Thus, the oocyte-like cells appear to be derived from SSCs in culture. Furthermore, we used ovarian sections from E The formation of embryos was further confirmed by the expression of genes from preimplantation embryos, such as Hmgpi and Trim43a [ 20 , 21 ] Figure 3 B. Overall, with 53 attempts of ICSI, we obtained 5 embryos of early developmental stages after artificial activation with none beyond 4-cells. Derivation of Oocyte-like cells from SSCs. D Immunofluorescence of ovarian sections of E Characterization of mature oocytes and embryos derived from spermatogonial stem cells SSCs in culture.
L Schematic representation of the reprogramming conditions from SSCs to oocyte-like cells. Because ESCs can develop into oocytes [ 10 ] while cultured SSCs can be reverted to pluripotent cells [ 5 — 9 ], it has been proposed that cultured SSCs may be converted to pluripotent cells that subsequently develop into oocytes. Moreover, the cultured SSCs can directly transdifferentiate into oocytes.
To understand the potential mechanisms underlying the conversion of SSCs into oocyte-like cells, we examined the expression of genes related to PGC development in the early cultured SSCs. Notably, Nanog expression was gradually lost before day 7, while Nobox expression started between day 5 and day 7 Figure 4 A and Additional file 1 : Figure S3. We further confirmed the presence of oocytes by co-staining of multiple markers of oocytes.
Moreover, compact colonies of embryonic stem-like cells were never observed in our cultures.
In addition, E-cadherin , which is highly expressed in mouse ESCs,was not detectable in these cells, and teratoma could not form from them data not shown. These results indicate that ESC-like cells were less likely to form in this culture but PGCs and oocytes were produced. Examination of the markers of PGCs and oocytes by immunofluorescent staining. A Time course immunofluorescence analysis of the expression of Nanos2, Nanos3, Nanog, Blimp1, and Nobox, the percentage of positive cells for each corresponding marker at different days of culture was statistized.
Parental imprints are established during gametogenesis and are essential for the function of gametes and the normal development of embryos. Thus, we were interesting to learn if imprinting reversals can be induced during the conversion of SSCs into oocytes. These results indicate that the epigenetic switching of imprints was associated with the transdifferentiation of SSCs into oocytes. Analysis of Sex-specific imprint pattern and sex chromosome-linked gene activation.
The activity of X chromosome-linked genes in male germ cells is different from that of female germ cells. The Y chromosome genes have been reported to be essential for spermatogenesis but not for oogenesis. Therefore, we addressed the gene activity status of sex chromosomes in the SSC-Oocs. We examined the expression of sex-dependent X- and Y-linked genes [ 29 , 30 ] and found that the X-linked testis specific genes were significantly down-regulated or turned off Figure 5 C and Additional file 4 : Table S2 , while oocyte specific genes including GDF9 , X-linked BMP15 and Usp9x were turned on Figure 5 D.
These data indicate that the gene expression pattern of the X chromosome was changed in favor of the formation of oocytes from SSCs.
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This hypothesis was supported by the reversal expression of Usp9x Figure 5 D and Additional file 2 : Table S2 , which is X-linked and expressed in both male and female embryonic germ cells, but turned off in male germ cells after birth [ 31 ]. Therefore, along with the morphological changes during the conversion of SSCs into oocytes, the epigenetic network was converted into the female germ cell form.
In mice, cellular pluripotency reprogramming mostly relies on the extrinsic signaling of LIF and the intrinsic factor Oct4; LIF signaling is sufficient in reprogramming of epiblast cells, in which Oct4 is not expressed, into pluripotent ESCs [ 32 , 33 ]. Oct4 in a defined culture condition can reprogram somatic cells into pluripotent cells [ 34 ].
It has also been revealed that a reversible path from stem cells to differentiation in the germ cell lineage exists [ 35 — 37 ]. In addition, a few studies have demonstrated that a small fraction of mouse SSCs can be reprogrammed back to embryonic stem-like cells [ 5 — 9 ]. Based on these findings, we thought as in the reprogramming epiblast cells into ES-like cells [ 32 , 33 ], the LIF signaling might trigger the dedifferentiation of SSCs.
This observation is consistent with earlier findings concerning the reprogramming capability of the LIF signaling in the presence of 2i [ 13 , 14 ] and further indicates the remarkable plasticity of SSCs in culture. Thus, the isolated SSCs were unipotent. In situ, we can only observe green cells in testes of OG2 mice before postnatal day 6.
Despite of the presence of EGFP green cells that indicated they were positive of Oct4, we did not observe any ESC-like colonies formed from these cells. Furthermore, we found that E-cadherin expression was absent in them and teratoma could not form them in nude mice, indicating they were not ESCs but more like PGCs. This was further confirmed by the expression of Blimp1 and Nanog.
These cells continued to grow with increasing size, expressed Stella, Nobox, and GDF9, and demonstrated morphology resembling that of oocytes. With our characterization of gene expression and morphology, we have clearly demonstrated that oocyte-like cells can be derived from SSCs via PGCs intermediates.
The imprinting patterns are established during gametogenesis with paternal imprints occurring during spermatogenesis and maternal imprints occurring during oogenesis. Defects in imprinting in gametes can give rise to severe problem in embryogenesis and predispose affected individuals to associated diseases after birth. It turned out that all three of them were switched to maternal status following the induction of oocyte-like cells from SSCs. Thus, in contrast to early report of germline-derived pluripotent stem gPS cells, which retain their original imprinting status [ 38 ], the reprogramming from SSCs to oocyte-like cells was accompanied by imprinting reversal.
Therefore, SSCs possess both cellular and epigenetic plasticity and even give rise to oocyte-like cells in vitro in a manner similar to cases of sex reversal in vivo. Finally, we also demonstrated that a small number of SSC-Oocs were capable of being fertilized by sperm in vitro. Our study has demonstrated that SSCs possess the potential to be reprogrammed into oocyte-like cells in culture.yqemyxevym.tk/map7.php
Cell Biology of Early Mouse Development
If culture conditions are further optimized, for example, using 3D culture system with ideal supportive cell feeder [ 39 ], to develop oocytes of high quality from SSCs, such a germ cell fate switch system should provide a useful in vitro model to study epigenetic regulation in oogenesis and sex reversal, furthering our understanding of the mechanisms that establish imprinting during gametogenesis. Mice were euthanized by CO 2 inhalation to ameliorate any suffering throughout these experimental studies.
To retrieve sperm, seminiferous tubules were collected and put in Hepes-CZB. They were then cut into small pieces with a pair of fine scissors.
MicroRNA-deficient mouse embryonic stem cells acquire a functional interferon response
Sperm were collected and injected into SSC-Oocs. Adams IR, McLaren A: Sexually dimorphic development of mouse primordial germ cells: switching from oogenesis to spermatogenesis. McLaren A: Sex chimaerism and germ cell distribution in a series of chimaeric mice. J Embryol Exp Morphol. McLaren A: The fate of germ cells in the testis of fetal Sex-reversed mice. J Reprod Fertil. Stem Cells Dev. These findings are the basis to further study the conservation of the miRNA-mediated regulation of the IFN response in somatic cells and in the context of human pluripotency.
All these investigations will provide a deeper understanding and tool set on how to enhance the innate immunity of ESCs and their differentiated progeny, an especially relevant aspect in clinical applications. Blelloch lab University of California, San Francisco.
Cells were grown on plates coated with 0. NIH3T3 cell line was provided by A. Cell numbers were determined in triplicates after 12, 24, 36 and 48 hr using a CASY cell counter. For TMEV infections, cells were infected for 1 hr with the required dilution, followed by replacement with fresh medium and incubation for the desired time. After replacement of the inoculum with fresh serum containing medium the cells were incubated for the desired period. To differentiate mESCs, they were first cultured as hanging droplets to induce embryoid body formation.
The embryoid bodies were consequently washed from the lids and transferred to petri dishes to further differentiate, all in the absence of LIF. After another incubation time of 48 hr, medium was removed and replaced with fresh medium containing nM of retinoic acid Sigma-Aldrich and incubated for 7 days while replacing the medium every 48 hr. After this incubation time, the embryoid bodies were collected and plated on normal gelatine-coated cell culture plates which allowed the embryoid bodies to adhere to the plastic and the cells to migrate from the embryoid bodies.
Again, the medium was refreshed every 48 hr for the cells to further differentiate. After electrophoresis, gel was stained with SYBR gold for visualization of equal loading. Gel was transferred onto a positively charged Nylon membrane for 1 hr at mA. Oligonucleotides used are listed in Supplementary file 1. Cells were incubated for approximately 16 hr for poly I:C - and 8 hr for DNA-transfections before harvest and further processing.
Cells were transfected with 2. The same procedure was followed for the antagomirs Dharmacon , but at a concentration of nM. Data was analysed using the StepOne software package. Proteins were transferred to nitrocellulose membrane using semi-dry transfer iBlot2, ThermoFisher. Membranes were blocked for 1 hr at room temperature in PBS-T 0. Protein bands were quantified using ImageJ v1. Cells in well format were transfected with ng plasmid using Lipofectamine and incubated for 24 hr.
Cells were subsequently lysed and assayed using the Dual-Glo Luciferase assay system Promega. Luminescence was measured in a Varioskan flash ThermoFisher platereader. The pH was checked by spotting onto pH paper, and peptide concentration estimated using a NanoDrop. After washing with uL 0. MS1 was acquired with mz range — and resolution 70,, and top 12 ions were selected for fragmentation with normalised collision energy of 26, and an exclusion window of 30 s.
MS2 were collected with resolution 17, Finally, the data were analysed using MaxQuant v 1. Average expression levels were calculated for each protein and significant differences identified using a two tailed t-test assuming equal variance homoscedasticity with a p-value lower than 0.
Verified plasmids containing the genes of interest were transfected in mESCs using Lipofectamine and selected with the appropriate antibiotic. After several weeks of selection, colonies were isolated, expanded and tested for expression by qRT-PCR and Western blot.
The mitochondria specific dye Rhodamine Sigma-Aldrich was used to measure mitochondrial activity. Cas9 protein and tracrRNAs were transfected with the Neon Transfection System followed by cell sorting to create single cell clones. Total RNA ng was used to quantify mmu-mmiR—5p levels. All processed Mass spectrometry data is provided as Figure 3—source data 1 , including LFQ intensity values for each protein detected in each of the samples.
All raw data are available from corresponding author upon request. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. Thank you for sending your article entitled "MicroRNA-deficient embryonic stem cells acquire a functional Interferon response" for peer review at eLife. Your article has been evaluated by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Tadatsugu Taniguchi as the Senior Editor.
Given the list of essential revisions, including some new experiments, the editors and reviewers invite you to respond within the next two weeks with an action plan and timetable for the completion of the additional work. In doing so, please describe point-by-point response to the issues raised by the reviewers. We plan to share your responses with the reviewers and then issue a binding recommendation. Biological relevance should be strengthened. Furthermore, more mechanistic study is needed, for examples, whether the ESCs have abnormal epigenetic regulation of miR and whether miR expression was suppressed through ESC differentiation.
This is an interesting study, and adds potential new understanding in this area. However, the data are not strongly supportive of the conclusion, and there are some conclusions need modification or additional experimental evidence to support. This is illogical, and correlation is not causation. MicroRNAs may target other anti-viral factors e. They still haven't explained the reason. Whether the ESCs have abnormal epigenetic regulation of miR? Which transcript factors caused this epigenetic change Oct4? Whether the reduced virus in these ESCs were caused by abnormities in cell proliferation or apoptosis?
We can't draw the conclusion from Figure 3A and Figure 3—figure supplement 1A. There lacks a vehicle control. In this manuscript Witteveldt et al. The experiments in this manuscript substantiate this conclusions and in my opinion there is no requirement for additional ones. We agree that the global disruption of miRNA expression can potentially lead to changes in other cellular processes that might influence the antiviral response.
However, our experiments clearly demonstrate that manipulating a single miRNA, using multiple approaches miR mimics, antagomirs or miR KO cell lines Figure 5 or, directly the MAVS expression levels Figure 4 is enough to recapitulate the phenotype observed in the absence of all miRNAs. Factors marked with a red asterisk are those that do not display differential expression in the absence of miRNAs, as shown by Western blot analyses Figure 3—figure supplement 1.
Those indicated with a blue asterisk are not differentially expressed in the absence of miRNAs, as determined by Mass spectrometry data analyses Figure 3—source data 1 , including those relevant ISGs that seem to confer antiviral protection in human ESCs 1. MAVS green asterisk is the only factor in these pathways that is significantly upregulated in the absence of miRNAs, as shown by Mass spectrometry analyses, and confirmed by Western blot Figure 3C.
Based on all these data, we have concluded there is a negligible contribution of other antiviral factors in the cellular models studied here. We agree that this is an interesting aspect that would benefit from further experimentation. We thank the reviewer for this suggestion. Indeed, miR is expressed during pluripotency and it becomes silenced during differentiation see new Figure 5G.
Both ESCs lines used in this study were differentiated using retinoic acid, and miR expression was assessed by qRT-PCR after 2 or 10 days of the differentiation protocol. All these suggest that miR expression and IFN-proficiency are incompatible. We feel that studying the epigenetic regulation of miR expression is beyond the scope of this manuscript, even though it is a very interesting aspect to follow-up. So far, there is no annotation for the promoter that drives expression of this miRNA, which challenges any approach to assess its epigenetic regulation.
The miR expression data during differentiation is included in new Figure 5G. We thank the reviewer for highlighting this possibility. This is an aspect of miR biology that we have already addressed. Transient reintroduction of miR in miRNA-deficient ECSs does not significantly affect the relative expression of pluripotency markers Nanog and Oct4 see left graph in Author response image 2. More importantly, miR knock-out cell lines also express similar levels of the pluripotency markers Nanog and Oct-4 in comparison to wild-type ESCs, whereas in mouse fibroblasts NIH3T3 they are absent as expected see right graph in Author response image 2.
These data suggest that differences in antiviral resistance upon miR manipulation are not due to a spontaneous loss of pluripotency.
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We are currently measuring the proliferation capacity of the CRISPR cell lines see point 5 , in addition to providing pictures of these cultures to demonstrate their ability to form colonies and phenotypical similarities to the wild-type counterparts. Disruption of miRNA expression by knocking-out Dgcr8 or Dicer genesimpairs the ability of ESCs to differentiate 4,5 and to efficiently transition from G1 to S phase of their cell cycle 6,7.
A genome-wide screening revealed that the reintroduction of only 14 single miRNAs rescued this phenotype 6. This seed sequence is not conserved in any of the miRNAs tested in this manuscript miRa-5p, miRb-5p, miRp and miRp , suggesting that they may not be involved in the direct regulation of Cdkn1a level, or cell cycle progression see Author response image 3. We apologize if these graphs were not clear. Both inhibitors have been extensively tested before for their action and targets in several cell lines, as shown for BX 8,9 and BMS 10, Controls for inhibitors are included as new Figure 3—figure supplement 1B and C.
We apologize for not including the references to previous studies showing the expression levels of both these miRNAs. Besides the data we show in Figure 5, early publications showed that both miR and miR are well expressed in mouse ESCs in a Dicer and Dgcr8- dependent manner 12, Later publications confirmed the expression of both miRNAs in ESCs and also found that especially miR decreased in expression upon differentiation 14,15,16, Besides cell lines, both miRNAs are also readily detected in the early stages of embryonic development 18,19, New references have been added.
Generating stable cell lines overexpressing proteins always result in variable expression levels, depending on insertion location and effect of the protein on the cell. This is one of the reasons why we have included two additional approaches to manipulate MAVS expression levels in ESCs and assess their effects on antiviral defence. J Virol. Nat Genet. Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. Genes Dev. Characterization of Dicer-deficient murine embryonic stem cells. J Biol Chem. Use of the pharmacological inhibitor BX to study the regulation and physiological roles of TBK1 and IkappaB kinase epsilon: a distinct upstream kinase mediates Ser phosphorylation and activation.
BMS is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappa B-dependent transcription in mice. Clin Cancer Res. MicroRNA expression profiling of single whole embryonic stem cells.
Related Mouse Development: From Oocyte to Stem Cells
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