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    Topic review

    Germ Cell Derivation from PSCs

    Subjects: Cell Biology
    View times: 3
    Submitted by: Jeong Tae Do

    Definition

    Assisted reproductive technologies (ARTs) have developed considerably in recent years; however, they cannot rectify germ cell aplasia, such as non-obstructive azoospermia (NOA) and oocyte maturation failure syndrome. In vitro gametogenesis is a promising technology to overcome infertility, particularly germ cell aplasia. Early germ cells, such as primordial germ cells, can be relatively easily derived from pluripotent stem cells (PSCs); however, further progression to post-meiotic germ cells usually requires a gonadal niche and signals from gonadal somatic cells.

    1. Introduction

    Mammalian cells are divided into somatic and germ cells according to their roles. Somatic cells are involved in the homeostasis and survival of the body, whereas germ cells are dispensable for survival; however, they are needed only for reproduction. The ancestors of germ cells are unipotent and undergo a process called gametogenesis, to differentiate into either spermatozoa or oocytes. In vivo gametogenesis is a very sophisticated process that is difficult to recapitulate in an in vitro system. The development of stem cell differentiation protocols has made it possible to obtain many functional cell types in vitro, including somatic and germ cells. In particular, since approximately two decades, in vitro gametogenesis from pluripotent stem cells (PSCs) has been focused on in developmental biology and stem cell research [1][2]. In mice, PSCs can differentiate into functional germ cells, which can be fertilized and developed to term after implantation [3][4]. However, mature germ cells capable of forming embryos after fertilization have yet to be derived from human PSCs.
    Studies on human germ cells generally involve ethical issues. The limited use of human origin materials for research impedes the identification of mechanisms of human germ cell development. Therefore, information regarding human germ cell development pathways has been deduced from the data obtained from animal models. The in vitro PSC differentiation approach can overcome the difficulties of using human materials and provide insight into the mechanism of human gametogenesis in vivo and in vitro [2].
    According to the United Nations World Population Prospects, the global fertility rate is continuously declining. The current fertility rate in 2021 is 2.438 births per woman, which is a 0.41% decline compared to that in the 2020 data of 2.448 births per woman [5]. Although the etiology of infertility varies, most cases of infertility originate from the poor production of sperm or eggs. As the donation of gametes poses various ethical and legal issues, gamete derivation via differentiation of patient-origin PSCs is considered a feasible option for solving infertility without gamete donation [6]. If functional gamete formation from human PSCs were possible, male and female infertility, such as non-obstructive azoospermia and oocyte maturation failure syndrome, would be curable.

    2. Germ Cell Differentiation from Pluripotent Stem Cells In Vitro

    2.1. In Vitro Derivation of PGC-Like Cells (PGCLCs) from Pluripotent Stem Cells

    In 2009, Ohinata et al. established the basic conditions for directed PGC specification in mouse pluripotent epiblast cells [7]. Pregastrula stage epiblasts were treated with BMP4, BMP8b, SCF, EGF, and LIF to induce the expression of Blimp1 and Prdm14. These epiblast-derived PGC-like cells (epiPGCs) expressing Blimp1-mVenus and Stella-ECFP showed genetic and epigenetic characteristics consistent with in vivo PGCs. When these epiPGCs were transplanted into neonatal testes, they developed into mature spermatozoa, which could fertilize oocytes and give rise to healthy offspring [7]. This research showed the basis of signal interactions essential for PGC specification from pluripotent cells and proved that the epiblast is the direct precursor of PGCs. Ohinata’s study prompted researchers to use PSCs to derive PGC-like cells (PGCLCs) in vitro, since PSCs are the in vitro derivatives of the peri-implantation epiblast.
    Initial studies on in vitro germ cell specification from PSCs have mainly focused on the derivation of PGCs through embryoid body (EB) formation from ESCs or EGCs [1][8][9]. Culture in the absence of LIF and other growth factors associated with self-renewal [1] and culture with BMP4 [10] or retinoic acid [11] could induce the differentiation of PSCs into PGC-like cells (PGCLCs). Toyooka et al. isolated PGCLCs expressing Mvh (DEAD box helicase 4) during PGC differentiation and cocultured them with BMP4-producing cells for 1 d, followed by aggregation with gonadal cells isolated from E12.5 to 15.5 mice [9]. Transplantation of these aggregates into the testis capsule resulted in the formation of testicular tubules, wherein ESC-derived mature sperm were formed. Geijsen et al. also suggested that differentiation through EB formation resulted in meiotic male germ cells that could fertilize oocytes and develop the blastocyst stage [11]. However, since these germ cell derivation protocols were random differentiation strategies using EB formation, the PGC specification was very heterogeneous and inefficient (approximately 0.5–3.6% per EB), raising the need for a directed induction method for PGC specification. To increase the PGC specification from PSCs, PSCs should be maintained in a homogenous population that is germline competent. Ying et al. suggested that germline competent naïve PSCs could be maintained under culturing with MAPK and GSK inhibitors (referred to as 2i) and LIF, called the ground state [12]. The ground state of pluripotency is equivalent to that of the E4.5 epiblast of preimplantation blastocysts in gene expression patterns. Thus, the 2iL condition in mice enables the induction of germline competent ESCs/iPSCs from all mouse strains tested [12][13][14].
    Hayashi et al. first proposed the stepwise differentiation of mouse PSCs into PGCLCs in 2011 [3]. They showed that mouse PSCs in ground state pluripotency were differentiated into epiblast-like cells (EpiLCs), an intermediate precursor for PGC formation, in the presence of activin A and bFGF for 2 days [3]. After culturing under conditions similar to those in Ohinata’s experiment (derivation of epiblast to PGCLCs), EpiLCs were subsequently differentiated into PGCLCs [14]. Global transcriptional profiles revealed that specification of PGCLCs from EpiLCs recapitulated the in vivo specification of epiblast to PGCs, suggesting that EpiLCs were germline competent precursors corresponding to pre-gastrulating epiblasts at around E5.75, and PGCLCs had properties similar to those of E9.5 PGCs [3]. Transplanting these male or female PGCLCs into germ cell-deficient mice results in the formation of a gonad-like structure containing gamete-like cells. In males, PGCLCs were further differentiated in an in vivo environment, such as seminiferous tubules of W/Wv mice lacking endogenous sperm, in which PGCLCs could resume spermatogenesis [3][15]. PGCLC-derived sperm developed in host testes could give rise to fertile offspring after intracytoplasmic injection (ICSI) into wild-type oocytes and transplantation into surrogate mothers [3]. Similar to the in vitro derivation of sperm, female ovary-like structures and oocyte-like cells could be derived from EpiLC-derived PGCLCs, which were differentiated from mouse ESCs and iPSCs [4]. Female PGCLCs also develop into oocytes, contributing to fertile offspring after in vitro maturation and fertilization with normal sperm [4]. These in vitro (EpiLC and PGCLC formation) and in vivo (transplantation of PGCLCs into host gonads) conjugated methods serve as bridges for the complete in vitro gametogenesis technology and provide important information for signaling cascades during germ cell fate transition.
    Human PSCs can differentiate into human PGCLCs (hPGCLCs) spontaneously [16][17] or under defined culture conditions [18][19]. Gkountela et al. differentiated hESCs via serum-induced random differentiation in EBs for 9 days and sorted TRA-1-81+/cKIT+ hPGCLCs (0.3% per sorted living population), which also expressed BLIMP1 and OCT4 with or without NANOS3 expression [16]. Kee et al. used another germ cell marker, VASA-GFP reporter, for hPGCLC isolation from differentiating hESCs [17]. VASA-GFP+ cells expressed other early germ cell markers, DAZL, BLIMP1, and STELLA. They also suggested that overexpression of BOULE alone or together with DAZL could enhance hPGCLC specification and meiotic progression, suggesting the importance of BOULE and DAZL for germ cell progression in humans [17]. For hPGCLC enrichment, Yu et al. treated the EBs formed from hESCs and iPSCs with activin A, BMP4, and retinoic acid, by which PGCLCs expressing DDX4, folliculogenesis-related markers, and postnatal oocyte-specific markers were derived [20]. Aggregates of PGCLCs with human granulosa cells gave rise to ovarian follicle-like structures in xenografted mice [20].
    Irie et al. suggested a defined culture condition for the specification of hPGCLCs from hESCs and hiPSCs. To efficiently derive hPGCLCs, they used distinct states of PSCs that were preconditioned in a medium containing four inhibitors (4i), including CHIR99021 (GSK-3 inhibitor), PD0325901 (MEK inhibitor), SB203580 (p38 MAPK inhibitor), and SP600125 (JNK inhibitor), sustaining cells in near ground state pluripotency and aiding in easy differentiation into germ cell lineage [18]. To induce hPGCLCs, hPSCs in 4i medium were cultured in bFGF/TGFβ-containing medium for 2 days, followed by a suspension culture in the presence of BMP2 (or BMP4), LIF, stem cell factor (SCF), and epidermal growth factor (EGF). The derived hPGCLCs were similar to human PGCs (hPGCs) in terms of gene expression and epigenetic patterns. They showed that SOX17, which is known as a transcription factor for endoderm specification, might be important for hPGCLC specification and maintenance since SOX17 began to be expressed as early as day 1 after suspension culture and was associated with BLIMP1, both of which are highly expressed in hPGCs; BLIMP alone repressed somatic differentiation and enhanced germ cell specification together with SOX17 [18]. Although the preconditioned hPSC state was sufficient for producing hPGCs, Sasaki et al. pointed out that hPSCs kept in 4i medium were not genuinely at the ground state, as cells did not show consistent upregulation in naïve pluripotency markers [19]. Instead, Sasaki et al. proposed the induction of PGCs from primed hPSCs by introducing new cell types, incipient mesoderm-like cells (iMeLCs), as intermediate cells in the process of inducing hPGCLCs from hiPSCs [19]. Double transgenic hiPSCs bearing BLIMP1-tdTomato and TFAP2C-EGFP were used as reporters for human fetal germ cells [21][22]. As a first step for germ cell differentiation, hiPSCs were stimulated with activin A and CHIR99021, which produced iMeLC-expressing genes for pluripotency and the mesoderm (T, EOMES, SP5, and MIXL1). After suspension culturing with BMP4, SCF, EGF, and LIF for 8 days, these iMeLCs were converted into hPGCLCs (BLIMP1-2A-tdTomato+ and TFAP2C-2A-EGFP+) expressing early human germ cell markers, such as OCT4, NANOG, BLIMP1, TFAP2C, and SOX17. GO term analysis after RNA sequencing revealed that potential regulators of hPGCLC specification from iMeLCs include BLIMP1 and SOX17, which were also suggested by Irie et al. [18][19].
    hPGCLC derivation using hESCs was facilitated by the stimulation of activin A [23] or by adding vitamin C to the induction medium [24]. Activin A is also a well-known inducer of fetal and postnatal oogenesis, indicating that it is involved in germ cell development [25]. Epigenetic changes induced by vitamin C through enhancing the expression of ten-eleven translocation (TET) may positively affect germ cell differentiation [24].

    2.2. In Vitro Derivation of Spermatogonia and Oogonia from Pluripotent Stem Cells

    PSC-derived PGCLCs were injected into neonatal testes to generate mature sperm from PSCs. However, PGCLCs are not suitable cell types for transplantation into the testis, since the niche for PGCs is not the postnatal testis, but the prenatal proximal epiblast, hindgut, and genital ridge. After birth, the earliest germ cell types in the testis are spermatogonia that reside in the basement membrane of the seminiferous epithelium. These spermatogonia are male germ line stem cells, so-called spermatogonial stem cells (SSCs), which continuously self-renew and differentiate into spermatocytes and haploid gametes by passing between two Sertoli cells [26]. Currently, long-term in vitro culture of SSCs is possible, although PGCs cannot be cultured in vitro [27][28]. Undifferentiated SSCs established in an in vitro culture system expressed Vasa, Pax7, Plzf, Gfra1, Etv5, and Bcl6b [29]. In contrast, female germline stem cells, or oogonia, transiently exist (between E12.5 and E13.5) and undergo meiosis during embryonic development [30]. Thus far, an in vitro culture of oogonia is not possible.
    Nayernia et al. first attempted to generate in vitro-derived SSCs using Stra8-EGFP and Prm1-DsRed double transgenic mouse ESCs [31]. They suggested that Stra8-EGFP+ cells were ESC-derived SSCs and that Prm1-DsRed+ could differentiate into haploid germ cells. However, Stra8-EGFP+ cells express PGC-specific genes; therefore, this differentiation protocol may yield heterogeneous germ cell populations, including SSCs. In addition, the ESC-derived spermatids they suggested are not fully characterized; therefore, it is not clear whether the cells are true spermatids. Ishikura et al. generated well-characterized in vitro-derived SSCs differentiated from PSCs using reconstituted testes formed by aggregating ESC-derived PGCLCs with E12.5 testicular somatic cells [32]. Aggregates of PGCLCs and testicular somatic cells formed seminiferous tubule-like structures, in which PLZF-positive SSC-like cells were detected within 21 days of culture [32]. SSC-like cells showed similar gene expression profiles and DNA methylomes to those of in vivo SSCs. In addition, after transplantation of these SSC-like cells into adult W/Wv mouse testes instead of neonatal testes, they developed into spermatids and spermatozoa, which could form embryos after an injection into oocytes and developed to term after transfer of the embryos to the surrogate mother.
    In human, Sasaki and colleagues et al. generated prospermatogonia-like cells from hiPSCs through long-term culturing of the xenogeneic aggregates of hiPSC-derived human germ cells with mouse testicular somatic cells [33]. hPGCLCs were obtained from xenogeneic aggregates after 14 days of culture on an air–liquid interface (ALI) system where xenogeneic aggregates were cultured on the surface of the porous filter. In the ALI culture system, hiPSC-derived hPGCLCs were differentiated into multiplying (M)-prospermatogonia on day 77 and finally differentiated into primary transitional (T1)-prospermatogonia at day 120. Although they did not test the functionality of the hiPSC-derived hPGCLCs, single-cell RNA sequencing analysis revealed that the gene expression patterns of in vivo M- and T1-prospermatogonia were very similar to those of in vivo M- and T1-prospermatogonia, respectively. As hiPSC-derived T1-prospermatogonia expressed a fraction of genes associated with “spermatogenesis” and “spermatogenic failure” these cells can be used as an in vitro platform for research on the mechanism of human spermatogenesis and male infertility [33].
    Recently, human female germline stem cells (oogonia) were generated in vitro from hiPSCs (BLIMP1-tdTomato and TFAP2C-EGFP double transgenic) using Sasaki’s approach, by which hiPSC-derived hPGCLCs were generated via iMeLCs [19][34]. BLIMP1-tdTomato+/TFAP2C-EGFP+ hPGCLCs were sorted by fluorescence-activated cell sorting (FACS) and cultured for further specification in xenogeneic ovary structures formed by aggregation of hiPSC-derived hPGCLCs with mouse ovarian somatic cells, as in a prospermatogonia-like cell-inducing protocol [33]. At day 7 after culture, aggregates began to form cyst-like structures and TFAP2C-EGFP+ cells developed into oogonia-like cells, which expressed DAZL and DDX4 and were delineated by mouse granulosa cells at culture day 77. On culture day 120, BLIMP1-tdTomato+/TFAP2C-EGFP+ cells appeared to differentiate into BLIMP1-tdTomato+/TFAP2C-EGFP- cells, which correspond to fetal germ cells expressing genes for meiosis initiation (STRA8) except for meiotic recombination (SPO1l, PRDM9, DMC1, or SYCP1). Transcriptome assays revealed that hiPSC-derived oogonia-like cells (TFAP2C-EGFP+) were similar to those of week 7 and 9 oogonia and gonocytes of human embryos [34][35].
    These reports suggest that male and female germline stem cells can be established from PSCs in vitro within reconstituted testes and ovary structures, where embryonic gonadal somatic cells were aggregated with PSC-derived PGCLCs. Of note, in vitro oogonia may provide a useful tool for elucidating the mechanism of female germline stem cell development and female infertility, which has not been studied well due to ethical issues and limitations in using human-origin materials.

    2.3. In Vitro Derivation of Haploid Spermatid-Like Cells from Pluripotent Stem Cells

    Haploid spermatid-like cells were also derived from mouse ESCs using the aggregation method [36]. Zhou et al. isolated Blimp1-Venus+ and Stella-ECFP+ PGCLCs, aggregated with testicular somatic cells in a medium containing retinoic acid for meiotic induction and BMP-2/4/7 and activin A for germ cell development and self-renewal. Under meiotic-inductive signals, including retinoic acid, BMP2/4/7, and activin A, PGCLCs in the aggregates developed beyond PGCs and underwent meiosis, confirmed by the formation of chromosome synapsis. Furthermore, hormonal stimulation, such as the follicle-stimulating hormone (FSH), testosterone, and bovine pituitary extract, induces post-meiotic haploid spermatid-like cells (SLCs) containing distinct acrosome structures and imprinted patterns in H19 and Snrpn loci. After intracytoplasmic injection of SLCs into normal oocytes, fertilized embryos were successfully developed. However, to date, mature spermatozoa have not yet been derived in vitro from PSCs. Thus, in vitro spermiogenesis from haploid spermatids is currently the only obstacle in the process of complete in vitro male gametogenesis, from PSCs to mature spermatozoa.

    2.4. In Vitro Oogenesis from Pluripotent Stem Cells

    Female germ cells were formed within ovarian follicles, which are an in vivo niche for the maturation of female gametes. One follicle contains one meiotically arrested oocyte surrounded by multilayered granulosa cells at puberty [37]. During follicular maturation in vivo, primary oocytes complete meiosis I, begin meiosis II, and arrest at the metaphase of meiosis II (MII). Primordial follicles can mature in vitro by stimulating oocyte growth and meiotic competency by EGF [38] and cAMP [39][40], respectively. In vitro oogenesis could also be recapitulated in PGCs obtained from E12.5 gonads by stimulation of follicle formation using α-MEM-based medium supplemented with fetal bovine serum (FBS) and an estrogen-receptor antagonist (ICI 187, 780) [41]. These studies prompted an alternative method to derive female gametes from PSCs, where germline specification is not started.
    The first published report showing germ cell differentiation from PSCs in vitro was on female gamete derivation from mouse ESCs [1]. Hübner et al. generated oocyte-like structures, which were randomly formed through EB formation, followed by two-dimensional culture. These non-directed differentiation protocols, or random differentiation, allowed even male ES cells to differentiate into oocyte-like structures. Similarly, Lacham-Kaplan et al. obtained presumptive ovary-like structures from male ESC-derived EBs using the conditioned medium from neonatal testis cells. The obtained structures contained oocyte-like cells expressing oocyte markers (Fig-α and ZP3), indicating the possible production of female germ cells from male PSCs [42]. However, these results were inconsistent with the proposed methods and lacked demonstration of functionality [11]; thus, further research is required. As previously mentioned, Hayashi et al. showed that in vitro-derived PGCLCs could further differentiate into functional oocytes in reconstituted ovaries after transplantation into female mice [4]. Combining these methods, Hayashi and colleagues, in 2016, finally derived MII oocytes from mouse ESCs in an entirely in vitro system [43]. They derived EpiLCs from mouse ESCs and further differentiated the EpiLCs into PGCLCs, which were then aggregated with E12.5 gonadal somatic cells. These aggregates were cultured for approximately 5 weeks in three steps: in vitro differentiation, in vitro growth, and in vitro maturation [43][44]. The aggregates of PGCLCs and gonadal somatic cells formed a follicular structure that successfully simulated the in vivo ovarian environment and performed the key events of oogenesis in vitro, including the formation of cumulus-oocyte complexes and development of MII oocytes [43][44]. These in-vitro-generated oocytes could be fertilized in vitro with wild-type sperm and developed into 2-cell embryos, giving rise to healthy fertile pups after transfer to pseudopregnant surrogate mice. However, the successful full-term development rate was as low as 3.5%. This in-vitro-oocyte generation method was also valid for iPSC lines reprogrammed from mouse embryonic fibroblasts [43]. Overall, this platform for in vitro gametogenesis by aggregating PSC-derived PGCLCs with gonadal somatic cells faithfully reconstitutes the in vivo-like gonadal environment in both males and females [32][36][43][44]. However, this approach still shows meiosis and epigenetic imperfections compared with their in vivo counterparts and requires embryonic somatic cells [32][43], which cannot be used in the clinic. Nevertheless, this differentiation method provides a useful in vitro system for studying the interaction between gametes and gonadal somatic cells [45][46].
    Recently, oocyte-like cells were directly induced without specification into PGCLCs [47]. Hamazaki et al. generated follicle structures from mouse ESCs and iPSCs aggregated with E12.5 female gonadal somatic cells through forced expression of eight transcription factors important for primordial-to-primary-follicle transition (PPT), such as Nobox, Figla, Tbpl2, Sohlh1, Stat3, Dynll1, Sub1, and Lhx8. Oocyte-like cells of the follicle structures could develop into MII oocytes, which were competent for fertilization with sperm and early embryonic development until the 8-cell stage. Overexpression of these eight transcription factors was not sufficient for the induction of oocyte-like cells from somatic cells (MEFs), suggesting that a pluripotent state is required for the induction of germ cells by overexpression of the eight transcription factors. However, it is still unknown whether different transcription factor combinations may induce germ cell specification in somatic cells.
    Furthermore, follicle-like cells (FLCs) were first derived from human PSCs by forced expression of DAZL and BOULE, which are involved in in vivo germ cell specification as discussed above [17][48]. Germ cell commitment from hESCs was first induced by BMP4 and BMP8a treatment followed by meiosis induction by lentiviral overexpression of DAZL and BOULE with the subsequent addition of GDF9 and BMP15. Follicle-like structures with oocyte-like cells surrounded by multilayered cell aggregates appeared on day 9 after differentiation. These oocyte-like cells expressed ZP2, NOBOX, AMH, and VASA and were found to have primordial oocyte identity, as confirmed by transcriptome analysis. In addition, transplantation of FLCs into kidney capsules resulted in the formation of primordial follicle-like structures, indicating that hESC-derived FLCs are functional ovarian follicles. This in vitro FLC derivation system could be used to elucidate the early mechanism of human germline development and folliculogenesis, the details of which are still elusive.

    3. Germ Cell Derivation via Synthetic Embryo Formation

    In addition to direct differentiation from PSCs, synthetic embryo formation is an emerging technique for the derivation of germ cells in vitro. Aggregation of two or three types of blastocyst-derived stem cells, including ESCs, trophoblast stem cells (TSCs), and extra-embryonic endoderm (XEN) cells, can form early embryo-like structures or synthetic embryos, mimicking natural embryos [49][50][51][52]. In 2017, Zernicka-Goetz and colleagues first generated synthetic embryos, ETS-embryos, by aggregating ESCs and TSCs, which were then cultured in a 3D-extracellular matrix (ECM) scaffold [49]. ETS-embryos successfully formed a proamniotic cavity and underwent key spatiotemporal morphogenesis of peri- and post-implantation stages, as seen at E6.5 natural embryos, leading to the specification of PGCs expressing Stella, Prdm14, Tfap2c, Nanos3, and Ddx4. PGC specification in ETS-embryos was found to be induced by BMP-SMAD signaling, similar to natural embryogenesis.
    Moreover, ETX embryos formed by the incorporation of ESCs, TSCs, and XEN cells developed to form gastruloid-like natural E7.0 mid-gastrular embryos [50]. ETX gastruloids showed more efficient mesodermal specification than ETS-embryos and asymmetric patterning, that is, anterior and posterior axis followed by PGC specification as shown by Stella- and Prdm14-positive populations at the posterior portion of the boundary of ESC and TSC compartments [50]. Similarly, ETX embryos generated under a nonadherent-suspension-shaking culture system also showed asymmetric patterning of PGC specification [51]. However, these synthetic embryos showed limited developmental potential only in the early post-implantation stage. Therefore, it is not yet known whether subsequent germline differentiation beyond early PGC specification through synthetic embryo formation is possible. However, these studies demonstrated that differentiation via synthetic embryo formation can be another way to generate PGCs from PSCs in vitro.
    Recently, Liu et al. reported the successful generation of a human blastocyst-like structure (iBlastoid) via reprogramming of fibroblasts [53]. During pluripotential reprogramming, unwanted cell types, such as trophoblasts and primitive endoderm and epiblast cells, emerged in the same dish by day 21 post-induction. After 3D culture of these heterogeneous cell populations in the AggreWell system for 6 days, aggregates developed into E5–7 human blastocyst-like structures, that is, iBlastoids. Under attachment culture of iBlastoids to test peri-implantation development, iBlastoids did not show any morphological signs of implantation or gastrulation. Although human PGCs have not been derived through synthetic embryo (iBlastoid) formation, we cannot rule out the possibility of this method as an alternative method to generate PGCs in vitro by reprogramming somatic cells.

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    The entry is from 10.3390/cells10081889

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