The role of CXC receptors signaling in early stages of mouse embryonic stem cell differentiation

Stem Cell Research 41 (2019) 101636

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The role of CXC receptors signaling in early stages of mouse embryonic stem cell differentiation

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Kamil Kowalski, Edyta Brzoska, Maria A. Ciemerych

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Department of Cytology, Faculty of Biology, University of Warsaw, Miecznikowa 1, Warsaw 02-096, Poland

ARTICLE INFO

ABSTRACT

Keywords: CXCR4 CXCR7 Differentiation Embryonic stem cells Mesoderm

Interplay between CXCR7 and other CXC receptors, namely CXCR4 or CXCR3, binding such ligands as SDF-1 or ITAC, was shown to regulate multiple cellular processes. The developmental role of signaling pathways mediated by these receptors was proven by the phenotypes of mice lacking either functional CXCR4, or CXCR7, or SDF-1, showing that formation of certain lineages relies on these factors. In this study, using in vitro differentiating mouse embryonic stem cells that lacked the function of CXCR7, we asked the question about the role of CXCR mediated signaling during early steps of differentiation. Our analysis showed that interaction of SDF-1 or ITAC with CXC receptors is necessary for the regulation of crucial developmental regulators expression and that CXCR7 is involved in the control of ESC pluripotency and differentiation into mesodermal lineages.

1. Introduction

mammalian development. For example, endoderm-specific markers, such α-fetoprotein, or transthyretin, are expressed with the similar timing as in mouse embryo (Abe et al., 1996). Expression of genes, such as the ones encoding embryonic ectoderm markers OCT4, FGF5, primitive endoderm marker GATA4, mesodermal markers BRACHYURY and NODAL, and those ones expressed in more specialized cells, i.e. FLK1, NKX2.3, EKLF, and MSX3, recapitulates the spatiotemporal changes observed in early embryos (Leahy et al., 1999). Leahy and coworkers concluded that first 3 days of in vitro culture covered pregastrulation stage and the period from day 3 to day 5 of differentiation corresponded to mouse embryo gastrulation (Leahy et al., 1999). Also the analyses of EBs at the level of single cell transcriptomes precisely characterized the changes in developmental markers expression (Spangler et al., 2018). Studies focusing at human EBs formation also revealed sequential expression of embryonic markers specific for certain cellular lineages, i.e., neurofilament 68 kDa marking ectoderm cells, α-FETOPROTEIN - for endoderm, α-cardiac actin - for mesoderm or γ-GLOBIN characteristic for mesodermal hematopoietic cells (Itskovitz-Eldor et al., 2000). Further culture of EBs and their outgrowths (EBOs) leads to the formation of various more specialized cell lineages, such as for example myogenic ones (Czerwinska et al., 2016a,b, Karbanova and Mokry, 2002). Thus, EBs and EBOs can serve as convenient model to test pluripotency and differentiation of ESCs derived from mouse [e.g. (Bem et al., 2018; Czerwinska et al., 2016a,b; Spangler et al., 2018)], human (Cowan et al., 2004; ItskovitzEldor et al., 2000), rat (Demers et al., 2011), or embryos of other

Embryonic stem cells (ESCs) are the pluripotent cells derived from the mammalian blastocysts (Evans et al., 1981, Martin, 1981). They are able to self-renew their population and differentiate, both in vitro and in vivo, into any given cell type, including germ cells. In vitro, under the defined culture conditions, ESCs preserve their undifferentiated character and are able to undergo nearly unlimited propagation. However, pluripotency and ability to self-renew of mouse ESCs require the interplay of variety of signaling pathways, including the ones activated by leukemia inhibitory factor (LIF) [for review see (Suwinska and Ciemerych, 2011)]. Removal of LIF from the culture medium provokes differentiation of mouse ESCs leading to the decrease in the expression of pluripotency markers, such as NANOG or OCT4, and upregulation of proteins driving differentiation. Many lines of evidence document specific culture conditions securing derivation of ESCs into required cell types. Among the commonly used methods allowing to induce ESCs differentiation is the generation of tridimensional structures composed of cells spontaneously forming ecto-, endo-, and mesoderm, i.e. embryoid bodies (EBs). Initial stages of differentiation occurring within EBs mimic the processes taking place in periimplatation mouse embryo. Thus, endoderm located at the surface of EBs is underlined by the mesoderm layer which surrounds the central mass of ectoderm. Detailed analyzes revealed that the sequence of germ-layer specific gene expression closely reflects the one observed during early

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Corresponding author. E-mail address: [email protected] (M.A. Ciemerych).

https://doi.org/10.1016/j.scr.2019.101636 Received 28 May 2019; Received in revised form 27 September 2019; Accepted 21 October 2019 Available online 31 October 2019 1873-5061/ © 2019 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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mammalian species. EBs are also analyzed in the studies on induced pluripotent stem cells (iPSCs) differentiation [(Ezashi et al., 2009; Sumer et al., 2011; Takahashi et al., 2007, Takahashi and Yamanaka, 2006)]. Importantly, EBs were used in many studies to investigate the phenotype of ESCs in that certain genes were silenced [e.g. (Czerwinska et al., 2016a,b; Desbaillets et al., 2000; Myer et al., 2001; Weitzer et al., 1995)]. EBs were also used to analyze the impact of gene overexpression [e.g. (Prelle et al., 2000; Rohwedel et al., 1995)] or treatment with extracellular factors, such as Wnt, on cellular differentiation (ten Berge et al., 2008) on differentiation. Among extracellular factors that can impact ESC differentiation are growth factors and chemokines, i.e. those ones that induce various cellular processes including differentiation or migration. Amid the ones which are best studied are those belonging to such families as TGF-β (tumor growth factor-β), Wnt or FGF (fibroblast growth factor; Wang and Chen, 2016). The ones that function in differentiating ESCs and remains pretty obscure include SDF-1 (stromal cell derived factor-1), encoded by Cxcl12, also known as chemokine CXCL12, i.e. the factor which is ubiquitously expressed in embryonic and adult tissues (Guo et al., 2005; Yu et al., 2006). SDF-1 participates in hematopoiesis, immune cell trafficking, cardiogenesis, angiogenesis, neurogenesis, and cancer metastasis (Teixido et al., 2018). SDF-1 action on the cells is executed by binding to receptors belonging to CXCR family, i.e. CXCR4 and CXCR7 (Sanchez-Martin et al., 2013; Singh et al., 2013). However, CXCR7 and also CXCR3 are able to interact with ligands other than SDF1, i.e. ITAC (IFN-inducible T cell α-chemoatractant), encoded by Cxcl11, and also known as CXCL11, which is expressed in peripheral blood leukocytes, neurons, mesenchymal stem cells, cell building pancreas, liver, thymus, spleen, and lungs (Sanchez-Martin et al., 2013; Singh et al., 2013). Moreover, CXCR4 and CXCR7 also interact with MIF (Migration Inhibitory Factor). Signaling mediated by CXCR4 plays important role in stem and progenitor cells migration during embryonic development and also in adult tissues (Miller et al., 2008). The phenotype of mice lacking CXCR4 is lethal, as well as of those ones lacking SDF-1. Both of mutant mice die in utero and present abnormalities in B lymphopoiesis, myelopoiesis, as well as cardiogenesis, vascular and cerebellar development (Ma et al., 1998; Nagasawa, 2007; Tachibana et al., 1998; Zou et al., 1998). Next, in developing embryos these factors mediate migration of neural stem cells, primordial germ cells (PGCs), cardiac as well as neural crest cells, hemangioblast cells (Miller et al., 2008). CXCR7 receptor (also known as atypical chemokine receptor 3, ACKR3), binds SDF-1, ITAC, and MIF (Singh et al., 2013). During mouse embryogenesis CXCR7 is expressed in heart, neural tube, brain, and septum (Sanchez-Martin et al., 2013). The CXCR7-deficient mice show abnormalities in developing heart and vascular system but not in hematopoiesis (Sanchez-Martin et al., 2013). CXCR3, on the other hand, is expressed by T- and B cells, monocytes, granulocytes, endothelial cells, fibroblasts, and pericytes (GarciaLopez et al., 2001; Lasagni et al., 2003; Loetscher et al., 1996; Murakami et al., 2013). CXCR3-deficient mice are viable, fertile, and show no obvious developmental abnormalities (Panzer et al., 2007). Except ITAC, CXCR3 also binds CXCL9 (MIG, monokine activated by interferon-γ), CXCL10 (IP10, INF-γ-inducible 10 kDa protein), and CXCL4 (PF-4) mediating chemotactic migration, cell proliferation, and cell death (Singh et al., 2013). CXCRs belong to the family of seven trans-membrane domain G proteins coupled receptors (GPCRs) which activate multiple signaling pathways (Murphy and Heusinkveld, 2018). The CXCR4 acting via Gproteins activates RAS-ERK, PLC, phosphatidyl-inositol-3-kinase (PI3K)-AKT-mTOR. Next, after the assotiation of SDF-1 CXCR4 is phosphorylated by G-protein coupled receptor kinase (GRK) what allows binding of β-arrestin (Pozzobon et al., 2016). The β-arrestin recruitment leads to dissociation of G-proteins from CXCR4 and induces receptor endocytosis. The CXCR4 could also signal independently of Gproteins for example by activating JAK-STAT pathway (Scala, 2015). CXCR7 was shown to be atypical receptor, i.e. it does not transmit the

signal via G-proteins. It was rather suggested to function as a scavenger for SDF-1 and ITAC (Teixido et al., 2018). Interestingly, CXCR7 affinity towards SDF-1 was shown to be ten times higher than in case of CXCR4. However, it was also shown that in some types of cells CXCR7 acting via G proteins activates the AKT, ERK, and PLC pathways (Sanchez-Martin et al., 2013). CXCR7, similarly to CXCR4, could also acts through β-arrestin causing either self-internalization or ERK pathway activation. CXCR3 activates the extracellular signal-regulated kinase (ERK), the protein kinase B (AKT), and phospholipases C (PLC) pathways (Bonacchi et al., 2001). Importantly, CXC receptors have been documented to form homo- or hetero-oligomeric complexes that could be formed in a ligand-inducible manner, resulting in complex networking and crosstalk with other signaling pathways. For example, CXCR4 and CXCR7 form complexes that constitutively recruit β-arrestin and impair G-protein mediated signaling (Decaillot et al., 2011). Moreover, in CXCR4/CXCR7 heterodimers CXCR7 acts as β-arrestin signaling enhancer (Decaillot et al., 2011). On the other hand, activation of CXCR3 pathway strengthens the function of CXCR4 (Jin et al., 2018; Murakami et al., 2013). It was show that CXCR3 forms heterodimers with CXCR4 and prevents CXCR4 internalization (Jin et al., 2018). The role of CXCRs was studied so far in many cells types, including cancer cells, as well as in developing embryo. However, analyzes of the mutant mouse did not uncover function of CXCRs and their ligands at the very early stages of cell differentiation, which could be mimicked in vitro using ESCs. To dissect the role of CXCR7 in ESCs we took advantage of CRISP-Cas9 method and obtained the cells that either lacked functional CXCR7 or expressed CXCR7 unable to bind SDF-1. Having such cells we analyzed how CXCR7 and their ligands – SDF-1 and ITAC, impact formation of ecto-, endo-, and mesoderm. 2. Materials and methods 2.1. The culture of embryonic stem cells Undifferentiated mouse ESCs (D3 line) were cultured on mitomycin C-inactivated mouse embryonic fibroblast feeder layer (MEFs) in knockout Dulbeco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% serum replacement (Gibco), 0.1 mM nonessential amino acid (Gibco), 0.1 mM β-merkaptoethanol (Sigma-Aldrich), 2 mM L-glutamine (Gibco), 100 U/ml penicillin/streptomycin (Gibco) and 1000 U/ ml of leukemia inhibitory factor (LIF) (Millipore). Cells were cultured at 37 °C and 5% CO2 in humidified air. Medium was changed daily and ESCs and were passaged every 2 days. 2.2. CRISPR-Cas9 mediated mutation of Ackr3 (CXCR7) locus in ESCs pSpCas9(BB)−2A-Puro (PX459) carrying puromycin resistance gene was a gift from Feng Zhang (Addgene plasmid # 48139). gRNA inserts directed to Ackr3 gene, encoding CXCR7, were designed using crispr.mit.edu platform. gRNAs were designed to acts on N-terminus region of CXCR7. Plasmid was digested using BbsI and designed oligonucleotides (Sigma-Aldrich) were cloned. The sequences of oligonucleotides aimed to the site of mutagenesis were as follows: 5′-AAACG ACGGGGATGGTGATGACG 5′-CACCGTCGTCATCACCATCCCCG. Next, plasmids were amplified, isolated, and purified prior to transfection procedures. ESCs were freed from MEFs by pre-plating technique. Briefly, ESC cultures were incubated in 0.05% trypsin/EDTA for 3–5 min, cells were suspended in a culture medium, plated again onto a culture dish covered with 1% gelatin, and then were incubated at 37 °C for 20 min, which allowed MEFs to attach to the dish. The medium containing ESC was transferred to another gelatin covered culture dish. Pre-plating was performed 3 times. Next, ESCs were cultured on 10% Matrigel (Becton Dickinson) as described above for 24 h. Plasmid encoding CRISPR-Cas9 carrying designed gRNA and puromycin resistance gene (250 ng/ml) was added to 2

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OptiMEM (Gibco) and ESCs were transfected using Lipofectamine™ LTX Reagent with PLUS™ Reagent (Thermo Fisher Sci.), according to manufacturer's instruction. Cas9 enzyme based on designed gRNA should result in small deletion in selected sequence. Two days after the transfection selection of successfully transfected cells was performed using puromycin (0.5 ug/ml). Three days later puromycin-resistant clones of cells were picked, transferred to 24-wells plates coated with Matrigel, and cultured in culture medium with 10% Serum Replacement (SR, Gibco) for 5 days. The use of Serum Replacement instead of FBS was caused by the necessity to culture cells under control conditions without SDF-1 and ITAC. Expanded ESC clones were collected and subjected to analyze confirming mutation in both alleles of Ackr3 gene. DNA was extracted by PureLink Genomic DNA Kit (Invitrogen). Fragment of Ackr3 gene was amplified using primers: 5′-CCCAAAGAGGTTGATGGAGA-3′ and 5′-TGTGGTGGTCTGGGTGA ATA-3′ and mutation was detected using Surveyor kit (IDTDNA Tech.), according to manufacturer's instruction. Clones of ESCs carrying mutation were selected for the second round of transfection with the same plasmid aiming at the second allele of Ackr3 gene. After second transfection ESC colonies were collected and DNA was extracted using Qiagen kit (Qiagen), then fragment of Ackr3 gene was amplified by PCR using the same starters as described above. Obtained products were sequenced (Genomed S.A.) and analyzed by comparison of the sequence between sequence in Pubmed BLAST, wild type ESC and ESCs after transfection. The cells with deletions in Ackr3 sequence were selected. ESCs with confirmed mutations were seeded onto MEFs, expanded in the medium containing LIF, and used for analyses. Three types of ESC lines were selected for experiments: wild type - ESC-WT, and ESCs carrying mutations - ESC-CXCR7(L-), i.e. modified receptor without ability to bind SDF-1, and ESC-CXCR7-, i.e. cells lacking recepto

obtained results were analyzed using GelDoc2000 using Quantity One software (BioRad). Analyzes were repeat 3 times. All antibodies used are listed in supplementary information (Figure S2). 2.5. SDF-1 and I-TAC treatment of embryoid bodies (EBs) and embryoid bodies outgrowths (EBOs) ESCs of selected lines were freed from MEFs, as described above, plated on 10% Matrigel coated 35 mm dishes, and cultured for 2 days. Next, ESC colonies were disaggregated into single-cell suspension using 0.25% trypsin. One thousand of ESCs were placed in 30 µl hanging drops of LIF-free DMEM medium - either control one or containing SDF1 (100 ng/ml) or ITAC (100 ng/ml). After 2 days of culture cells formed EBs. Single EBs were transferred to non-adhesive 96 well plates and cultured in suspension either in control medium or medium supplemented with SDF-1 or ITAC, for next 5 days without changing the medium. Single EBs were also placed in 24 well plates to induce formation of the outgrowths and cultured in LIF-free DMEM medium either control one or containing SDF-1 (100 ng/ml) or ITAC (100 ng/ ml) for another 21 days. ESCs, EBs (at 7 day of culture, starting from EB formation) or EBOs (at 21 day of culture, starting from EB plating) were collected for mRNA and protein analyses. 2.6. The real time polymerase chain reaction Total RNA was isolated from undifferentiated ESCs, EBs cultured for 7 days or EBOs cultured for 21 days using mirVana reagent (Thermo Fisher Sci.), according to the manufacturer's instructions. Total RNA was then treated with DNase (turboDNase, Thermo Fisher Sci.). The reverse transcription (RT) of total RNA was done using SuperScript VILO cDNA synthesis kit (Thermo Fisher Sci.), according to the manufacturer's instructions, using 1 µg RNA and random primers. Real time PCR profile consisted 120 s of preincubation at 50 °C, 10 min of initial activation at 95 °C, followed by 50 cycles of 15 s denaturation at 95 °C and 1 min annealing and extension at 60 °C. The relative gene expression data was analyzed by comparative Cq method (∆∆Cq). Results were normalized against the Hypoxanthine-guanine phosphoribosyltransferase (HPRT) expression, as an internal control. All gene expression TaqMan used are listed in supplementary information (Figure S2A). All assays were repeated five times.

2.3. Karyotype analysis To determine if procedure of Ackr3 gene disruptions does not affect the ESCs karyotype Giemsa staining of metaphase chromosomes was performed. ESC-WT, ESC-CXCR7(L-), and ESC-CXCR7- lines were cultured on Matrigel coated 35 mm dish for 48 h. Next, they were incubated in medium containing colchicine (10 µg/ml) at 37 °C for 2 h. Next cells were incubated in 0.56% calcium chloride at room temperature for 20 min and then fixed with methanol:acetic acid solution (3:1) for 10 min at 4 °C. Finally, cells were dropped onto slides, air dried, and stained with Giemsa solution (Merck). The number of chromosome were counted, i.e. 100 metaphase plates for each ESC line was analyzed.

2.7. Immunocytochemistry ESCs cultured on cover slips placed in 35 mm dishes were washed in PBS and fixed in 3% PFA for 10 min, incubated in 0.05% Triton/PBS for 3 min, then in 0.25% glycine for 20 min. After washing in PBS cells were blocked in PBS containing 3% bovine serum albumin (BSA) and 2% donkey serum (Gibco) for 30 min before incubation with primary antibodies (1:100) overnight at 4 °C. Specimens were then incubated with secondary antibodies conjugated with fluorochrome (1:200) at room temperature for 2 h, counterstained with DRAQ5 1:1000 in PBS at room temperature for 5 min, and mounted using Fluorescent Mounting Medium (Dako Cytomation). All antibodies used are listed in supplementary information (Figure S2B).

2.4. Immunoprecipitation and western blotting ESCs were cultured and expanded without MEFs on 10% Matrigel, as described above. Next, medium was changed to the one containing SDF-1 (150 ng/ml). After 15 min of incubation with SDF-1 ESCs were collected and whole protein lysates were obtained using cOmplete Lysis buffer (Roche Applied Science). Then, according with manufacture's instruction, CXCR7-SDF-1 complexes were immunoprecipitated using anti CXCR7 antibody and Immunoprecipitation Kit Dynabeads Protein G (Thermo Fisher Sci.). Wester blotting using anti-SDF-1, anti-ITAC, and anti-CXCR7 antibodies was used to confirm the interaction between CXCR7 and SDF-1, as well as with ITAC. To do so, 50 µg of total protein lysate were denatured by boiling in Laemmli buffer, separated using SDS-Page, and transferred to PVDF membranes (Roche Applied Science). After blocking with 5% non- fatty milk in TBS for 1 h membranes were incubated with primary antibodies diluted 1:2000 in 5% non- fatty milk in TBS at 4 °C overnight, and then in secondary antibodies diluted 1:20000 at room temperature, for 2 h. Protein bands were visualized with SuperSignalWest Pico Chemiluminescent Substrate (Thermo Fisfer Sci.) after the exposition to chemiluminescence positive film (Amersham Hyperfilm ECL, GE Healthcare). The

2.8. Cell cycle analysis To determine whether CXCR7 disruption affected the cell cycle of ESCs two methods were used - CFSE (carboxyfluorescein succinimidyl ester, Thermo Fisher Sci.) or propidium iodide staining. ESCs (ESC-WT, ESC-CXCR7(L-), and ESC-CXCR7-) were freed from MEFs and then incubated in 0.5 mM CFSE in PBS at 37 °C for 10 min. Next, ESCs were seeded on Matrigel coated 6-well plates at the density 1 × 106 cells per well and cultured in LIF-containing DMEM. Two hours after plating some cells were treated with SDF-1 (100 ng/ml) or ITAC (100 ng/ml) for 6 h. After 8 h of culture ESCs in control or SDF-1 or ITAC containing 3

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medium were washed in PBS, disaggregated with 0.25% tripsin (Gibco), rinsed in PBS, and subjected to flow cytometry analysis (BD FACSCalibur, BD Biosciences) using CellQuestPro software. ESCs that were not subjected to incubation in CFSE served as negative control. ESCs that were analyzed directly after CFSE labeling served as positive control. For propidium iodide staining ESC-WT, ESC-CXCR7(L-), and ESC-CXCR7- were cultured on 10% Matrigel coated dishes in LIF-containing DMEM, washed in PBS and fixed in 66% ethanol, followed by next PBS wash and incubation in propidinium iodide and RNase for 30 min, according to manufacturer's instruction (Abcam). Cells were analyzed using FACSCallibur (BD Bioscience). For each analysis three independent experiments were performed. 2.9. Flow cytometry analysis 7 days old EBs were disaggregated in StemPro Accutase Cell Dissociation Reagent (Gibco) by gently shaking at 37 °C for 30 min. Obtained cell suspension was filtered through 40 µm cell strainer. Then, cells were incubated in 0.5% BSA supplemented with 1% FBS in PBS, at room temperature, for 30 min. Next, cells were transferred to fluorochrome-conjugated primary antibody in PBS with 0.5% BSA and incubated in dark at 4 °C for 30 min, washed in PBS, and then fixed in 3% PFA in PBS, at room temperature for 10 min. Samples were analyzed using a FACSCalibur flow cytometer (BD Bioscience). Control samples were incubated with isotype fluorochrome matched immunoglobulin antibodies, all antibodies used are listed in supplementary information (Figure S2 B). Each analysis was repeated 3 times.

Fig. 1. Expression of CXCR4 and CXCR7 in differentiating ESCs. (A) The level of Cxcr7 and Cxcr4 transcripts in ESCs, EBs cultured in hanging drops for 2 (EB2) or 7 days (EB7), and EBOs cultured for 21 days. (B) The level of CXCR4 and CXCR7 protein in ESCs and EB2 and EB7. (C) The co-immunoprecipitation of CXCR7 and its ligands: the level of SDF-1, ITAC and CXCR7 in immunoprecypitates obtained using anti CXCR7 antibody from ESC-WT, ESCCXCR7(L-), and ESC-CXCR7-. (D) The localization of CXCR4 and CXCR7 in ESCWT, ESC-CXCR7(L-), and ESC-CXCR7-. Blue - nuclei, green CXCR4 or CXCR7 immunolocalization using specific antibodies. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2.10. Statistical analysis All data are represented as a mean ± SD. Statistical analyses were performed using GraphPad Prism 5. Data was analyzed using t-test. The differences were considered statistically significant when p< 0.05 (marked on charts with asterisks).

made the CXCR7 unable to bind SDF-1 (Fig. 1C). Second type of ESC lines, described as ESC-CXCR7-, was characterized by the frame shift started from aminoacid in the 33 position (glutamine) (Figure S1B), what affected CXCR7 expression and resulted in CXCR7-null cells (Fig. 1C). Next, we analyzed the karyotype of “maternal” wild type, i.e. D3 ESC, wild type cells described as ESC-WT, i.e. the cells carrying wild type alleles of Ackr3 gene and transfected with “empty” plasmid, ESCCXCR7(L-), and ESC-CXCR7- lines (Figure S1C). For further studies we have chosen the lines in which proportion of cells characterized by normal karyotype (calculated from 100 metaphase plates) was 68% for ESC-WT, 58% for ESC-CXCR7(L-), and 62% for ESC-CXCR7-, i.e. comparable to non-manipulated D3 ESC line in that 64% of cells contained correct chromosome number (Figure S1C). We also compared proliferation potential of wild-type and mutant ESC lines using CSFE method and propidium iodide staining. CSFE analysis showed that ESCs of each cell lines analyzed undergo similar number of cell division during 8 h long culture (Figure S1D). Similarly, these ESCs did not differ in the proportion of the cells in G1, S, and G2/M cell cycle phases (Figure S1E). Thus, introduced mutations did not affect the proliferation of ESCs. Finally, we compared the expression of crucial marker of pluripotency, i.e. Nanog, in non-manipulated D3 ESCs with ESC-WT, ESC-CXCR7(L-) and ESC-CXCR7- and found that it did not differ between the cell lines analyzed (Figure S1F). Co-immunoprecipitation and western blotting analyzes confirmed that the CXCR7 was present in ESC-WT, as well as in ESC-CXCR7(L-) and absent in ESC-CXCR7- (Fig. 1C). Moreover, it showed that SDF-1 and ITAC were able to bind the CXCR7 in control while they failed to do so in mutant cells (Fig. 1C). Immunolocalization of CXCR7 showed that ESC-WT, as well as ESC-CXCR7(L-), expressed while ESC-CXCR7lacked CXCR7 protein (Fig. 1D). At the same time all 3 ESC lines tested expressed CXCR4 (Fig. 1D).

3. Results 3.1. Derivation of embryonic stem cells lacking functional CXCR7 The levels of CXCR4 and CXCR7 encoding mRNAs were analyzed in undifferentiated mouse embryonic stem cells (ESCs) and embryoid bodies (EBs), i.e. cellular aggregates composed of ESCs differentiating into ecto-, endo-, and mesoderm, cultured for 2 (EB2) or 7 days (EB7) as well as in embryonic body outgrowths (EBOs) cultured for 21 days (Fig. 1A). The protein of these receptors was assessed in ESCs, EB2 and EB7 (Fig. 1B). The expression of both receptors increased during differentiation. We also followed the levels of mRNA encoding CXCR3, SDF-1 (Cxcl12), and ITAC (Cxcl11) and showed that also these transcripts were upregulated (Figure S1A). To determine the role of CXCR7 during ESC differentiation we used CRISPR-Cas9 technology and obtained cells in that function of CXCR7 was affected (Figure S1B). CXCR7 is a 362 amino acids protein localized in cell membrane and composed of seven transmembrane α-helixes with intracellular and extracellular loops. Extracellular N terminal tail (46 amino acids domain coding by 138 nucleotides) is responsible for ligand binding. CXCR7 protein conformation model is unknown but on the basis of the high sequence similarity with CXCR4 the site of SDF-1 or ITAC binding was predicted. Designed gRNA aimed at nucleotide 92–112 to either disturb protein conformation or prevent the synthesis of functional protein. Altogether, we generated 18 ESC lines carrying mutations in the Ackr3 locus encoding CXCR7, then we selected 3 lines for detailed analyses (Figure S1B). First type of ESC lines, described as ESCCXCR7(L-), was characterized by the expression of CXCR7 in that, according to sequence analysis, 4 amino acid, i.e. proline, threonine, methionine, proline, were deleted (Figure S1B). Such modification 4

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Thus, in obtained ESCs, i.e. ESC-CXCR7(L-) and ESC-CXCR7-, SDF-1 and ITAC were not able to activate signaling pathways mediated by this receptor. Importantly, in such ESC-CXCR7(L-) the ability to form complexes between CXCR7 and CXCR4 or CXCR3 should not be affected. Therefore, under physiological conditions SDF-1 should bind CXCR4 and CXCR7 receptors, while ITAC interact with CXCR7 and CXCR3, activating appropriate signaling pathways [(Burns et al., 2006); for review see (Cencioni et al., 2012)]. As a result, in ESC-CXCR7(L-) and ESC-CXCR7- changes caused by SDF-1 would result from CXCR4 signaling, while those mediated by ITAC from CXCR3 activation. Thus, by creating cells lacking CXCR7 function we were able to uncover which differentiation decisions depend on the presence of functional CXCR7 and which ones on the presence of complexes formed by CXCR7 and other receptors.

elevated in both type of mutant cells, (Fig. 2B). Analyzes of mutant cells revealed that under control conditions T was upregulated in ESC-CXCR7(L-), i.e. the cells in that CXCR7 was able to form complexes with CXCR4 or CXCR3, but not in ESC-CXCR7- (Fig. 2B). Such phenotype implicated that physical presence and function of CXCR7 is necessary for the expression of T and that CXCR3 cannot substitute for lacking receptor. However, when inactive CXCR7 was present within the cells the signaling appeared to be redirected to other CXCRs which action was apparently more potent, as compared to CXCR7. In case of other mesoderm markers, Nodal and Tbx, ablation of CXCR7 function led to the upregulation of their expression. Interestingly, the presence of active CXCR7 was necessary for the expression of mRNAs encoding Kdr, Twist1, and Bmp2 (Fig. 2B). Observed upregulation of T mRNA was accompanied by the higher number of cells expressing BRACHYURY. Moreover, also the number of cells expressing another mesoderm marker – SNAIL - increased (Carver et al., 2001) (Fig. 2C). Finally, we studied the phenotype of mutant cells in EBOs, i.e. after 21 days of differentiation. Again, we did not notice differences in the levels of mRNAs encoding CXCR4, CXCR3, SDF-1 or ITAC, as well as of other genes studied: 1). neuroectoderm and neural markers Pax6, Otx (Acampora et al., 2001), Tubb3 (Jiang and Oblinger, 1992), Neurog1 encoding neurogenin (Sun et al., 2001); 2) endoderm derived cell markers Alb encoding albumin (Cascio and Zaret, 1991), Afp encoding alpha-fetoprotein (Hay et al., 2008), Pdx (Gu et al., 2002), Dcx (Gu et al., 2002); mesoderm derivatives markers, Pdgfr (Kataoka et al., 1997), cTnTn (Siedner et al., 2003), Pax3, Myf5 (Buckingham and Relaix, 2015). Surprisingly, we did not detect any significant differences between analyzed cell lines (Fig. 2D). Thus, we concluded that the absence of functional CXCR7 affects very early stages of ESC differentiation, even in the absence of elevated levels of SDF-1 or ITAC in the cell environment.

3.2. CXCR7 regulates early stages of ESC differentiation Comparison of ESC-WT and cells lacking CXCR7 function allowed us to closely analyze the impact of CXC receptors and their ligands, such as SDF-1 and ITAC, on the formation of ecto-, endo-, and mesoderm lineages, assessed by the expression of appropriate markers. First, we compared the expression of Cxcr4, Cxcr7, Cxcl12, and Cxcl11 in ESCs, EBs, and EBOs formed from three types of cells, i.e. ESC-WT, ESC-CXCR7(L-), and ESC-CXCR7- cultured in control medium, i.e. without the stimulation with SDF-1 or ITAC. We showed that the level of all analyzed transcripts increased during differentiation (Figure S1G). Next, we focused at the EBs and EBOs and apart the expression of mRNAs encoding CXCR4, CXCR3, SDF-1 (Cxcl12), ITAC (Cxcl11), we also compared those of pluripotency and ectodermal lineages markers, such as OCT4 (Pou5f1) and SOX2 (Avilion et al., 2003; Chew et al., 2005; Rodda et al., 2005), BMP4 involved in the ectoderm specification (Davis et al., 2004; Harvey et al., 2010), and postimplantation epiblast/ ectoderm marker - FGF5 (Haub and Goldfarb, 1991, Li et al., 2013) (Fig. 2A). In EBs cultured under control conditions ablation of CXCR7 function impacted at the expression of mRNAs encoding neither CXCR4, CXCR3, SDF-1, ITAC, nor SOX2. It decreased, however, the levels of Pou5f1 and Bmp4 mRNAs (Fig. 2A). In case of Fgf5 expression of this gene was significantly increased only in differentiating ESC-CXCR7-. Next, we focused at the expression of mRNAs encoding the primitive/visceral endoderm markers such as GATA4 (Duncan 2005; Jacobsen et al., 2002; Soudais et al., 1995) and SOX17 (Artus et al., 2011; Igarashi et al., 2018; Viotti et al., 2014). Presence of CXCR7 unable to bind the ligand affected the expression of mRNA encoding GATA4. Sox17 mRNA level was comparable between EBs derived from ESC-WT and ESC-CXCR7(L-) but significantly reduced in those ones originating from ESC-CXCR7- cultured under control conditions, suggesting that presence of inactive CXCR7 forming complexes with other CXCRs is sufficient for the expression of this factor (Fig. 2A). FACS analysis revealed that the number of GATA4+ cells was similar between all cell lines analyzed. Also the number of PAX6 cells, i.e. expressing the factor characteristic for neuroectoderm development (Osumi et al., 2008), was comparable in all cell lines studied which were cultured under control conditions (Fig. 2C). Expression of several genes involved in the formation of mesoderm and more specialized cells derived from this germ layer was also analyzed. Among chosen genes were primitive streak markers T (encoding BRACHYURY) (Martin and Kimelman, 2008, Martin and Kimelman, 2010) and Nodal (Shen, 2007), Kdr (FLK-1) which product marks proximal lateral mesoderm (Kataoka et al., 1997), Tbx1 and Bmp2 that are involved in the specification of several lineages, including cardiac mesoderm [e.g. (Francou et al., 2014; Pane et al., 2018; Schlange et al., 2000)], and Twist1 regulating cranial neural crest and mesoderm development [e.g. (Bildsoe et al., 2016)]. Comparison of EB7, generated from all analyzed cell lines and cultured under control conditions, showed that the presence of functional CXCR7 is necessary for the expression of Kdr, Twist1, and Bmp2. Interestingly, Nodal expression was

3.3. SDF-1 or ITAC regulate early stages of ESC differentiation Since we observed that lack of functional CXCR7 results in the modification of the expression of certain factors we asked the question whether stimulation of CXCR with either SDF-1 or ITAC will uncover the role of these receptors in ESC differentiation. First we analyzed the cells at early stages of differentiation, i.e. in EB7. Stimulation with SDF-1 or ITAC dramatically upregulated the levels of mRNAs encoding CXCR4 but not CXCR3 in ESC-WT but not in ESC-CXCR7(L-) and ESC-CXCR7-, suggesting that CXCR7 receptor mediated signaling controls expression of CXCR4 during ESC differentiation. On the other hand, the level of mRNA encoding SDF-1 decreased after SDF-1 or ITAC treatment in ESC-WT but not in ESC-CXCR7(L-) and ESC-CXCR7-, suggesting that CXCR7 receptor also regulated expression of Cxcl12 during ESC differentiation. Both ligands negatively regulated the levels of Cxcl12 mRNA in control but not in mutant cells (Fig. 3A).The level of mRNAs encoding ITAC were not sensitive to SDF-1 or ITAC treatment. Culture of EBs derived from ESC-WT in the presence of SDF-1 or ITAC led to decrease in the expression of Pou5f1 and Sox2, suggesting involvement of CXCR4 or CXCR7 mediated signaling (Fig. 3B). Analysis of mutant cells showed that SDF-1 and ITAC stimulation resulted in significant downregulation of mRNAs encoding Pou5f1 and Sox2 in CXCR7 dependent manner (Fig. 3B). mRNAs encoding ectoderm markers, i.e. Fgf5 and Bmp4, decreased in ESC-WT treated with SDF-1 (Fig. 3B). However, in the mutant cells the changes in Fgf5 and Bmp4 level were opposite. Interestingly, Fgf5 expression level was upregulated and Bmp4 was downregulated in mutant cells cultured under control conditions as well as in the presence of SDF-1 or ITAC. Importantly, CXCR7 was necessary to sustain expression of Bmp4, since absence of its function led to dramatic decrease in mRNA encoding this gene (Fig. 3B). Moreover, in mutant cells treated with ITAC the levels of Bmp4 mRNA decreased, suggesting the CXCR3 engagement. Thus, CXCR7 mediated signaling appeared to be involved in the regulation of 5

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Fig. 2. CXCRs receptors, their ligands, and differentiating cell markers in EB7 and EBOs formed by ESC-WT, ESC-CXCR7(L-), or ESC-CXCR7- cultured in control medium. (A) Expression of Cxcr4, Cxcr3, Cxcl12 and Cxcl11, pluripotency/ectoderm markers - Pouf5f1, Sox2, Fgf5, Bmp4, endoderm markers Gata4 and Sox17 in EB7. (B) Expression of mesodermal markers T, Nodal, Kdr, Tbx1, Twist1, Bmp2 in EB7. (C) Proportion of GATA4 and PAX6 positive cells established by flow cytometry of cells isolated from EB7. (D) Expression of Cxcr4, Cxcr3, Cxcl12 and Cxcl11, ectoderm and neuroectoderm markers - Pax6, Otx, Tubb3, Neurog1, endodermal cells markers - Alb, Afp, Pdx, Dcx, mesodermal markers Kdr, Pdgfrα, cTnTn, Pax3, Myf5 in EBOs.

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Fig. 3. CXCRs receptors, their ligands, and differentiating cell markers in EB7 formed by ESC-WT, ESC-CXCR7(L-), or ESC-CXCR7- subjected to SDF-1 or ITAC treatment (A) Expression of Cxcr4, Cxcr3, Cxcl12 and Cxcl11. (B). Expression of pluripotency/ectoderm markers - Pouf5f1, Sox2, Fgf5, Bmp4 (C) Expression of endoderm markers Gata4 and Sox17 (D) Proportion of GATA4 and PAX6 positive cells established by flow cytometry.

pluripotency and early ectoderm markers and may stimulate differentiation of ESCs. In case of endoderm markers expressed in EBs derived from ESC-WT that was SDF-1 but not ITAC that dramatically downregulated Gata4 expression. Sox17 mRNA levels, on the other hand, increased in the reaction to SDF-1 or ITAC (Fig. 3C). Gata4 expression was downregulated by SDF-1 in CXCR4-dependent manner. ITAC, however, did not change the level of Gata4 in ESC-WT and ESC-CXCR7(L-), but decreased it in ESC-CXCR7-. On the other hand, Sox17 expression was upregulated by ITAC in differentiating ESCs in CXCR7-dependent manner (Fig. 3C). Again, we tested the proportion of the GATA4 positive cells within EBs and showed that it reflected the expression of mRNAs encoding that marker in all analyzed cell lines cultured in the presence of SDF-1 or ITAC (Fig. 3D). Thus, the number of GATA4+ cells was decreased by SDF-1 in CXCR4-dependent manner. ITAC reduced the number of GATA4+ cells in the population of differentiating ESC-CXCR7-. SDF-1 and ITAC treatment resulted in lower number of PAX6+ in the populations of cells lacking CXCR7 signaling (Fig. 3D). Analysis of mesodermal markers in EB7 showed that the treatment of ESC-WT with SDF-1 or ITAC upregulated expression of mRNAs encoding Nodal, Kdr, and Tbx1. T expression was sensitive only to ITAC mediated signaling. Twist1 and Bmp2 mRNA levels did not changed, regardless of the treatment (Fig. 4A). Assessment of the cells expressing

BRACHYURY showed that SDF-1 treatment did not change the number of BRACHYURY+ cell in none of the cell lines analyzed (Fig. 4B). However, ITAC increased proportion of such cells in CXCR7-dependent manner. Analysis of the proportion of cells expressing SNAIL showed that SDF-1 and ITAC driven increase in the proportion of SNAIL+ cells relied on the CXCR7 presence (Fig. 4B). In EBOs analyzed at day 21 of differentiation Cxcr4 was upregulated but only by ITAC treatment. SDF-1 reduced the levels of mRNA encoding this gene in all analyzed cell types. Similarly, as it was shown for EBs, also in case of EBOs ITAC driven upregulation of Cxcr4 expression depended on functional CXCR7. Cxcr3 expression was modulated neither by SDF-1 nor ITAC. Moreover, these ligands did not affect the levels of Cxcl12 regardless of cell genotype. However, expression of Cxcl11 was downregulated in mutant cells (Fig. 5). Expression of the markers characteristic for ectoderm, neuroectoderm, endoderm and mesoderm derived cells was also studied. Pax6 expression, similarly as it was observed for EBs, was downregulated in CXCR7 depended manner. Tubb3, on the other hand, was upregulated in the absence of functional CXCR7. Changes in other neuroectoderm derived cell markers, Otx or Neurog 1, were either minimal or relying on the type of treatment (Fig. 6A). Among the endoderm-derived cell markers studied, i.e. Alb, Afp, Pdx, and Dcx, only Afp and Dcx expression seemed to be regulated by SDF-1/CXCR7 interaction (Fig. 6B). Similarly, as it was 7

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Fig. 4. Mesoderm markers in EB7 formed by ESC-WT, ESC-CXCR7(L-), or ESC-CXCR7- cultured in control medium and subjected to SDF-1 or ITAC treatment. (A) Expression of T, Nodal, Kdr, Tbx1, Twist1, Bmp2. (B) proportion of BRACHYURY and SNAIL positive cells established by flow cytometry.

shown for EBs also in EBOs Kdr expression was upregulated by SDF-1 in CXCR7 dependent manner. Neither Pdgfrα, nor cTnTn, nor Myf5 expression, on the other hand, required active CXCR7. SDF-1 driven Pax3 induction was, however, depending on CXCR7 active and able to form complexes (Fig. 6C).

4. Discussion Embryonic stem cells are widely used to study processes occurring during animal and human embryonic lineages differentiation, both in vitro and in vivo. Since their derivation they were used in myriad experiment and analyzes aiming to understand molecular background of developmental processes. Since 2006 such studies are also possible using another pluripotent stem cell type, i.e. iPSCs (Takahashi et al.,

Fig. 5. CXCRs receptors and their ligands in EBOs formed by ESC-WT, ESC-CXCR7(L-), or ESC-CXCR7- subjected to SDF-1 or ITAC treatment. Expression of Cxcr4, Cxcr3, Cxcl12 and Cxcl11. 8

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Fig. 6. Differentiating cell markers in EBOs formed by ESC-WT, ESC-CXCR7(L-), or ESC-CXCR7- and subjected to SDF-1 or ITAC treatment. (A) Expression of ectoderm and neuroectodermal cells markers Pax6, Otx, Tubb3, Neurog1. (B) Expression of endodermal cells markers - Alb, Afp, Pdx, Dcx. (C) Expression of mesodermal markers Kdr, Pdgfr, cTnTn, Pax3, Myf5 in EBOs.

2007, Takahashi, and Yamanaka, 2006). Genetic manipulations of pluripotent stem cells resulted in the precise discrimination of multiple gene functions first by generating traditional and then conditional knock-out cells or animals [e.g. (Doyle et al., 2012; Mak, 2007)]. Finally, CRISPR/Cas technique entered the stage allowing genome editing, i.e. introduction of point mutations, modifications or deletion of selected loci [e.g. (Horii and Hatada, 2016, Hotta and Yamanaka, 2015, Oji et al., 2016)]. This technique was also used by us to genetically modify mouse ESCs by introducing mutations into the Ackr3 locus encoding CXCR7 receptor. In the past the role of CXCR4, CXCR7, and CXCR3 during embryonic development has been directly analyzed using knock-out mice. These studies showed that CXCR3-null mice do not present any obvious developmental phenotype (Panzer et al., 2007), however, CXCR4 and

CXCR7 mutant animals were characterized by multiple defects caused by abnormal cell migration and mesodermal lineages development. Thus, lack of CXCR4 resulted in embryonic lethality due to abnormal lymphopoiesis, myelopoiesis, cardiogenesis, vascular, and cerebellar development, as well as abnormal homing of HSCs to bone marrow (Ma et al., 1998; Nagasawa, 2007; Tachibana et al., 1998; Zou et al., 1998). Ablation of CXCR7 function affected cardio- and vasculogenesis (Sanchez-Martin et al., 2013). Apparently, CXCR4 and CXCR7 were not able to compensate for each other, or to be compensated by CXCR3, which appeared to be dispensable for development. Such inability of CXCR4 or CXCR3 to compensate for CXCR7 function we also observed in our study, in which we analyzed the impact of CXCR7 deficiency in differentiating ESCs. We also confirmed previously published data showing that the levels of Cxcr4 and Cxcl12 expression increased during 9

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their differentiation within EBs (Guo et al., 2005), and in our case also in EBOs. Moreover, SDF-1 was shown to increase the survival and migration ability of ESC colonies (Guo et al., 2005; Kowalski et al., 2016). In the current study we followed the impact of the lack of CXCR7 mediated signaling at ESC differentiation. We generated two types of mutant ESCs. First, expressed mutated form of CXCR7 which was not able to bind its ligands, i.e. SDF-1 and ITAC, but able to form complexes with other CXCR family members. Second type of cells lacked CXCR7 protein. By analyzing the phenotype of mutant ESCs we were also able to draw conclusions on the CXCR4 and CXCR3 role in early embryonic signaling. All of CXC receptors we studied were expressed in differentiating ESCs. Under control conditions, i.e. in the absence of exogenous SDF-1 or ITAC, lack of functional CXCR7 did not affect the expression of Cxcr3, Cxcl12 or Cxcl11 in EBs. Both, SDF-1 and ITAC stimulation upregulated the expression of Cxcr4 in EBs, ITAC did so at the later stages of differentiation, i.e. in EBOs. Both, ligands decrease the expression of Cxcl12 but only in EBs, not in EBOs, suggesting that such negative feedback control does not act at the later stages of differentiation. CXCR7 dysfunction prevented ligand-stimulated expression of Cxcr4 in EBs and EBOs. We showed that CXCR7 function was necessary to sustain expression such pluripotency/ectoderm related factors, such as Pou5f1 or Bmp4. Exogenous SDF-1 or ITAC dramatically downregulate the expression of Pou5f1, Sox2 or Fgf5 only in wild-type cells but not in those ones lacking CXCR7 function, strongly suggesting that activation of this receptor is crucial for the pluripotency-exit (see schematic diagram). These results were to some extent in agreement with the observation showing that knock-down of CXCR4 and/or CXCR7 in human iPSC led to the increase in the expression of OCT4 and SOX2 but at the first day of differentiation - at later stages such differences was not detectable (Ceholski et al., 2017). The fact that, in our hands, the Fgf5 increase the expression of this factor only in ESC-CXCR7- derived EBs suggested that the formation of the complex between CXCR7 and CXCR4 or CXCR3 is sufficient to transmit signal necessary to increase expression of this gene. There are many lines of evidence showing that the signal transduction differs depending on the compositions of CXCR heterodimers (Sanchez-Martin et al., 2013). Moreover, the reaction of cells expressing functional or nonfunctional CXCR7 versus those ones lacking this protein supporting the notion that such interaction may occur. Thus, we concluded that SDF-1 and ITAC induced downregulation of Pou5f, Sox2, and Fgf5 is controlled by activation of CXCR7 signaling. Expression of Bmp4 in EBs, on the other hand, requires CXCR7 function. At the later stages of differentiation, i.e. in EBOs, both SDF-1 and ITAC downregulated Pax6 in CXCR7dependent manner, and SDF-1 had such effect for Neurog1. Primitive endoderm markers were also sensitive to CXCR signaling in cells differentiating in EBs. Under control conditions lack of CXCR7 function led to Gata4 as well as Sox17 downregulation, however, to different extent, depending on cell line tested. SDF-1 treatment dramatically downregulated Gata4 expression in wild-type cells, however, such effect was also visible in mutant cell lines. In the absence of CXCR7 signaling expression of this gene was significantly higher, as compared to wild-type cells treated with SDF-1. Under control conditions Sox17 expression was downregulated only in Cxcr7-null cells. In EBs, both, SDF-1 and ITAC upregulated this gene but only in wild-type cells showing that its expression also relied on CXCR7 activity and presence. Interestingly, mesodermal markers required CXCR7 function and SDF-1 or ITAC treatment uncovered that T and Bmp2 expression could be upregulated by CXCR7/ITAC action while Kdr, Nodal, Tbx1 reacted either to CXCR4/SDF-1 or CXCR3/ITAC. Thus, by using SDF-1 or ITAC ESCs differentiation could be modulated. The absence of functional CXCR7 in differentiating mouse ESCs led to the drop in Kdr, Twist1, and Bmp2 expression what might affect the further specification of mesodermal lineages. This notion was supported by analyzes of EBOs which confirmed the role of CXCR7 in the expression of Kdr. Downregulation of CXCR7 in human iPSCs affected cardiomyocyte generation (Ceholski et al., 2017). However, we did not

observed any impact of this receptor dysfunction on cTnTn expression. Also, expression of myogenic markers were not affected in in the absence of functional CXCR7. Nevertheless, we concluded that SDF-1 by decreasing pluripotency as well as endoderm and increasing mesoderm markers could be used to stimulate ESC differentiation towards the mesoderm-derived lineages. We previously showed, that in vitro coculture of ESCs in the presence of SDF-1 increased their migration as well as ability to fuse with myoblasts (Brzoska et al., 2015; Kowalski et al., 2016). Transplantation of SDF-1 pretreated ESCs into regenerating skeletal muscle also improved their migration but not the participation in the formation of new muscle fibers (Kowalski et al., 2016). However, in this study ESCs which were not induced to differentiate in the presence of SDF-1 were transplanted into the mouse muscle. Thus, it is possible that the application of cells differentiating within EBs treated with SDF-1 would enhance the myogenic potential of ESCs. The positive impact of SDF-1 at survival and migration was shown in other studies focusing at hematopoietic or vascular differentiation of ESCs (Chen et al., 2007; Guo et al., 2005). There is, to our knowledge, no available data concerning ITAC impact at pluripotent stem cells differentiation. We show here, however, that in mouse differentiating ESCs ITAC dramatically increased expression of T and number of cells synthesizing its product, i.e. BRACHURY, which is crucial for the primitive streak formation. It also impacted at the level of other mesoderm associated factors, i.e. Tbx1, or number of cells expressing SNAIL. Thus, both factors SDF-1 and ITAC, as well as CXC receptors activated by them could be involved in early steps of pluripotent stem cells differentiation. 5. Conclusion Presented results suggest that different CXCR are involved in the regulation of different developmental markers (graphical abstract). Using CXCR7 deficient cells we show that CXCR7 mediated signaling is necessary for the downregulation of mRNAs encoding pluripotency markers OCT4, SOX2, and also FGF5 allowing initial steps of differentiation. We also show that SDF-1 and ITAC mediated signaling is crucial for the mesoderm formation. Interestingly, upregulation of mRNA encoding BRACHYURY can be mediated in the presence of CXCR7 able to form complexes with other CXCRs but not to transmit the signal. Summarizing, SDF-1, ITAC, and CXCR activated by them play crucial role during early steps of ESC differentiation. Authors' contribution Kamil Kowalski: conception and design, financial support, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; Edyta Brzoska: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript; Maria A. Ciemerych: data analysis and interpretation, manuscript writing, final approval of manuscript. Availability statement The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Declaration of Competing Interest None. Acknowledgements This work was supported by grant provided by budget funds from National Science Centre, Poland (Narodowe Centrum Nauki) for Kamil Kowalski, PRELUDIUM 2013/11/N/NZ3/00186. 10

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Authors would like to thank Andrzej Dziembowski and Jakub Gruchota for help with introducing us to CRISPR-Cas9 technology.

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