p38 MAPK as a gatekeeper of reprogramming in mouse migratory primordial germ cells

Abstract

Mammalian germ cells are derived from primordial germ cells (PGCs) and ensure species continuity through generations. Unlike irreversible committed mature germ cells, migratory PGCs exhibit a latent pluripotency characterized by the ability to derive embryonic germ cells (EGCs) and form teratoma. Here, we show that inhibition of p38 mitogen-activated protein kinase (MAPK) by chemical compounds in mouse migratory PGCs enables derivation of chemically induced Embryonic Germ-like Cells (cEGLCs) that do not require conventional growth factors like LIF and FGF2/Activin-A, and possess unique naïve pluripotent-like characteristics with epiblast features and chimera formation potential. Furthermore, cEGLCs are regulated by a unique PI3K-Akt signaling pathway, distinct from conventional naïve pluripotent stem cells described previously. Consistent with this notion, we show by performing ex vivo analysis that inhibition of p38 MAPK in organ culture supports the survival and proliferation of PGCs and also potentially reprograms PGCs to acquire indefinite proliferative capabilities, marking these cells as putative teratoma-producing cells. These findings highlight the utility of our ex vivo model in mimicking in vivo teratoma formation, thereby providing valuable insights into the cellular mechanisms underlying tumorigenesis. Taken together, our research underscores a key role of p38 MAPK in germ cell development, maintaining proper cell fate by preventing unscheduled pluripotency and teratoma formation with a balance between proliferation and differentiation.

Keywords: EGCs; gatekeeper; migratory primordial germ cells; p38 MAPK; pluripotency; reprogramming; teratoma.

FIGURE 1

p38 MAPK, which is exclusively expressed in its phosphorylated form in migratory mouse PGCs, induces reprogrammed colony-forming cells through its inhibition (A) Schematic representation of the experimental workflow in induction of reprogramming in mouse migratory PGCs with p38 MAPK inhibitor. Isolated hindgut encapsulating Oct4-GFP-expressing migratory PGCs at E8.75 is dissociated, and then plated on STO-feeder cells with p38 MAPK inhibitor-contained medium. Colony-forming cells which are positive for Oct4-GFP expression are emerged after 12–14 days in culture. (B) Graphical representation of the change in the number of ALP (alkaline phosphatase)-positive cultured PGCs with or without the p38 MAPK inhibitor, SB239063, and the images at the indicated culture date. Error bars indicate s.d. (n = 3, biological replicates). The two-way ANOVA results show that there is a significant effect of the group (p = 0.0031). Scale bar, 100 μm. (C) Fluorescence images of dispersed (control) and clustered (with SB239063) Oct4-GFP-positive cultured PGCs on day 5. Scale bar, 100 μm. (D) Images of Oct4-GFP-positive colony-forming cells that appeared approximately 12–14 days after plating. White arrowheads indicate Oct4-GFP-positive cells that formed a colony at the edge of the well. Scale bar, 200 μm. (E) Immunofluorescence of phosphorylated p38 MAPK in cryosection of hindgut isolated from Oct4-GFP embryos at E8.75. White arrowheads indicate Oct4-GFP-positive migrating PGCs that exclusively express the phosphorylated p38 MAPK in the hindgut (filled arrowheads), and not express (empty arroheads). Scale bar, 50 μm.

FIGURE 2

Pluripotency of cEGLC in naïve state. (A) Images of Oct4-GFP-positive EGCs (Embryonic Germ Cells) and cEGLCs-23 (chemically induced EG-like cells with SB239063) at the indicated passage number in images. Scale bar, 100 μm. (B) Cloning efficiency in EGC, cEGLCs-20 (chemically induced EG-like cells with SB203580) and cEGLCs-23. Error bars indicate s.d. (n = 3, independent experiments). (C) Graphical representation of relative cell number in proliferation of cultured EGC, cEGLC-20 and -23. The ANOVA test reveals that there is no significant difference among the three groups (p = 0.6577). (D) Quantitative PCR analysis of expression of pluripotent markers in EGCs, cEGLC-s20 and -23. Error bars indicate s.d. (n = 3, biological replicates). t-test, **p < 0.01. *p < 0.05. Images of OCT4 (upper) and SOX2 (bottom) protein expression (pluripotent markers) in colonies of EGCs and cEGLCs-23. Scale bar, 100 μm. (E) Representative images of hematoxylin and eosin-stained sections of the subcutaneously formed teratoma. Scale bar, 100 μm. (F) Bioinformatic analysis of RNA-sequencing data with mouse pluripotent stem cells including cEGLCs. MDS (Multidimensional scaling) plot and hierarchical clustering dendrogram under optimal rank (c) based on differentially expressed genes (DEGs) among 8 cell types. Replicates of the same conditions are indicated by same color, cEGLCs-20 and −23 (black), ESCs (orange), EGCs (red), EpiLCs (green line, published data set (Shirane et al., 2016)), ECCs (P19) (purple, published data set (Dai et al., 2022)), EpiSCs (light blue) and rsEpiSCs (blue) published data set (Wu J. et al., 2015).

FIGURE 3

Epiblast-like characteristics in cEGLCs despite naïve state. (A) Differential intensity of alkaline phosphatase (ALP) activity between EGCs and cEGLCs-23. Scale bar, 100 μm. (B) Immunofluorescence images of OTX2 protein expression (a definitive marker of the epiblast) in colonies of EGCs and cEGLCs-23. Nuclei were counterstained with DAPI. The encircling dotted lines indicate a portion of cells in the colony of cEGLCs-23 that are clearly expressed for OTX2 protein. Scale bar, 50 μm. (C) Quantitative PCR analysis of expression of epiblast markers in EGCs, cEGLCs-20 and −23. Error bars indicate s.d. (n = 3, biological replicates). t-test, **p < 0.01. *p < 0.05. (D) Analysis of definitive endoderm formation in embryoid bodies. Representative images showing the formation of embryoid bodies derived from EGCs and cEGLCs-23 on day 4 in culture. Middle images correspond to square dotted lines in top images at higher magnification. White filled and empty arrowheads indicate a thick and no apparent layer of primitive endoderm respectively. Immunohistochemistry images for GATA4 protein expression detected by DAB staining in embryoid bodies derived from EGCs and cEGLCs-23. Scale bar in top and bottom images, 300 and 200 µm respectively.

FIGURE 4

Differential signaling requirements for maintenance of pluripotency between EGCs and cEGLCs. (A) Representative images of Oct4-GFP and OCT4 protein expression (markers for the undifferentiated state) in colonies of EGCs, cEGLCs-23 on day 4 with administration of drugs, JAKi (A Janus kinase inhibitor), SU54 (FGFR inhibitor), SB431542 (TGF-β/Activin inhibitor), a combination of JAKi/SU54. Nuclei were counterstained with DAPI. The white circled dotted line indicates colonies of EGCs that lose OCT4 expression. Scale bar, 100 μm. (B) Gene Set Enrichment Analysis (GSEA) of DEGs between ESC-EGC and cEGLCs groups. Cnetplot shows enriched KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. (C) Representative images of Oct4-GFP and OCT4 protein expression in colonies of cEGLCs-23 on day 4 with LY294002, a PI3K kinase inhibitor. Nuclei were counterstained with DAPI. The white circled dotted line indicates colonies of cEGLCs-23. Scale bar, 100 μm.

FIGURE 5

Supplementation of 2iLIF elicits the competence of chimera formation in cEGLCs. (A) Chimera formation analysis by blastocyst injection with cEGLCs-23 in different culture conditions. Schematic of the blastocyst injection with cEGLC-23 adapted in 2iLIF supplementation, and image of mouse pup showing the partially coat color-altered chimera in the head and tail. Table for chimera formation efficiencies in different media conditions with the actual number performed. (B) Images of Oct4-GFP expression and immunofluorescence of OCT4 and OTX2 protein expression in cEGLCs-23 adapted with 2iLIF supplementation. Nuclei were counterstained with DAPI. Scale bar in left and right images, 100 and 50 µm respectively. (C) The effect of 2iLIF supplementation on alkaline phosphatase (ALP) activity compared with that of SB239063. Scale bar, 100 μm. (D) Quantitative PCR analysis of naïve and epiblast markers expression in cEGLCs treated with SB239063 or 2iLIF. Error bars indicate s.d. (n = 3, biological replicates). t-test, **p < 0.01. *p < 0.05. (E) Bioinformatic analysis of RNA-sequencing data. (left panel) The dendrogram with all detected genes shows the hierarchical relationship between RNA-sequencing samples across cell lines and culture conditions. (right panel) PCA plot based on differentially expressed genes (DEGs) among cell types and different cultured conditions. cEGLCs-20 and -23 maintained treated with each of p38 MAPK inhibitors (circled with black line), cEGLCs-20 and −23 adapted with 2iLIF supplementation (circled with gray line), ESCs (circled with orange line) and EGCs (circled with red line).

FIGURE 6

Ex vivo analysis of reprogramming competence in migrating PGCs by inhibition of p38 MAPK. (A) Schematic of the experimental workflow for induction of ex vivo reprogramming of migrating PGCs in an isolated hindgut encapsulating Oct4-GFP expressing PGCs is isolated from Oct4-GFP mouse embryo at E8.75 with SB239063. (B) Image of the outgrowth of plated hindgut encapsulating the dispersed Oct4-GFP-expressing PGCs in control (the empty arrowheads in left images) and those clustered in SB239063-treated (the filled arrowheads in middle images). Images of Oct4-GFP-positive colony-forming cells appeared after dissociated replating even after withdrawal of SB239063 (right images). The edge of plated hindgut outgrowth is depicted with dotted outline. Scale bar, 100 μm. (C) Schematic summary of in vitro and ex vivo reprogramming of mouse migratory PGCs with p38 MAPK inhibitor(s) for cEGLC derivation.