Efficient generation of marmoset primordial germ cell-like cells using induced pluripotent stem cells
- University of Pennsylvania, United States
- The University of Texas at San Antonio, United States
Abstract
Reconstitution of germ cell fate from pluripotent stem cells provides an opportunity to understand the molecular underpinnings of germ cell development. Here, we established robust methods for pluripotent stem cell (iPSC) culture in the common marmoset (Callithrix jacchus, cj), allowing stable propagation in an undifferentiated state. Notably, iPSCs cultured on a feeder layer in the presence of a WNT signaling inhibitor upregulated genes related to ubiquitin-dependent protein catabolic processes and enter a permissive state that enables differentiation into primordial germ cell-like cells (PGCLCs) bearing immunophenotypic and transcriptomic similarities to pre-migratory cjPGCs in vivo. Induction of cjPGCLCs is accompanied by transient upregulation of mesodermal genes, culminating in the establishment of a primate specific germline transcriptional network. Moreover, cjPGCLCs can be expanded in monolayer while retaining the germline state. Upon co-culture with mouse testicular somatic cells, these cells acquire an early prospermatogonia-like phenotype. Our findings provide a framework for understanding and reconstituting marmoset germ cell development in vitro, thus providing a comparative tool and foundation for a preclinical modeling of human in vitro gametogenesis.
Data availability
Accession number for RNA-seq and whole genome bisulfite sequencing generated in this study is GSE210576. The Exome sequence data generated in this study are available as SRA BioProject PRJNA856282.
-
Efficient generation of marmoset primordial germ cell-like cells using induced pluripotent stem cellsNCBI Gene Expression Omnibus, GSE210576.
-
Sequencing of Callithrix jacchus iPSC linesSRA BioProject, PRJNA856282.
Article and author information
Author details
Funding
Open Philanthropy Project (2019-197906)
- Kotaro Sasaki
Open Philanthropy Project (10080664)
- Kotaro Sasaki
National Institute on Drug Abuse (U01DA054170)
- Brian P Hermann
Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD090007)
- Brian P Hermann
National Institute on Aging (P51OD011133)
- Corinna N Ross
National Institute on Minority Health and Health Disparities (G12MD007591)
- Sean Vargas
National Science Foundation (DBI-1337513)
- Sean Vargas
National Science Foundation (DBI-2018408)
- Sean Vargas
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All procedures were reviewed and approved by the Texas Biomedical Research Institute IACUC (1772CJ).
Copyright
© 2023, Seita et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,628
- views
-
- 403
- downloads
-
- 20
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Efficient generation of marmoset primordial germ cell-like cells using induced pluripotent stem cells
eLife 12:e82263.
https://doi.org/10.7554/eLife.82263
Further reading
-
- Developmental Biology
The morphogen FGF8 establishes graded positional cues imparting regional cellular responses via modulation of early target genes. The roles of FGF signaling and its effector genes remain poorly characterized in human experimental models mimicking early fetal telencephalic development. We used hiPSC-derived cerebral organoids as an in vitro platform to investigate the effect of FGF8 signaling on neural identity and differentiation. We found that FGF8 treatment increases cellular heterogeneity, leading to distinct telencephalic and mesencephalic-like domains that co-develop in multi-regional organoids. Within telencephalic regions, FGF8 affects the anteroposterior and dorsoventral identity of neural progenitors and the balance between GABAergic and glutamatergic neurons, thus impacting spontaneous neuronal network activity. Moreover, FGF8 efficiently modulates key regulators responsible for several human neurodevelopmental disorders. Overall, our results show that FGF8 signaling is directly involved in both regional patterning and cellular diversity in human cerebral organoids and in modulating genes associated with normal and pathological neural development.
-
- Developmental Biology
Wnt signaling plays crucial roles in embryonic patterning including the regulation of convergent extension (CE) during gastrulation, the establishment of the dorsal axis, and later, craniofacial morphogenesis. Further, Wnt signaling is a crucial regulator of craniofacial morphogenesis. The adapter proteins Dact1 and Dact2 modulate the Wnt signaling pathway through binding to Disheveled. However, the distinct relative functions of Dact1 and Dact2 during embryogenesis remain unclear. We found that dact1 and dact2 genes have dynamic spatiotemporal expression domains that are reciprocal to one another suggesting distinct functions during zebrafish embryogenesis. Both dact1 and dact2 contribute to axis extension, with compound mutants exhibiting a similar CE defect and craniofacial phenotype to the wnt11f2 mutant. Utilizing single-cell RNAseq and an established noncanonical Wnt pathway mutant with a shortened axis (gpc4), we identified dact1/2-specific roles during early development. Comparative whole transcriptome analysis between wildtype and gpc4 and wildtype and dact1/2 compound mutants revealed a novel role for dact1/2 in regulating the mRNA expression of the classical calpain capn8. Overexpression of capn8 phenocopies dact1/2 craniofacial dysmorphology. These results identify a previously unappreciated role of capn8 and calcium-dependent proteolysis during embryogenesis. Taken together, our findings highlight the distinct and overlapping roles of dact1 and dact2 in embryonic craniofacial development, providing new insights into the multifaceted regulation of Wnt signaling.
