Human Primordial Germ Cells Are Specified from Lineage-Primed Progenitors

doi:10.1016/j.celrep.2019.11.083

. 2019 Dec 24;29(13):4568-4582.e5.

doi: 10.1016/j.celrep.2019.11.083.

Human Primordial Germ Cells Are Specified from Lineage-Primed Progenitors

Di Chen¹, Na Sun², Lei Hou², Rachel Kim³, Jared Faith¹, Marianna Aslanyan¹, Yu Tao¹, Yi Zheng⁴, Jianping Fu⁵, Wanlu Liu⁶, Manolis Kellis⁷, Amander Clark⁸

Affiliations

¹ Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
² MIT Computer Science and Artificial Intelligence Laboratory, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
³ Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁴ Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
⁵ Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA; Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
⁶ Zhejiang University-University of Edinburgh Institute, Zhejiang University School of Medicine, 310058 Hangzhou, PR China.
⁷ MIT Computer Science and Artificial Intelligence Laboratory, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address: manoli@mit.edu.
⁸ Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: clarka@ucla.edu.

PMID: 31875561
PMCID: PMC6939677
DOI: 10.1016/j.celrep.2019.11.083

Human Primordial Germ Cells Are Specified from Lineage-Primed Progenitors

Di Chen et al. Cell Rep. 2019.

. 2019 Dec 24;29(13):4568-4582.e5.

doi: 10.1016/j.celrep.2019.11.083.

Authors

Di Chen¹, Na Sun², Lei Hou², Rachel Kim³, Jared Faith¹, Marianna Aslanyan¹, Yu Tao¹, Yi Zheng⁴, Jianping Fu⁵, Wanlu Liu⁶, Manolis Kellis⁷, Amander Clark⁸

Affiliations

¹ Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
² MIT Computer Science and Artificial Intelligence Laboratory, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
³ Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁴ Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
⁵ Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA; Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
⁶ Zhejiang University-University of Edinburgh Institute, Zhejiang University School of Medicine, 310058 Hangzhou, PR China.
⁷ MIT Computer Science and Artificial Intelligence Laboratory, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address: manoli@mit.edu.
⁸ Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: clarka@ucla.edu.

PMID: 31875561
PMCID: PMC6939677
DOI: 10.1016/j.celrep.2019.11.083

Abstract

In vitro gametogenesis is the process of making germline cells from human pluripotent stem cells. The foundation of this model is the quality of the first progenitors called primordial germ cells (PGCs), which in vivo are specified during the peri-implantation window of human development. Here, we show that human PGC (hPGC) specification begins at day 12 post-fertilization. Using single-cell RNA sequencing of hPGC-like cells (hPGCLCs) differentiated from pluripotent stem cells, we discovered that hPGCLC specification involves resetting pluripotency toward a transitional state with shared characteristics between naive and primed pluripotency, followed by differentiation into lineage-primed TFAP2A⁺ progenitors. Applying the germline trajectory to TFAP2C mutants reveals that TFAP2C functions in the TFAP2A⁺ progenitors upstream of PRDM1 to regulate the expression of SOX17. This serves to protect hPGCLCs from crossing the Weismann's barrier to adopt somatic cell fates and, therefore, is an essential mechanism for successfully initiating in vitro gametogenesis.

Keywords: TFAP2A; TFAP2C; germ cells; pluripotency; single cell RNA-sequencing; stem cells.

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Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

**Figure 1.. Tracking the Specification of Human PGCs**
(A) Schematic illustration of human embryos used in this study. Frozen embryos at day 5 or day 6 post-fertilization were thawed and allowed to recover for 24 h to become day 6 or day 7, staining or culturing until day 12 as human embryo attachment cultures. Human embryos at weeks 4–5 were stained as positive controls for identifying hPGCs. (B) Human embryo attachment culture at day 12 stained for TFAP2C (purple), SOX17 (yellow), and NANOG (cyan). Two TFAP2C/SOX17/NANOG triple-positive cells are highlighted by a white arrowhead, one of which is enlarged in inset. The number in the 4′,6-diamidino-2-phenylindole (DAPI) (gray) panel indicates triple-positive cells were identified in two day 12 embryos from a total of 26 embryos examined at this time point. (C) Schematic illustration of hPGCLC differentiation. The UCLA1 and UCLA2 hESC lines are induced to differentiate into iMeLCs for 24 h, followed by differentiation as 3D disorganized aggregates for 4 days. Samples were collected at all six time points from each hESC line in biological duplicate for 10x Genomics scRNA-seq. (D) Identification of ITGA6/EPCAM double-positive hPGCLCs by flow cytometry from UCLA1 and UCLA2. (E) Percentage of ITGA6/EPCAM double-positive hPGCLCs isolated from UCLA1 and UCLA2 at day 4 of differentiation. ***, indicates statistically significant difference comparing more than 10 biological replicates of UCLA1 and UCLA2 at day 4 of differentiation (shown is mean and SEM) (F) Immunofluorescence of TFAP2C (purple), SOX17 (yellow), and NANOG (cyan) in day 1–4 aggregates from UCLA1 (top) and UCLA2 (bottom). The percentage of TFAP2C/SOX17/NANOG triple-positive cells at each day is shown. Percentages were calculated from five or more biological replicates at each day. (G and H) UMAP display of all cells in the UCLA1 (G) and UCLA2 (H) dataset in biological duplicate (n = 12 samples for each cell line). The expression of *NANOS3* indicates hPGCLCs. Scale bars: 50 μM. See also Figure S1.

**Figure 2.. Highly Germline-Competent hESC Lines Specify More hPGCs within 48 h of BMP4 Exposure**
(A and B) The emergence of *TFAP2C*/*SOX17*/*NANOG*/*NANOS3* quadruple-positive cells in UCLA1 (A) and UCLA2 (B) by scRNA-seq. UMAP was applied to visualize all single cells at each stage. The percentage of *TFAP2C*/*SOX17*/*NANOG*/*NANOS3* quadruple-positive cells (red) is shown. Cutoff: log-transformed-normalized UMI counts of >0.5 for *TFAP2C*, *SOX17*, *NANOG*, and *NANOS3*. (C and D) URD tree showing the developmental trajectories from hESC and iMeLCs through 4 days of aggregate differentiation from UCLA1 (C) and UCLA2 (D). The expression of *NANOS3* marks the end of the germline trajectory and establishment of hPGCLCs in (C) and (D). Cells with endoderm (Endo) identity are outside of the germline trajectory, indicating hPGCLCs do not originate from *SOX17*-positive endoderm. VE/YE, visceral/yolk-sac endoderm; EXMC, extra-embryonic mesenchyme; Pre-EPIs, pre-implantation epiblasts; PostE-EPI, post-implantation early epiblasts; Post-TE, post-implantation trophectoderm; Gast, gastrulating cells; AMLCs, amnion-like cells (Nakamura et al., 2016; Zheng et al., 2019). See also Figure S2.

**Figure 3.. hPGCLC Specification through a Translational Germinal Pluripotent State**
(A) The expression patterns of *TFAP2C*, *NANOG*, *SOX2*, *POU5F1*(*OCT4*), *SOX17*, and *NANOS3* within the germline trajectory of UCLA2. Dash lines outline iMeLCs in germline trajectory in (A)–(C). (B) Relative average expression of signature genes from cynomolgus ICM (inner cell mass), Pre-EPIs, PostE-EPIs, and PostL-EPI (post-implantation late epiblast) (Nakamura et al., 2016) in germline trajectory of UCLA2. (C) Relative average expression of signature genes from primed and naive hESCs (Messmer et al., 2019) in germline trajectory of UCLA2. Signature score bar is same as (B). (D) Heatmap showing the expression of differentially expressed genes in naive, primed, and transitional germinal pluripotent cells. (E) UMAP displaying the single cells from day 12 human embryonic cells. (F) Relative expression of signature genes in the 13 epiblasts cells from (E) compared to primed and naive hESCs (Messmer et al., 2019) and cells from iMeLCs, day 1 to day 4 cells in germline trajectory. See also Figure S3.

**Figure 4.. hPGCLC Specification Involves a Trajectory through a Transient TFAP2A-Positive State with a Signature of Both Amnion and Gastrulating Cell Fates**
(A and B) The expression patterns of *EMOES*, T, *CDX2*, *GATA3*, and *TFAP2A* within germline trajectory of UCLA1 (A) and UCLA2 (B). (C) Relative average expression of signature genes from cynomolgus Gast (gastrulating cells), ePGC (early PGCs) (Nakamura et al., 2016) and human AMLCs and hPGCLC at day 2 (Zheng et al., 2019) in germline trajectory of UCLA2. (D) Expression of *TFAP2A*, T, *EOMES*, and *MIXL1* in day 1 progenitor population from UCLA2. See also Figure S4.

**Figure 5.. TFAP2C Acts Upstream of SOX17**
(A) Heatmap showing the differentially expressed genes in the UCLA2 germline trajectory. Signature transcription factors (TFs) and predicted regulators are shown for each cluster. (B) Network showing the regulatory role of TFAP2C and its putative targets in the germline trajectory of UCLA2. Red arrows highlight the predicted direct regulation of SOX17 by TFAP2C. (C) Tracks showing the ATAC-seq, TFAP2C ChIP-seq, H3K27ac ChIP-seq, and RNA-seq peaks around *SOX17* site at each stage. See also Figure S5.

**Figure 6.. TFAP2C Mutant Cells Exit the Germline Trajectory to Become Primitive Streak or Amnion-like Somatic Cells**
(A) Experimental design comparing scRNA-seq libraries of *TFAP2C* mutant (*T2C*^−/−) hESCs, iMeLCs, day 1, day 2, day 3, and day 4 to wild-type cells of the same genetic background (UCLA1). (B) UMAP showing wild-type and *T2C*^−/− single cells. (C) Overlay from wild-type UCLA1 (blue) and *T2C*^−/− cells (pink) from each of the six time points. Clusters that are largely pink are enriched in mutant cells; clusters that are largely blue are enriched in wild-type cells. (D) *NANOS3* expression indicates hPGCLCs. (E) URD trees showing the differentiation trajectories of all cells combined (wild-type and *T2C*^−/− mutant). (F) Overlay of *TFAP2C*^−/− cells (pink) and wild-type UCLA1 (blue) cells in the URD trees illustrates cell trajectories enriched in either mutant (pink) or control cells (blue) cells. Percentages of *TFAP2C* ^−/− cells in each of the blades of the trajectories. (G and H) *NANOS3* (G) and *SOX17* (H) highlight the germline trajectory ending in hPGCLCs (G) and hPGCLCs and VE/YE (H) Red box indicates the mutant-enriched trajectory that originates from the common progenitor with hPGCLCs. (I and J) URD tree showing the expression of somatic (primitive-streak or early amnion genes) *MIXL1* (I) and T (J), in the *TFAP2C*^−/− enriched blade. (K) Comparison of cynomolgus Pre-EPI, PostL-EPI, and ePGCs (Nakamura et al., 2016) signatures in germline trajectory between wild-type and *TFAP2C* mutant cells. See also Figure S5.

**Figure 7.. Proposed Model**
Human PGCs are specified beginning at day 12. Based on the *in vitro* germline trajectory, we propose that hPGCs originate from a lineage-primed *TFAP2A*⁺ progenitor at around day 11 which is specified from a transitional pluripotent state that shares characteristics with pre- and post-implantation epiblasts that we have called ‘‘germinal pluripotency.’’ *TFAP2A*⁺ progenitors have the potential for hPGC specification and also for differentiation of somatic cells, including amnion and gastrulating cells. TFAP2C functions at the critical point of hPGC specification to directly regulate expression of the human germ cell fate determinant SOX17, while simultaneously preventing germline cells from crossing Weismann’s barrier to become somatic cells.

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References

1. Blakeley P, Fogarty NME, Del Valle I, Wamaitha SE, Hu TX, Elder K, Snell P, Christie L, Robson P, and Niakan KK (2015). Defining the three cell lineages of the human blastocyst by single-cell RNA-seq. Development 142, 3151–3165. - PMC - PubMed
1. Butler A, Hoffman P, Smibert P, Papalexi E, and Satija R (2018). Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol 36, 411–420. - PMC - PubMed
1. Chen D, Liu W, Lukianchikov A, Hancock GV, Zimmerman J, Lowe MG, Kim R, Galic Z, Irie N, Surani MA, et al. (2017). Germline competency of human embryonic stem cells depends on eomesodermin. Biol. Reprod 97, 850–861. - PMC - PubMed
1. Chen D, Liu W, Zimmerman J, Pastor WA, Kim R, Hosohama L, Ho J, Aslanyan M, Gell JJ, Jacobsen SE, and Clark AT (2018). The TFAP2C-Regulated OCT4 Naive Enhancer Is Involved in Human Germline Formation. Cell Rep 25, 3591–3602.e5. - PMC - PubMed
1. Deglincerti A, Croft GF, Pietila LN, Zernicka-Goetz M, Siggia ED, and Brivanlou AH (2016). Self-organization of the in vitro attached human embryo. Nature 533, 251–254. - PubMed

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[1] Blakeley P, Fogarty NME, Del Valle I, Wamaitha SE, Hu TX, Elder K, Snell P, Christie L, Robson P, and Niakan KK (2015). Defining the three cell lineages of the human blastocyst by single-cell RNA-seq. Development 142, 3151–3165. - PMC - PubMed

[2] Blakeley P, Fogarty NME, Del Valle I, Wamaitha SE, Hu TX, Elder K, Snell P, Christie L, Robson P, and Niakan KK (2015). Defining the three cell lineages of the human blastocyst by single-cell RNA-seq. Development 142, 3151–3165. - PMC - PubMed

[3] Butler A, Hoffman P, Smibert P, Papalexi E, and Satija R (2018). Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol 36, 411–420. - PMC - PubMed

[4] Butler A, Hoffman P, Smibert P, Papalexi E, and Satija R (2018). Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol 36, 411–420. - PMC - PubMed

[5] Chen D, Liu W, Lukianchikov A, Hancock GV, Zimmerman J, Lowe MG, Kim R, Galic Z, Irie N, Surani MA, et al. (2017). Germline competency of human embryonic stem cells depends on eomesodermin. Biol. Reprod 97, 850–861. - PMC - PubMed

[6] Chen D, Liu W, Lukianchikov A, Hancock GV, Zimmerman J, Lowe MG, Kim R, Galic Z, Irie N, Surani MA, et al. (2017). Germline competency of human embryonic stem cells depends on eomesodermin. Biol. Reprod 97, 850–861. - PMC - PubMed

[7] Chen D, Liu W, Zimmerman J, Pastor WA, Kim R, Hosohama L, Ho J, Aslanyan M, Gell JJ, Jacobsen SE, and Clark AT (2018). The TFAP2C-Regulated OCT4 Naive Enhancer Is Involved in Human Germline Formation. Cell Rep 25, 3591–3602.e5. - PMC - PubMed

[8] Chen D, Liu W, Zimmerman J, Pastor WA, Kim R, Hosohama L, Ho J, Aslanyan M, Gell JJ, Jacobsen SE, and Clark AT (2018). The TFAP2C-Regulated OCT4 Naive Enhancer Is Involved in Human Germline Formation. Cell Rep 25, 3591–3602.e5. - PMC - PubMed

[9] Deglincerti A, Croft GF, Pietila LN, Zernicka-Goetz M, Siggia ED, and Brivanlou AH (2016). Self-organization of the in vitro attached human embryo. Nature 533, 251–254. - PubMed

[10] Deglincerti A, Croft GF, Pietila LN, Zernicka-Goetz M, Siggia ED, and Brivanlou AH (2016). Self-organization of the in vitro attached human embryo. Nature 533, 251–254. - PubMed

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Human Primordial Germ Cells Are Specified from Lineage-Primed Progenitors

Affiliations

Human Primordial Germ Cells Are Specified from Lineage-Primed Progenitors

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