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. 2018 Apr 11;233(9):6944–6951. doi: 10.1002/jcp.26483

Tfcp2l1 safeguards the maintenance of human embryonic stem cell self‐renewal

Hongwei Sun 1, Yu You 1, Mengmeng Guo 1, Xiaohu Wang 1, Yan Zhang 1, Shoudong Ye 1,✉
PMCID: PMC13482437  PMID: 29323720

Abstract

Tfcp2l1 is a transcription factor critical for mouse embryonic stem cell (mESC) maintenance. However, its role in human ESCs (hESCs) remains unclear. Here, we investigated the role of Tfcp2l1 in controlling hESC activity and showed that Tfcp2l1 is functionally important in the maintenance of hESC identity. Tfcp2l1 expression is highly enriched in hESCs and dramatically decreases upon differentiation. Forced expression of Tfcp2l1 promoted hESC self‐renewal. Functional analysis of the mutant forms of Tfcp2l1 revealed that both the CP2‐ and SAM‐like domains are indispensable for Tfcp2l1 to maintain the undifferentiated state of hESCs. Notably, the CP2‐like domain is closely related to the suppression of definitive endoderm and mesoderm commitment. Accordingly, knockdown of Tfcp2l1 significantly induced differentiation preferentially into definitive endoderm and mesoderm. Further studies found that inhibition of Wnt/β‐catenin signaling pathway by IWR1 is able to eliminate the differentiation caused by Tfcp2l1 downregulation. Taken together, these findings reveal the unique and crucial role of Tfcp2l1 in the determination of hESC fate and will expand our understanding of the self‐renewal and differentiation circuitry in hESCs.

Keywords: human embryonic stem cells, self‐renewal, Tfcp2l1, Wnt

Tfcp2l1 expression is highly enriched in hESCs and dramatically decreases upon differentiation. Forced expression of Tfcp2l1 promoted hESC self‐renewal, while knockdown of Tfcp2l1 induced differentiation.Our data reveal the unique and crucial role of Tfcp2l1 in hESC maintenance.

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1. INTRODUCTION

Embryonic stem cells (ESCs) are derived from the inner cell mass of mammalian blastocysts (Evans & Kaufman, 1981; Martin, 1981; Thomson et al., 1998). They are capable of infinite self‐renewal and retain the developmental potential to differentiate into any kind of cell type. Mouse ESCs (mESCs) have long been used to explore the mechanism for the self‐renewal of ESCs, and numerous factors, such as Klf2, Tfcp2l1, Sp5, Gbx2, and Esrrb, have been identified as important ESC pluripotency factors (Huang, Ye, Zhou, Liu, & Ying, 2015; Martello, Bertone, & Smith, 2013; Martello et al., 2012; Qiu et al., 2015; Wang, Tang, Liu, Ying, & Ye, 2017; Ye, Li, Tong, & Ying, 2013; Ye et al., 2016). Although human ESCs (hESCs) share many similarities with mESCs, there are significant differences between these ESCs including expression of different sets of surface markers and distinct growth factor requirements (Huang et al., 2015; Ye, Liu, & Ying, 2014). hESCs are difficult to culture, displaying slow growth and poor survival, especially upon cellular detachment and dissociation (Thomson et al., 1998). Thus, hESCs were originally cultured in clusters on supporting feeder layers, like mouse embryonic fibroblasts (MEF) (Thomson et al., 1998). Feeder‐free culture is possible if hESCs are grown on Matrigel in chemically defined medium (Ludwig et al., 2006). These properties increase the difficulty of exploring the molecular mechanisms underline the maintenance of hESC identity.

Although some factors, including Oct4, Sox2, and Nanog, share similar functions in the self‐renewal of mouse and human ESCs (Huang et al., 2015), others do not appear to, such as Stat3 and β‐catenin, which are able to support mESC self‐renewal (Niwa, Burdon, Chambers, & Smith, 1998), but not hESC self‐renewal (Daheron et al., 2004). Our and other groups’ recent studies demonstrated that Tfcp2l1 functions as an important common downstream target of STAT3 and β‐catenin to support mESC self‐renewal (Martello et al., 2013; Qiu et al., 2015; Ye et al., 2013) and also plays a crucial role in the sustaining of the naïve‐like human pluripotent stem cells (Takashima et al., 2014; Wang et al., 2014). In addition, Tfcp2l1 is highly expressed in the inner cell mass of human blastocysts (O'Leary et al., 2012). Besides, it is noteworthy that human Tfcp2l1 protein is 93% identical to mouse Tfcp2l1, implying conservative inputs to the maintenance the self‐renewal of pluripotent stem cells. However, the physiological role of Tfcp2l1 in hESCs has not yet been clarified.

Here, we hypothesized that hESCs, like mESCs, may also have to maintain normal level of Tfcp2l1. To test this hypothesis, we used gain‐ and loss‐of‐function approaches, coupled with transcriptome profiling. The results reveal that Tfcp2l1 plays a crucial role in the regulation of hESC identity. Elevated expression of Tfcp2l1 delays hESC differentiation while Tfcp2l1 inhibition directly disrupts self‐renewal and associates with the Wnt/β‐catenin signaling pathway to induce definitive endodermal and mesodermal commitment. These data uncover previously unrecognized functions of Tfcp2l1 in modulating hESC stemness.

2. MATERIALS AND METHODS

2.1. Culture of hESCs

HES2 hESCs were kindly provided by Qi‐Long Ying (University of Southern California). Human induced pluripotent stem cells (ZSSY‐001) were kindly provided by the NuwaCell.Ltd, China. The basal media for hESC culture is N2B27 plus 10% KSR (10828, Life Technology, Tarrytown, NY). N2B27: DMEM/F12 (11330–032, Life Technology) and Neurobasal medium (21103–049, Life Technology) mixed at a 1:1 ratio, 1 × N2 (17502–048, Life Technology), 1 × B27 (17504–044, Life Technology), 2 mM Glutamax (35050–061, Life Technology), 1 × NEAA (11140–050, Life Technology), and 0.1 mM β‐mercaptoethanol (21985, Gibco). HES2 hESCs were cultured on plates pre‐coated with Matrigel (BD Biosciences, San Jose, CA) in basal media supplemented with 10 ng/ml bFGF and 10 ng/ml Activin A. Y27632 (1 μM; Y0503, Sigma, Darmstadt, Germany) was added when hESCs were passaged. For passaging, hESCs were dissociated with a Calcium Trypsin KSR (CTK) solution (Hasegawa, Fujioka, Nakamura, Nakatsuji, & Suemori, 2006) every 3–5 days and replated on Matrigel‐coated plates. Four Micrometr IWR1 (I0161, Sigma) were added in cultured media for supporting Tfcp2l1 shRNA hESCs.

2.2. Plasmid construction

The coding region of the Tfcp2l1 gene was inserted into PiggyBac transposon vector and then was introduced into hESCs with the LTX reagent. Overlapping PCR was used to generate Tfcp2l1 mutations. For RNA interference in hESCs, short hairpin RNA (shRNA) constructs were designed to target 21‐base specific regions of Tfcp2l1 and were then cloned into the pLKO.1‐TRC plasmid. The targeted sequence was as follow: GCTACAATGGTTCTCCAAACA.

2.3. Quantitative real time PCR (qRT‐PCR)

Total RNA was extracted with a TransZol Up plus RNA Kit (ER501‐01, TRANSGEN BIOTECH, Beijing, China). cDNA was synthesized from 1 μg of total RNA using TransScript All‐in‐One First‐Strand cDNA Synthesis SuperMix for qPCR (One–Step gDNA Removal) (AT341‐02, TRANSGEN BIOTECH) according to the manufacturer's instructions. qRT‐PCR was performed with TransStart Top Green qPCR SuperMix (AQ131‐03, TRANSGEN BIOTECH) in a PikoReal Real‐time PCR machine (Thermo Scientific, Finland, NY). Gene expression was normalized to human GAPDH expression. The primers used are listed in Supplementary Table S1.

2.4. Alkaline phosphatase activity assay

Cells were fixed in 4% paraformaldehyde for 2 min at room temperature, washed in PBS, and incubated in AP staining reagent (85L3R‐1KT, Sigma) for 45 min at room temperature in the dark. After being washed twice with PBS, cells were visualized under a Leica DMI8 microscope.

2.5. Western blotting

Cells were lysed in ice‐cold RIPA cell buffer (P0013B, Beyotime Biotechnology, China) supplemented with Protease Inhibitor Cocktail (DI111‐02, TRANSGEN BIOTECH). Proteins were separated with a 10% PAGE gel and electrotransferred to a PVDF membrane. Probing was performed with specific primary antibodies and HRP‐conjugated secondary antibodies. The primary antibodies used were Flag (M2; Sigma, 1:2000) and α‐tubulin (SC‐8035, Santa Cruz, Dallas, TX, 1:2000).

2.6. Immunofluorescence staining

Cells were fixed in 4% paraformaldehyde for 20 min at room temperature, washed in PBS, blocked for 1 hr at 37 °C in blocking buffer (PBS containing 5% BSA and 0.2% Triton X‐100), and incubated overnight at 4 °C with the primary antibody Oct4 (SC‐5279, Santa Cruz, 1:200). Alexa Fluor 488 (Invitrogen, Grand Island, NY, 1:1000) conjugated secondary antibody was used at 1:1,000. Nuclei were stained with Hoechst (Invitrogen, 1:5000).

2.7. Statistical analysis

All data are reported as the mean ± SD. Student's t‐test was used to determine the significance of differences in comparisons. Values of p < 0.05 were considered as statistically significant.

3. RESULTS

3.1. Overexpression of Tfcp2l1 promotes hESC self‐renewal

Tfcp2l1 expression is robust in undifferentiated mESCs, but declines abruptly upon differentiation (Ye et al., 2013). Furthermore, Tfcp2l1 transcript becomes significantly downregulated during derivation of hESCs from the inner cell mass of human blastocysts (O'Leary et al., 2012). To investigate whether the expression of Tfcp2l1 will be further decreased during hESC differentiation, we performed monolayer differentiation. After cultured in basal media for 8 days, differentiated HES2 cells expressed lower level of Tfcp2l1 than the undifferentiated hESCs (Figure 1a). This change in expression was similar to that seen for the pluripotency genes Oct4, Nanog, Sox2, and Prdm14 (Figure 1a), indicating that Tfcp2l1 is also a pluripotency marker of hESCs. Previous studies have shown that Tfcp2l1 positively regulates mESC self‐renewal (Martello et al., 2013; Ye et al., 2013). However, whether such a role is conserved in hESCs remains obscure. Here, we first generated a flag‐tagged Tfcp2l1‐overexpressing hESC line using a PiggyBac (PB) vector (PB‐Tfcp2l1) in which Tfcp2l1 expression was efficiently enhanced (Figure 1b). Interestingly, PB‐Tfcp2l1 hESCs became dome‐shape (Figure 1c). To examine whether these PB‐Tfcp2l1 hESCs were converted into naïve‐like cells, we digested these cells into single cells and incubated them in N2B27/2i/LIF culture conditions, a classical media for culturing naïve pluripotent stem cells, but all of them died or differentiated (data are not shown), indicating that they are still primed pluripotent stem cells. We then want to test the ability of Tfcp21l to support hESC self‐renewal. After cultured in hESC basal media without Activin A and bFGF for 6 days, PB cells differentiated, while PB‐Tfcp2l1 cells maintained an undifferentiated morphology and retained alkaline phosphatase (AP)‐positive staining (Figure 1c). Immunofluorescence showed positive expression of the pluripotency marker OCT4 (Figure 1c). At the transcriptional level, as assessed by quantitative real‐time PCR (qRT–PCR), overexpression of Tfcp2l1 maintained pluripotency genes (Oct4 and Nanog), but suppressed the differentiation markers (Gata6 and Mixl1), when compared to the PB empty vector (Figure 1d). To confirm that the effect observed upon Tfcp2l1 overexpression was not unique to one cell line, we enforced Tfcp2l1 in human induced pluripotent stem cells (hiPSCs) (Supplementary Figure S1a). Although Tfcp2l1 could not induce dome‐shape colonies in hiPSCs, like in HES2 hESCs, PB‐Tfcp2l1 hiPSCs still maintained the undifferentiated phenotype in the absence of exogenous addition factors (Supplementary Figures S1b). Notably, HES2 hESCs overexpressed Tfcp2l1 only could be split for five passages and then differentiated (Supplementary Figure S2). Therefore, overexpressing Tfcp2l1 enables short‐term self‐renewal of hESC. Although enforced Tfcp2l1 could partially recapitulate the self‐renewal‐promoting effect of Activin A and bFGF, Tfcp2l1 transcript could not be induced by either component (Supplementary Figure S3), suggesting that other signaling pathways were involved in Tfcp2l1 expression in hESCs.

Figure 1.

Figure 1

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Enforced Tfcp21 promotes hESC self‐renewal. (a) comparison of Tfcp2l1 expression between undifferentiated HES2 hESCs and differentiated cells cultured in basal media for 8 days. **p < 0.01 versus Undifferentiated. (b) Western blot analysis of FLAG in HES2 hESCs with stable Flag tagged Tfcp2l1 transgene expression (PB‐Tfcp2l1). (c) alkaline phosphatase (AP) staining of PB and PB‐Tfcp2l1 HES2 hESCs cultured in basal media for 6 days. Scare bar: 100 μM. (d) Immunofluorescence analysis of OCT4 expression in PB and PB‐Tfcp2l1 cells. OCT4 is a marker for undifferentiated ESCs. The nuclei were counterstained with Hoechst 33342 (Hoechst). Scare bar: 100 μM. (e) quantitative real‐time PCR (qRT‐PCR) analysis of self‐renewal (Oct4 and Nanog) and differentiation (Gata6 and Mixl1) gene expression levels. **p < 0.01 versus PB. (a,e) data represent mean ± s.d. of three biological replicates

3.2. Cp2‐ and SAM‐like domains are critical for Tfcp2l1 activity in hESCs

Tfcp2l1 contains two distinct domains, the CP2‐like domain at the N‐terminus and the SAM‐like domain at the C‐terminus (Kim, Jang, & Park, 2016). Tfcp2l1 is hexamerized in solution via the SAM‐like domain, while binds directly to DNA via the CP2‐like domain (Kim et al., 2016). To define which domain is indispensable for the ability of Tfcp2l1 to maintain pluripotency, we generated PB system‐mediated expression constructs encoding full‐length (FL) and two different mutant human Tfcp2l1 proteins lacking the CP2‐like domain (ΔCP2) or SAM‐like domain (ΔSAM) (Figure 2a). These Flag‐tagged mutated forms of Tfcp2l1 were successfully overexpressed in HES2 hESCs (Figure 2b). Transgenic clones were then cultured in basal medium without Activin A and bFGF for two passages. The morphology of the Tfcp2l1ΔCP2 and Tfcp2l1ΔSAM transgenic hESCs appeared similar to PB transfected cells (Figures 2c and 2d). They acquired an enlarged, flattened, and differentiated morphology in the edge of colonies (Figures 2c and 2d). Accordingly, they lost AP‐positive activity, accompanied by low expression of the pluripotent markers Oct4 and Nanog (Figure 2C–E). Therefore, the Cp2‐ and SAM‐like domains are suggested to be necessary for Tfcp2l1 to promote hESC self‐renewal.

Figure 2.

Figure 2

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Effect of different Tfcp2l1 mutants on hESC maintenance. (a) schematic outline of the Tfcp2l1 domains. (b) Western blot analysis of different mutated TFCP2L1 proteins. (c) different Tfcp2l1 mutants transfectants were cultured in basal media for two passages. Scare bar: 100 μM. (d) Immunofluorescence analysis of OCT4 expression in different Tfcp2l1 mutants overexpressing cells. Scare bar: 100 μM. (e) qRT–PCR analysis of self‐renewal (Oct4 and Nanog) and differentiation (Gata6 and Mixl1) markers in different Tfcp2l1 mutant transfectants. Data represent mean ± s.d. of three biological replicates. **p < 0.01 vs PB

3.3. Suppression ofTfcp2l1 induces differentiation toward definitive endoderm and mesoderm specification

As mentioned above, Tfcp2l1ΔCP2 failed to maintain hESC self‐renewal under basal media (Figures 2c and 2d). However, the Tfcp2l1ΔCP2 transfectants, but not Tfcp2l1ΔSAM overexpressing cells, generated a lot of spontaneous differentiation (as indicated by the red arrow in the figure), even in the presence of Activin A and bFGF (Figure 3a). They appeared AP‐negative morphology (Figure 3a). This difference may be because Tfcp2l1ΔCP2 competitively inhibit the function of endogenous Tfcp2l1 through direct binding Tfcp2l1 target locus to trigger hESC differentiation. To investigate lineage induction, Tfcp2l1ΔCP2‐specific differentiation signature was analyzed by RNA‐sequence to assess the gene expression pattern (GEO Number: GSE102718). Compared with PB hESCs, PB‐Tfcp2l1ΔCP2 hESCs showed an upregulation of a panel of definitive endodermal (FoxA2, Sox17, and Gata6) and mesoderm (Eomes, T and Mixl1) markers, while expressed low levels of self‐renewal markers (Dppa3 and Zfp42) (Figure 3b). This expression pattern was further validated by qRT‐PCR (Figure 3c), suggesting that inhibition of endogenous Tfcp2l1 might initiate hESC exit from naïve pluripotency and exhibits enhanced spontaneous differentiation toward the fates of definitive endoderm and mesoderm.

Figure 3.

Figure 3

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Suppression of Tfcp2l1 impairs HES2 hESC self‐renewal. (a) AP staining of HES2 hESCs overexpressing different Tfcp2l1 mutants cultured in basal media supplemented with Activin A and bFGF for 2 passages. Scare bar: 100 μM. (b) Heat map showed the indicated gene expression pattern in PB and PB‐Tfcp2l1ΔCP2 HES2 hESCs cultured in basal media supplemented with Activin A and bFGF for 2 passages. (c) qRT–PCR was used to confirm those gene expression as shown in (b). **p < 0.01 versus PB. (d) qRT–PCR analysis of Tfcp2l1 expression in Tfcp2l1 shRNA (shTfcp2l1) knockdown cells. The transcript level was normalized against a scramble shRNA control. **p < 0.01 versus Scramble. (e) Morphology and AP staining of scramble control and shTfcp2l1 hESCs cultured in basal media in the presence of Activin A and bFGF for three passages. Scare bar: 100 μM. (f) qRT–PCR analysis of hESC pluripotency (Oct4 and Nanog) and differentiation (Gata6, FoxA2, Mixl1, and T) marker expression in Tfcp2l1 shRNA hESCs cultured in basal media with Activin A and bFGF for 3 passages. **p < 0.01 versus Scramble. (g) Immunostaining for OCT4 in the scramble control and Tfcp2l1 shRNA hESCs. Scare bar: 100 μM. (h) Heat map showed the indicated gene expression pattern in scramble and Tfcp2l1 shRNA HES2 hESCs cultured in basal media supplemented with Activin A and bFGF for three passages. FoxA2, Gata6, T, Cxcr4, Sox17, Gata4, and Hnf4a are definitive endodermal markers. Mixl1, Eomes, T, Gsc, and Twist1 are mesodermal genes. (c, d, f) data represent mean ± s.d. of three biological replicates

We then utilized an RNA interference approach to complement the results obtained by overexpression of Tfcp2l1ΔCP2 . Initially, a shRNA targeting the human Tfcp2l1 transcript (Tfcp2l1 shRNA) was placed into the pLko.1 lentiviral vector. Additionally, we used a construct containing a non‐specific shRNA sequence (scramble shRNA) as a control. Scramble and Tfcp2l1 shRNA lentiviral particles were then used to infect HES2 hESCs. Tfcp2l1 expression was reduced by 90 % at the mRNA level (Figure 3d) compared to scramble shRNA‐infected control cells. After three passages, scramble shRNA hESCs retained a typical hESC‐like morphology and high expression of pluripotency markers, whereas Tfcp2l1 shRNA hESCs lost their characteristic morphology and differentiated, demonstrated by reduced AP activity, down‐regulation of pluripotency markers (Oct4 and Nanog), while up‐regulation of differentiation markers, namely Gata6 and FoxA2 for definitive endoderm, T and Mixl1 for mesoderm (Figure 3e–g). We also performed RNA‐sequence assay to confirm the differentiation potential of the Tfcp2l1 shRNA hESCs (GEO Number: GSE102798) (Figure 3h). Similar results were obtained in hiPSCs (Supplementary Figure S4a–b). Collectively, these data indicate that suppression of the activity of endogenous Tfcp2l1 will initiate commitment to definitive endoderm and mesoderm.

3.4. Inhibition of Wnt/β‐catenin signaling pathway eliminatesTfcp2l1 knockdown‐induced differentiation

To gain insight into how suppression of Tfcp2l1 induced definitive endoderm and mesoderm, we set out to check the gene expression profile of RNA‐sequence (GEO Number: GSE102798) and found that the expression levels of many members of Wnt/β‐catenin signaling pathway, such as the Wnt proteins (Wnt3a, Wnt5b, and Wn8a), receptor (Fzd8), β‐catenin‐interactors (Lef1 and Tcf7) and Wnt targets (Cdx1, Axin2, and Sp5) were increased in Tfcp2l1 shRNA and Tfcp2l1ΔCP2 cells (Figures 3b and 4a). We next used qRT‐PCR to confirm the expression of Sp5, a classical direct target of Wnt/β‐catenin signaling pathway. As seen in Figure 4b–d, Sp5 transcript was significantly upregulated in Tfcp2l1 shRNA and PB‐Tfcp2l1ΔCP2 hESCs, whereas its expression was repressed by Tfcp2l1FL . Together, these results mean that suppression of Tfcp2l1 triggers hESC differentiation through activation of Wnt/β‐catenin signaling pathway. To test this hypothesis, IWR1, an inhibitor of Wnt/β‐catenin signaling (Chen et al., 2009), was added into hESC culture medium. After cultured in basal media supplemented with Activin A and bFGF for two passages, Tfcp2l1 shRNA hESCs had a flat morphology and lost AP expression, while IWR1‐treated Tfcp2l1 shRNA cells exhibited compact morphology seen in scramble shRNA hESCs (Figure 4e), maintained AP activity and expressed high levels of pluripotency genes (Oct4 and Nanog), but low levels of differentiation associated markers (FoxA2, Gata6, Mixl1, Gata4, and T) (Figures 4e and g). Cdx1, a classical direct target of Wnt/β‐catenin signaling pathway, is suppressed upon IWR1 stimulation as expected (Figure 4g). Overall, these results suggest that the cellular changes observed upon down‐regulation of Tfcp2l1 expression occur, in part, due to the increased activity of the Wnt/β‐catenin signaling pathway, and inhibition of the latter can block the Tfcp2l1 shRNA‐induced differentiation.

Figure 4.

Figure 4

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Knockdown of Tfcp2l1 is associated with Wnt/β‐catenin signaling to induce differentiation. (a) Heat map showed the gene expression of Wnt family members in scramble and Tfcp2l1 shRNA HES2 hESCs cultured in basal media supplemented with Activin A and bFGF for three passages. (b–d) qRT–PCR analysis of Sp5 expression in Tfcp2l1 shRNA, PB‐Tfcp2l1ΔCP2 , and PB‐Tfcp2l1 hESCs cultured in basal media supplemented with Activin A and bFGF for three passages. **p < 0.01 versus Scramble or PB. (e) Morphology and AP staining of scramble control and shTfcp2l1 hESCs cultured in basal media in the presence of Activin A and bFGF with or without 4 µM IWR1 for two passages. Scare bar: 100 μM. (f) Immunostaining for OCT4 in the scramble control and Tfcp2l1 shRNA hESCs in the presence or absence of IWR1. Scare bar: 100 μM. (g) qRT–PCR analysis of hESC pluripotency (Oct4 and Nanog) and differentiation (Gata6, FoxA2, Gata4, Mixl1, and T) marker expression in Tfcp2l1 shRNA hESCs with or without IWR1. Cdx1 is a direct target of β–catenin. *p < 0.05, **p < 0.01 vs Scramble. (b–d, f) Data represent mean ± s.d. of three biological replicates

4. DISCUSSION

The molecular mechanisms underlying the maintenance of hESCs have yet to be clearly established. This work provides evidence for novel activities of the Tfcp2l1 as a gatekeeper in the maintenance of hESC self‐renewal. We demonstrate that Tfcp2l1 is one of the pluripotency markers and that overexpression of Tfcp2l1 can substitute for Activin A and bFGF for the short‐term maintenance of hESC identity, while inhibition of the endogenous activity of Tfcp2l1 triggers hESC differentiation into definitive endoderm and mesoderm partially through associating with the activation of the Wnt/β‐catenin signaling pathway. Our study therefore established Tfcp2l1 as a critical mediator of hESC self‐renewal status.

The extrinsic self‐renewal signals and morphology differ between human and mouse ESCs, but both have ability to proliferate identically while maintain pluripotency, it is therefore critical to identify the self‐renewal regulators that are conserved between the two species. To resolve this issue, we focus on Tfcp2l1 and provide evidence for the functional conservation of Tfcp2l1 in both mouse and human ESCs, because Tfcp2l1 has been previously described in mESCs and is one major common mediator downstream of mESC self‐renewal pathways, such as LIF/Stat3 and Wnt/β‐catenin signaling (Martello et al., 2013; Qiu et al., 2015; Ye et al., 2013). Tfcp2l1 expression is enriched in the undifferentiated mESCs, but declines markedly upon differentiation (Ye et al., 2013). The similar expression pattern also can be observed in hESCs (Figure 1a), suggesting that Tfcp2l1 is a pluripotency maker for both mESCs and hESCs. Moreover, knockdown of Tfcp2l1 in mESCs leads to a decrease in the expression of pluripotency‐associated transcription factors, as well as an increase in the expression of gene markers associated with differentiation (Martello et al., 2013; Ye et al., 2013). Parallel experiments conducted in hESCs demonstrate that hESCs also require Tfcp2l1 (Figures 3d–h). Thus, it is evident that Tfcp2l1 is essential for maintaining the self‐renewal and pluripotency of mESCs and hESCs. However, it is unclear why enforced Tfcp2l1 can only maintain short‐term self‐renewal of hESCs (Figures 1b–e). This might be due to the different stages of development of mESCs and hESCs, and/or the differences in the signaling pathways active in them (Huang et al., 2015). It will be of great interesting to investigate and compare the molecular mechanism by which Tfcp2l1 promotes mESC and hESC self‐renewal respectively, for example, whether Nanog is capable of mediating the self‐renewal‐promoting effect of Tfcpl21 in hESCs as in mESCs (Ye et al., 2013), because Nanog is also capable of supporting hESC identity when overexpressed (Xu et al., 2008).

Importantly, our studies also demonstrate an important function of the canonical Wnt/β‐catenin signaling pathway in the process of Tfcp2l1 shRNA‐induced differentiation, and this is likely the major contribution of Tfcp2l1 shRNA to the emergence of definitive endoderm and mesoderm (Figures 4a–e). This is unsurprising, given that Wnt/β‐catenin signaling pathway has been shown to play a key role in development events in different species and ESC differentiation (Huang et al., 2015; Valenta, Hausmann, & Basler, 2012). For examples, human induced pluripotent stem cells or hESCs, treated with different concentrations of Wnt/β‐catenin pathway activator or modulating this pathway in different stages of differentiation process, could be induced differentiation into many types of definitive cells (Matsuno et al., 2016; Touboul et al., 2016). Likewise, Wnt signaling also can collaborate with OCT4 and TCF1 to drive definitive endoderm induction by triggering endodermal genes in hESCs (Ying, Mills, French, & Gadue, 2015; Sun et al., 2017). On the other hand, activation of Wnt/β‐catenin signaling pathway is also important for mesoderm formation. Upon early and transient treatment of hESCs with Wnt3a, mesoderm formation is enhanced, leading to greater differentiation toward cardiomyocytes (Tran et al., 2009). In contrast, blockade of endogenously produced Wnts markedly inhibits mesoderm formation in hESCs, the endogenous Wnt/β‐catenin signaling thus is required for cardiac differentiation (Paige et al., 2010). In line with these results, constitutive activation of Wnt signaling results in loss of self‐renewal and induction of mesoderm in hESCs even under pluripotency maintenance conditions, whereas inhibition of Wnt/β‐catenin signaling promotes hESC self‐renewal (Davidson et al., 2012). Future work will explore the precise mechanism that how Tfcp2l1 modulates Wnt/β‐catenin signaling to regulate the formation of definitive endoderm and mesoderm.

In summary, these findings show that the self‐renewal‐promoting function of Tfcpl21 is conserved in the mouse and human ESCs, and that a restricted window of Tfcp2l1 activity is necessary for the maintenance of ESCs. How the expression of Tfcp2l1 in hESCs is regulated by various extrinsic factors and how Tfcp2l1 integrates with the core regulators of pluripotency, however, remain to be further discovered. These mechanistic insights obtained will facilitate to better understand the regulatory network of pluripotency and could favor wider utilization of human pluripotent stem cells in basic and clinical applications.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ACCESSION NUMBERS

The accession numbers for the RNA‐seq dataset reported in this paper are Gene Expression Omnibus (GEO): GSE102718 and GSE102798.

Supporting information

Additional Supporting Information may be found online in the supporting information tab for this article.

Figure S1. Enforced Tfcpl21 promotes hiPSC self‐renewal.

Figure S2. Enforced Tfcpl21 fails to maintain long‐term self‐renewal of hESCs.

Figure S3. Activin A and bFGF fail to induce Tfcp2l1 expression.

Figure S4. Knockdown of Tfcpl21 induces hiPSC differentiation.

Table S1.List of primers used for qRT‐PCR analysis.

JCP-233-6944-s001.pdf (314.7KB, pdf)

ACKNOWLEDGMENTS

This work was supported by the Natural Science Foundation of China (grant numbers 31671535, 31501191) and Anhui Province (grant number 1508085SQC204], and the Scientific Research Startup Fund of Anhui University (grant numbers J01006068, J01006045, J10118520411).

AUTHORS’ CONTRIBUTIONS

HWS, YY, and SDY conceived and designed experiments. HWS, YY, and MMG performed the experiments. XHW and YZ analyzed the data. SDY wrote the paper.

Sun H, You Y, Guo M, Wang X, Zhang Y, Ye S. Tfcp2l1 safeguards the maintenance of human embryonic stem cell self‐renewal. J Cell Physiol. 2018;233: 6944–6951. 10.1002/jcp.26483

Hongwei Sun, Yu You, and Mengmeng Guo contributed equally to this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional Supporting Information may be found online in the supporting information tab for this article.

Figure S1. Enforced Tfcpl21 promotes hiPSC self‐renewal.

Figure S2. Enforced Tfcpl21 fails to maintain long‐term self‐renewal of hESCs.

Figure S3. Activin A and bFGF fail to induce Tfcp2l1 expression.

Figure S4. Knockdown of Tfcpl21 induces hiPSC differentiation.

Table S1.List of primers used for qRT‐PCR analysis.

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