Scalable and efficient generation of mouse primordial germ cell-like cells

Xinbao Ding, Liangdao Li, Jingyi Gao, Dain Yi, John C Schimenti

Preprint posted on 15 February 2024 https://pubmed.ncbi.nlm.nih.gov/38405756/

A reliable and cost-effective method to produce germ cells at scale… what’s not to love?

Selected by Carly Guiltinan

Categories: bioengineering, cell biology, developmental biology

Background

Primordial germ cells (PGCs), the precursors of oocytes and sperm, are specified during early embryonic development and represent the genetic link between generations. Recent studies have examined the transcriptional program that supports PGC specification – as well as the potential to replicate this process in vitro from embryonic stem cells (ESCs) – in several mammalian species, but the majority of progress has been made in the mouse. Namely, reconstitution of the entire cycle of oogenesis and formation of spermatid-like cells capable of generating offspring have been shown in mice^1,2. The generation of PGC-like cells (PGCLCs) from ESCs for these reports has relied upon exogenous cytokines in the medium³, which recapitulate the signaling cascade initiated by extraembryonic structures that support PGC formation in vivo. Notably, long-term maintenance and expansion of PGCLCs have been difficult, which limits the feasibility of large-scale germline development studies.

Germline specification occurs in the peri-gastrula from a subset of cells that are initially destined for a somatic mesodermal fate. In the mouse, PGC specification initiates by selective activation of Prdm1 and Prdm14, and downstream initiation of Tfap2c expression⁴. Cells with this profile comprise the founder population of PGCs, which then migrate to the forming gonads while proliferating and undergoing extensive epigenetic reprogramming crucial for later stages of germline development. Uniquely, PGCs retain pluripotency (i.e., Nanog), which is important for repressing somatic cell lineages and acquiring markers of the germ cell program.

Rather than utilizing cytokines to modulate cell signaling, another approach to enable efficient induction of a specific cell type is by overexpression of transcription factors. One study showed that overexpressing Prdm1, Prdm14, and Tfap2c in mouse ESCs was sufficient to induce the germ cell lineage, but these cells began to differentiate shortly after induction⁵. Recent progress understanding the functions of other genes, including Nanog, in maintaining early PGC identity⁶, as well as the establishment of conditions that support formative ESCs that are permissive to direct germline induction⁷, have warranted further studies to increase the scalability of production and long-term expansion of PGCLCs.

Objectives

This study aimed to produce a scalable and efficient system to produce ample mouse PGCLCs without the use of cytokines in the medium. To do so, the authors simultaneously overexpressed the PGC master regulator transcription factors (Prdm1, Prdm14, and Tfap2c) along with a pluripotency gene important for retaining PGC identity (Nanog) – hereafter called the 4TF system – in formative mouse ESCs and epiblast-like cells (EpiLCs). Then they assessed the profile and functional capacity of the resulting cells to give context of their developmental status, and tested the potential scale-up and long-term culture of 4TF-induced PGCLCs.

Key findings

(1) The 4TF system supports efficient PGCLC generation and long-term culture without cytokines. First, the authors sought to uncover the effect of Nanog overexpression in PGCLC induction outcomes, alone or in combination with the PGC master regulators. They found that the addition of the factors increased the efficiency from ~30% for Nanog alone to ~80% for all four factors (Fig. 1), compared to ~70% described for cytokine-based PGCLC induction in formative mouse ESCs⁷. This system worked with either mouse EpiLCs, which have been a widely used intermediate for PGCLC induction from naïve ESCs, or recently described formative ESCs. Interestingly, the authors found an inverse relationship between size of the 3D cell aggregate (embryoid body) and PGCLC differentiation efficiency, highlighting the utility of smaller aggregates in larger well sizes for scalability purposes. When cultured in the presence of Dox and passaged every 4 days, 4TF-PGCLCs could be maintained long-term, with >70% of cells expressing characteristic PGC markers at day 43, which was likely aided by the role of Nanog in retaining early germline identity.

**Figure 1**. Percentage of PGCLCs (inferred by Stella-eGFP) for combinations of Nanog and PGC master regulator gene overexpression (Preprint figure 1A)

(2) 4TF-induced PGCLCs resemble E10.5-11.5 PGCs and show potential for continued in vitro development. RNA-sequencing analysis of 4TF-PGCLCs at day 2, 4, and 6-post induction was compared against published data from cytokine-induced PGCLCs, E9.5-13.5 in vivo PGCs, as well as ESCs and EpiLCs (Fig. 2). Interestingly, 4TF-PGCLCs clustered closely to E10.5-11.5 PGCs while cytokine-PGCLCs were more similar to E9.5-10.5 PGCs, suggesting that the overexpression system captured PGCLCs with a more developmentally advanced phenotype. In addition, the day 2 cytokine-induced samples were distinct from cells induced by the 4TF system or collected at day 4-6 following cytokine induction, indicating delayed initiation of the germ cell program compared to 4TF which was relatively homogeneous across the three timepoints. Culturing 4TF-PGCLCs in the presence of neonatal testicular tissue led to expression of later germline markers, including DDX4 and GCNA, suggesting their capacity to be used for in vitro gametogenesis.

**Figure 2**. Principal component analysis of RNA sequencing data from PGCLCs induced by 4TF- or cytokine (Ck)-system at day 2, 4, and 6, in vivo PGC at E9.5-13.15, and starting cell type (ESC, EpiLC; Preprint figure 2A)

Importance

Several uses of PGCLCs, including genomic screening and in vitro gametogenesis, require a high number of cells and therefore a method that can reproducibly and affordably produce cells at scale. Overexpression of 4 transcription factors permitted efficient mouse PGCLC induction without the need for cytokines, providing a cost-effective alternative. Moreover, the resulting cells could be maintained long-term in the presence of Dox. Collectively, these findings indicate that the system used in this study can reliably produce and maintain PGCLCs that can be used for large-scale studies of germ cell development.

Questions for the authors

Were you surprised by or do you have theories behind the 4TF-induced PGCLCs having a more developmentally advanced phenotype compared to cytokine-induced PGCLCs?
During the long-term culture period (day 6-43), were the PGCLCs highly proliferative as would be expected for ~E11 PGCs? Did you examine the transcriptomic profile of cells upon long-term culture, or do you plan to?
To speak to the scalability of this system, could you loosely approximate the cost of 4TF- vs. cytokine-based PGCLC induction for any given starting number of cells?
What are you hoping to test next using 4TF-induced PGCLCs?

References

Hikabe, O. et al. Reconstitution in vitro of the entire cycle of the mouse female germ line. Nature 539, 299–303 (2016).
Zhou, Q. et al. Complete meiosis from embryonic stem cell-derived germ cells in vitro. Cell Stem Cell 18, 330–340 (2016).
Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S. & Saitou, M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146, 519–532 (2011).
Magnúsdóttir, E. et al. A tripartite transcription factor network regulates primordial germ cell specification in mice. Nat Cell Biol 15, 905–915 (2013).
Nakaki, F. et al. Induction of mouse germ-cell fate by transcription factors in vitro. Nature 501, 222–226 (2013).
Murakami, K. et al. NANOG alone induces germ cells in primed epiblast in vitro by activation of enhancers. Nature 529, 403–407 (2016).
Yu, L. et al. Derivation of intermediate pluripotent stem cells amenable to primordial germ cell specification. Cell Stem Cell 28, 550-567.e12 (2021).

Posted on: 5 March 2024 , updated on: 7 March 2024

doi: https://doi.org/10.1242/prelights.36667

Read preprint

(No Ratings Yet)

Author's response

Xinbao Ding & John Schimenti shared

1) Were you surprised by or do you have theories behind the 4TF-induced PGCLCs having a more developmentally advanced phenotype compared to cytokine-induced PGCLCs?

We were indeed surprised on the developmentally advanced phenotype exhibited by 4TF-induced PGCLCs. Our analysis indicated that some pivotal downstream genes, such as DMRT1 and DAZL, play a facilitating role in the transition of PGCLC from early to late stages.

2) During the long-term culture period (day 6-43), were the PGCLCs highly proliferative as would be expected for ~E11 PGCs? Did you examine the transcriptomic profile of cells upon long-term culture, or do you plan to?

We are planning to characterize the long-term cultured PGCLCs and compare them to in vivo germ cells and PGCLCs at days 2/4/6. It will be very convenient if they are comparable to day 6 PGCLCs.

3) To speak to the scalability of this system, could you loosely approximate the cost of 4TF- vs. cytokine-based PGCLC induction for any given starting number of cells?

For 10ml of PGCLC differentiation media, the cost of cytokines is up to $456 (for small aliquots of BMP4, for instance). Given the difference in differentiation efficiency of the standard procedure compared to the 4TF system, we estimate that using cytokines to produce the same number of PGCLCs is >500-fold higher.

4) What are you hoping to test next using 4TF-induced PGCLCs?

We developed the platform with the ultimate goals of using it to define 1) genetic and 2) environmental factors (namely those causing epigenetic disruptions) affecting fertility. For the latter, we’ve already used the system to conduct a CRISPRi screen of epigenetic genes that impact mouse PGCLC differentiation. It is posted on bioRxiv (https://doi.org/10.1101/2024.02.26.582097). Regarding #1, we aim to use the system for functional evaluation of genetic variants (i.e., SNPs) in vitro (we’ve done a lot of work making mouse models, but that is expensive and not highly scalable). Accordingly, we are working towards further differentiating the PGCLCs through the spermatogonial and meiosis stages, which would expand the types of SNPs/genes that would be screenable. Additionally, we are working on porting the system into human iPSCs.

Have your say Cancel reply

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Also in the bioengineering category:

Generalized Biomolecular Modeling and Design with RoseTTAFold All-Atom

Rohith Krishna, Jue Wang, Woody Ahern, et al.

Selected by Saanjbati Adhikari

Multi-pass, single-molecule nanopore reading of long protein strands with single-amino acid sensitivity

Keisuke Motone, Daphne Kontogiorgos-Heintz, Jasmine Wee, et al.

Selected by Benjamin Dominik Maier, Samantha Seah

Discovery and Validation of Context-Dependent Synthetic Mammalian Promoters

Adam M. Zahm, William S. Owens, Samuel R. Himes, et al.

Selected by Jessica L. Teo

Also in the cell biology category:

Fetal brain response to maternal inflammation requires microglia

Bridget Elaine LaMonica Ostrem, Nuria Dominguez Iturza, Jeffrey Stogsdill, et al.

Selected by Manuel Lessi

Alteration of long and short-term hematopoietic stem cell ratio causes myeloid-biased hematopoiesis

Katsuyuki Nishi, Taro Sakamaki, Akiomi Nagasaka, et al.

Selected by Manuel Vicente, Anaisa Ferreira

Clusters of lineage-specific genes are anchored by ZNF274 in repressive perinucleolar compartments

Martina Begnis, Julien Duc, Sandra Offner, et al.

Selected by Silvia Carvalho

Also in the developmental biology category:

Alteration of long and short-term hematopoietic stem cell ratio causes myeloid-biased hematopoiesis

Katsuyuki Nishi, Taro Sakamaki, Akiomi Nagasaka, et al.

Selected by Manuel Vicente, Anaisa Ferreira

Temporal constraints on enhancer usage shape the regulation of limb gene transcription

Raquel Rouco, Antonella Rauseo, Guillaume Sapin, et al.

Selected by María Mariner-Faulí

OGT prevents DNA demethylation and suppresses the expression of transposable elements in heterochromatin by restraining TET activity genome-wide

Hugo Sepulveda, Xiang Li, Xiaojing Yue, et al.

Selected by Mansi

preLists in the bioengineering category:

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

Scalable and efficient generation of mouse primordial germ cell-like cells

Background

Objectives

Key findings

Importance

Questions for the authors

References

Share this:

Have your say Cancel reply

Sign up to customise the site to your preferences and to receive alerts

Also in the bioengineering category:

Generalized Biomolecular Modeling and Design with RoseTTAFold All-Atom

Multi-pass, single-molecule nanopore reading of long protein strands with single-amino acid sensitivity

Discovery and Validation of Context-Dependent Synthetic Mammalian Promoters

Also in the cell biology category:

Fetal brain response to maternal inflammation requires microglia

Alteration of long and short-term hematopoietic stem cell ratio causes myeloid-biased hematopoiesis

Clusters of lineage-specific genes are anchored by ZNF274 in repressive perinucleolar compartments

Also in the developmental biology category:

Alteration of long and short-term hematopoietic stem cell ratio causes myeloid-biased hematopoiesis

Temporal constraints on enhancer usage shape the regulation of limb gene transcription

OGT prevents DNA demethylation and suppresses the expression of transposable elements in heterochromatin by restraining TET activity genome-wide

preLists in the bioengineering category:

CSHL 87th Symposium: Stem Cells

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

3D Gastruloids

ASCB EMBO Annual Meeting 2019

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Lung Disease and Regeneration

Advances in microscopy

Also in the cell biology category:

BSCB-Biochemical Society 2024 Cell Migration meeting

‘In preprints’ from Development 2022-2023

preLights peer support – preprints of interest

The Society for Developmental Biology 82nd Annual Meeting

CSHL 87th Symposium: Stem Cells

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

9th International Symposium on the Biology of Vertebrate Sex Determination

Alumni picks – preLights 5th Birthday

CellBio 2022 – An ASCB/EMBO Meeting

Fibroblasts

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

FENS 2020

Planar Cell Polarity – PCP

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

Cell Polarity

TAGC 2020

3D Gastruloids

ECFG15 – Fungal biology

ASCB EMBO Annual Meeting 2019

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Autophagy

Lung Disease and Regeneration

Cellular metabolism

BSCB/BSDB Annual Meeting 2019

MitoList

Biophysical Society Annual Meeting 2019

ASCB/EMBO Annual Meeting 2018

Also in the developmental biology category:

BSDB/GenSoc Spring Meeting 2024

GfE/ DSDB meeting 2024

‘In preprints’ from Development 2022-2023

preLights peer support – preprints of interest

The Society for Developmental Biology 82nd Annual Meeting

CSHL 87th Symposium: Stem Cells

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

9th International Symposium on the Biology of Vertebrate Sex Determination

Alumni picks – preLights 5th Birthday

CellBio 2022 – An ASCB/EMBO Meeting

2nd Conference of the Visegrád Group Society for Developmental Biology

Fibroblasts

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

EMBL Conference: From functional genomics to systems biology

Single Cell Biology 2020

Society for Developmental Biology 79th Annual Meeting

FENS 2020

Planar Cell Polarity – PCP

Cell Polarity

TAGC 2020

3D Gastruloids