Single cell analysis of malignant stem cell clones in myelofibrosis
Aims: To develop a new method to allow parallel mutational analysis, RNA sequencing and lineage tracing of single stem cell clones. To apply this technology to explore biological heterogeneity of single stem/progenitor populations in patients with myelofibrosis.
Haematopoiesis is an ideal tractable model for the application of pioneering techniques in single cell genomics. Over the last few years, the Haematopoietic Stem Cell (HSC) Biology Laboratory at the WIMM has established state of the art techniques to study gene expression in single haematopoietic stem and progenitor cells. However, major challenges for the application of single cell transcriptome analysis in malignant stem cell biology remain. One of these relates to a lack of sensitivity for mutation detection using standard RNA-sequencing approaches due to a combination of technical "drop outs" in the data as well as true biological heterogeneity of expression of mutations at the single cell level. A second limitation of single-cell RNA-sequencing of stem cells is that this only provides a “snapshot” of the cells at a certain point in time, with no indication of their output in terms of clonal progeny.
The purpose of this studentship will make use of a new technique we have developed in the laboratory which allows high-sensitivity mutation detection with parallel analysis of the whole transcriptome of single stem cells. This involves targeted gDNA and global RNA analysis from the same single cells through refined procedures for generating single cell RNA-seq libraries in order to minimise technical drop outs in the data. This technique will allow lineage tracing of stem cell clones using either the specific mutations detected in patients with blood cancers or through lentiviral based cellular barcoding. This will provide excellent experience in state of the art molecular and stem cell biology techniques as well as training in the use of high throughput robotics platforms and bioinformatics.
In this project, this approach will be used for the analysis of stem cells from patients with myelofibrosis, a poor prognosis form of blood cancer which is propagated by rare malignant stem cell populations. Myelofibrosis is an ideal tractable model system to apply the above single cell approach, with broad applicability for cancer biology. Normal and myelofibrosis HSCs will be analysed using a combination of in vitro and in vivo stem cell assays combined with the above single cell analysis technique in order to characterise driver mutations in single malignant stem cells and to correlate this mutational information with whole transcriptome analysis of stem cells and readout of their clonal progeny. The analysis will focus on disruption of megakaryocytic differentiation pathways during the development of myelofibrosis. This information will be correlated with clinical outcome and response to targeted therapies in patients, with potential to provide unique insights into the biology of myelofibrosis, with potential for clinical benefit in patients through biomarker and therapeutic target discovery.
The studentship will provide an excellent training in the exciting emerging field of single cell analysis using state of the art molecular and stem cell biology approaches. The Mead laboratory has clearly defined protocols to support training in specific experimental techniques. Standard operating procedures are regularly updated to ensure that methods are optimal. The student will undergo a formalised training and assessment of each technique either by Dr Mead or other members of the laboratory as appropriate. The above project utilises a wide range of state of the art molecular and cell biological techniques, in vivo stem cell assays and bioinformatics analysis and will consequently provide an excellent foundation for a research career.
As well as the specific training detailed above, students will have access to a wide-range of seminars and training opportunities through the many research institutes and centres based in Oxford. Students are also able to attend the Methods and Techniques course run by the MRC Weatherall Institute of Molecular Medicine. This course runs through the year, ensuring that students have the opportunity to build a broad-based understanding of differing research techniques. There are also courses on Immunology and Bioinformatics and others may be added. Institute Seminars are held on a weekly basis and regularly attract world-class scientists in haematopoiesis research. Informal exchange of ideas in the coffee area is encouraged and is an attractive feature of the MRC WIMM.
Generic skills training is offered through the Medical Sciences Division's Skills Training Programme. This programme offers a comprehensive range of courses covering many important areas of researcher development: knowledge and intellectual abilities, personal effectiveness, research governance and organisation, and engagement, influence and impact. Students are actively encouraged to take advantage of the training opportunities available to them.
The department has a successful mentoring scheme, open to graduate students, which provides an additional possible channel for personal and professional development outside the regular supervisory framework. We hold an Athena SWAN Silver Award in recognition of our efforts to support the careers of female students and staff.