Snap29 Is Dispensable for Self-Renewal Maintenance but Required for Proper Differentiation of Mouse Embryonic Stem Cells

Pluripotent embryonic stem cells (ESCs) can self-renew indefinitely and are able to differentiate into all three embryonic germ layers. Synaptosomal-associated protein 29 (Snap29) is implicated in numerous intracellular membrane trafficking pathways, including autophagy, which is involved in the maintenance of ESC pluripotency. However, the function of Snap29 in the self-renewal and differentiation of ESCs remains elusive. Here, we show that Snap29 depletion via CRISPR/Cas does not impair the self-renewal and expression of pluripotency-associated factors in mouse ESCs. However, Snap29 deficiency enhances the differentiation of ESCs into cardiomyocytes, as indicated by heart-like beating cells. Furthermore, transcriptome analysis reveals that Snap29 depletion significantly decreased the expression of numerous genes required for germ layer differentiation. Interestingly, Snap29 deficiency does not cause autophagy blockage in ESCs, which might be rescued by the SNAP family member Snap47. Our data show that Snap29 is dispensable for self-renewal maintenance, but required for the proper differentiation of mouse ESCs.


Introduction
Embryonic stem cells (ESCs) are pluripotent cells that can self-renew indefinitely under optimal culture conditions and retain the potential to generate any somatic lineage as well as germ cells [1]. Thus, ESCs are promising donor cell sources for regenerative medicine and cellular agriculture. The pluripotency of ESCs is mainly maintained by feed-forward networks formed by the transcription factors Oct4, Sox2, and Nanog [2,3]. Quantitative balances of these factors are necessary for the self-renewal and differentiation of ESCs [4]. Using a standard test of the embryoid body (EB) formation, ESCs are able to spontaneously differentiate into three embryonic germ layers: the ectoderm, mesoderm, and endoderm [5]. Hence, ESCs are regarded as an appropriate in vitro model for investigating gene regulation, signal transduction, and embryogenesis [6,7].
Snap29 is a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) that mediates membrane fusion of vesicles and target membranes in eukaryotic cells [8]. In mammals, the SNAP29 gene is expressed in many tissues and is involved in various membrane trafficking processes, including synaptic transmission [9], endocytic recycling [10], and autophagy [11][12][13]. Autophagy is a highly conserved catabolic process with well-organized membrane dynamics [14]. In this process, the isolation membrane of the phagophore first recruits multiple proteins and expands to form a double-membrane autophagosome, which fuses with lysosomes to generate autolysosomes, where sequestrated materials are degraded [14]. As identified previously, Snap29 acts as a Qbc-SNARE and promotes autophagosome-lysosome fusion by interacting with the Qa-SANRE Stx17 and R-SNARE Vamp7/8 [11,15] or separately forming another SNARE complex with Ykt6 and Stx7 [12,16]. Based on the above findings, Snap29 is indispensable for SNARE complex formation and the fusion of autophagosomes and lysosomes. Furthermore, autophagic homeostasis is required for the maintenance of ESC pluripotency [17,18]. However, the role of Snap29 in the self-renewal and differentiation of ESCs remains elusive.
In this study, the Snap29 gene was depleted via CRISPR/Cas9 in mouse ESCs, and the self-renewal capacity, differentiation potential, and autophagic flux were measured in Snap29 +/+ and Snap29 −/− ESCs. Our data reveal that the self-renewing capacity of Snap29 −/− ESCs is well-maintained, while the differentiation potential is severely impaired in ESCs with Snap29 deficiency.

Snap29 Deficiency Does Not Impair the Self-Renewal of ESCs
To explore the function of Snap29 in ESCs, we attempted to knock out Snap29 with the CRISPR/Cas9 system. As exon 1 of the mouse Snap29 gene partially overlaps with the Pi4ka gene (Ensemble database), Cas9 sgRNA targeting exon 2 of Snap29 was designed ( Figure S1A). Next, 2 knockout clones separately with a 28 bp deletion (KO-1) and a 1 bp insertion (KO-2) were obtained (Figure S1B), and the disruption of the Snap29 protein was confirmed using Western blot analysis ( Figure S1C). Then, we investigated the self-renewal ability of Snap29 +/+ and Snap29 −/− ESCs. Snap29-deficient ESCs formed compact cell clones with distinct boundaries and high alkaline phosphatase activity, which were comparable to those of wild-type ESCs ( Figure 1A). Interestingly, Snap29 depletion promoted the proliferative capacity of ESCs ( Figure 1B and Figure S1D). Although the expression levels of Klf4 and Esrrb were moderately increased in Snap29 −/− KO-2 cells through quantitative real-time PCR (qPCR), we did not observe significant expression changes in the core pluripotent markers Oct4, Nanog and Sox2 between Snap29 +/+ and Snap29 −/− ESCs (Figure 1C). Western blot analysis and immunofluorescence staining confirmed the retained expression of pluripotency-associated factors upon Snap29 deficiency ( Figure 1E-H). Furthermore, representative lineage marker genes were generally not activated in Snap29 −/− ESCs ( Figure 1D).
To exclude potential off-target effects of the CRISPR/Cas system, Snap29 was silenced in ESCs using two separate short hairpin RNAs (shRNAs). Consistently, Snap29 knockdown did not alter clonal patterns, alkaline phosphatase activity, proliferation, or the expression levels of core pluripotent markers of ESCs ( Figure S2A-C).
Next, we performed RNA sequencing (RNA-seq) to analyze the systemic effects of Snap29 depletion on ESCs. Because they displayed more differences than WT ESCs ( Figure 1 and Figure S1), KO-2 cells were selected for transcriptome analysis. Compared with Snap29 +/+ ESCs, 424 genes were upregulated and 514 genes were downregulated in Snap29 −/− ESCs (Figure 2A, Padj < 0.05, fold change ≥ 2). Consistent with the qPCR results, the expression levels of the pluripotency genes Oct4, Sox2, Klf4, and Esrrb were similar between Snap29 +/+ and Snap29 −/− ESCs ( Figure 2B). We took advantage of previously reported lists of genes required for pluripotency maintenance [6], and found no significantly altered pluripotency scores between Snap29 +/+ and Snap29 −/− ESCs ( Figure 2C). Meanwhile, representative lineage marker genes were expressed at quite low levels overall (based on low FPKM < 5.0) in ESCs regardless of whether they had a Snap29 deficiency or not ( Figure 2D). We then performed Gene Ontology (GO) analysis using differentially expressed genes (DEGs) and found numerous membrane-related cellular components ( Figure 2E), which is consistent with the membrane trafficking role of Snap29 [8,10]. Furthermore, DEGs were enriched in several pathways, including breast cancer, basal cell carcinoma, transcription misregulation in cancer, gastric cancer, and others ( Figure 2F).  (F) Quantity using ImageJ software of pluripotent protein levels relative to β-tubulin as a loading control. (G,H) Immunofluorescence of the pluripotency-associated markers Oct4, Nanog, Sox2, and SSEA-1. Scale bar, 25 µm. Mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant (p > 0.05), unpaired two-tailed Student's t-test, compared with WT groups. Taken together, we successfully established the Snap29 knockout ESC lines and found that the disruption of Snap29 did not influence the self-renewal capacity or the maintenance of pluripotent markers of ESCs. Taken together, we successfully established the Snap29 knockout ESC lines and found that the disruption of Snap29 did not influence the self-renewal capacity or the maintenance of pluripotent markers of ESCs.

Snap29 Depletion Promotes ESC Differentiation into Heart-like Beating Cells
To examine whether Snap29 is essential for the differentiation of mouse ESCs, we performed in vitro differentiation using a standard embryoid body (EB) formation assay. Snap29 +/+ and Snap29 −/− ESCs could form EBs with smooth and round edges, while EBs formed with Snap29 −/− cells were significantly larger than those of the control group ( Figure 3A). Upon differentiation, all ESCs showed a significantly reduced expression of pluripotency markers, including Oct4, Nanog and Sox2 ( Figure S3A,B), indicating that the repression of pluripotency factors is not affected by Snap29 knockout. During differentiation, heart-like beating cells emerged from the EB-derived cell cultures. Surprisingly, while approximately 60% of EBs formed from Snap29 −/− ESCs exhibited heart-like beating, only 42% of the control cells showed visible beating ( Figure 3C). The beating frequency (times per minute) was similar between Snap29 +/+ and Snap29 −/− ESC-derived cells ( Figure 3D). Moreover, differentiation of Snap29 +/+ and Snap29 −/− ESCs both yielded cells representing three embryonic germ layers, as indicated by the tissue-specific immunofluorescence staining of βIII-tubulin (neurons, ectoderm), AFP (liver, endoderm), and α-SMA (smooth muscle, mesoderm) ( Figure 3E-G). Nevertheless, Snap29 depletion improved the expression of α-SMA in differentiated cells ( Figure 3G,H), suggesting the enhanced mesoderm specification of ESCs without Snap29.

Snap29 Deficiency Leads to Defective Differentiation of ESCs
To further reveal the role of Snap29 in the differentiation of mouse ESCs, we performed RNA-seq analysis using Snap29 +/+ and Snap29 −/− ESC-derived differentiated cells at day 14. Notably, 291 genes were upregulated and 545 genes were downregulated in Snap29 −/− cells when compared with Snap29 +/+ cells ( Figure 4A, Padj < 0.05, fold change ≥ 2). Interestingly, numerous myogenesis-related terms were top-ranked in GO analysis by all DEGs, including myotube differentiation, cardiac muscle contraction, regulation of myotube differentiation, striated muscle contraction in Biological Process, contractile fiber, striated muscle thin filament, myofibril, myofilament, contractile fiber part in Cellular Component, extracellular matrix structural constituent, and tropomyosin binding in Molecular Function ( Figure 4B). We then measured the FPKMs of GO-enriched genes and found that the majority of them were significantly upregulated in Snap29 −/− cells, and representative genes are shown in Figure 4C. Consistently, the expression levels of the genes with the highest FPKM values, including Acta1, Col5a2, Jam2, Cxcl10, and Trim54, were significantly increased in Snap29 −/− cells through qPCR ( Figure 4D). These results indicate that Snap29 deficiency significantly promotes the expression of genes associated with muscle lineage differentiation and formation, which is consistent with the increased incidence of heart-like cells upon Snap29 depletion ( Figure 3). Moreover, adhesion molecules, cytokine receptor interaction, focal adhesion, PI3K-Akt signaling pathways, and ECM-receptor interaction were included in enriched pathways by KEGG analysis (Figure 4E), and might be involved in the regulation of myogenesis.   The enriched KEGG pathways of DEGs between Snap29 +/+ and Snap29 −/− differentiated cells. Mean ± SEM from three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant (p > 0.05), unpaired two-tailed Student's t-test.
Next, we investigated the functional role of 545 genes downregulated in Snap29 −/− cells ( Figure 4A). Taking advantage of previously reported lists of the genes essential for the germ layer differentiation of pluripotent stem cells (PSCs) [19], we found that 23 downregulated genes were required for the definitive endoderm differentiation of PSCs, including Ccnd2, Hesx1, Flt1, and Lonrf3; 8 genes for early mesoderm formation, including Tubb4A, Kif21B, Cdx2, and Cacna1H; and 16 genes for neuroectoderm differentiation, including Snap29 itself, Fstl3, Thyh3, and II3Ra ( Figure 5A). The FPKMs of related genes are shown in Figure 5B and verified using a qPCR assay ( Figure 5C). In GO analysis, embryonic organ development was top-ranked in the enriched biological process using downregulated genes upon Snap29 disruption ( Figure 5D). Consistently, pathways regulating cell differentiation and organ development, including the PI3K-Akt pathway, Ras signaling pathway, and MAPK signaling pathway, were enriched in KEGG pathways ( Figure 5E). Together, transcriptome analysis strongly suggests that Snap29 deficiency significantly disturbs the expression of the genes related to embryonic germ layer differentiation and further embryonic organ development. onic organ development was top-ranked in the enriched biological process using downregulated genes upon Snap29 disruption ( Figure 5D). Consistently, pathways regulating cell differentiation and organ development, including the PI3K-Akt pathway, Ras signaling pathway, and MAPK signaling pathway, were enriched in KEGG pathways ( Figure  5E). Together, transcriptome analysis strongly suggests that Snap29 deficiency significantly disturbs the expression of the genes related to embryonic germ layer differentiation and further embryonic organ development.

Snap29 Depletion Does Not Cause Autophagic Blockage in ESCs and Differentiated Cells
Next, the role of the autophagy modulation of Snap29 in ESCs was measured. In contrast to previous studies in nonembryonic cells [11,15], Snap29 knockout or knockdown in mouse ESCs did not lead to the accumulation of the autophagosome marker LC3-II or the universal autophagic substrate SQSTM1/p62 ( Figure 6A and Figure S2D). Upon differentiation, less LC3-II turnover and retained p62 protein were observed in Snap29 knockout cells at day 7 ( Figure 6B). However, at day 14 of differentiation, the LC3-II/LC3-I ratio was increased in Snap29 −/− cells in comparison with Snap29 +/+ cells ( Figure 6C). These results suggest that Snap29 is not necessarily required for autophagosome-lysosome fusion in mouse ESCs. Given the essential role of the SNARE complex in autophagy regulation, we speculate that other SNAP proteins might compensate in Snap29-deficient ESCs. The SNAP family contains four known members in mammals: Snap23, Snap25, Snap29, and Snap47, all of which contain two separate SNARE motifs [8]. Based on our RNA-seq data, we found that the expression of Snap25 was extremely low in mouse ESCs and differentiated cells (FPKM < 0.1) and that the expression of Snap47 was upregulated in Snap29 knockout cells ( Figure 6D). Then, we knocked down Snap47 and Snap23 using small interfering RNAs (siRNAs) in Snap29 +/+ and Snap29 −/− ESCs ( Figure 6E). Interestingly, Snap47 knockdown, but not Snap23 knockdown, resulted in the obvious accumulation of LC3-II and p62 in the Snap29 knockout ESCs (Figure 6F), indicating autophagic blockage in ESCs with dual disruption of Snap29 and Snap47. Taken together, these data suggest that Snap29 knockout does not lead to defective autophagic flux in ESCs and differentiated cells, which might be due to the rescue effect mediated by paralogous Snap47.

Snap29 Depletion Does Not Cause Autophagic Blockage in ESCs and Differentiated Cells
Next, the role of the autophagy modulation of Snap29 in ESCs was measured. In contrast to previous studies in nonembryonic cells [11,15], Snap29 knockout or knockdown in mouse ESCs did not lead to the accumulation of the autophagosome marker LC3-II or the universal autophagic substrate SQSTM1/p62 (Figures 6A and S2D). Upon differentiation, less LC3-II turnover and retained p62 protein were observed in Snap29 knockout cells at day 7 ( Figure 6B). However, at day 14 of differentiation, the LC3-II/LC3-I ratio was increased in Snap29 −/− cells in comparison with Snap29 +/+ cells ( Figure 6C). These results suggest that Snap29 is not necessarily required for autophagosome-lysosome fusion in mouse ESCs. Given the essential role of the SNARE complex in autophagy regulation, we speculate that other SNAP proteins might compensate in Snap29-deficient ESCs. The SNAP family contains four known members in mammals: Snap23, Snap25, Snap29, and Snap47, all of which contain two separate SNARE motifs [8]. Based on our RNA-seq data, we found that the expression of Snap25 was extremely low in mouse ESCs and differentiated cells (FPKM < 0.1) and that the expression of Snap47 was upregulated in Snap29 knockout cells ( Figure 6D). Then, we knocked down Snap47 and Snap23 using small interfering RNAs (siRNAs) in Snap29 +/+ and Snap29 −/− ESCs ( Figure 6E). Interestingly, Snap47 knockdown, but not Snap23 knockdown, resulted in the obvious accumulation of LC3-II and p62 in the Snap29 knockout ESCs ( Figure 6F), indicating autophagic blockage in ESCs with dual disruption of Snap29 and Snap47. Taken together, these data suggest that Snap29 knockout does not lead to defective autophagic flux in ESCs and differentiated cells, which might be due to the rescue effect mediated by paralogous Snap47.

Discussion
Snap29 plays a role in membrane trafficking and autophagy modulation [8,13]. Here we show that in pluripotent mouse ESCs, Snap29 knockout or knockdown does not impair cell proliferation, colony formation, alkaline phosphatase activity, or the expression of pluripotent markers (Figure 1 and Figure S2). Furthermore, transcriptome analysis confirmed that the core pluripotent factors are well-maintained in Snap29 −/− ESCs ( Figure 2). Interestingly, the DEGs between Snap29 +/+ and Snap29 −/− ESCs were enriched in several cancer-related pathways ( Figure 2F). The evidence demonstrates that the altered Snap29 activity might contribute to the pathogenesis of cancers [20]. Recent evidence from a breast cancer cell line and a human melanoma cell line suggests that Snap29 plays a role in the TNFα-NF-κB-FOXP3 signaling axis, and silencing Snap29 promotes tumor cell migration [21]. Thus, Snap29 might represent a novel safeguard to counteract the pathogenic processes involved in cancer.
As a Qbc SNARE, the function of Snap29 in promoting autophagosome-lysosome fusion is well-established. Unexpectedly, unlike many other studies [11,13,15,22], Snap29 depletion in mouse ESCs did not lead to defective autophagosome-lysosome fusion or the accumulation of autophagosomes ( Figure 6). We speculate that this unusual outcome might be caused by the O-linked β-N-acetylglucosamine (O-GlcNAc) modification of Snap29 in mouse ESCs. Guo et al. demonstrated that O-GlcNAcylated Snap29 inhibits the formation of a Snap29-containing SNARE complex, decreases the fusion of autophagosomes and endosomes/lysosomes, and blocks autophagic flux [13]. UDP-GlcNAc is a key substrate for the O-GlcNAcylation of proteins, and is produced primarily through glycolysis and hexosamine biosynthetic pathways [23]. As has been widely studied, PSCs, such as mouse ESCs, prefer glycolysis over oxidative phosphorylation to increase glucose uptake and energy supply [24]. Supplementation with glutamine in cell culture media (see Methods section) may also increase the production of UDP-GlcNAc [25]. O-GlcNAc modification is balanced by two enzymes: O-GlcNAc transferase (OGT) adds the modification and O-GlcNAcase (OGA) removes it [26]. As reported, mouse ESCs exhibit high expression of OGT, and Oct4 and Sox2 are modified with O-GlcNAc in ESCs, which is required for the activity of the pluripotency network [27]. Taken together, it is reasonable to speculate that Snap29 is O-GlcNAcylated and fails to form the SNARE complex with the associated factors in mouse ESCs. Further investigations should be performed to verify the O-GlcNAcylation of Snap29 and its biological functions in pluripotent ESCs.
In light of the fundamental role of Snap29-containing SNARE complexes in membrane trafficking, unknown Qbc SNARE domain-containing proteins may exist in mouse ESCs. In this study, preliminary findings suggest that Snap47 may compensate for Snap29 to promote autophagosome and lysosome fusion ( Figure 6). Snap47 protein contains two separate SNARE domains and its silencing in HeLa cells leads to impaired autophagic flux [28]. Moreover, Snap47 can interact with ATG14 to promote viral capsid protein conjugation and coxsackievirus propagation, while its role in autophagy is undetermined [29]. Overall, the function of Snap47 has not been extensively investigated. It would be intriguing to examine the role and mechanism of Snap47 in SNARE complex formation, autophagy regulation, and pluripotency maintenance in ESCs and other types of cells.
In this study, we performed standard in vitro differentiation of Snap29 +/+ and Snap29 −/− ESCs by EB formation. This method has been extensively used to investigate regulatory factors or signaling pathways that mediate ESC differentiation toward numerous cell lineages [30,31]. Moreover, the expression of tissue-specific genes of ESCs during in vitro differentiation recapitulates the early process of in vivo development [32]. Interestingly, Snap29 −/− ESCs formed significantly larger EBs than Snap29 +/+ cells ( Figure 3A,B). Meanwhile, Snap29 deficiency significantly promoted the generation of heart-beating cardiac cells (Figure 3). The size of EBs has been demonstrated to influence the differentiation potential of ESCs in this system [33], and larger EBs are associated with the enhanced cardiogenesis of ESCs [34]. Furthermore, the transcriptome analysis confirmed that the differentiation of ESCs into muscle lineage cells was enhanced by Snap29 knockout (Figure 4). We then examined the expression of the genes related to myogenesis, and found that the majority of them were significantly upregulated in Snap29 −/− cells (Figure 4). Representatively, Myod1 and Cav3 are essential transcription factors characterizing myogenic precursors; the Acta1 gene encodes Actin-alpha 1, a skeletal muscle protein, and the Tnnt gene encodes the Troponin T (TnT) protein, both of which are fundamental proteins found in muscle tissues; the Col4a and Col5a genes encode several types of collagens, which are flexible proteins that are important in the structure of many tissues, including muscles [35]. Our data uncover the role of Snap29 in muscle lineage differentiation and myofiber formation. Nonetheless, the causal link between Snap29, myogenic factors, and myogenesis requires further investigation.
Additionally, numerous genes that are required for germ layer differentiation are significantly misregulated in Snap29 −/− cells compared to control cells, especially the genes for definitive endoderm and neuroectoderm ( Figure 5). AFP is extensively used as a marker indicating the endoderm differentiation of PSCs. However, AFP + cells emerged from both the Snap29 +/+ and Snap29 −/− ESC-derived cell populations (Figure 3 F,H). The disagreement can be explained by the versatile expression of AFP, which is secreted by the primitive and definitive endoderm lineages, including the visceral endoderm, its derivative yolk sac endoderm, fetal liver hepatocytes, and the gut epithelium of the developing embryos [36]. Taken together, we assume that, during in vitro differentiation, although some AFP + cells emerge without Snap29, the ability of Snap29 −/− cells to differentiate into functional progenitors or somatic cells in the endoderm might be insufficient.
ESCs are an adequate in vitro model for studying embryogenesis [6,7]. In humans, the loss of functional Snap29 results in CEDNIK syndrome (cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma), a neurocutaneous disease characterized by severe developmental abnormalities of the nervous system and aberrant differentiation of the epidermis [37]. In addition, CEDNIK patients show signs of microcephaly and facial dysmorphism, hypoplastic optic disk, sensorineural deafness, and severe cachexia [37], indicating multisystem defects caused by Snap29 deficiency. In this study, we reveal that numerous genes that are required for neuroectoderm differentiation are significantly downregulated upon Snap29 knockout, including Fstl3, Ttyh3, and Il3Ra ( Figure 5). In the immunoassays, the expression of βIII-tubulin in Snap29 −/− cells was comparable to that in control cells ( Figure 3). As reported previously, detecting only the expression of βIII-tubulin is inadequate to measure the neuroectoderm differentiation of PSCs [38]. In addition to βIII-tubulin and the DEGs analyzed in RNA-seq, other essential regulatory factors and signaling pathways need to be tested in the following studies. Furthermore, although several animal models have been established to recapitulate CEDNIK syndrome [39][40][41], cell models are rather scarce [42]. Our ESC model lacking Snap29 may serve as a powerful tool for investigating the embryonic differentiation process of CEDNIK syndrome and seeking compounds that might ameliorate traits of the disease.
In this study, we uncovered that Snap29 is not required for the self-renewal maintenance of mouse ESCs. Snap29 depletion does not impair cell proliferation or the expression of pluripotency-associated markers in ESCs. However, Snap29 is indispensable for the proper differentiation of mouse ESCs. Snap29 deficiency enhances the differentiation of ESCs into cardiomyocytes, as indicated by heart-like beating cells. Moreover, transcriptome analysis revealed that Snap29 depletion significantly decreased numerous genes required for embryonic germ layer differentiation. Furthermore, Snap29 deficiency does not cause autophagy blockage in ESCs, which might be rescued by the SNAP family member Snap47. Our study reveals the role of Snap29 in the maintenance of ESC pluripotency and provides insights for further investigation of the complicated pathogenic mechanisms of CEDNIK syndrome caused by Snap29 deficiency.

Generation of Snap29 Knockout ESCs
Snap29 knockout ESCs were generated using the CRISPR/Cas9 system. Single-guide RNAs (sgRNAs) targeting exon 2 of the mouse Snap29 gene were designed using the online design tool available at http://crispr.genome-engineering.org/ (accessed on 28 December 2020). SgRNAs were cloned into the pSpCas9(BB)-2A-Puro (PX459) vector, and transfected into ESCs with lipofectamine TM transfection reagent (Invitrogen), according to the manufacturer's recommendation. Then, knockout ESC clones were identified through Sanger sequencing and confirmed using western blot analysis.

Cell Proliferation Analysis by Accumulative Growth Curve
2 × 10 5 WT, Snap29 KO-1, and KO-2 ESCs were initially seeded into 6-wells and cultured for 2 days. Then, ESCs were digested using Typsin-EDTA, counted by the hemocytometers, and passaged at certain splitting ratios of 1:5-1:12. ESCs were passaged for 6 days. The accumulative growth curve was drawn using the product of the cell numbers and the splitting ratios.

MTT Assay and AP Staining
The methyl thiazolyl tetrazolium (MTT) assay was used to measure cell viability, and committed using an MTT Cell Proliferation and Cytotoxicity Assay Kit (Beyotime), according to the manufacturer's instructions. Alkaline phosphatase (AP) staining was performed with a BCIP/NBT Alkaline Phosphatase Colour Development Kit (Beyotime), according to the manufacturer's instructions.

In Vitro Differentiation of ESCs
For ESC differentiation, three-dimensional colonies known as embryoid bodies (EBs) were generated [30]. Briefly, undifferentiated ESCs were trypsinized to obtain a single cell suspension, and EBs were formed in ESC culture medium without LIF, in a definite number of cells in hanging drops for 3 days. Then, EBs were transferred to 24-well culture plates with 1 EB per well. Daily microscopic observations were conducted to detect beating EBs. In total, 20 to 30 EBs were transferred to 6-well microwell plates per well for protein, RNA, and sample collection.

Gene Expression by Quantitative Real-Time PCR
Total RNA was isolated from samples using the MiniBEST Universal RNA Extraction Kit (Takara), according to the manufacturer's protocol. The purity and concentration of RNA were checked using Nanodrop technology. Then, 1 µg RNA was subjected to cDNA synthesis using PrimeScript TM RT Master Mix (Takara). Quantitative real-time PCR (qPCR) reactions were set up in duplicate with the ChamQ SYBR qPCR Master Mix (Vazyme) and run on the QuantStudio TM 5 Real-time PCR Instrument (Applied Biosystems). Each sample was repeated at least twice and analyzed with GAPDH serving as the internal control. Quantification of gene expression was based on the Ct (cycle threshold) value. Melting curve analysis was performed to control PCR product specificities and exclude nonspecific amplification. Primers for qPCR are listed in Supplementary Table S1.

Western Blot
Cells were collected and resuspended in NP40 cell lysis buffer containing 1 mM PMSF (Beyotime) and protease inhibitor cocktail (Bimake). Twenty micrograms of protein was separated on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat milk or 5% BSA in 1× TBST at room temperature for 2 h. Samples were probed with primary antibodies overnight at 4 • C (for antibody details, see Supplementary Table S2). HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies were diluted at 1:5000. Protein bands were detected using Super Sensitive ECL Luminescent reagents (Qinxiang). The band intensity was measured using ImageJ software and normalized to the intensity of β-tubulin. The relative expression level was calculated from the results of at least three independent experiments or samples.

Immunofluorescence
ESCs were grown on gelatin-treated coverslips and washed twice in PBS, then fixed in freshly prepared 4% paraformaldehyde for overnight at 4 • C, permeabilized in 0.1% Triton X-100 in blocking solution (3% goat serum in PBS) for 30 min, washed three times, and left in blocking solution for 1.5 h. Samples were incubated overnight at 4 • C with primary antibodies (for antibody details, see Supplementary Table S2), washed three times, and incubated for two hours with the secondary antibodies, goat anti-mouse IgG (H +L) FITC or goat anti-rabbit IgG (H + L) Alexa Fluor 594 (Jackson). Samples were washed twice with PBS, stained with 0.5 µg/mL DAPI, and mounted in Vectashield mounting medium. Fluorescence was detected and imaged using a Leica Microsystems CMS microscope.

RNA Sequencing and Bioinformatics Analysis
ESCs or differentiated cells were harvested and total RNA was extracted using TRIzol reagent and according to the manufacturer's instructions. The quality control method was mainly performed using an Agilent 2100 bioanalyzer. The initial RNA for library construction was total RNA, and the mRNA with a poly A tail was enriched with oligo (dT) magnetic beads. Using fragmented mRNA as a template and random oligonucleotides as primers, the first strand of cDNA was synthesized in the M-MuLV reverse transcriptase system, which was followed by second strand cDNA synthesis. The cDNA fragments were added to a single "A" base and subsequently ligated with the adapter. The products were then purified and enriched with PCR amplification. The PCR yield was quantified and samples were pooled together to make a single-stranded DNA circle (ssDNA circle), generating the final library. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina NovaSeq platform and 150 bp paired-end reads were generated.
For bioinformatics analysis, the clean reads were mapped to the Mus musculus mm10 reference genome using HISAT2 v2.0.5. Gene quantification (read counts) was calculated by FeatureCounts (1.5.0-p3). Then the normalized gene expression (FPKM) was calculated with read counts and gene lengths. DESeq2 was used to identify differentially expressed genes. Functional enrichment (GO annotation, KEGG) of gene sets with different expression patterns was performed using cluster Profiler.

Knockdown by shRNA or siRNA
For shRNA-mediated knockdown, interfering oligonucleotides were synthesized and cloned into the pSIREN-RetroQ vector, according to the manufacturer's instructions. The packaging cell line PT67 was transfected with 2 µg plasmid using lipofectamine TM transfection reagent (Invitrogen). After 48 h, the supernatant of the recombinant retrovirus was collected and used to infect ESCs. The clones were selected by 2 µg/mL puromycin, and surviving ones were picked. Subsequently, the knockdown efficiency was identified using qPCR. For siRNA-mediated knockdown, synthesized oligonucleotides were transfected into ESCs with lipofectamine TM transfection reagent (Invitrogen). Forty-eight hours later, ESCs were collected and analyzed for qPCR or western blotting. Sequences of oligonucleotides for shRNA and siRNA are listed in Supplementary Table S3.

Statistical Analysis
Data were analyzed by two-tailed Student's t-test or χ 2 test depending on specific experiments, and the p value was calculated. Statistical significance was defined as * p < 0.05, ** p < 0.01 or *** p < 0.001.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms24010750/s1. Author Contributions: R.G. contributed to the conception and design and manuscript writing. Y.J. contributed to collection and assembly of data, data analysis and interpretation, and manuscript writing. Z.G., J.Z., G.Q. and W.S. contributed to the collection and assembly of data. Y.Y. and H.W. contributed to discussion of the experiments and revision of the manuscript. All authors have read and agreed to the published version of the manuscript.