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Investigating the function of developmental regulators in early human embryogenesis and stem cells

  • Full or part time
    Dr K Niakan
  • Application Deadline
    Tuesday, November 12, 2019
  • Funded PhD Project (Students Worldwide)
    Funded PhD Project (Students Worldwide)

Project Description

This 4-year PhD studentship is offered in Dr Kathy Niakan’’s Group based at the Francis Crick Institute (the Crick).

This project will characterise key regulators of human embryogenesis. We have identified transcription factors that are highly expressed in pluripotent epiblast cells of the developing human embryo [1]. The pluripotent epiblast has the unique potential to give rise to the entire fetus in vivo and can self-renew indefinitely as embryonic stem cells (hESCs) in vitro. Understanding the molecular basis of pluripotency in human cells is of fundamental biological importance and has significant clinical implications for the use of hESCs to treat diseases. Importantly, the transcription factors we identified as enriched in human embryos are not expressed in mouse embryos at the equivalent developmental stage [1], further suggesting differences in pluripotency mechanisms between these species. The aim of the project is to functionally test these putative regulators of human pluripotency and embryogenesis. The specific objectives of the project are:


1. The student will evaluate protein expression of putative pluripotency factors in human embryos by immunofluorescence and confocal microscopy. Proteins highly expressed specifically in epiblast cells will be good candidates for future investigation. We have validated some of the human epiblast-enriched factors [1]. However, other candidates have yet to be tested, such as ARGFX or VENTX. This first objective is essential for subsequent objectives and only candidates that have been validated will be further investigated.


2. The student will test the functional requirement of the putative pluripotency factors in recently established ‘naïve’ hESCs [2]. Some of the factors we identified (i.e. ARGFX or VENTX) are also expressed in ‘naïve’ hESCs that more closely resemble the in vivo embryonic epiblast [2], compared to conventional hESCs [3]. To test their requirement for the establishment and maintenance of pluripotency in naïve hESCs, CRISPR/Cas9 mutagenesis will be used to disrupt the gene. Established stem cell self-renewal and pluripotency assays [4] will be used to comprehensively characterize the mutant hESCs. Transcriptome analysis will be performed to investigate gene expression changes resulting from CRISPR-induced null mutations. This experiment will not only allow the student to screen for guide RNA efficiency and specificity before they are used in human embryos, but also to test the functional role of the candidate gene in hESC pluripotency or self-renewal.

3. Depending on the outcome above, established methods for ChIP-sequencing analysis [4] will be used to investigate how the factor fits into the well-defined human pluripotency gene regulatory network. A number of publicly available ChIP-sequencing and transcriptome databases will be integrated together with data generated from these studies.

4. Time permitting, the student will test the function of these genes in human embryos (with assistance from an SLRS in the lab) and determine if they are required for the establishment and maintenance of pluripotent epiblast progenitor cells using techniques optimised in the lab [5]. Well-established transcriptome, genome and immunofluorescence assays will be used to assess the phenotype and genotype of the targetted embryos.

Through these experiments the student will provide fundamental insights into human biology with direct relevance to stem cell biology.

The Niakan lab has an HFEA licence to genetically modify human embryos for research and has established collaborations with several UK-based IVF clinics to consent embryos for the licenced research project that would cover all of the objectives outlined. We currently have human 1-cell zygotes consented for this project. We have MRC stem cell steering committee approval to use human embryonic stem cells and have a GMO licence to conduct gene manipulation on human cells using a variety of methods. All of the standard methods used to microinject embryos, target genes using the CRISPR/Cas9 system and to evaluate the effect of any factor on the establishment or maintenance of pluripotency in human embryos or stem cells are well established in the lab. We have tested qRT-PCR primers, antibodies and standard reagents required for all of the experiments outlined above.

Talented and motivated students passionate about doing research are invited to apply for this PhD position. The successful applicant will join the Crick PhD Programme in September 2020 and will register for their PhD at one of the Crick partner universities (Imperial College London, King’s College London or UCL).

APPLICATIONS MUST BE MADE ONLINE VIA OUR WEBSITE (ACCESSIBLE VIA THE ‘APPLY NOW’ LINK ABOVE) BY 12:00 (NOON) 13 NOVEMBER 2019. APPLICATIONS WILL NOT BE ACCEPTED IN ANY OTHER FORMAT.

Funding Notes

Successful applicants will be awarded a non-taxable annual stipend of £22,000 plus payment of university tuition fees. Students of all nationalities are eligible to apply.

Applicants should hold or expect to gain a first/upper second-class honours degree or equivalent in a relevant subject and have appropriate research experience as part of, or outside of, a university degree course and/or a Masters degree in a relevant subject.

References

1. Blakeley, P., Fogarty, N. M. E., del Valle, I., Wamaitha, S. E., Hu, T. X., Elder, K., . . . Niakan, K. K. (2015)

Defining the three cell lineages of the human blastocyst by single-cell RNA-seq.

Development 142: 3151-3165. PubMed abstract

2. Takashima, Y., Guo, G., Loos, R., Nichols, J., Ficz, G., Krueger, F., . . . Smith, A. (2014)

Resetting transcription factor control circuitry toward ground-state pluripotency in human.

Cell 158: 1254-1269. PubMed abstract

3. Ludwig, T. E., Bergendahl, V., Levenstein, M. E., Yu, J., Probasco, M. D. and Thomson, J. A. (2006)

Feeder-independent culture of human embryonic stem cells.

Nature Methods 3: 637-646. PubMed abstract

4. Wamaitha, S. E., Del Valle, I., Cho, L. T. Y., Wei, Y., Fogarty, N. M. E., Blakeley, P., . . . Niakan, K. K. (2015)

Gata6 potently initiates reprograming of pluripotent and differentiated cells to extraembryonic endoderm stem cells.

Genes & Development 29: 1239-1255. PubMed abstract

5. Fogarty, N. M. E., McCarthy, A., Snijders, K. E., Powell, B. E., Kubikova, N., Blakeley, P., . . . Niakan, K. K. (2017)

Genome editing reveals a role for OCT4 in human embryogenesis.

Nature 550: 67-73. PubMed abstract



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