G-banding and, increasingly commonly, massively parallel sequencing (MPS), which enable the detection of chromosomal abnormalities and even, in the case of MPS, potentially harmful single nucleotide changes. Incidentally, the first clinical trial using hPSC-derived retinal pigmented epithelium (RPE) was halted after potentially harmful mutations were identified in both the hPSCs and the RPE cells derived from this line 92 . Unfortunately, standard methods for genetic screening cannot detect abnormalities present as low-grade mosaics in the hPSC culture, which are known to be common 93 – 95 . If any of the cells carry an abnormality that specifically impairs the differentiation to the chosen lineage, their presence could lead to a cell product containing a subpopulation of poorly differentiated or mis-specified cells and, in the worst-case scenario, with tumor-initiating capacity. This risk further highlights the need for more detailed knowledge of the impacts of specific abnormalities on the properties of hPSCs so that targeted screening approaches that can detect potentially small populations of abnormal cells in cultures can be developed. In conclusion, in this study, we have characterized the differentiation capacity of hESCs with 18q loss, one of the recurrent, albeit less common, genetic abnormalities found in hPSC cultures. We found that these cells are characterized by abnormal differentiation into cardiac progenitors and an impaired capacity for neuroectoderm commitment, the latter driven by the loss of one copy of SALL3 . This gene is an inhibitor of DNMT3A , and its downregulation results in changes in the expression of genes involved in the maintenance of pluripotency and in hESC differentiation. Further research will be needed to assess whether other cell-type specific effects of this abnormality exist and might be revealed by longer differentiation protocols, beyond the progenitor stage, as well as the potential consequences of 18q loss for oncogenic potential. Materials and methods hESC maintenance and passaging All hESC lines were derived and characterized as reported previously 96 , 97 and are also registered in the EU hPSC registry ( https://hpscreg.eu/ ). They were cryopreserved in freezing medium composed of 90% knock-out serum (Thermo Fisher Scientific) mixed with 10% dimethyl sulfoxide (Sigma-Aldrich). The hESCs were maintained in NutriStem hESC XF medium (NS medium; Biological Industries) with 100 U/mL penicillin/streptomycin (P/S) (Thermo Fisher Scientific) in a 37°C incubator with 5% CO 2, and the culture medium was changed daily. The tissue culture dishes and plates (Thermo Scientific) were coated with 10 µg/mL Biolaminin 521 (Biolamina®) at 4°C and then incubated at 37°C for at least 20 min before the cells were seeded. The cells were passaged as single cells using TrypLE Express (Thermo Fisher Scientific) and split at a ratio of 1:10 to 1:100 as needed at 70–90% confluence. The medium was supplemented with 10 µM Rho kinase (ROCK) inhibitor Y-27632 (ROCKi, Tocris) for the first 24 h after passaging. Copy number variant (CNV) analysis The genetic content of the hESCs was assessed through shallow whole-genome sequencing by the BRIGHTcore of UZ Brussels, Belgium, as previously described 98 . We also conducted copy number variant analysis using quantitative real-time PCR (qRT-PCR) at regular intervals, particularly before and after performing lentiviral transduction and starting differentiation. DNA was extracted with a DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturers' protocol. qPCR was performed with the subsequent copy number assays: RNaseP (Thermo Fisher Scientific) as a reference and KIF14 , NANOG , NMT1 , and ID1 (Thermo Scientific) representing the 1q, 12p, 17q and 20q regions, respectively, which are commonly subject to CNV in hESCs. The reaction systems were prepared by mixing RNaseP , TaqMan 2× Mastermix Plus – Low ROX (Eurogentec), and the related TaqMan assays together with 8 µl of the diluted DNA samples (n = 3). qPCR was performed on a ViiA7 thermocycler (Thermo Fisher Scientific), and Applied Biosystems Copy Caller v.2.1 was used to analyze the CNVs. Total RNA isolation and cDNA synthesis Total RNA was isolated using RNeasy Mini and Micro kits (Qiagen) following the manufacturer’s guidelines, including on-column DNase I treatment. The isolated RNAs were collected in nuclease-free water and analyzed for quality and quantity using UV spectrophotometry. The final RNA was stored at − 80°C. A minimum of 500 ng of mRNA was reverse-transcribed into biotinylated cDNA using the First-Strand cDNA Synthesis Kit (Cytiva) with the NotI-d(T)18 primer, and the resulting cDNA was stored at -20°C for subsequent analysis. Quantitative real-time PCR (qRT-PCR) for gene expression analysis Quantitative real-time PCR (qRT-PCR) was carried out using TaqMan mRNA expression assays (Thermo Fisher Scientific) and TaqMan 2× Mastermix Plus – Low ROX (Eurogentec) on a ViiA 7 thermocycler (Thermo Fisher Scientific) using the standard cycling protocol provided by the manufacturer. The relative expression of target genes was quantified using the comparative threshold cycle (Ct) method and normalized to the TaqMan GUSB transcript (Applied Biosystems) as the endogenous housekeeping gene. All the samples were run in triplicate, and the related TaqMan assays used in the present study are listed in Supplementary Table 5. Immunostaining Differentiated cells were first fixed in a solution of PBS containing 3.7% formaldehyde (Sigma-Aldrich) for 15 min, permeabilized in 0.1% Triton X for 10 min (Sigma-Aldrich) and then blocked with 10% fetal bovine serum (ThermoFisher Scientific) for 1 h at room temperature (RT). Sequentially, primary antibodies appropriately diluted in blocking solution (1:200 dilution in 10% FBS) were incubated overnight at 4°C. Thereafter, secondary antibodies conjugated to Alexa 488, Alexa 594 or Alexa 647 (1:200 dilutions in 10% FBS, Thermo Fisher Scientific) and Hoescht (1:1000 dilution, ThermoFisher Scientific) were applied for 1–2 h at room temperature in the dark. PBS was used to wash the cells three times between steps. Confocal images were acquired with an LSM800 confocal microscope (Carl Zeiss) with a 10× or 20× objective. For quantification, the fixed differentiated cells stained with respective antibodies were counted and compared to the number of Hoescht-stained nuclei to determine the percent positivity using Zen 2 (blue edition) imaging software. The areas are randomly selected (n = 3–6) within a single well based on the Hoescht channel, and the positive cells are quantified by calculating the ratio of total positive cells to the total number of cells selected within those areas. The lists with antibodies can be found in supplementary Table 6. In vitro differentiation of iPSCs Definitive neuroectoderm specification The protocol for inducing neuroectoderm differentiation was adapted from the protocol described by Douvaras and colleagues 99 . In brief, hESCs were seeded on Biolaminin 521 (Biolamina®)-coated 24-well plates at a ratio of 100,000 cells per cm 2 and grown to 90% confluence. Then, differentiation was induced by incubating the hESCs with neural induction specification medium for up to 8 days with daily medium changes. The neural induction specification medium consisted of basal medium supplemented with the following differentiation factors: 100 nM retinoic acid (Sigma-Aldrich), 10 µM SB431542 (Tocris), and 250 nM LDN193189 (STEMCELL Technologies). The basal medium was prepared by mixing DMEM/F12 (Thermo Scientific) basic medium supplemented with 1x NEAA (Thermo Scientific), 1x GlutaMAX (Thermo Scientific), 1x 2-mercaptoethanol (Thermo Scientific), 25 µg/mL insulin (Sigma-Aldrich) and 1x penicillin/streptomycin (P/S) (Thermo Scientific). Cardiac progenitor differentiation The induction of cardiac progenitor differentiation was initiated using a slightly modified version of the protocol from a previous publication 100 . hESCs were seeded on Biolaminin-521-coated 24-well plates at a density of 100,000 cells/cm 2 and allowed to reach 80–90% confluence. At this point, differentiation was initiated by treating the hESCs with cardiomyocyte differentiation basal medium (CDBM) with 5 mM CHIR99021 (Tocris) to activate Wnt/β-catenin signaling and form the mesendoderm layer. After 24 h, CHIR99021 was removed, and the cells were cultured in CDBM with 0.6 U/mL heparin (Sigma-Aldrich) for 24 h. Subsequently, the CDBM medium was supplemented with 0.6 U/ml heparin and 3 mM IWP2 (Tocris) for another 3-day incubation with medium refreshed daily. The cardiomyocyte differentiation basal medium (CDBM) was composed of 10 µg/ml transferrin (Sigma Aldrich), 1x chemically defined lipid concentrate (Thermo Scientific) and E8 basal medium. The E8 medium was composed of DMEM/F12 (Thermo Scientific) basic medium with 64 mg/L L-ascorbic acid (Sigma-Aldrich) and 13.6 µg/L sodium selenium (Sigma-Aldrich). Hepatoblast differentiation Differentiation of hESCs into hepatoblasts was conducted using a protocol based on 101 . The hESCs were seeded at a density of 5x10 4 cells/cm 2 on 24-well plates precoated with Biolaminin-521. Upon reaching approximately 40–50% confluency, the hESCs were treated with liver differentiation medium (LDM) along with 50 ng/mL Activin A (STEMCELL Technologies), 50 ng/mL WNT3A (PeproTech) and 6 µL/mL DMSO (Sigma-Aldrich) for 48 h. The cells were incubated for an additional 48 h in the same medium without WNT3A. Then, the medium was changed to LDM with 50 ng/mL BMP4 (STEMCELL Technologies) and 6 µL/mL DMSO for the following 4 days, with the medium changed every two days. The samples were collected on day 8. LDM medium was created by adding MCDB 201 medium (pH = 7.2, Sigma-Aldrich), L-ascorbic acid (Sigma-Aldrich), insulin-transferrin-selenium (ITS-G, Thermo Scientific), linoleic acid-albumin from bovine serum albumin (LA-BSA, Sigma-Aldrich), 2-mercaptoethanol (Thermo scientific), and dexamethasone (Sigma-Aldrich) to DMEM high glucose medium (Westburg Life Sciences). Generation of SALL3 knock-down and overexpression cell lines hESC WT_ SALL3 KD cells were generated by infecting hESC WT with lentiviral particles expressing SALL3 -targeted shRNAs. To generate lentiviral particles, we transfected HEK 293T cells with individual clones from a SigmaMISSION shRNA targeting set (TRCN0000019754, TRCN0000417790) or control shRNA plasmid along with packaging plasmids (plasmid pMDG, encoding VSV G, and plasmid pCMV∆R8.9, encoding gag-pol). hESC del18q_ SALL3 OE cells were generated by infecting hESC del18q with lentiviral particles expressing SALL3 . Lentiviral particles were generated as described above. The pLVSIN-EF1α puromycin vector expressing SALL3 was a gift from Yoji Sato from the Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Japan. For transduction, hESCs were seeded at a density of 50,000/cm 2 and then transduced with a 2:1 mix of NutriStem and lentivirus-containing medium in the presence of 10 mg/mL protamine sulfate (LEO Pharma) for 4 h. Cells were then washed with PBS before adding fresh NutriStem medium. Twenty-four hours later, the cells were again washed with PBS and then selected by puromycin. RNA Sequencing RNA-seq library preparation was performed using QuantSeq 3′ mRNA-Seq Library Prep Kits (Lexogen) following Illumina protocols. Sequencing was performed on a high-throughput Illumina NextSeq 500 flow cell. On average, 13.9 x 10 6 ± 7.1 x 10 6 paired-end reads per sample were uniquely mapped, with an average coverage of 101 paired reads. The FastQC algorithm 102 was used to perform quality control on the raw sequence reads prior to the downstream analysis. The raw reads were aligned to the new version of the human Ensembl reference genome (GRCh38.p13) with Ensembl (GRCh38.83gtf) annotation using STAR version 2.5.3 in 2-pass mode 103 .The aligned reads were then quantified, and transcript abundances were estimated using RNA-seq by expectation maximization (RSEM, version 1.3.3) 104 . The count matrices were imported into R software (version 3.3.2) for further processing. The edgeR 105 package was utilized to identify differentially expressed genes (DEGs) between groups. Transcripts with a count per million (cpm) greater than 1 in at least two samples were considered for the downstream analysis. Genes with a log2-fold change greater than 1 or less than − 1 and a false discovery rate (FDR)-adjusted P-value less than 0.05 were considered significantly differentially expressed. Volcano plots of DEGs were generated using the ggplot2 106 package in R, while Venn diagrams using VennDiagram function were used to visualize the overlap of the DEGs among different individuals. Principal component analysis (PCA) and heatmap clustering were performed using normalized counts and R packages. The heatmap was generated using the heatmap.2 funtion. PCA was performed using the prcomp function and plotted using ggplot2. Gene set enrichment analysis (GSEA) was applied to detect the enrichment of pathways using the GSEA function in R with the MSigDB C2 and H databases. Values are ranked by sign(logFC)*(-log10(FDR)). |NES| > 1 and p-value < 0.05 were considered the thresholds for significance for the gene sets. Statistics All differentiation experiments were carried out in at least triplicate (n ≥ 3). All data are presented as the mean ± standard error of the mean (SEM). Statistical evaluation of differences between 2 groups was performed using unpaired two-tailed t tests in GraphPad Prism9 software, with p < 0.05 determined to indicate significance. Declarations Data availability The RNA sequencing counts per million tables are provided in the supplementary material. The raw sequencing data are available upon request. AUTHOR CONTRIBUTIONS Y.L. carried out all of the experiments and bioinformatics analysis unless stated otherwise and co-wrote the manuscript. D.A. D. cowrote the manuscript. N.K. packaged the lentivirus and assisted in transduction. E.C.D.D. assisted with the bioinformatics analysis. M.R. assisted in microscopy and cell counting. 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Bioinformatics 26, 139–140 (2010). Wickham, H. ggplot2. (2016) doi: 10.1007/978-3-319-24277-4 . Additional Declarations (Not answered) Supplementary Files SupplementaryDataLeietal.docx SupplementaryTable2.xlsx Supplemental table 2 SupplementaryTable3.xlsx Supplemental Table 3 SupplemntaryTable4.xlsx Supplemental Table 4 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3100381","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":221276806,"identity":"106bf7dc-0f5b-4a14-b445-99e980a8b60a","order_by":0,"name":"Claudia Spits","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYDACdiDmMQCxGBsYGCoYGAwYeAhoYUZoaWxgOEO0FgiTsYGxjQgt/M3MzyTeFNgxyM9ubn/4c97hxO0MvAcf4NMicZjNTHKOQTKDwZ2Djc282w4n7mzgSzbAp8WAmcFMmgdIGkgkNjYzbktL3HCAx0wCvxb2b0At9QzyMxIbG3/OAWsx/4FfCw/IlsMMDDcSGxt4G2zAtuDTAfQLT7HlHIPjPCC/zOY5ZmO8s5kvGa/D+NvbN95486daTn52+4OPP2okZLez9x78gNcaKOBhgJvMTIx6iBOJVjkKRsEoGAUjDQAAN2JEuvHXoecAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0187-5138","institution":"Vrije Universiteit Brussel, Belgium","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Spits","suffix":""},{"id":221276807,"identity":"2b6d7703-8a21-4aab-9933-27ab5b41dfea","order_by":1,"name":"Yingnan Lei","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yingnan","middleName":"","lastName":"Lei","suffix":""},{"id":221276808,"identity":"1ddd6880-a5fb-4ab9-95d0-29ee91bbcc56","order_by":2,"name":"Diana Al Delbany","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Diana","middleName":"Al","lastName":"Delbany","suffix":""},{"id":221276809,"identity":"4a44df3f-afc8-4c64-be0d-4f07dca19ae6","order_by":3,"name":"Nuša Krivec","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nuša","middleName":"","lastName":"Krivec","suffix":""},{"id":221276810,"identity":"fb6517b1-eeb4-46bd-bbaf-28bc2ab8943d","order_by":4,"name":"Marius Regin","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marius","middleName":"","lastName":"Regin","suffix":""},{"id":221276811,"identity":"c3f66ea6-391d-4a0c-92d8-107b9f7115d2","order_by":5,"name":"Edouard Couvreu de Deckersberg","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Edouard","middleName":"Couvreu","lastName":"de Deckersberg","suffix":""},{"id":221276812,"identity":"732731fe-8391-43bb-bc0d-d83b8d386bf3","order_by":6,"name":"Charlotte Janssens","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Charlotte","middleName":"","lastName":"Janssens","suffix":""},{"id":221276813,"identity":"28c88f42-e68d-4051-9328-db4239a1699a","order_by":7,"name":"Manjusha Ghosh","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Manjusha","middleName":"","lastName":"Ghosh","suffix":""},{"id":221276814,"identity":"a91d19aa-2e3d-4623-893e-ac7f9c69a9a6","order_by":8,"name":"Karen Sermon","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Karen","middleName":"","lastName":"Sermon","suffix":""}],"badges":[],"createdAt":"2023-06-23 12:35:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3100381/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3100381/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":40734832,"identity":"6df1cb79-473c-45c2-bfc2-d487a0a8bce9","added_by":"auto","created_at":"2023-07-28 17:01:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":596166,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe minimal common region of 18q loss spans 14 genes expressed in undifferentiated hESCs, including \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSALL3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(A) Graphical representation of the regions of chromosome 18 loss in all VUB hESC\u003csup\u003edel18q\u003c/sup\u003e lines and showing the normalized expression of the genes located in the minimal common region of loss examined by bulk-RNA sequencing to compare the transcriptome of hESC\u003csup\u003edel18q\u003c/sup\u003e and hESC\u003csup\u003eWT\u003c/sup\u003e as well as the genetically modified counterparts.\u003cstrong\u003e \u003c/strong\u003e(B) Overview of the experimental setup. We used two lines carrying a loss of 18q and three genetically balanced lines. The WT lines were also genetically modified to down-regulate \u003cem\u003eSALL3\u003c/em\u003e, while 18q loss lines were modified to over-express \u003cem\u003eSALL3\u003c/em\u003e. All lines were subjected to differentiate to neuroectoderm, hepatoblasts and cardiac progenitors, all differentiation experiments were carried out at least in triplicate. qRT-PCR and immunostaining were used to assess the identity of the differentiated cells. AA: Activin A; Lenti-sh\u003cem\u003e-SALL3\u003c/em\u003e: lentiviral transduction for a short-hairpin RNA against \u003cem\u003eSALL3.\u003c/em\u003e Lenti-O3-\u003cem\u003eSALL3\u003c/em\u003e: lentiviral transduction with a construct for the transgenic expression of \u003cem\u003eSALL3.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3100381/v1/12e1029b8ed8a0c43c2a0f12.png"},{"id":40735493,"identity":"0b1c2884-e94b-45ea-8b62-7e81c25f9abd","added_by":"auto","created_at":"2023-07-28 17:09:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":910291,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehESCs with 18q loss show impaired neuroectoderm differentiation. \u003c/strong\u003e(A) Relative mRNA expression as measured by qRT-PCR for ectoderm markers \u003cem\u003ePAX6\u003c/em\u003e, \u003cem\u003eNES\u003c/em\u003e and \u003cem\u003eSOX1\u003c/em\u003e (n = 3–6). Data are shown asthe means ± SEM. Different patterns indicate different cell lines, and the horizontal bars with asterisks *, **, *** and **** represent statistical significance between samples at 5%, 1%, 0.1% and 0.01% respectively (unpaired t-test). (B) Immunostaining for PAX6 (green) and POU5F1 (red) in mutant and control lines after 8 days of neuroectoderm differentiation, all scale bars are 50 µm. (C) Percentage of PAX6-positive cells in the immunostainings shown in B.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3100381/v1/2630c4eb60fd31ef30f41548.png"},{"id":40733812,"identity":"3f270d65-d9a1-47ef-a968-dd7048a4a617","added_by":"auto","created_at":"2023-07-28 16:53:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":754722,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehESC\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003edel18q\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cells show abnormal cardiomyocyte progenitor differentiation. \u003c/strong\u003e(A) Relative mRNA expression as measured by qRT-PCR for the cardiac progenitor markers \u003cem\u003eGATA4\u003c/em\u003e, \u003cem\u003eISL1\u003c/em\u003e, \u003cem\u003eNKX2-5\u003c/em\u003e and \u003cem\u003ePDGFRA \u003c/em\u003e(n = 3–6). Data are shown as the means ± SEM; different patterns indicate different cell lines, and the horizontal bars with asterisks *, **, *** and **** represent statistical significance between samples at 5%, 1%, 0.1% and 0.01% respectively (unpaired t-test). (B) Immunostaining for NKX2-5 (green) and POU5F1 (red) in mutant and control lines after 5 days of cardiac progenitor differentiation, all scale bars are 100 µm. (C) Percentage of GATA4-positive cells in the immunostainings shown in B.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3100381/v1/5c1cd9851a3db9c6dcb55036.png"},{"id":40733807,"identity":"6e701af6-da6e-4f31-9bf9-bcf91b43925b","added_by":"auto","created_at":"2023-07-28 16:53:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":804642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehESC\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003edel18q\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e and their genetically balanced counterparts differentiate equally well into hepatoblasts. \u003c/strong\u003e(A) Relative mRNA expression of the cardiac progenitor markers \u003cem\u003eHNF4A\u003c/em\u003e, \u003cem\u003eAFP\u003c/em\u003e, \u003cem\u003eALB \u003c/em\u003eand \u003cem\u003eFOXA2\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eas measured by qRT-PCR (n = 3). Data are shown as the means ± SEM; different patterns indicate different cell lines, and the horizontal bars with asterisks *, **, *** and **** represent statistical significance between samples at 5%, 1%, 0.1% and 0.01% respectively (unpaired t-test). (B) Immunostaining for HNF4A (green) and POU5F1 (red) in mutant and control lines after 8 days of hepatoblast differentiation, all scale bars are 100 µm. (C) Percentage of HNF4A-positive cells in the immunostainings shown in B.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3100381/v1/69cb3e984ab1dc9298e8ac18.png"},{"id":40733810,"identity":"5b62d974-647c-4d5f-8491-0b24d59ea957","added_by":"auto","created_at":"2023-07-28 16:53:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1168926,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDownregulation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSALL3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edrives the impaired neuroectoderm differentiation of hESCs with 18q loss. \u003c/strong\u003e(A) Relative mRNA expression as measured by qRT-PCR for the ectoderm marker \u003cem\u003ePAX6\u003c/em\u003e (n = 3). Data are shown as the means ± SEM; different patterns indicate different cell lines, and the horizontal bars with asterisks *, **, *** and **** represent statistical significance between samples at 5%, 1%, 0.1% and 0.01% respectively (unpaired t-test). (B) Immunostaining for PAX6 (green) and POU5F1 (red) in mutant and control lines after 8 days of neuroectoderm differentiation, the scale bars in knock down groups are 50 µm and the scale bars in over-expression groups are 100 µm. (C) Percentage of PAX6-positive cells in the immunostainings shown in B.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3100381/v1/b356a33b155759d5c3532920.png"},{"id":40734836,"identity":"016de237-35d9-4b20-ad9e-d907e53e09f3","added_by":"auto","created_at":"2023-07-28 17:01:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1030757,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSALL3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expression do not regulate cardiac progenitor differentiation. \u003c/strong\u003e(A) Relative mRNA expression as measured by qRT-PCR for the cardiac progenitor marker \u003cem\u003eGATA4\u003c/em\u003e (n = 3). Data are shown as the means ± SEM; different patterns indicate different cell lines, and the horizontal bars with asterisks *, **, *** and **** represent statistical significance between samples at 5%, 1%, 0.1% and 0.01% respectively (unpaired t-test).\u003cstrong\u003e \u003c/strong\u003e(B) Immunostaining for GATA4 (green) and POU5F1 (red) in mutant and control lines after 5 days of cardiac progenitor differentiation, all scale bars are 100 µm.\u003cstrong\u003e \u003c/strong\u003e(C) Percentage of GATA4-positive cells in the immunostainings shown in B.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3100381/v1/15d211c2433277aa8e36d979.png"},{"id":40733814,"identity":"899bee44-1a7d-432b-82d5-409fbfa46233","added_by":"auto","created_at":"2023-07-28 16:53:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1176193,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSALL3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expression do not impact hepatoblast differentiation \u003c/strong\u003e(A) Relative mRNA expression of the ectoderm marker \u003cem\u003eHNF4A\u003c/em\u003e as measured by qPCR (n = 3). Data are shown as the means ± SEM; different patterns indicate different cell lines, and the horizontal bars with asterisks *, **, *** and **** represent statistical significance between samples at 5%, 1%, 0.1% and 0.01% respectively (unpaired t-test).\u003cstrong\u003e \u003c/strong\u003e(B) Immunostaining for HNF4A (green) and POU5F1 (red) in mutant and control lines after 8 days of hepatoblast differentiation, all scale bars are 100 µm.\u003cstrong\u003e \u003c/strong\u003e(C) Percentage of HNF4A positive cells.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3100381/v1/4608a6d0f556141b4296ee82.png"},{"id":40734834,"identity":"9e643d0e-447f-4d32-a86e-4d6d98c3dd87","added_by":"auto","created_at":"2023-07-28 17:01:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":416479,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDownregulation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSALL3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and loss of 18q result in the deregulation of genes in pathways associated with pluripotency and differentiation. \u003c/strong\u003e(A,B) Unsupervised clustering heatmap(A) and Principal component analysis (PCA) (B) of the coding genes with a count per million greater than one in at least two samples. (C, D, E) Volcano plots of differential gene expression analysis for VUB13\u003csup\u003edel18q \u003c/sup\u003eversus hESC\u003csup\u003eWT \u003c/sup\u003eand hESC\u003csup\u003eWT-NT\u003c/sup\u003e(C), hESC\u003csup\u003eWT_\u003c/sup\u003e\u003csup\u003e\u003cem\u003eSALL3\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eKD\u003c/sup\u003e versus hESC\u003csup\u003eWT\u003c/sup\u003e and hESC\u003csup\u003eWT-NT\u003c/sup\u003e(D), and VUB13\u003csup\u003edel18q_\u003c/sup\u003e\u003csup\u003e\u003cem\u003eSALL3\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eOE\u003c/sup\u003e versus VUB13\u003csup\u003edel18q\u003c/sup\u003e (E) with a cutoff value of |log\u003csub\u003e2\u003c/sub\u003efold change|\u0026gt; 1 and FDR \u0026lt; 0.05. \u0026nbsp;(F) Venn diagrams showing the genes downregulated in VUB13\u003csup\u003edel18q_\u003c/sup\u003e\u003csup\u003e\u003cem\u003eSALL3\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eOE\u003c/sup\u003e and upregulated in VUB13\u003csup\u003edel18q \u003c/sup\u003eand hESC\u003csup\u003eWT_\u003c/sup\u003e\u003csup\u003e\u003cem\u003eSALL3\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eKD\u003c/sup\u003e. (G) Venn diagrams showing the genes upregulated in VUB13\u003csup\u003edel18q_\u003c/sup\u003e\u003csup\u003e\u003cem\u003eSALL3\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eOE\u003c/sup\u003e and downregulated in VUB13\u003csup\u003edel18q\u003c/sup\u003e and hESC\u003csup\u003eWT_\u003c/sup\u003e\u003csup\u003e\u003cem\u003eSALL3\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eKD\u003c/sup\u003e. (H) Venn diagrams of the pathways in the H library common among VUB13\u003csup\u003edel18q\u003c/sup\u003e, hESC\u003csup\u003eWT_\u003c/sup\u003e\u003csup\u003e\u003cem\u003eSALL3\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eKD\u003c/sup\u003e, VUB13\u003csup\u003edel18q_\u003c/sup\u003e\u003csup\u003e\u003cem\u003eSALL3\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eOE\u003c/sup\u003e and hESC\u003csup\u003eWT\u003c/sup\u003e. (I) Venn diagrams of the pathways in the C2 library common among VUB13\u003csup\u003edel18q\u003c/sup\u003e, hESC\u003csup\u003eWT_\u003c/sup\u003e\u003csup\u003e\u003cem\u003eSALL3\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eKD\u003c/sup\u003e, VUB13\u003csup\u003edel18q_\u003c/sup\u003e\u003csup\u003e\u003cem\u003eSALL3\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eOE\u003c/sup\u003e and hESC\u003csup\u003eWT\u003c/sup\u003e. (J) Pathways commonly deregulated among the different samples are associated with pluripotency and differentiation.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3100381/v1/01756720ddd63b1043bf0688.png"},{"id":42713612,"identity":"50733888-b4e4-481b-9763-36e68d952af8","added_by":"auto","created_at":"2023-09-06 13:34:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6002077,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3100381/v1/35cd1773-1149-4c85-9525-db54134081e5.pdf"},{"id":40733817,"identity":"996d22a7-1f7e-4f72-b72e-7533c3d30725","added_by":"auto","created_at":"2023-07-28 16:53:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4198990,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryDataLeietal.docx","url":"https://assets-eu.researchsquare.com/files/rs-3100381/v1/afe08683e77b649d8c79cbed.docx"},{"id":40733806,"identity":"375f43ca-ba70-4033-aa5d-3b98f1dd0426","added_by":"auto","created_at":"2023-07-28 16:53:31","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":33384,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental table 2\u003c/p\u003e","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3100381/v1/75f4552264370273cca1857a.xlsx"},{"id":40735494,"identity":"d7ee77a4-fe65-4e09-b78f-736a8ad1d15f","added_by":"auto","created_at":"2023-07-28 17:09:32","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":74135,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Table 3\u003c/p\u003e","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3100381/v1/e2684292d285818c0c130c8c.xlsx"},{"id":40734835,"identity":"7dd94cda-41b3-45e3-a879-206b20b3a568","added_by":"auto","created_at":"2023-07-28 17:01:31","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":21015,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Table 4\u003c/p\u003e","description":"","filename":"SupplemntaryTable4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3100381/v1/ed9761318cb85c5e4d1264e5.xlsx"}],"financialInterests":"(Not answered)","formattedTitle":"SALL3 mediates the loss of neuroectodermal differentiation potential in human embryonic stem cells with chromosome 18q loss","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuman pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), are self-renewing cells that can give rise to any cell type originating from any of the three embryonic germ layers\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This makes hPSCs an attractive resource for \u003cem\u003ein vitro\u003c/em\u003e disease modelling, developmental biology research, drug discovery, and cell transplantation therapy. A substantial number of clinical trials are underway using hPSC-derived cell products, including for the treatment of age-related macular degeneration, spinal cord injury and type 1 diabetes\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. An important hurdle for both safe clinical translation and the reliable use of hPSCs as \u003cem\u003ein vitro\u003c/em\u003e research models is the occurrence of cell culture drift due to the acquisition of genetic abnormalities\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. A subset of these genetic aberrations are highly recurrent and are found in hPSC lines worldwide. These recurrent changes vary in size from single-nucleotide point mutations to large chromosome structural variants, the most common being gains of chromosomes 1q, 12p, 17, 20, and X and losses of 10p, 18q and 22p, as well as mutations in TP53\u003csup\u003e6,7,9\u0026ndash;12\u003c/sup\u003e. Other genetic changes include epigenetic variations, including erosion of X-chromosome inactivation\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, mutations in the mitochondrial genome\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and an array of other point mutations and structural variants spread throughout the genome\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThese recurrent genetic changes arise from the pool of common variations in hPSC cultures via different cell competition mechanisms. hPSCs are prone to replication stress, leading to DNA damage, which in turn is a source of de novo genetic variation\u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. For instance, up to 20% of cells in an hESC culture carry de novo structural variants, but only a minority of them have the potential to confer a selective advantage to the cells\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, leading to the mutant cells rapidly outcompeting their genetically balanced counterparts\u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The exact traits that the different chromosomal abnormalities confer on undifferentiated cells, as well as the specific driver genes of these traits, are only well established for the gain of 20q11.21\u003csup\u003e28\u003c/sup\u003e. This abnormality confers a decreased sensitivity to apoptosis-inducing events due to increased expression of the gene \u003cem\u003eBCL2L1\u003c/em\u003e, located in the minimal gained region\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. For gains of 12p, it is considered that \u003cem\u003eNANOG\u003c/em\u003e drives at least part of the growth advantage of the cells\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, and cells with a complex karyotype carrying all the most common abnormalities (gains of 1, 12, 17 and 20q) can outcompete the other cells by corralling and mechanical compression\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAn important concern about these genetic variants is whether and how they alter the differentiation capacity of hPSCs and potentially prime differentiated cells for malignant transformation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The gain of 1q is common in cancers, particularly lung adenocarcinoma, breast invasive carcinoma and liver hepatocellular carcinoma\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and appears to confer a growth advantage to cells during differentiation from hESCs to neural precursors\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Moreover, variants in 1q21.1 can alter neurodevelopmental trajectories upon hiPSC differentiation, with the deletion of 1q21.1 accelerating neuronal production and its duplication delaying the transition from neural progenitor cell to neuron\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Gains in chromosome 12 are frequently found in testicular germ cell tumors\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. hPSCs with trisomy 12 display a reduced tendency toward spontaneous differentiation\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003c/sup\u003e and gain of 12p13.31 results in an overall reduction in trilineage differentiation capacity and foci of residual pluripotent cells during hepatic differentiation\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The highly recurrent gain of 20q11.21 impairs the neuroectodermal lineage commitment of hPSCs\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003c/sup\u003e and hESCs with 20q11.1q11.2 amplification have a reduced propensity to differentiate down the hematopoietic lineage, maintain more immature phenotypes along the neural differentiation trajectory and generate teratomas with foci of undifferentiated cells\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The gain of chromosome 17 is common in neuroblastomas, testicular germ cell tumors and breast cancers\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. hPSC lines with a gain of chromosome 17 show altered differentiation patterns in embryoid bodies\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, and the amplification of \u003cem\u003eWNT3A\u003c/em\u003e and \u003cem\u003eWNT9\u003c/em\u003e on chromosome 17q21.31 alters neuronal differentiation\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDeletions of chromosome 18q are one of the rarer recurrent structural chromosomal abnormalities in hPSCs. This deletion was first reported as a single event by Maitra \u003cem\u003eet al.\u003c/em\u003e in 2005\u003csup\u003e45\u003c/sup\u003e, and our group later found 18q deletions in three different hESC lines at relatively early passages, always as part of a derivative chromosome 18\u003csup\u003e28\u003c/sup\u003e. A large study by Amps \u003cem\u003eet al\u003c/em\u003e. in 2011 revealed 5 instances of this deletion\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, A large study by Amps \u003cem\u003eet al\u003c/em\u003e. in 2011 revealed 5 instances of this deletion, and WiCell has reported that 4% of the 7300 hPSC cultures evaluated over nearly eight years carried an 18q deletion (WiCell Cytogenetics Lab, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.wicell.org/media.acux/29102c0e-e88e-426b-ab7d-bac4c2a9ec6a\u003c/span\u003e\u003cspan address=\"https://www.wicell.org/media.acux/29102c0e-e88e-426b-ab7d-bac4c2a9ec6a\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Chromosome 18q loss is common in cancers, especially gastrointestinal tract cancers\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and is linked to several disorders, including congenital malformations, developmental delays, and intellectual disability\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. However, the impact of 18q loss on the functional properties of hPSCs is unknown. Therefore, the aim of this work was to examine the functional effects of 18q deletions during hPSC differentiation into the three embryonic germ layers and to determine the molecular mechanisms involved.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eThe minimal common region of 18q loss spans 14 genes expressed in undifferentiated\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ehESCs\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and includes \u003cem\u003eSALL3\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe initially identified\u0026nbsp;18q losses in the hESC lines VUB04 and VUB26, in the form of a derivative chromosome 18. VUB04 presented a deletion at 18q21.2qter and a duplication at 5q14.2qter,\u0026nbsp;and VUB26 showed the minimal 18q loss region (18q23qter) and a duplication at 7q33qter (Fig 1A. Sup.\u0026nbsp;Fig.\u0026nbsp;1 and Sup. Table 1)\u003csup\u003e48\u003c/sup\u003e. These two hESC lines were not used in the present work because they further genetically drifted and acquired gain of 1q and 20q11.21, but their analysis helped to\u0026nbsp;narrow\u0026nbsp;down the common 18q deletion region. In this study, we used two other hESC lines bearing derivative chromosomes 18 involving a loss of 18q losses (hESC\u003csup\u003edel18q\u003c/sup\u003e), VUB14\u003csup\u003edel18q\u003c/sup\u003e and VUB13\u003csup\u003edel18q\u003c/sup\u003e, as well as three chromosomally balanced lines (hESC\u003csup\u003eWT\u003c/sup\u003e), VUB14\u003csup\u003eWT\u003c/sup\u003e, and VUB04\u003csup\u003eWT\u003c/sup\u003e and VUB03\u003csup\u003eWT\u003c/sup\u003e, which served as controls\u0026nbsp;for VUB13\u003csup\u003edel18q\u003c/sup\u003e since VUB13\u003csup\u003eWT\u003c/sup\u003e was lost (details on the karyotypes of the lines and their characterization are shown in Sup.\u0026nbsp;Fig.\u0026nbsp;1 and Sup. Table 1). We used shallow genome sequencing to confirm the karyotypes before starting the experiments and all lines were routinely inspected with qPCR assays targeting recurrent chromosomal abnormalities (1q, 12p, 20q11.21 and 17q) to confirm their genomic stability for the duration of the different experiments.\u003c/p\u003e\n\u003cp\u003eThe 18q losses exhibited a common loss region from bp 75,773,285 to 80,373,285, spanning 37 loci. Bulk RNA sequencing of undifferentiated hESCs indicated that 14 genes within this region are expressed in undifferentiated hESCs, with counts per million greater than one in at least two samples (Fig. 1A). Of these coding genes, \u003cem\u003eADNP2\u003c/em\u003e, \u003cem\u003eSALL3\u003c/em\u003e and \u003cem\u003eTXNL4A\u003c/em\u003e had the highest expression and showed decreased transcript levels in mutant cells. \u003cem\u003eADNP2\u003c/em\u003e and \u003cem\u003eTXNL4A\u003c/em\u003e have no known function in hPSC. \u003cem\u003eADNP2\u003c/em\u003e is predicted to be a transcription factor, and its silencing increases oxidative stress-mediated cell death\u003csup\u003e49\u003c/sup\u003e. \u003cem\u003eTXNL4A\u003c/em\u003e is a component of the U5 small ribonucleoprotein particle, which is involved in pre-mRNA splicing and is associated with Burn-McKeown syndrome\u003csup\u003e50\u003c/sup\u003e. \u003cem\u003eSALL3\u003c/em\u003e was more promising as candidate driver gene as it has previously been reported to regulate the differentiation propensity of hiPSC lines\u003csup\u003e51\u003c/sup\u003e. Kuroda \u003cem\u003eet al\u003c/em\u003e. showed that hiPSC lines expressing high levels of \u003cem\u003eSALL3\u003c/em\u003e differentiated preferentially into ectoderm, while hiPSC lines expressing lower levels of \u003cem\u003eSALL3\u003c/em\u003e tended to differentiate into mesoderm and endoderm\u003csup\u003e51\u003c/sup\u003e. It has also been shown that \u003cem\u003eSALL3\u003c/em\u003e interacts with the Mediator complex in neural stem cells\u003csup\u003e52\u003c/sup\u003e and is related to the development of the nervous system\u003csup\u003e53\u003c/sup\u003e. Considering these previous findings, we hypothesized that decreased expression of \u003cem\u003eSALL3\u003c/em\u003e as a result of a loss of one copy of the gene could alter the differentiation capacity of hESCs\u003csup\u003edel18q\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ehESCs\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;with 18q loss show impaired neuroectoderm\u0026nbsp;differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs a first step, we investigated the effect of 18q deletion on\u0026nbsp;hESC\u0026nbsp;ectoderm lineage commitment.\u0026nbsp;hESC\u003csup\u003eWT\u0026nbsp;\u003c/sup\u003eand hESC\u003csup\u003edel18q\u003c/sup\u003e were subjected to neuroectoderm differentiation for 8 days using LDN193189 (LDN), SB431542 (SB) and retinoic acid (RA)\u003csup\u003e54\u003c/sup\u003e (Fig. 1B). We measured the mRNA levels of different neuroectoderm markers to evaluate neuroectoderm differentiation efficiency and the expression of the undifferentiated state markers \u003cem\u003eNANOG\u003c/em\u003e and \u003cem\u003ePOUF51\u003c/em\u003e (Fig. 2A and Sup Fig. 2A). VUB13\u003csup\u003edel18q\u003c/sup\u003e and VUB14\u003csup\u003edel18q\u003c/sup\u003e had significantly lower mRNA levels of \u003cem\u003ePAX6\u003c/em\u003e, \u003cem\u003eNES\u003c/em\u003e and \u003cem\u003eSOX1\u003c/em\u003e, which were decreased by 3-fold, 15-fold, and 25-fold, respectively, compared to the levels in hESC\u003csup\u003eWT\u003c/sup\u003e (p\u0026le;0.0001 unpaired t-test), indicating a decreased neuroectodermal differentiation efficiency in hESC\u003csup\u003edel18q\u003c/sup\u003e. \u003cem\u003ePOU5F1\u003c/em\u003e and \u003cem\u003eNANOG\u003c/em\u003e mRNA expression levels were almost undetectable for all differentiated cells\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(Sup Fig. 2A). We also evaluated the differentiation of hESC\u003csup\u003eWT\u003c/sup\u003e and hESC\u003csup\u003edel18q\u0026nbsp;\u003c/sup\u003ecells by immunostaining (Fig.\u0026nbsp;2B-C). We observed\u0026nbsp;a\u0026nbsp;lower percentage of PAX6 positive\u003csup\u003e\u0026nbsp;\u003c/sup\u003ecells in differentiated hESC\u003csup\u003edel18q\u0026nbsp;\u003c/sup\u003ethan in\u0026nbsp;differentiated hESC\u003csup\u003eWT\u0026nbsp;\u003c/sup\u003ecells\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(45% vs 70%, p = 0.0114, unpaired t-test, Fig. 2C), which was consistent with the decrease in the levels of \u003cem\u003ePAX6\u003c/em\u003e mRNA. Taken together, these results show that hESCs\u003csup\u003edel18q\u003c/sup\u003e differentiation into neuroectoderm is impaired, and rather than to remain undifferentiated state, they miss-specify.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ehESC\u003csup\u003edel18q\u003c/sup\u003e readily\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003edifferentiates into\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;mesendoderm\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ederivatives\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;but\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eshows\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;abnormal cardiomyocyte progenitor differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe further investigated the impact of 18q deletion on the\u0026nbsp;mesendoderm\u0026nbsp;differentiation capacity of hESCs by\u0026nbsp;differentiating\u0026nbsp;hESC\u003csup\u003eWT\u003c/sup\u003e and hESC\u003csup\u003edel18q\u0026nbsp;\u003c/sup\u003einto, in this case cardiac\u0026nbsp;progenitors\u0026nbsp;and hepatoblasts. Thus,\u0026nbsp;we\u0026nbsp;induced the differentiation of\u0026nbsp;hESC\u003csup\u003eWT\u003c/sup\u003e and hESC\u003csup\u003edel18q\u003c/sup\u003e into mesoderm using a 5-day cardiac progenitor induction protocol as described previously\u003csup\u003e55\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(Fig. 1B). We evaluated the mRNA levels of the cardiac progenitor markers \u003cem\u003eGATA4\u003c/em\u003e, \u003cem\u003eISL1\u003c/em\u003e, \u003cem\u003eNKX2-5\u003c/em\u003e and \u003cem\u003ePDGFRA\u0026nbsp;\u003c/em\u003e(Fig.\u0026nbsp;3A).\u0026nbsp;hESC\u003csup\u003edel18q\u0026nbsp;\u003c/sup\u003eshowed 2-fold lower levels of \u003cem\u003eGATA4\u003c/em\u003e mRNA (p = 0.0012 unpaired t- test)\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(Fig.\u0026nbsp;3A). hESC\u003csup\u003edel18q\u003c/sup\u003e lines expressed \u003cem\u003eISL1\u003c/em\u003e at 15-fold higher levels, on average (p=0.0016, unpaired t-test), than hESC\u003csup\u003eWT\u003c/sup\u003e lines. We found no significant difference in the expression levels of \u003cem\u003eNKX2-5\u0026nbsp;\u003c/em\u003eor \u003cem\u003ePDGFRA\u003c/em\u003e between the control and mutant cardiac progenitor groups (p=0.11 and p=0.42, unpaired t-test, Fig. 3A). We also evaluated the proportion of differentiated cardiac progenitor and undifferentiated cells by immunostaining for GATA4 and POU5F1. The percentage of GATA4-positive cells was not statistically significantly different in hESC\u003csup\u003edel18q\u003c/sup\u003e than in hESC\u003csup\u003eWT\u003c/sup\u003e (p=0.2942, unpaired t-test, Fig. 3B-C), mainly due to the low differentiation efficiency of VUB03\u003csup\u003eWT\u003c/sup\u003e, while the cells\u0026nbsp;were\u0026nbsp;all POU5F1 negative. Taken together, and bearing in mind the temporal expression of these markers during cardiac differentiation\u003csup\u003e56,57\u003c/sup\u003e, the results suggest that hESC\u003csup\u003edel18q\u003c/sup\u003e may experience differentiation delays or arrest, reaching an ISL1\u003csup\u003ehigh\u003c/sup\u003e GATA4\u003csup\u003elow\u003c/sup\u003e stage at day 5 but remaining less mature than their hESC\u003csup\u003eWT\u003c/sup\u003e counterparts.\u003c/p\u003e\n\u003cp\u003eNext, we differentiated hESC\u003csup\u003eWT\u003c/sup\u003e and hESC\u003csup\u003edel18q\u003c/sup\u003e into hepatoblasts by applying the modified differentiation protocol for 8 days as described previously\u003csup\u003e58\u003c/sup\u003e (Fig. 1B). We measured the expression levels of the hepatoblast markers \u003cem\u003eHNF4A\u003c/em\u003e, \u003cem\u003eAFP\u003c/em\u003e, \u003cem\u003eALB\u0026nbsp;\u003c/em\u003eand \u003cem\u003eFOXA2\u003c/em\u003e (Fig. 4A). We found no significant difference in the mRNA expression levels of \u003cem\u003eHNF4A\u003c/em\u003e, \u003cem\u003eALB\u003c/em\u003e, and \u003cem\u003eFOXA2\u003c/em\u003e between hESC\u003csup\u003eWT\u003c/sup\u003e and hESC\u003csup\u003edel18q\u003c/sup\u003e (p\u0026gt;0.05, unpaired t-test), while AFP had a lower expression in hESC\u003csup\u003edel18q\u003c/sup\u003e (Fig. 4A, p=0.0095, unpaired t-test). We further evaluated hepatoblast differentiation by immunostaining for HNF4A and found that the percentages of HNF4A\u003csup\u003e-\u003c/sup\u003epositive cells in hESC\u003csup\u003edel18q\u003c/sup\u003e cells were similar to those in the wild type (Fig. 4B-C, p=0.2514, unpaired t-test).\u003c/p\u003e\n\u003cp\u003eFor both\u0026nbsp;mesodermal and endodermal\u0026nbsp;lineage\u0026nbsp;commitment, all\u0026nbsp;hESC\u003csup\u003eWT\u003c/sup\u003e and hESC\u003csup\u003edel18q\u003c/sup\u003e lines displayed low mRNA and protein levels of undifferentiated state markers (Sup Fig. 2B-C), indicating a loss of pluripotency for all cell lines during differentiation. Overall, our results indicate that hESC\u003csup\u003eWT\u003c/sup\u003e and hESC\u003csup\u003edel18q\u003c/sup\u003e differentiate into definitive mesoderm and endoderm\u0026nbsp;and that\u0026nbsp;there may be a delay or impairment in the progression of hESC\u003csup\u003edel18q\u003c/sup\u003e toward the cardiac progenitor stage. The differences in gene expression observed during hepatoblast differentiation are likely due to between-line variation in differentiation propensity rather than to the 18q deletion itself.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDownregulation of \u003cem\u003eSALL3\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;impairs neuroectoderm differentiation but does not affect differentiation into\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;mesoderm and endoderm\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWe next examined\u0026nbsp;\u003c/strong\u003e\u003cem\u003eSALL3\u003c/em\u003e mRNA\u0026nbsp;expression levels in\u0026nbsp;undifferentiated cells and found that \u003cem\u003eSALL3\u003c/em\u003e expression\u0026nbsp;was\u0026nbsp;significantly lower in hESC\u003csup\u003edel18q\u003c/sup\u003e than in\u0026nbsp;hESC\u003csup\u003eWT\u0026nbsp;\u003c/sup\u003e(Sup Fig. 3A), supporting the notion that \u003cem\u003eSALL3\u003c/em\u003e could be a key gene in the altered differentiation capacity of hESCs\u003csup\u003edel18q\u003c/sup\u003e\u003cstrong\u003e. W\u003c/strong\u003ee \u003cstrong\u003efirst\u0026nbsp;\u003c/strong\u003egenerated \u003cem\u003eSALL3\u003c/em\u003e knockdown (KD) lines from \u003cstrong\u003et\u003c/strong\u003ehree hESC\u003csup\u003eWT\u003c/sup\u003e lines (VUB02\u003csup\u003eWT\u003c/sup\u003e, VUB03\u003csup\u003eWT\u003c/sup\u003e and VUB04\u003csup\u003eWT\u003c/sup\u003e) by transducing\u0026nbsp;a lentiviral vector containing shRNA\u0026nbsp;targeting the \u003cem\u003eSALL3\u003c/em\u003e transcript\u0026nbsp;(hESC\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u003c/sup\u003e)\u0026nbsp;or a\u0026nbsp;nontargeting\u0026nbsp;shRNA as\u0026nbsp;a\u0026nbsp;control (hESC\u003csup\u003eWT-NT\u003c/sup\u003e). We confirmed the knockdown efficiency of the generated hESC\u003csup\u003eWT\u003c/sup\u003e\u003cem\u003eSALL3\u003c/em\u003e\u003csup\u003eKD\u003c/sup\u003e lines by measuring the \u003cem\u003eSALL3\u003c/em\u003e mRNA expression levels and found that they were reduced by 30%, 40% and 20% in VUB02\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u003c/sup\u003e, VUB03\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u003c/sup\u003e and VUB04\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u0026nbsp;\u003c/sup\u003erespectively, compared to\u0026nbsp;the\u0026nbsp;controls (Sup Fig. 3B). Next, \u003cstrong\u003ewe generated\u0026nbsp;\u003c/strong\u003ehESC\u003csup\u003edel18q\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;with stable overexpression of \u003cem\u003eSALL3\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e(hESC\u003csup\u003edel18q_\u003cem\u003eSALL3\u003c/em\u003eOE\u003c/sup\u003e) by transducing\u0026nbsp;VUB13\u003csup\u003edel18q\u003c/sup\u003e and VUB14\u003csup\u003edel18q\u003c/sup\u003e with\u0026nbsp;the\u0026nbsp;\u003cem\u003eSALL3\u003c/em\u003e lentiviral vector,\u0026nbsp;and we verified the\u0026nbsp;overexpression\u0026nbsp;by measuring\u0026nbsp;\u003cem\u003eSALL3\u003c/em\u003e mRNA levels, which were\u0026nbsp;significantly increased in\u0026nbsp;VUB13\u003csup\u003edel18q_\u003cem\u003eSALL3\u003c/em\u003eOE\u0026nbsp;\u003c/sup\u003e(3-fold) and VUB14\u003csup\u003edel18q_\u003cem\u003eSALL3\u003c/em\u003eOE\u003c/sup\u003e (5-fold) compared to controls\u0026nbsp;(Sup Fig. 3C).\u003c/p\u003e\n\u003cp\u003eTo investigate the role of \u003cem\u003eSALL3\u003c/em\u003e in regulating hESC differentiation propensity, we first induced neuroectodermal differentiation in hESC\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u003c/sup\u003e, hESC\u003csup\u003edel18q_\u003cem\u003eSALL3\u003c/em\u003eOE\u003c/sup\u003e, and the corresponding control cells. All three hESC\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u003c/sup\u003e lines had lower levels of all NE markers than nontarget controls (Fig. 5A and Sup Fig. 4A-B). PAX6 protein levels were also reduced in hESC\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u003c/sup\u003e (Fig. 5B-C), with only 20% of the cells expressing PAX6,\u003csup\u003e\u0026nbsp;\u003c/sup\u003ecompared to 80% of PAX6 positive cells in the control group (Fig. 5B-C). Our results show that \u003cem\u003eSALL3\u003c/em\u003e suppression in hESC\u003csup\u003eWT\u003c/sup\u003e recapitulates the impaired NE differentiation seen in hESC\u003csup\u003edel18q\u003c/sup\u003e lines. In contrast, hESC\u003csup\u003edel18q_\u003cem\u003eSALL3\u003c/em\u003eOE\u003c/sup\u003e cells efficiently differentiated into neuroectoderm cells, accompanied by a significant increase in the mRNA levels of \u003cem\u003ePAX6\u003c/em\u003e, \u003cem\u003eSOX1\u003c/em\u003e, and \u003cem\u003eNES\u0026nbsp;\u003c/em\u003e(Fig.\u0026nbsp;5A and Sup Fig. 4A-B); moreover,\u0026nbsp;hESC\u003csup\u003edel18q\u0026shy;\u003cem\u003eSALL3\u003c/em\u003eOE\u0026nbsp;\u003c/sup\u003ecultures included more PAX6\u003csup\u003e\u0026nbsp;\u003c/sup\u003epositive cells than hESC\u003csup\u003edel18q\u0026nbsp;\u003c/sup\u003ecultures (60% vs 20%, respectively)\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(Fig. 5B-C). These results indicate that exongenous \u003cem\u003eSALL3\u003c/em\u003e expression can rescue the impairment of ectoderm differentiation caused by 18q loss.\u003c/p\u003e\n\u003cp\u003eNext, we induced\u0026nbsp;the\u0026nbsp;differentiation of the different hESC lines into cardiac progenitors.\u0026nbsp;The three\u0026nbsp;hESC\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u003c/sup\u003e lines differentiated inconsistently toward mesoderm fates. VUB04\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u003c/sup\u003e showed lower levels of \u003cem\u003eGATA4\u0026nbsp;\u003c/em\u003e(Fig.\u0026nbsp;6A, p=0.0003, unpaired t-test), whereas compared to the controls, VUB03\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u003c/sup\u003e and\u003csup\u003e\u0026nbsp;\u003c/sup\u003eVUB02\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u003c/sup\u003e showed no differences in \u003cem\u003eGATA4\u003c/em\u003e expression (Fig. 6A). Additionally, the percentage of GATA4\u003csup\u003e+\u003c/sup\u003e cells detected by immunostaining in hESC\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u003c/sup\u003e followed the same pattern, consistent with the mRNA levels of each line (Fig. 6B-C). The hESC\u003csup\u003edel18q_\u003cem\u003eSALL3\u003c/em\u003eOE\u003c/sup\u003e cells exhibited variable marker profiles, with increases in GATA4 expression at both the mRNA (Fig. 6A, p=0.004, unpaired t-test) and protein levels for VUB13\u003csup\u003edel18q_\u003cem\u003eSALL3\u003c/em\u003eOE\u003c/sup\u003e but no difference in \u003cem\u003eGATA4\u0026nbsp;\u003c/em\u003emRNA levels (Fig.\u0026nbsp;6A, p=0.3622, unpaired t-test)\u0026nbsp;and a slight decrease\u0026nbsp;in\u0026nbsp;GATA4 protein levels\u0026nbsp;in\u0026nbsp;VUB14\u003csup\u003edel18q_\u003cem\u003eSALL3\u003c/em\u003eOE\u003c/sup\u003e (Fig. 6B-C). Similarly, the mRNA expression levels of other markers, \u003cem\u003eNKX2-5\u003c/em\u003e, \u003cem\u003eISL1\u003c/em\u003e, and \u003cem\u003ePDGFRA\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e showed no consistent trend in the hESC\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u0026nbsp;\u003c/sup\u003egroups and exhibited no consistent differences in hESCs\u003csup\u003edel18q\u003c/sup\u003e and hESC\u003csup\u003edel18q\u003c/sup\u003e\u003cem\u003eSALL3\u003c/em\u003e\u003csup\u003eOE\u003c/sup\u003e (Sup Fig. 5A-C).\u003c/p\u003e\n\u003cp\u003eWhen hESCs were differentiated into hepatoblast, \u003cem\u003eSALL3\u003c/em\u003e downregulation in hESC\u003csup\u003eWT\u003c/sup\u003e resulted in increased \u003cem\u003eHNF4A\u003c/em\u003e, \u003cem\u003eALB\u003c/em\u003e, and \u003cem\u003eFOXA2\u003c/em\u003e mRNA expression (Fig. 7A and Sup Fig. 4B-C). The percentage of HNF4A\u003csup\u003e\u0026nbsp;\u003c/sup\u003epositive cells\u0026nbsp;was\u0026nbsp;higher in\u0026nbsp;VUB03\u003csup\u003e\u0026nbsp;WT\u003c/sup\u003e\u003cem\u003eSALL3\u003c/em\u003e\u003csup\u003eKD\u003c/sup\u003e cells but not in VUB04\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u003c/sup\u003e and VUB02\u003csup\u003eWT_S\u003cem\u003eALL3\u003c/em\u003eKD\u003c/sup\u003e cells compared to controls (Fig. 7B-C). The mRNA expression of another marker, \u003cem\u003eAFP\u003c/em\u003e, also did not show consistent changes (Sup Fig. 4A). Upon overexpression of \u003cem\u003eSALL3\u003c/em\u003e, the differentiation profiles into hepatoblast did not show the expected mirroring effect. The mRNA expression of all the hepatoblast markers \u003cem\u003eHNF4A\u003c/em\u003e, \u003cem\u003eALB\u003c/em\u003e, \u003cem\u003eAFP\u003c/em\u003e and \u003cem\u003eFOXA2\u003c/em\u003e increased in VUB14\u003csup\u003edel18q_\u003cem\u003eSALL3\u003c/em\u003eOE\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ecells,\u003csup\u003e\u0026nbsp;\u003c/sup\u003econsistent with the changes observed in\u0026nbsp;the\u0026nbsp;HNF4A protein level, but this same effect was not observed in\u0026nbsp;VUB13\u003csup\u003edel18q_\u003cem\u003eSALL3\u003c/em\u003eOE\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ecells (Fig. 7 and Sup Fig. 4).\u003c/p\u003e\n\u003cp\u003eOverall, these results suggest that the effect of \u003cem\u003eSALL3\u003c/em\u003e on mesoderm and endoderm differentiation may be line-specific and is not the reason for the delayed progression seen during cardiac differentiation\u0026nbsp;in\u0026nbsp;hESCs\u003csup\u003e18q\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDownregulation of \u003cem\u003eSALL3\u003c/em\u003e and loss of 18q result in the deregulation of genes in pathways associated\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ewith\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;pluripotency and differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo gain\u0026nbsp;deeper insight\u0026nbsp;into\u0026nbsp;the effect of\u0026nbsp;\u003cem\u003eSALL3\u003c/em\u003e downregulation on\u0026nbsp;the\u0026nbsp;global transcriptomic profile of cells with 18q loss, we carried out\u0026nbsp;bulk RNA sequencing of hESC\u003csup\u003eWT-NT\u003c/sup\u003e (N=6),\u0026nbsp;hESC\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u0026nbsp;\u003c/sup\u003e(N=6), hESC\u003csup\u003eWT\u003c/sup\u003e (N=6), VUB13\u003csup\u003edel18q\u003c/sup\u003e (N=4) and\u0026nbsp;VUB13\u003csup\u003edel18q_\u003cem\u003eSALL3\u003c/em\u003eOE\u003c/sup\u003e (N=5)\u0026nbsp;cells.\u0026nbsp;To estimate the similarity among the samples, we\u0026nbsp;generated\u0026nbsp;a distance\u0026nbsp;clustering heatmap with global row\u0026nbsp;scaling (Fig.\u0026nbsp;8A) and performed principal component analysis (Fig.\u0026nbsp;8B). The heatmap shows that while\u0026nbsp;VUB13\u003csup\u003edel18q\u003c/sup\u003e and hESC\u003csup\u003eWT_\u003c/sup\u003e\u003cem\u003e\u003csup\u003eSALL3\u003c/sup\u003e\u003c/em\u003e\u003csup\u003eKD\u003c/sup\u003e cluster together, they cluster apart from the WT cell lines, as well as from\u0026nbsp;VUB13\u003csup\u003edel18q_\u003cem\u003eSALL3\u003c/em\u003eOE\u003c/sup\u003e. The samples in this last group\u0026nbsp;clustered\u0026nbsp;more closely to the WT cell lines than to their unmodified VUB13\u003csup\u003edel18q\u0026nbsp;\u003c/sup\u003e(Fig.\u0026nbsp;8A). This pattern was also reflected in the first dimension of the PCA (Fig.\u0026nbsp;8B), where hESC\u003csup\u003eWT\u003c/sup\u003e, hESC\u003csup\u003eWT-NT\u0026nbsp;\u003c/sup\u003eand\u0026nbsp;VUB13\u003csup\u003edel18q_\u003cem\u003eSALL3\u003c/em\u003eOE\u003c/sup\u003e clustered more closely with each other than with\u0026nbsp;VUB13\u003csup\u003edel18q\u003c/sup\u003e and hESC\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u0026nbsp;\u003c/sup\u003e(Sup Fig.\u0026nbsp;6A). These results suggest that the downregulation of \u003cem\u003eSALL3\u003c/em\u003e in WT cells is sufficient to alter the transcriptome such that its profile is closer to that of\u0026nbsp;an\u0026nbsp;hESC line with a loss of 18q, and \u003cem\u003eSALL3\u003c/em\u003e overexpression in hESC\u003csup\u003edel18q\u003c/sup\u003e can restore the transcriptome to a near-WT state. Taken together,\u0026nbsp;these findings support\u0026nbsp;the our hypothesis that the differences between hESC\u003csup\u003eWT\u003c/sup\u003e and hESC\u003csup\u003edel18q\u003c/sup\u003e are mostly driven by the downregulation of \u003cem\u003eSALL3\u003c/em\u003e due to the loss of one copy of this gene. For this reason,\u0026nbsp;we pooled the hESCs\u003csup\u003eWT-NT\u003c/sup\u003e with the untreated hESCs\u003csup\u003eWT\u003c/sup\u003e for further analysis.\u003c/p\u003e\n\u003cp\u003eNext, we carried out differential gene expression analysis to gain further insight\u0026nbsp;into\u0026nbsp;which genes and pathways form the basis of the differences between hESC\u003csup\u003eWT\u003c/sup\u003e and hESC\u003csup\u003edel18q\u003c/sup\u003e and which of these are driven by \u003cem\u003eSALL3\u003c/em\u003e. Figure 8B-D shows volcano plots of the differentially expressed genes in the different\u0026nbsp;groups. We considered this differential expression to be significant at a |log\u003csub\u003e2\u003c/sub\u003efold-change| \u0026gt; 1.0 and false discovery rate (FDR)\u0026lt; 0.05.\u0026nbsp;VUB13\u003csup\u003edel18q\u003c/sup\u003e shows 891 and 448 genes that are significantly upregulated and downregulated in\u0026nbsp;hESC\u003csup\u003edel18q\u003c/sup\u003e, respectively, compared to the WT cells (Fig 8C). Downregulation of \u003cem\u003eSALL3\u003c/em\u003e in WT cells\u0026nbsp;led\u0026nbsp;to the differential expression of\u0026nbsp;745 genes,\u0026nbsp;of which 399\u0026nbsp;were\u0026nbsp;upregulated and 346\u0026nbsp;were\u0026nbsp;downregulated (Fig 8D).\u0026nbsp;Overexpression\u0026nbsp;of \u003cem\u003eSALL3\u003c/em\u003e in VUB13\u003csup\u003edel18q\u0026nbsp;\u003c/sup\u003eresulted in the upregulation of 121 genes and the downregulation of 605 genes (Fig 8E).\u003c/p\u003e\n\u003cp\u003eTo elucidate which of the transcriptional differences between hESC\u003csup\u003eWT\u003c/sup\u003e and\u0026nbsp;hESC\u003csup\u003edel18q\u003c/sup\u003e are mediated by \u003cem\u003eSALL3\u003c/em\u003e, we investigated the overlaps in up-\u0026nbsp;and downregulated genes across conditions. First, we found that 231 and 93 genes were commonly up-\u0026nbsp;and downregulated, respectively, between VUB13\u003csup\u003edel18q\u003c/sup\u003e and\u0026nbsp;hESC\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u003c/sup\u003e, representing\u0026nbsp;26% of the differentially expressed genes in\u0026nbsp;hESCs\u0026nbsp;with\u0026nbsp;an\u0026nbsp;18q deletion (Fig 8F and G). Next, we compared these subsets of genes to the genes with altered gene expression in\u0026nbsp;hESC\u003csup\u003edel18q\u003c/sup\u003e upon\u0026nbsp;overexpression\u0026nbsp;of \u003cem\u003eSALL3\u003c/em\u003e. We compared the genes that\u0026nbsp;were\u0026nbsp;upregulated by \u003cem\u003eSALL3\u003c/em\u003e knockdown and by loss of 18q to those downregulated by\u0026nbsp;overexpression\u0026nbsp;of \u003cem\u003eSALL3\u003c/em\u003e in hESC\u003csup\u003e18q\u003c/sup\u003e, and vice\u0026nbsp;versa (Fig 8F and G). Out of the 231 upregulated genes shared by\u0026nbsp;the\u0026nbsp;VUB13\u003csup\u003edel18q\u003c/sup\u003e and\u0026nbsp;hESC\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u0026nbsp;\u003c/sup\u003egroups,\u0026nbsp;173 genes showed increased expression in\u0026nbsp;VUB13\u003csup\u003edel18q_\u003cem\u003eSALL3\u003c/em\u003eOE\u0026nbsp;\u003c/sup\u003e(Fig 8F).\u0026nbsp;Additionally, 18 of the 93 downregulated genes shared by VUB13\u003csup\u003edel18q\u003c/sup\u003e and\u0026nbsp;hESC\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u0026nbsp;\u003c/sup\u003edisplayed increased expression in\u0026nbsp;VUB13\u003csup\u003edel18q\u003c/sup\u003e\u003cem\u003eSALL3\u003c/em\u003e\u003csup\u003eOE\u0026nbsp;\u003c/sup\u003e(Fig 8G).\u0026nbsp;This further refined the gene set to a core of 191 genes that are the most strongly regulated by \u003cem\u003eSALL3\u003c/em\u003e in\u0026nbsp;hESCs, both by the loss of a copy of the gene itself\u0026nbsp;and\u0026nbsp;by the modulation of its expression (Supplementary Table 2). The other differences in gene\u0026nbsp;expression are likely associated\u0026nbsp;with\u0026nbsp;the loss of other genes in 18q or\u0026nbsp;with\u0026nbsp;the\u0026nbsp;overexpression\u0026nbsp;of genes in the duplicated regions of chromosomes 5 and 7,\u0026nbsp;which\u0026nbsp;are part of the derivative chromosome 18 in hESC\u003csup\u003edel18q\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFinally, to identify potential molecular targets and elucidate the underlying functional mechanisms that contribute to the observed impairment of differentiation capacity in hESC\u003csup\u003edel18q\u003c/sup\u003e,\u0026nbsp;we analyzed the differential gene expression of the three groups using Gene Set Enrichment Analysis (GSEA)\u0026nbsp;and the\u0026nbsp;MSigDB database. Specifically, we focused on the Kyoto Encyclopedia of Genes and Genomes (KEGG) and WikiPathways\u0026nbsp;databases\u0026nbsp;within the C2 library and the pathways of the H library.\u0026nbsp;We filtered the significant pathways based on a normalized enrichment score |NES| \u0026gt; 1, a p-value \u0026lt; 0.05, and the proportion of leading-edge genes accounting for over 30% of the entire gene set involved in the pathway.\u003c/p\u003e\n\u003cp\u003eFigures 8H and I show Venn diagrams of the overlap between significantly enriched pathways in the C2 and H libraries, respectively, for each of the three groups. The full list can be found in the Supplementary Table 3. In total, we found 13 pathways that overlapped among the three groups, all 13 of which were positively enriched in both\u0026nbsp;VUB13\u003csup\u003edel18q\u003c/sup\u003e and hESC\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u0026nbsp;\u003c/sup\u003eand negatively enriched in\u0026nbsp;VUB13\u003csup\u003edel18q_\u003cem\u003eSALL3\u003c/em\u003eOE\u003c/sup\u003e (Fig 8J shows the cell-type relevant pathways; the whole set can be found in the Supplementary Table 4). Because of the critical role that these pathways play in pluripotency maintenance and differentiation, we analyzed the expression of pluripotency-associated genes, and we found that \u003cem\u003eNANOG, POU5F1, LIN28, SOX2, PODXL, SUSD2, MYC, FOXD3\u003c/em\u003e and \u003cem\u003eDPPA3\u003c/em\u003e are overexpressed in VUB13\u003csup\u003edel18q\u003c/sup\u003e, with all but \u003cem\u003eDPPA3\u003c/em\u003e and \u003cem\u003ePOU5F1\u003c/em\u003e also being overexpressed upon \u003cem\u003eSALL3\u003c/em\u003e\u003csup\u003eKD\u003c/sup\u003e and downregulated by transgenic \u003cem\u003eSALL3\u0026nbsp;\u003c/em\u003eoverexpression in\u0026nbsp;VUB13\u003csup\u003edel18q\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(Sup Fig. 7A).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we examined the repercussions of the loss of chromosome 18q on the differentiation capacity of hESCs. For this, we used an early-stage differentiation approach to generate lineage-specific cell types representing the three germ layers: neuroectoderm, hepatoblast and cardiac progenitors. Our \u003cem\u003ein vitro\u003c/em\u003e lineage commitment studies indicated that the deletion of 18q in hESCs impaired neuroectodermal differentiation and delayed cardiac progenitor differentiation, while no consistent differences were observed in the commitment toward hepatoblasts. To study the mechanistic basis for these changes in differentiation, we looked at the genes located in the minimal region of loss. We found that decreased \u003cem\u003eSALL3\u003c/em\u003e expression due to the loss of one copy of the gene was sufficient to result in the observed decreased neuroectoderm differentiation but not to modulate cardiac and hepatoblast differentiation. In this sense, our results are only partially aligned with those obtained by Kuroda \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. These authors found that downregulating \u003cem\u003eSALL3\u003c/em\u003e resulted not only in decreased neuroectoderm differentiation, similar to our results, but also in increased cardiac progenitor differentiation. While we can only speculate about the reasons for these differences, it is likely that the genetic background of the cell lines plays an important role\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Also, given that \u003cem\u003eSALL3\u003c/em\u003e is a modulator of DNMT3A activity\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, the preexisting epigenetic marks in each of the lines, particularly histone modifications, may influence the recruitment of DNMT3A to methylate the DNA\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Furthermore, it is possible that other genes located in 18q, or in the gain regions of chromosomes 5 and 7, cause cell-line specific effects.\u003c/p\u003e \u003cp\u003eIn line with this reasoning, the gene expression analysis revealed that only part of the divergence between hESCs with 18q loss and their chromosomally normal isogenic counterparts was related to the differential expression of \u003cem\u003eSALL3\u003c/em\u003e. Interestingly, the core set of deregulated genes were associated with pathways involved in maintenance of and exit from the undifferentiated pluripotent state, and key regulators of pluripotency were both upregulated in hESCs\u003csup\u003e18q\u003c/sup\u003e and regulated by \u003cem\u003eSALL3\u003c/em\u003e expression. For instance, TGF-beta signaling is key to the maintenance of the primed pluripotent state\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. TGF-beta and BMP4 signaling are also core genes in regulating the balance between neuroectoderm differentiation and mesendoderm in both humans and mice\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Notch activation mediates TGF-β signaling during hESC and mesenchymal stem cell differentiation into smooth muscle cells\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, and its inhibition supports na\u0026iuml;ve state consolidation in rodent models\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. The cytokine tumor necrosis factor alpha has been shown to negatively regulate the differentiation of various cell types, including cardiomyocytes\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e, embryoid bodies\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e and osteoblasts\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Additionally, nuclear factor-κB inhibition has been found to mediate na\u0026iuml;ve pluripotency in mice\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Overall, these results suggest that downregulation of \u003cem\u003eSALL3\u003c/em\u003e due to the loss of 18q alters undifferentiated-state maintenance in hESCs by affecting pluripotency-associated pathways, and these changes have profound effects on the differentiation capacity of the cells.\u003c/p\u003e \u003cp\u003eWith hPSCs steadily moving into clinical trials\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e and being broadly used as a cell source for \u003cem\u003ein vitro\u003c/em\u003e modelling of, for instance, developmental processes and diseases, determining the impact of recurrent genetic abnormalities is critical\u003csup\u003e\u003cspan additionalcitationids=\"CR73\" citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. Work from our group and others is beginning to generate a detailed picture showing how these genetic abnormalities affect differentiation in a cell lineage-specific manner. For instance, 20q11.21 gain impairs neuroectoderm commitment without affecting mesendoderm induction\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e,\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e, and recently, it has been shown that cells with an isochromosome 20q are not able to survive RPE differentiation and display overall disruptions in the ability to correctly differentiate\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. In this work, we show that 18q loss specifically impairs neuroectoderm commitment and appears to delay cardiac differentiation. Taken together, these results highlight the importance of the genetic screening of hPSC cultures to ensure that these abnormalities do not pass unnoticed. In a research setting, chromosomal abnormalities could lead to confounding effects that decrease the reliability and reproducibility of the work, and in a clinical setting, they could lead at best to decreased therapeutic efficacy and at worst to tumorigenesis\u003csup\u003e72\u0026ndash;7478,79\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLosses of chromosome 18q are recurrently found in cancers \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and have been shown to be early events in midgut carcinoids\u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e,\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. Loss of heterozygosity on chromosome 18q is associated with significantly decreased survival in head and neck squamous cell carcinoma patients, and the methylation status of the \u003cem\u003eSALL3\u003c/em\u003e promoter correlates with shortened disease-free survival\u003csup\u003e\u003cspan additionalcitationids=\"CR83\" citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. Furthermore, three cancer suppressor genes have been identified in 18q, PIGN, MEX3C and ZNF516, which play roles in replication stress and in cervical neoplasia\u003csup\u003e\u003cspan additionalcitationids=\"CR86\" citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this context, several studies have found that even when chromosomally abnormal hPSCs are capable of differentiating, they display altered gene expression patterns suggestive of malignant transformation\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan additionalcitationids=\"CR89 CR90\" citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e. Furthermore, the abnormalities seen in hPSCs could be regarded as a first hit in cancerous transformation. Cells that are already genetically abnormal upon transplantation are not cancerous yet but may have a higher chance of undergoing oncogenic transformation as they could require fewer additional genetic hits to initiate the process\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e,\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. With this in mind, centers involved in clinical work subject their hPSC cultures and hPSC-based products to genetic screening prior to their use in patients. This typically involves the use of G-banding and, increasingly commonly, massively parallel sequencing (MPS), which enable the detection of chromosomal abnormalities and even, in the case of MPS, potentially harmful single nucleotide changes. Incidentally, the first clinical trial using hPSC-derived retinal pigmented epithelium (RPE) was halted after potentially harmful mutations were identified in both the hPSCs and the RPE cells derived from this line\u003csup\u003e\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e. Unfortunately, standard methods for genetic screening cannot detect abnormalities present as low-grade mosaics in the hPSC culture, which are known to be common\u003csup\u003e\u003cspan additionalcitationids=\"CR94\" citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e\u003c/sup\u003e. If any of the cells carry an abnormality that specifically impairs the differentiation to the chosen lineage, their presence could lead to a cell product containing a subpopulation of poorly differentiated or mis-specified cells and, in the worst-case scenario, with tumor-initiating capacity. This risk further highlights the need for more detailed knowledge of the impacts of specific abnormalities on the properties of hPSCs so that targeted screening approaches that can detect potentially small populations of abnormal cells in cultures can be developed.\u003c/p\u003e \u003cp\u003eIn conclusion, in this study, we have characterized the differentiation capacity of hESCs with 18q loss, one of the recurrent, albeit less common, genetic abnormalities found in hPSC cultures. We found that these cells are characterized by abnormal differentiation into cardiac progenitors and an impaired capacity for neuroectoderm commitment, the latter driven by the loss of one copy of \u003cem\u003eSALL3\u003c/em\u003e. This gene is an inhibitor of \u003cem\u003eDNMT3A\u003c/em\u003e, and its downregulation results in changes in the expression of genes involved in the maintenance of pluripotency and in hESC differentiation. Further research will be needed to assess whether other cell-type specific effects of this abnormality exist and might be revealed by longer differentiation protocols, beyond the progenitor stage, as well as the potential consequences of 18q loss for oncogenic potential.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003ehESC maintenance and passaging\u003c/h2\u003e\n \u003cp\u003eAll hESC lines were derived and characterized as reported previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e96\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e and are also registered in the EU hPSC registry (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hpscreg.eu/\u003c/span\u003e\u003c/span\u003e). They were cryopreserved in freezing medium composed of 90% knock-out serum (Thermo Fisher Scientific) mixed with 10% dimethyl sulfoxide (Sigma-Aldrich). The hESCs were maintained in NutriStem hESC XF medium (NS medium; Biological Industries) with 100 U/mL penicillin/streptomycin (P/S) (Thermo Fisher Scientific) in a 37\u0026deg;C incubator with 5% CO\u003csub\u003e2,\u003c/sub\u003e and the culture medium was changed daily. The tissue culture dishes and plates (Thermo Scientific) were coated with 10 \u0026micro;g/mL Biolaminin 521 (Biolamina\u0026reg;) at 4\u0026deg;C and then incubated at 37\u0026deg;C for at least 20 min before the cells were seeded. The cells were passaged as single cells using TrypLE Express (Thermo Fisher Scientific) and split at a ratio of 1:10 to 1:100 as needed at 70\u0026ndash;90% confluence. The medium was supplemented with 10 \u0026micro;M Rho kinase (ROCK) inhibitor Y-27632 (ROCKi, Tocris) for the first 24 h after passaging.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eCopy number variant (CNV) analysis\u003c/h2\u003e\n \u003cp\u003eThe genetic content of the hESCs was assessed through shallow whole-genome sequencing by the BRIGHTcore of UZ Brussels, Belgium, as previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e98\u003c/span\u003e\u003c/sup\u003e. We also conducted copy number variant analysis using quantitative real-time PCR (qRT-PCR) at regular intervals, particularly before and after performing lentiviral transduction and starting differentiation. DNA was extracted with a DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturers\u0026apos; protocol. qPCR was performed with the subsequent copy number assays: \u003cem\u003eRNaseP\u003c/em\u003e (Thermo Fisher Scientific) as a reference and \u003cem\u003eKIF14\u003c/em\u003e, \u003cem\u003eNANOG\u003c/em\u003e, \u003cem\u003eNMT1\u003c/em\u003e, and \u003cem\u003eID1\u003c/em\u003e (Thermo Scientific) representing the 1q, 12p, 17q and 20q regions, respectively, which are commonly subject to CNV in hESCs. The reaction systems were prepared by mixing \u003cem\u003eRNaseP\u003c/em\u003e, TaqMan 2\u0026times; Mastermix Plus \u0026ndash; Low ROX (Eurogentec), and the related TaqMan assays together with 8 \u0026micro;l of the diluted DNA samples (n\u0026thinsp;=\u0026thinsp;3). qPCR was performed on a ViiA7 thermocycler (Thermo Fisher Scientific), and Applied Biosystems Copy Caller v.2.1 was used to analyze the CNVs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eTotal RNA isolation and cDNA synthesis\u003c/h2\u003e\n \u003cp\u003eTotal RNA was isolated using RNeasy Mini and Micro kits (Qiagen) following the manufacturer\u0026rsquo;s guidelines, including on-column DNase I treatment. The isolated RNAs were collected in nuclease-free water and analyzed for quality and quantity using UV spectrophotometry. The final RNA was stored at \u0026minus;\u0026thinsp;80\u0026deg;C. A minimum of 500 ng of mRNA was reverse-transcribed into biotinylated cDNA using the First-Strand cDNA Synthesis Kit (Cytiva) with the NotI-d(T)18 primer, and the resulting cDNA was stored at -20\u0026deg;C for subsequent analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eQuantitative real-time PCR (qRT-PCR) for gene expression analysis\u003c/h2\u003e\n \u003cp\u003eQuantitative real-time PCR (qRT-PCR) was carried out using TaqMan mRNA expression assays (Thermo Fisher Scientific) and TaqMan 2\u0026times; Mastermix Plus \u0026ndash; Low ROX (Eurogentec) on a ViiA 7 thermocycler (Thermo Fisher Scientific) using the standard cycling protocol provided by the manufacturer. The relative expression of target genes was quantified using the comparative threshold cycle (Ct) method and normalized to the TaqMan GUSB transcript (Applied Biosystems) as the endogenous housekeeping gene. All the samples were run in triplicate, and the related TaqMan assays used in the present study are listed in Supplementary Table\u0026nbsp;5.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunostaining\u003c/h2\u003e\n \u003cp\u003eDifferentiated cells were first fixed in a solution of PBS containing 3.7% formaldehyde (Sigma-Aldrich) for 15 min, permeabilized in 0.1% Triton X for 10 min (Sigma-Aldrich) and then blocked with 10% fetal bovine serum (ThermoFisher Scientific) for 1 h at room temperature (RT). Sequentially, primary antibodies appropriately diluted in blocking solution (1:200 dilution in 10% FBS) were incubated overnight at 4\u0026deg;C. Thereafter, secondary antibodies conjugated to Alexa 488, Alexa 594 or Alexa 647 (1:200 dilutions in 10% FBS, Thermo Fisher Scientific) and Hoescht (1:1000 dilution, ThermoFisher Scientific) were applied for 1\u0026ndash;2 h at room temperature in the dark. PBS was used to wash the cells three times between steps. Confocal images were acquired with an LSM800 confocal microscope (Carl Zeiss) with a 10\u0026times; or 20\u0026times; objective. For quantification, the fixed differentiated cells stained with respective antibodies were counted and compared to the number of Hoescht-stained nuclei to determine the percent positivity using Zen 2 (blue edition) imaging software. The areas are randomly selected (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;6) within a single well based on the Hoescht channel, and the positive cells are quantified by calculating the ratio of total positive cells to the total number of cells selected within those areas. The lists with antibodies can be found in supplementary Table\u0026nbsp;6.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eIn vitro differentiation of iPSCs\u003c/h2\u003e\n \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n \u003ch2\u003eDefinitive neuroectoderm specification\u003c/h2\u003e\n \u003cp\u003eThe protocol for inducing neuroectoderm differentiation was adapted from the protocol described by Douvaras and colleagues\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e99\u003c/span\u003e\u003c/sup\u003e. In brief, hESCs were seeded on Biolaminin 521 (Biolamina\u0026reg;)-coated 24-well plates at a ratio of 100,000 cells per cm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and grown to 90% confluence. Then, differentiation was induced by incubating the hESCs with neural induction specification medium for up to 8 days with daily medium changes. The neural induction specification medium consisted of basal medium supplemented with the following differentiation factors: 100 nM retinoic acid (Sigma-Aldrich), 10 \u0026micro;M SB431542 (Tocris), and 250 nM LDN193189 (STEMCELL Technologies). The basal medium was prepared by mixing DMEM/F12 (Thermo Scientific) basic medium supplemented with 1x NEAA (Thermo Scientific), 1x GlutaMAX (Thermo Scientific), 1x 2-mercaptoethanol (Thermo Scientific), 25 \u0026micro;g/mL insulin (Sigma-Aldrich) and 1x penicillin/streptomycin (P/S) (Thermo Scientific).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eCardiac progenitor differentiation\u003c/h2\u003e\n \u003cp\u003eThe induction of cardiac progenitor differentiation was initiated using a slightly modified version of the protocol from a previous publication\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e100\u003c/span\u003e\u003c/sup\u003e. hESCs were seeded on Biolaminin-521-coated 24-well plates at a density of 100,000 cells/cm\u003csup\u003e2\u003c/sup\u003e and allowed to reach 80\u0026ndash;90% confluence. At this point, differentiation was initiated by treating the hESCs with cardiomyocyte differentiation basal medium (CDBM) with 5 mM CHIR99021 (Tocris) to activate Wnt/\u0026beta;-catenin signaling and form the mesendoderm layer. After 24 h, CHIR99021 was removed, and the cells were cultured in CDBM with 0.6 U/mL heparin (Sigma-Aldrich) for 24 h. Subsequently, the CDBM medium was supplemented with 0.6 U/ml heparin and 3 mM IWP2 (Tocris) for another 3-day incubation with medium refreshed daily. The cardiomyocyte differentiation basal medium (CDBM) was composed of 10 \u0026micro;g/ml transferrin (Sigma Aldrich), 1x chemically defined lipid concentrate (Thermo Scientific) and E8 basal medium. The E8 medium was composed of DMEM/F12 (Thermo Scientific) basic medium with 64 mg/L L-ascorbic acid (Sigma-Aldrich) and 13.6 \u0026micro;g/L sodium selenium (Sigma-Aldrich).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eHepatoblast differentiation\u003c/h2\u003e\n \u003cp\u003eDifferentiation of hESCs into hepatoblasts was conducted using a protocol based on\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e101\u003c/span\u003e\u003c/sup\u003e. The hESCs were seeded at a density of 5x10\u003csup\u003e4\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e on 24-well plates precoated with Biolaminin-521. Upon reaching approximately 40\u0026ndash;50% confluency, the hESCs were treated with liver differentiation medium (LDM) along with 50 ng/mL Activin A (STEMCELL Technologies), 50 ng/mL WNT3A (PeproTech) and 6 \u0026micro;L/mL DMSO (Sigma-Aldrich) for 48 h. The cells were incubated for an additional 48 h in the same medium without WNT3A. Then, the medium was changed to LDM with 50 ng/mL BMP4 (STEMCELL Technologies) and 6 \u0026micro;L/mL DMSO for the following 4 days, with the medium changed every two days. The samples were collected on day 8. LDM medium was created by adding MCDB 201 medium (pH\u0026thinsp;=\u0026thinsp;7.2, Sigma-Aldrich), L-ascorbic acid (Sigma-Aldrich), insulin-transferrin-selenium (ITS-G, Thermo Scientific), linoleic acid-albumin from bovine serum albumin (LA-BSA, Sigma-Aldrich), 2-mercaptoethanol (Thermo scientific), and dexamethasone (Sigma-Aldrich) to DMEM high glucose medium (Westburg Life Sciences).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGeneration of\u003c/strong\u003e \u003cstrong\u003eSALL3\u003c/strong\u003e \u003cstrong\u003eknock-down and overexpression cell lines\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ehESC\u003csup\u003eWT_\u003cem\u003eSALL3\u003c/em\u003eKD\u003c/sup\u003e cells were generated by infecting hESC\u003csup\u003eWT\u003c/sup\u003e with lentiviral particles expressing \u003cem\u003eSALL3\u003c/em\u003e-targeted shRNAs. To generate lentiviral particles, we transfected HEK 293T cells with individual clones from a SigmaMISSION shRNA targeting set (TRCN0000019754, TRCN0000417790) or control shRNA plasmid along with packaging plasmids (plasmid pMDG, encoding VSV G, and plasmid pCMV∆R8.9, encoding gag-pol). hESC\u003csup\u003edel18q_\u003cem\u003eSALL3\u003c/em\u003eOE\u003c/sup\u003e cells were generated by infecting hESC\u003csup\u003edel18q\u003c/sup\u003e with lentiviral particles expressing \u003cem\u003eSALL3\u003c/em\u003e. Lentiviral particles were generated as described above. The pLVSIN-EF1\u0026alpha; puromycin vector expressing \u003cem\u003eSALL3\u003c/em\u003e was a gift from Yoji Sato from the Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Japan. For transduction, hESCs were seeded at a density of 50,000/cm\u003csup\u003e2\u003c/sup\u003e and then transduced with a 2:1 mix of NutriStem and lentivirus-containing medium in the presence of 10 mg/mL protamine sulfate (LEO Pharma) for 4 h. Cells were then washed with PBS before adding fresh NutriStem medium. Twenty-four hours later, the cells were again washed with PBS and then selected by puromycin.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eRNA Sequencing\u003c/h2\u003e\n \u003cp\u003eRNA-seq library preparation was performed using QuantSeq 3\u0026prime; mRNA-Seq Library Prep Kits (Lexogen) following Illumina protocols. Sequencing was performed on a high-throughput Illumina NextSeq 500 flow cell. On average, 13.9 x 10\u003csup\u003e6\u003c/sup\u003e \u0026plusmn; 7.1 x 10\u003csup\u003e6\u003c/sup\u003e paired-end reads per sample were uniquely mapped, with an average coverage of 101 paired reads. The FastQC algorithm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e102\u003c/span\u003e\u003c/sup\u003e was used to perform quality control on the raw sequence reads prior to the downstream analysis. The raw reads were aligned to the new version of the human Ensembl reference genome (GRCh38.p13) with Ensembl (GRCh38.83gtf) annotation using STAR version 2.5.3 in 2-pass mode\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e103\u003c/span\u003e\u003c/sup\u003e.The aligned reads were then quantified, and transcript abundances were estimated using RNA-seq by expectation maximization (RSEM, version 1.3.3)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e104\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eThe count matrices were imported into R software (version 3.3.2) for further processing. The edgeR\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e105\u003c/span\u003e\u003c/sup\u003e package was utilized to identify differentially expressed genes (DEGs) between groups. Transcripts with a count per million (cpm) greater than 1 in at least two samples were considered for the downstream analysis. Genes with a log2-fold change greater than 1 or less than \u0026minus;\u0026thinsp;1 and a false discovery rate (FDR)-adjusted P-value less than 0.05 were considered significantly differentially expressed. Volcano plots of DEGs were generated using the ggplot2\u003csup\u003e106\u003c/sup\u003e package in R, while Venn diagrams using VennDiagram function were used to visualize the overlap of the DEGs among different individuals.\u003c/p\u003e\n \u003cp\u003ePrincipal component analysis (PCA) and heatmap clustering were performed using normalized counts and R packages. The heatmap was generated using the heatmap.2 funtion. PCA was performed using the prcomp function and plotted using ggplot2. Gene set enrichment analysis (GSEA) was applied to detect the enrichment of pathways using the GSEA function in R with the MSigDB C2 and H databases. Values are ranked by sign(logFC)*(-log10(FDR)). |NES| \u0026gt; 1 and p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered the thresholds for significance for the gene sets.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistics\u003c/h2\u003e\n \u003cp\u003eAll differentiation experiments were carried out in at least triplicate (n\u0026thinsp;\u0026ge;\u0026thinsp;3). All data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical evaluation of differences between 2 groups was performed using unpaired two-tailed t tests in GraphPad Prism9 software, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 determined to indicate significance.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA sequencing counts per million tables are provided in\u0026nbsp;the\u0026nbsp;supplementary material. The raw sequencing data\u0026nbsp;are\u0026nbsp;available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.L. carried out all of the experiments\u0026nbsp;and\u0026nbsp;bioinformatics analysis unless stated otherwise and co-wrote the manuscript. D.A. D.\u0026nbsp;cowrote\u0026nbsp;the manuscript. N.K.\u0026nbsp;packaged\u0026nbsp;the\u0026nbsp;lentivirus and assisted\u0026nbsp;in\u0026nbsp;transduction. E.C.D.D. assisted with the bioinformatics analysis. M.R. assisted\u0026nbsp;in\u0026nbsp;microscopy and cell counting. C.J. and M. G\u0026nbsp;assisted with the cell culture.\u0026nbsp;K.S. proofread the paper. C.S.\u0026nbsp;cowrote\u0026nbsp;the manuscript\u0026nbsp;and\u0026nbsp;designed and supervised the experimental work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Authors declare no Competing Financial or Non-Financial Interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank Yoji Sato from\u0026nbsp;the\u0026nbsp;Division of Cell-Based Therapeutic Products, National Institute of Health Sciences in Japan for kindly sharing the lentiviral construct for \u003cem\u003eSALL3\u003c/em\u003e overexpression. Y.L. is a predoctoral fellow supported by the China Scholarship Council (CSC), and M.R., C.J., N.K. and E.C.D.D. are predoctoral fellows supported by the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO). This research was supported by the FWO (grant number 1506617N) and the Methusalem Grant to Karen Sermon (Vrije Universitet Brussel).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eTakahashi, K. \u003cem\u003eet al.\u003c/em\u003e Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861\u0026ndash;72 (2007).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eThomson, J. A. \u003cem\u003eet al.\u003c/em\u003e Embryonic Stem Cell Lines Derived from Human Blastocysts. 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(2016) doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-319-24277-4\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3100381/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3100381/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHuman pluripotent stem cell (hPSC) cultures are prone to genetic drift, as cells that have acquired specific genetic abnormalities experience a selective advantage in vitro. These abnormalities are highly recurrent in hPSC lines worldwide, but currently their functional consequences in differentiating cells are scarcely described. An accurate assessment of the risk associated with these genetic variants in both research and clinical settings is therefore lacking. In this work, we established that one of these recurrent abnormalities, the loss of chromosome 18q, impairs neuroectoderm commitment and affects the cardiac progenitor differentiation of hESCs. We show that downregulation of \u003cem\u003eSALL3\u003c/em\u003e, a gene located in the common 18q loss region, is responsible for failed neuroectodermal differentiation. Knockdown of \u003cem\u003eSALL3\u003c/em\u003ein control lines impaired differentiation in a manner similar to the loss of 18q, while transgenic overexpression of \u003cem\u003eSALL3 \u003c/em\u003ein hESCs with 18q loss rescued the differentiation capacity of the cells. Finally, we show by gene expression analysis that loss of 18q and downregulation of \u003cem\u003eSALL3\u003c/em\u003e leads to changes in the expression of genes involved in pathways regulating pluripotency and differentiation, including the WNT, NOTCH, JAK-STAT, TGF-beta and NF-kB pathways, suggesting that these cells are in an altered state of pluripotency.\u003c/p\u003e","manuscriptTitle":"SALL3 mediates the loss of neuroectodermal differentiation potential in human embryonic stem cells with chromosome 18q loss","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-07-28 16:53:26","doi":"10.21203/rs.3.rs-3100381/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"94ebe94b-5d1a-425f-b283-9a698cfacd30","owner":[],"postedDate":"July 28th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":23527879,"name":"Biological sciences/Stem cells/Pluripotent stem cells/Embryonic stem cells"},{"id":23527880,"name":"Biological sciences/Stem cells/Stem-cell differentiation"}],"tags":[],"updatedAt":"2023-09-06T13:26:12+00:00","versionOfRecord":[],"versionCreatedAt":"2023-07-28 16:53:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3100381","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3100381","identity":"rs-3100381","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}