Heading towards a dead end: The role of DND1 in germ line differentiation of human iPSCs
Mall EM, Lecanda A, Drexler HCA, Raz E, Schöler HR, Schlatt S., 15.10.2021
Abstract
The DND microRNA-mediated repression inhibitor 1 (DND1) is a conserved RNA binding protein (RBP) that plays important roles in survival and fate maintenance of primordial germ cells (PGCs) and in the development of the male germline in zebrafish and mice. Dead end was shown to be expressed in human pluripotent stem cells (PSCs), PGCs and spermatogonia, but little is known about its specific role concerning pluripotency and human germline development. Here we use CRISPR/Cas mediated knockout and PGC-like cell (PGCLC) differentiation in human iPSCs to determine if DND1 (1) plays a role in maintaining pluripotency and (2) in specification of PGCLCs. We generated several clonal lines carrying biallelic loss of function mutations and analysed their differentiation potential towards PGCLCs and their gene expression on RNA and protein levels via RNA sequencing and mass spectrometry. The generated knockout iPSCs showed no differences in pluripotency gene expression, proliferation, or trilineage differentiation potential, but yielded reduced numbers of PGCLCs as compared with their parental iPSCs. RNAseq analysis of mutated PGCLCs revealed that the overall gene expression remains like non-mutated PGCLCs. However, reduced expression of genes associated with PGC differentiation and maintenance (e.g., NANOS3, PRDM1) was observed. Together, we show that DND1 iPSCs maintain their pluripotency but exhibit a reduced differentiation to PGCLCs. This versatile model will allow further analysis of the specific mechanisms by which DND1 influences PGC differentiation and maintenance.
Mall EM, Lecanda A, Drexler HCA, Raz E, Schöler HR, Schlatt S. Heading towards a dead end: The role of DND1 in germ line differentiation of human iPSCs. PLoS One. 2021 Oct 15;16(10):e0258427. doi: 10.1371/journal.pone.0258427. PMID: 34653201; PMCID: PMC8519482.
Publication: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0258427 Repository: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE174464
Disclaimer
The publication Heading towards a dead end: The role of DND1 in germ line differentiation of human iPSCs by Mall EM, Lecanda A, Drexler HCA, Raz E, Schöler HR, Schlatt S. is published under an open access license: https://creativecommons.org/licenses/by/4.0/. Permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
Curation by the MFGA team Relevant data sets presented in the publication have been identified. If possible, annotations (title, general information, conditions, processed tissue types and processed cell types) have been added based on information from the publication. Data tables and images that provide a good overview on the publication's findings on the data set have been extracted from the publication and/or supplement. If not stated otherwise, images are depicted with title and description exactly as in the publication. Tables have been adjusted to the MFGA table format. Conducted adjustments are explained in the detailed view of the tables. However, titles and descriptions have been adopted from the publication.
Data set 1: Heading towards a dead end: the role of DND1 in germ line differentiation of human iPSCs
Transcriptome: Bulk RNA-Sequencing
Species
| Species |
|---|
| Human |
Tissue Types
| BRENDA tissue ontology | Maturity | Description | Species | Replicates |
|---|---|---|---|---|
| BTO_0001363: testis | A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity. | Human | ||
| BTO_0000089: blood | The fluid that circulates in the heart, arteries, capillaries, and veins of a vertebrate animal carrying nourishment and oxygen to and bringing away waste products from all parts of the body. | Human |
Cell Types
| Cell ontology | Maturity | Description | Species | Replicates | Cells per replicate |
|---|---|---|---|---|---|
| CL_0002246: peripheral blood stem cell | A hematopoeitic stem cell found in the blood. Normally found in very limited numbers in the peripheral circulation | Human | |||
| CL_0002371: somatic cell | Adult | A cell of an organism that does not pass on its genetic material to the organism's offspring | Human |
Images
Figure 1. Characterisation of DND1-/- hiPSCs.
(A) End-point PCR showing presence of large deletions in DND1 genomic locus. (B) All lines exhibited similar doubling times of about 20 hours. Doubling times counted from 4 independent passages per line. (C) About 90% of TS hiPSCs were double positive for OCT4 and NANOG, irrespective of DND1 status. CB hiPSCs showed reduced NANOG expression with 52–81% double positive cells. (D) RT-qPCR showed similar expression of OCT4, SOX2, and DNMTB1 in all lines compared to H9 hESCs. CB hiPSCs showed about 50% reduction of NANOG expression, irrespective of DND1 status. DND1 is expressed in low levels in hESCs and DND1+/+ hiPSCs and mRNA levels are strongly reduced in DND1-/- hiPSCs. The data were normalised to two internal controls (ACTB and GAPDH) and are shown as fold change to H9 ESCs. N = 3 biological replicates.
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Figure 2. Differentiation towards PGCLCs.
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Figure 3. RNA expression analysis.
(A) PCA clustering showed that DND1 KO lines clustered together with DND1 WT lines. Main differences are associated with the differentiation status, with PGCLCs clustering distinct from iPSCs and iMeLCs. (B) Heatmap showing differentially expressed genes in PGCLCs. DND1 KO PGCLCs show decreased expression in known PGC differentiation regulators as PRDM1 and NANOS3 (cluster 1).
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