Monolayer platform to generate and purify primordial germ-like cells in vitro provides insights into human germline specification

Sivakamasundari Vijayakumar; Roberta Sala; Gugene Kang; Angela Chen; Michelle Ann Pablo; Abidemi Ismail Adebayo; Andrea Cipriano; Jonas L Fowler; Danielle L Gomes; Lay Teng Ang; Kyle M Loh; Vittorio Sebastiano

doi:10.1038/s41467-023-41302-w

Monolayer platform to generate and purify primordial germ-like cells in vitro provides insights into human germline specification

Nat Commun. 2023 Sep 14;14(1):5690. doi: 10.1038/s41467-023-41302-w.

Authors

Sivakamasundari Vijayakumar^#^{1

2}, Roberta Sala^#^{1

3}, Gugene Kang^#^{1

3}, Angela Chen^{1

2}, Michelle Ann Pablo^{1

3}, Abidemi Ismail Adebayo^{1

3

4}, Andrea Cipriano^{1

3}, Jonas L Fowler^{1

2}, Danielle L Gomes^{1

3}, Lay Teng Ang¹, Kyle M Loh^{5

6}, Vittorio Sebastiano^{7

8}

Affiliations

¹ Institute for Stem Cell Biology & Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, 94305, USA.
² Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA, 94305, USA.
³ Department of Obstetrics & Gynecology, Stanford University School of Medicine, Stanford, CA, 94305, USA.
⁴ Department of Mechanical Engineering, Stanford University, Stanford, CA, 94305, USA.
⁵ Institute for Stem Cell Biology & Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, 94305, USA. kyleloh@stanford.edu.
⁶ Department of Obstetrics & Gynecology, Stanford University School of Medicine, Stanford, CA, 94305, USA. kyleloh@stanford.edu.
⁷ Institute for Stem Cell Biology & Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, 94305, USA. vsebast@stanford.edu.
⁸ Department of Obstetrics & Gynecology, Stanford University School of Medicine, Stanford, CA, 94305, USA. vsebast@stanford.edu.

^# Contributed equally.

Abstract

Generating primordial germ cell-like cells (PGCLCs) from human pluripotent stem cells (hPSCs) advances studies of human reproduction and development of infertility treatments, but often entails complex 3D aggregates. Here we develop a simplified, monolayer method to differentiate hPSCs into PGCs within 3.5 days. We use our simplified differentiation platform and single-cell RNA-sequencing to achieve further insights into PGCLC specification. Transient WNT activation for 12 h followed by WNT inhibition specified PGCLCs; by contrast, sustained WNT induced primitive streak. Thus, somatic cells (primitive streak) and PGCLCs are related-yet distinct-lineages segregated by temporally-dynamic signaling. Pluripotency factors including NANOG are continuously expressed during the transition from pluripotency to posterior epiblast to PGCs, thus bridging pluripotent and germline states. Finally, hPSC-derived PGCLCs can be easily purified by virtue of their CXCR4⁺PDGFRA^-GARP^- surface-marker profile and single-cell RNA-sequencing reveals that they harbor transcriptional similarities with fetal PGCs.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Cell Differentiation
Embryonic Development*
Fetus
Germ Cells*
Humans
RNA

Substances

RNA

Grants and funding

DP5 OD024558/OD/NIH HHS/United States