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• W3100505853 abstract "•A new iPSC differentiation approach that mimics human embryonic testis development•Induction of bipotential markers by day 10 and testis markers by day 15•3D aggregation yields organoids with tissue structures and Sertoli cell markers Currently an in vitro model that fully recapitulates the human embryonic gonad is lacking. Here we describe a fully defined feeder-free protocol to generate early testis-like cells with the ability to be cultured as an organoid, from human induced pluripotent stem cells. This stepwise approach uses small molecules to mimic embryonic development, with upregulation of bipotential gonad markers (LHX9, EMX2, GATA4, and WT1) at day 10 of culture, followed by induction of testis Sertoli cell markers (SOX9, WT1, and AMH) by day 15. Aggregation into 3D structures and extended culture on Transwell filters yielded organoids with defined tissue structures and distinct Sertoli cell marker expression. These studies provide insight into human gonadal development, suggesting that a population of precursor cells may originate from a more lateral region of the mesoderm. Our protocol represents a significant advance toward generating a much-needed human gonad organoid for studying disorders/differences of sex development. Currently an in vitro model that fully recapitulates the human embryonic gonad is lacking. Here we describe a fully defined feeder-free protocol to generate early testis-like cells with the ability to be cultured as an organoid, from human induced pluripotent stem cells. This stepwise approach uses small molecules to mimic embryonic development, with upregulation of bipotential gonad markers (LHX9, EMX2, GATA4, and WT1) at day 10 of culture, followed by induction of testis Sertoli cell markers (SOX9, WT1, and AMH) by day 15. Aggregation into 3D structures and extended culture on Transwell filters yielded organoids with defined tissue structures and distinct Sertoli cell marker expression. These studies provide insight into human gonadal development, suggesting that a population of precursor cells may originate from a more lateral region of the mesoderm. Our protocol represents a significant advance toward generating a much-needed human gonad organoid for studying disorders/differences of sex development. During mammalian embryonic development, the bipotential gonads develop on the ventromedial surface of the mesonephros and differentiate into either testes or ovaries based on the presence or absence of the Y sex chromosome. Disruption to gonadal development or function can result in disorders/differences of sex development (DSD) in humans, congenital conditions in which the anatomical, gonadal, or chromosomal sex is atypical (Hughes et al., 2006Hughes I.A. Houk C. Ahmed S.F. Lee P.A. Lawson Wilkins Pediatric Endocrine Society/European Society for Paediatric Endocrinology Consensus GroupConsensus statement on management of intersex disorders.J. Pediatr. Urol. 2006; 2: 148-162Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). Currently only 40% of patients with a DSD receive a genetic diagnosis (Eggers et al., 2016Eggers S. Sadedin S. van den Bergen J.A. Robevska G. Ohnesorg T. Hewitt J. Lambeth L. Bouty A. Knarston I.M. Tan T.Y. et al.Disorders of sex development: insights from targeted gene sequencing of a large international patient cohort.Genome Biol. 2016; 17: 243Crossref PubMed Scopus (131) Google Scholar), suggesting that unknown genes and genomic regions must contribute. When genomic sequencing identifies a novel DSD candidate gene variant, establishing pathogenicity is often hampered by a lack of appropriate human embryonic gonad cell lines. Thus, a reproducible human in vitro system comprising embryonic gonadal lineages is urgently required. In recent years, directed differentiation of a wide range of cell lineages and tissues from human induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) has been achieved. Indeed, cardiac (Yang et al., 2008Yang L. Soonpaa M.H. Adler E.D. Roepke T.K. Kattman S.J. Kennedy M. Henckaerts E. Bonham K. Abbott G.W. Linden R.M. et al.Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population.Nature. 2008; 453: 524-528Crossref PubMed Scopus (1078) Google Scholar), intestinal (McCracken et al., 2011McCracken K.W. Howell J.C. Wells J.M. Spence J.R. Generating human intestinal tissue from pluripotent stem cells in vitro.Nat. Protoc. 2011; 6: 1920-1928Crossref PubMed Scopus (241) Google Scholar), cerebral (Lancaster and Knoblich, 2014Lancaster M.A. Knoblich J.A. Generation of cerebral organoids from human pluripotent stem cells.Nat. Protoc. 2014; 9: 2329-2340Crossref PubMed Scopus (545) Google Scholar), and renal lineages (Takasato et al., 2014Takasato M. Er P.X. Becroft M. Vanslambrouck J.M. Stanley E.G. Elefanty A.G. Little M.H. Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney.Nat. Cell Biol. 2014; 16: 118-126Crossref PubMed Scopus (412) Google Scholar, Takasato et al., 2015Takasato M. Er P.X. Chiu H.S. Maier B. Baillie G.J. Ferguson C. Parton R.G. Wolvetang E.J. Roost M.S. Chuva de Sousa Lopes S.M. et al.Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.Nature. 2015; 526: 564-568Crossref PubMed Scopus (648) Google Scholar) have provided important disease models and an understanding of human embryonic development. Such an approach has the potential to provide an in vitro model of the human gonad. To date, several studies have used pluripotent cells to induce gonad-like cells: bipotential gonads (Sepponen et al., 2017Sepponen K. Lundin K. Knuus K. Väyrynen P. Raivio T. Tapanainen J.S. Tuuri T. The role of sequential BMP signaling in directing human embryonic stem cells to bipotential gonadal cells.J. Clin. Endocrinol. Metab. 2017; 102: 4303-4314Crossref PubMed Scopus (7) Google Scholar), Sertoli or Leydig cells (Bucay et al., 2009Bucay N. Yebra M. Cirulli V. Afrikanova I. Kaido T. Hayek A. Montgomery A.M.P. A novel approach for the derivation of putative primordial germ cells and sertoli cells from human embryonic stem cells.Stem Cells. 2009; 27: 68-77Crossref PubMed Scopus (115) Google Scholar; Buganim et al., 2012Buganim Y. Itskovich E. Hu Y.-C. Cheng A.W. Ganz K. Sarkar S. Fu D. Welstead G.G. Page D.C. Jaenisch R. Direct reprogramming of fibroblasts into embryonic Sertoli-like cells by defined factors.Cell Stem Cell. 2012; 11: 373-386Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar; Chen et al., 2019Chen X. Li C. Chen Y. Xi H. Zhao S. Ma L. Xu Z. Han Z. Zhao J. Ge R. et al.Differentiation of human induced pluripotent stem cells into Leydig-like cells with molecular compounds.Cell Death Dis. 2019; 10: 220Crossref PubMed Scopus (9) Google Scholar; Rodríguez Gutiérrez et al., 2018Rodríguez Gutiérrez D. Eid W. Biason-Lauber A. A human gonadal cell model from induced pluripotent stem cells.Front. Genet. 2018; 9: 498Crossref PubMed Scopus (15) Google Scholar; Jadhav and Jameson, 2011Jadhav U. Jameson J.L. Steroidogenic factor-1 (SF-1)-driven differentiation of murine embryonic stem (ES) cells into a gonadal lineage.Endocrinology. 2011; 152: 2870-2882Crossref PubMed Scopus (26) Google Scholar; Kjartansdóttir et al., 2015Kjartansdóttir K.R. Reda A. Panula S. Day K. Hultenby K. Soder O. Hovatta O. Stukenborg J.-B. A combination of culture conditions and gene expression analysis can Be used to investigate and predict hES cell differentiation potential towards male gonadal cells.PLoS One. 2015; 10: e0144029Crossref PubMed Scopus (11) Google Scholar; Yang et al., 2015Yang Y. Su Z. Xu W. Luo J. Liang R. Xiang Q. Zhang Q. Ge R.-S. Huang Y. Directed mouse embryonic stem cells into leydig-like cells rescue testosterone-deficient male rats in vivo.Stem Cells Dev. 2015; 24: 459-470Crossref PubMed Scopus (25) Google Scholar; Yang et al., 2017Yang Y. Li Z. Wu X. Chen H. Xu W. Xiang Q. Zhang Q. Chen J. Ge R.-S. Su Z. et al.Direct reprogramming of mouse fibroblasts toward leydig-like cells by defined factors.Stem Cell Rep. 2017; 8: 39-53Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar; Yazawa et al., 2006Yazawa T. Mizutani T. Yamada K. Kawata H. Sekiguchi T. Yoshino M. Kajitani T. Shou Z. Umezawa A. Miyamoto K. Differentiation of adult stem cells derived from bone marrow stroma into Leydig or adrenocortical cells.Endocrinology. 2006; 147: 4104-4111Crossref PubMed Scopus (110) Google Scholar), and germ cell lineages (Hayashi et al., 2011Hayashi K. Ohta H. Kurimoto K. Aramaki S. Saitou M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.Cell. 2011; 146: 519-532Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar; Irie et al., 2015Irie N. Weinberger N. Tang W. Kobayashi T. Viukov S. Manor Y.S. Dietmann S. Hanna J. Surani M.A. SOX17 is a critical specifier of human primordial germ cell fate.Cell. 2015; 160: 253-268Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar; Hikabe et al., 2016Hikabe O. Hamazaki N. Nagamatsu G. Obata Y. Hirao Y. Hamada N. Shimamoto S. Imamura T. Nakashima K. Saitou M. Hayashi K. Reconstitution in vitro of the entire cycle of the mouse female germ line.Nature. 2016; 539: 299-303Crossref PubMed Scopus (250) Google Scholar; Shlush et al., 2017Shlush E. Maghen L. Swanson S. Kenigsberg S. Moskovtsev S. Barretto T. Gauthier-Fisher A. Librach C.L. In vitro generation of Sertoli-like and haploid spermatid-like cells from human umbilical cord perivascular cells.Stem Cell Res. Ther. 2017; 8: 37Crossref PubMed Scopus (17) Google Scholar; Yamashiro et al., 2018Yamashiro C. Sasaki K. Yabuta Y. Kojima Y. Nakamura T. Okamoto I. Yokobayashi S. Murase Y. Ishikura Y. Shirane K. et al.Generation of human oogonia from induced pluripotent stem cells in vitro.Science. 2018; 362: 356-360Crossref PubMed Scopus (68) Google Scholar; Gell et al., 2020Gell J.J. Liu W. Sosa E. Chialastri A. Hancock G. Tao Y. Wamaitha S.E. Bower G. Dey S.S. Clark A.T. An extended culture system that supports human primordial germ cell-like cell survival and initiation of DNA methylation erasure.Stem Cell Rep. 2020; 14: 1-14Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Some have overexpressed transcription factors to directly reprogram mouse or human fibroblasts into Sertoli and Leydig-like cells (Buganim et al., 2012Buganim Y. Itskovich E. Hu Y.-C. Cheng A.W. Ganz K. Sarkar S. Fu D. Welstead G.G. Page D.C. Jaenisch R. Direct reprogramming of fibroblasts into embryonic Sertoli-like cells by defined factors.Cell Stem Cell. 2012; 11: 373-386Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar; Jadhav and Jameson, 2011Jadhav U. Jameson J.L. Steroidogenic factor-1 (SF-1)-driven differentiation of murine embryonic stem (ES) cells into a gonadal lineage.Endocrinology. 2011; 152: 2870-2882Crossref PubMed Scopus (26) Google Scholar; Yang et al., 2015Yang Y. Su Z. Xu W. Luo J. Liang R. Xiang Q. Zhang Q. Ge R.-S. Huang Y. Directed mouse embryonic stem cells into leydig-like cells rescue testosterone-deficient male rats in vivo.Stem Cells Dev. 2015; 24: 459-470Crossref PubMed Scopus (25) Google Scholar, Yang et al., 2017Yang Y. Li Z. Wu X. Chen H. Xu W. Xiang Q. Zhang Q. Chen J. Ge R.-S. Su Z. et al.Direct reprogramming of mouse fibroblasts toward leydig-like cells by defined factors.Stem Cell Rep. 2017; 8: 39-53Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar; Yazawa et al., 2006Yazawa T. Mizutani T. Yamada K. Kawata H. Sekiguchi T. Yoshino M. Kajitani T. Shou Z. Umezawa A. Miyamoto K. Differentiation of adult stem cells derived from bone marrow stroma into Leydig or adrenocortical cells.Endocrinology. 2006; 147: 4104-4111Crossref PubMed Scopus (110) Google Scholar). The ectopic expression of the transcription factors GATA4 and NR5A1 appears to be sufficient to reprogram human fibroblasts into Sertoli-like cells (Liang et al., 2019Liang J. Wang N. He J. Du J. Guo Y. Li L. Wu W. Yao C. Li Z. Kee K. Induction of Sertoli-like cells from human fibroblasts by NR5A1 and GATA4.Elife. 2019; 8: 301Crossref Scopus (10) Google Scholar). Other protocols have used co-cultures of human and mouse cells (Rodríguez Gutiérrez et al., 2018Rodríguez Gutiérrez D. Eid W. Biason-Lauber A. A human gonadal cell model from induced pluripotent stem cells.Front. Genet. 2018; 9: 498Crossref PubMed Scopus (15) Google Scholar; Shlush et al., 2017Shlush E. Maghen L. Swanson S. Kenigsberg S. Moskovtsev S. Barretto T. Gauthier-Fisher A. Librach C.L. In vitro generation of Sertoli-like and haploid spermatid-like cells from human umbilical cord perivascular cells.Stem Cell Res. Ther. 2017; 8: 37Crossref PubMed Scopus (17) Google Scholar; Yamashiro et al., 2018Yamashiro C. Sasaki K. Yabuta Y. Kojima Y. Nakamura T. Okamoto I. Yokobayashi S. Murase Y. Ishikura Y. Shirane K. et al.Generation of human oogonia from induced pluripotent stem cells in vitro.Science. 2018; 362: 356-360Crossref PubMed Scopus (68) Google Scholar) or co-cultures of human iPSCs with NT2D1 cells to differentiate Sertoli-like cells (Rodríguez Gutiérrez et al., 2018Rodríguez Gutiérrez D. Eid W. Biason-Lauber A. A human gonadal cell model from induced pluripotent stem cells.Front. Genet. 2018; 9: 498Crossref PubMed Scopus (15) Google Scholar). Finally, bone morphogenetic protein (BMP) and WNT signaling drives bipotential gonad marker expression in human ESCs without the requirement for co-culture or transfection/transduction (Sepponen et al., 2017Sepponen K. Lundin K. Knuus K. Väyrynen P. Raivio T. Tapanainen J.S. Tuuri T. The role of sequential BMP signaling in directing human embryonic stem cells to bipotential gonadal cells.J. Clin. Endocrinol. Metab. 2017; 102: 4303-4314Crossref PubMed Scopus (7) Google Scholar). Despite these advances, differentiation of control or patient iPSCs into a stable population that accurately recapitulates the early human embryonic gonad without the need for co-culture or lentiviral induction remains elusive. Furthermore, culturing of these differentiated cells in a three-dimensional (3D) organoid to model the complex cellular structures and interactions of the embryonic gonad has not yet been demonstrated. This study aimed to generate testis lineages from human iPSCs. Specifically, we employed a stepwise directed differentiation approach to guide cells through developmental cell populations that ultimately give rise to the bipotential gonads and early Sertoli cells. We present a novel feeder-free protocol for the induction of these lineages from human iPSCs in approximately 15 days. Furthermore, culturing of these cells in 3D led to the development of defined tissue structures with distinct expression of Sertoli cell markers. This work has given us insight into the potential origin and regulatory interactions during human gonad development. It represents a significant step toward a human iPSC-derived in vitro model for embryonic gonad/testis development and DSD. During embryonic development, the posterior primitive streak (PS) gives rise to the intermediate mesoderm (IM). In mice, the coelomic epithelium overlaying the IM develops into the somatic cells of the bipotential gonad (Karl and Capel, 1998Karl J. Capel B. Sertoli cells of the mouse testis originate from the coelomic epithelium.Dev. Biol. 1998; 203: 323-333Crossref PubMed Scopus (306) Google Scholar) (Figure 1A). To characterize cell identity during differentiation of human iPSCs to gonadal cells, we devised a panel of marker genes for gonad development (Figure 1 and Table S1). Publicly available RNA-sequencing (RNA-seq) data from mouse, including bulk RNA-seq from embryonic day (E) 10.5–E13.5 gonads (Zhao et al., 2018Zhao L. Wang C. Lehman M.L. He M. An J. Svingen T. Spiller C.M. Ng E.T. Nelson C.C. Koopman P. Transcriptomic analysis of mRNA expression and alternative splicing during mouse sex determination.Mol. Cell Endocrinol. 2018; 478: 84-96Crossref PubMed Scopus (13) Google Scholar) and single-cell RNA-seq (scRNA-seq) of sorted gonadal cells (Stévant et al., 2019Stévant I. Kuhne F. Greenfield A. Chaboissier M.-C. Dermitzakis E.T. Nef S. Dissecting cell lineage specification and sex fate determination in gonadal somatic cells using single-cell transcriptomics.Cell Rep. 2019; 26: 3272-3283Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), were interrogated to confirm marker expression. The Lhx9, Nr5A1, Gadd45g, Wt1, Gata4, Fog2/Zfpm2, Nr0B1, Emx2, and Hsd3b2 genes were chosen to represent the bipotential gonad. These are expressed in mouse embryonic gonads between E10.5 and E11.5 and then decrease as the gonads differentiate. Of these, Lhx9, Wt1, Gata4, and Emx2 have the earliest expression, in the early pre-Sertoli and pre-granulosa cells. Expression was also assessed in human fetal gonad datasets, including an RNA-seq dataset from 7–19 weeks gestation (Guo et al., 2015Guo F. Yan L. Guo H. Li L. Hu B. Zhao Y. Yong J. Hu Y. Wang X. Wei Y. et al.The transcriptome and DNA methylome landscapes of human primordial germ cells.Cell. 2015; 161: 1437-1452Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar) and scRNA-seq data (weeks 4–26 gestation ovaries and testis) (Li et al., 2017Li L. Dong J. Yan L. Yong J. Liu X. Hu Y. Fan X. Wu X. Guo H. Wang X. et al.Single-cell RNA-seq analysis maps development of human germline cells and gonadal Niche interactions.Cell Stem Cell. 2017; 20: 858-873Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Analysis using the ReproGenomics Viewer (Darde et al., 2015Darde T.A. Sallou O. Becker E. Evrard B. Monjeaud C. Le Bras Y. Jégou B. Collin O. Rolland A.D. Chalmel F. The ReproGenomics Viewer: an integrative cross-species toolbox for the reproductive science community.Nucleic Acids Res. 2015; 43: W109-W116Crossref PubMed Scopus (28) Google Scholar, Darde et al., 2019Darde T.A. Lecluze E. Lardenois A. Stévant I. Alary N. Tüttelmann F. Collin O. Nef S. Jégou B. Rolland A.D. et al.The ReproGenomics Viewer: a multi-omics and cross-species resource compatible with single-cell studies for the reproductive science community.Bioinformatics. 2019; 35: 3133-3139Crossref PubMed Scopus (23) Google Scholar) confirmed early expression of these bipotential gonad markers, with the caveat that in humans, LHX9, WT1, and EMX2 are expressed more strongly in the early female tissues (Figures S1A–S1C). These data confirmed the expected specificity of our bipotential gonad markers to the somatic cells, with the exception of GATA4, which also has some low-level expression in the primordial germ cells (Figure S1F). For testis, we chose SOX9, FGF9, AMH, CLAUDIN11 (CLDN11), and DHH as Sertoli cell markers. Sox9 and Fgf9 are among the earliest markers of testis development, along with Sry, in the mouse gonad (peaking at E11.5) and in humans (peaking at 7–7.5 weeks gestation) (Belle et al., 2017Belle M. Godefroy D. Couly G. Malone S.A. Collier F. Giacobini P. Chédotal A. Tridimensional Visualization and analysis of early human development.Cell. 2017; 169: 161-173.e12Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). In mouse gonads, Sox9 activates the Amh gene at E12. In mouse, Amh expression is also observed in granulosa and endothelial cells, whereas in humans it appears more specific to the Sertoli cells, as does the mature Sertoli cell marker CLDN11 (Figures S1E and S1J). Expression of the Leydig markers Star and Hsd3b1 peaks at later time points in mouse, as Leydig cells differentiate in response to Sertoli cell signaling. In the mouse, these markers are highly specific to testis, with little expression in ovary. In human, STAR appears to be expressed in both testis and ovarian cells (Figure S1G), and HSD3B1 shows almost no expression in human gonads (Figure S1H). GATA4 and NR5A1 also show Leydig expression in humans (Figures S1F and S1I). To address gonadal specificity, marker gene expression was evaluated in the IM-derived kidney and adrenal cortex. Expression datasets for human fetal kidney (Lindström et al., 2018Lindström N.O. McMahon J.A. Guo J. Tran T. Guo Q. Rutledge E. Parvez R.K. Saribekyan G. Schuler R.E. Liao C. et al.Conserved and divergent features of human and mouse kidney organogenesis.J. Am. Soc. Nephrol. 2018; 29: 785-805Crossref PubMed Scopus (89) Google Scholar), human iPSC-derived kidney organoids (Phipson et al., 2019Phipson B. Er P.X. Combes A.N. Forbes T.A. Howden S.E. Zappia L. Yen H.-J. Lawlor K.T. Hale L.J. Sun J. et al.Evaluation of variability in human kidney organoids.Nat. Meth. 2019; 16: 79-87Crossref PubMed Scopus (69) Google Scholar; Takasato et al., 2015Takasato M. Er P.X. Chiu H.S. Maier B. Baillie G.J. Ferguson C. Parton R.G. Wolvetang E.J. Roost M.S. Chuva de Sousa Lopes S.M. et al.Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.Nature. 2015; 526: 564-568Crossref PubMed Scopus (648) Google Scholar), and embryonic mouse kidney (Combes et al., 2019Combes A.N. Phipson B. Lawlor K.T. Dorison A. Patrick R. Zappia L. Harvey R.P. Oshlack A. Little M.H. Single cell analysis of the developing mouse kidney provides deeper insight into marker gene expression and ligand-receptor crosstalk.Development. 2019; 146: dev178673Crossref PubMed Scopus (35) Google Scholar) (Figures S1O–S1Q) revealed that several markers exhibited widespread expression, such as EMX2 and WT1, whereas GATA4, AMH, LHX9, and NR5A1 show greater gonad specificity. A microarray study on human fetal adrenal gland, kidney, and gonads (Del Valle et al., 2017Del Valle I. Buonocore F. Duncan A.J. Lin L. Barenco M. Parnaik R. Shah S. Hubank M. Gerrelli D. Achermann J.C. A genomic atlas of human adrenal and gonad development.Wellcome Open Res. 2017; 2: 25Crossref PubMed Scopus (35) Google Scholar) indicated that NR5A1, NR0B1, HSD3B2, and STAR are also expressed in developing adrenal lineages (Figures S1K–S1N). Markers to screen for unwanted cell types, i.e., precursor populations (pluripotent stem cells, OCT4; IM cells, LHX1, PAX2, OSR1; lateral plate mesoderm [LPM], FOXF1), non-gonadal IM-derived tissues (adrenal, ARHGAP36, SULT2A1; kidney, NPHS2), and ovarian cells (FOXL2 and NR0B1), were also included (Figure 1A and Table S1). Several gonadal markers were validated using immunofluorescence (IF) staining of week 9 human fetal testes, some of which have not been previously reported (Figures 1B–1U). GATA4 had the highest expression in the testis cords, where it showed strong nuclear staining with some weak staining or background outside of the cords. This protein also appeared to have low-level cytoplasmic staining (Figures 1B, 1D, and 1E). The Sertoli cell markers SOX9 (Figures 1G, 1I, and 1J), WT1 (Figures 1L, 1N, and 1O), and AMH (Figures 1Q, 1S, and 1T) had testis cord-specific staining. SOX9 is strongly nuclear with some weak cytoplasmic staining (Figure 1J), while WT1 is nuclear (Figure 1O) and AMH is mostly cytoplasmic (Figure 1T). OCT4 is nuclear in germ cells (Figures 1B, 1C, 1G, 1H, 1Q, and 1R). Laminin marked epithelial basement membrane throughout the testis, with strong expression surrounding the cords—best visualized at the edge of the gonadal section (Figures 1L and 1M). In humans, the bipotential gonad is widely considered to arise from the IM between weeks 5 and 6 of embryonic development (Satoh, 1991Satoh M. Histogenesis and organogenesis of the gonad in human embryos.J. Anat. 1991; 177: 85-107PubMed Google Scholar). The trunk mesoderm, including the IM, forms from cells arising from the posterior PS as the body axis elongates (Figure 2A) (Takasato and Little, 2015Takasato M. Little M.H. The origin of the mammalian kidney: implications for recreating the kidney in vitro.Development. 2015; 142: 1937-1947Crossref PubMed Scopus (74) Google Scholar), giving rise to a host of different tissues depending upon the positioning of these cells along the streak at the time of exit. As the trunk elongates, differentiation into distinct regions of organ primordia is specified by gradients of morphogenic factors signaling along the various axes, including craniocaudal, dorsoventral, and mediolateral. Hence, the patterning to gonad requires precise control of fate initially with respect to position within the PS and the axes of the body plan. As both the mammalian kidney and the gonad arise from the IM (Karl and Capel, 1998Karl J. Capel B. Sertoli cells of the mouse testis originate from the coelomic epithelium.Dev. Biol. 1998; 203: 323-333Crossref PubMed Scopus (306) Google Scholar), we used a renal differentiation protocol (Takasato et al., 2015Takasato M. Er P.X. Chiu H.S. Maier B. Baillie G.J. Ferguson C. Parton R.G. Wolvetang E.J. Roost M.S. Chuva de Sousa Lopes S.M. et al.Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.Nature. 2015; 526: 564-568Crossref PubMed Scopus (648) Google Scholar) as a starting point to create gonadal cells in vitro. This stepwise differentiation guides iPSCs through the posterior PS using the GSKβ inhibitor/WNT signaling activator, CHIR99021 (CHIR). The WNT signaling pathway patterns the anterior-posterior axis of the PS (Figure 2A), which can be altered by changing CHIR concentration or duration (Takasato et al., 2015Takasato M. Er P.X. Chiu H.S. Maier B. Baillie G.J. Ferguson C. Parton R.G. Wolvetang E.J. Roost M.S. Chuva de Sousa Lopes S.M. et al.Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.Nature. 2015; 526: 564-568Crossref PubMed Scopus (648) Google Scholar). To form kidney organoids, the differentiating cultures were guided toward an IM lineage (OSR1/LHX1/PAX2+) with FGF9 (Takasato et al., 2015Takasato M. Er P.X. Chiu H.S. Maier B. Baillie G.J. Ferguson C. Parton R.G. Wolvetang E.J. Roost M.S. Chuva de Sousa Lopes S.M. et al.Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.Nature. 2015; 526: 564-568Crossref PubMed Scopus (648) Google Scholar), although changes in the initial CHIR duration also appeared to shift between more cranial (anterior IM) and caudal (posterior IM) identities (Takasato et al., 2015Takasato M. Er P.X. Chiu H.S. Maier B. Baillie G.J. Ferguson C. Parton R.G. Wolvetang E.J. Roost M.S. Chuva de Sousa Lopes S.M. et al.Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.Nature. 2015; 526: 564-568Crossref PubMed Scopus (648) Google Scholar). We aimed to identify the optimal conditions under which gonadal precursor cells (GPCs) would arise using a male control iPSC line. The kidney protocol used 8 μM CHIR (Takasato et al., 2015Takasato M. Er P.X. Chiu H.S. Maier B. Baillie G.J. Ferguson C. Parton R.G. Wolvetang E.J. Roost M.S. Chuva de Sousa Lopes S.M. et al.Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.Nature. 2015; 526: 564-568Crossref PubMed Scopus (648) Google Scholar), but as a lower CHIR concentration has previously been shown to induce gonadal differentiation of human ESCs (Sepponen et al., 2017Sepponen K. Lundin K. Knuus K. Väyrynen P. Raivio T. Tapanainen J.S. Tuuri T. The role of sequential BMP signaling in directing human embryonic stem cells to bipotential gonadal cells.J. Clin. Endocrinol. Metab. 2017; 102: 4303-4314Crossref PubMed Scopus (7) Google Scholar), we started by using 4 μM CHIR with varied duration (3, 4, or 5 days with 4 μM CHIR followed by 200 ng/mL FGF9). Bipotential gonad and testis marker expression was assessed in differentiated cells relative to day 0 iPSCs (Figure 2B). After 7 days, the bipotential gonad markers LHX9, WT1, GATA4, ZFPM2, NR0B1, EMX2, and HSD3B2 were induced under all three conditions. Low-level induction of the early Sertoli marker SOX9 was also observed (Figure 2B). For many of these markers, expression was similar between CHIR durations, with the exception of LHX9, WT1, and HSD3B2, for which a longer CHIR treatment induced higher expression, and NR0B1, for which a shorter duration induced higher expression (Figure 2B). NR5A1 showed generally low expression, with significant induction seen only with CHIR treatment for 5 days. Notably, GADD45g was also expressed in the day 0 iPSCs, and its expression did not increase after 7 days under any condition. AMH was not upregulated under any condition, indicating that additional factors may be necessary to induce Sertoli cell maturation. Given these results, an intermediate duration (4 days) of CHIR was used subsequently. Next, CHIR concentration was tested (3, 4, or 5 μM) with markers assessed at day 7 (Figure 2C) and day 12 (Figure 2D). At day 7, the bipotential gonad markers LHX9, GATA4, and EMX2 were highly induced and GADD45g and WT1 were induced at lower levels (Figure 2C). Of these markers, LHX9, EMX2, and WT1 responded best to 5 μM CHIR, whereas GATA4 and GADD45g responded best to 3 μM (Figure 2C). After 12 days of differentiation, continued induction of the bipotential gonad markers was observed, with significant induction of LHX9 under all three conditions (Figure 2D). GADD45g, WT1, GATA4, and EMX2 responded best to 3 μM CHIR. By day 12, evidence for the activation of the testis pathway (Sertoli cell markers) was also observed, with a 50- to 300-fold induction of SOX9 and a 20- to 50-fold increase in AMH expression relative to the day 0 control (Figure 2D). Hence, 3 μM CHIR for 4 days is optimal for gonadal/testis marker induction. Along the mediolateral axis, the mesoderm forms paraxial mesoderm, IM, and LPM from the center to the periphery, respectively (Figure 3A). Although often described as discrete regions, these likely represent a continuum patterned by growth factor gradients both mediolaterally and dorsoventrally. The best characterized of the mediolateral patterning factors is BMP4. BMP4 is produced within the LPM, and BMP pathway activity is hi" @default.
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• W3100505853 date "2020-12-01" @default.
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• W3100505853 title "An In Vitro Differentiation Protocol for Human Embryonic Bipotential Gonad and Testis Cell Development" @default.
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• W3100505853 doi "https://doi.org/10.1016/j.stemcr.2020.10.009" @default.
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