The North West Embryonic Stem Cell Centre

Professor Sue Kimber

BA, MA, PhD

PhD Projects

Development of iPSCs from patients with Urofacial Syndrome for disease modelling

Funding

Project available for individuals with self arranged funding.

Description

Peripheral neuropathies (PNs) affect 2% of the general population. Somatic PNs cause locomotor and sensory deficits, while autonomic PNs cause cardiac dysrhythmias and gastrointestinal and urinary bladder dysfunction. Despite that facts that PNs are, as a group, common and they cause considerable morbidity, specific treatments to cure or ameliorate them are lacking. PNs have varied aetiologies, including environmental and genetic. An example of the latter is the urofacial syndrome (UFS), and this motor and autonomic neuropathy can be used as a model for novel biological therapies. UFS pathobiology and therapies will act as paradigms for commoner PNs, including those which occur in type 1 diabetes mellitus.  One approach to this is to develop disease models for the neural element of UFS by making induced pluripotent stem cells (iPSCS) from somatic cells of patients with specified genetic mutations leading to UFS and differentiate these to relevant neuronal subtypes. In this project the student will optimise a neural differentiation protocol already developed in the lab from published protocols for neurepithelium and motor neurons.  The student will characterise wild type human iPSCS and hESCs differentiated to neurons and establish the range of phenotypes obtained from different genetic back grounds.  He/she will compare UFS iPSCs (HPSE2 and LRIG2 mutations) differentiated to neurons with wild type iPSCs and hESCs and where possible this will include iPSCs from unaffected siblings. In collaboration with postdoctoral researchers he/she will correct the mutation using gene editing technologies to produce a generically matched iPSC control. The research will be extended to use biochemical correction techniques potentially leading to new drug discovery if time allows. Techniques include pluripotent stem cell (PSP) culture, iPSP derivation and characterization by immunocytochemistry flow cytometry Q-PCR and embryoid body analysis. Western blotting, electophysiological neuron analysis, gene editing technologies.

Related Publications

  • Daly SB, Urquhart JE, Hilton E, McKenzie EA, Kammerer RA, Lewis M, Kerr B, Stuart H, Donnai D, Long DA, Burgu B, Aydogdu O, Derbent M, Garcia-Minaur S, Reardon W, Gener B, Shalev S, Smith R, Woolf AS,  Black GC, Newman WG. Mutations in HPSE2 cause urofacial syndrome. Am J Hum Genet 11:963-969, 2010.
  • McKay TR, Camarasa MV, Iskender B, Ye J, Bates N, Miller D, Fitzsimmons JC, Foxler D, Mee M, Sharp TV, Aplin J, Brison DR, Kimber SJ. Human feeder cell line for derivation and culture of hESc/hiPSc. Stem Cell Res 7:154-162, 2011.
  • Newman WG, Woolf AS, Stuart HM. Urofacial Syndrome. In: Pagon RA, Adam MP, Bird TD, et al, editors. GeneReviews™ [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2013. http://www.ncbi.nlm.nih.gov/books/NBK154138/
  • Stuart HM, Roberts NA, Bergu B, Daly SB, Urquhart JE, Bhaskar S, Dickerson J, Mermerkaya M, Silay MS, Lewis MA,  Olondriz BO, Gener B, Beetz C, Varga RE, Gülpinar O, Süer E, Yalçinkaya F, Gücük A, Yue WW, Erdogan F, Berry A, Hanley NA, McKenzie EA, Hilton EN, Woolf AS,  Newman WG. LRIG2 mutations cause urofacial syndrome. Am J Hum Genet 92:259-264, 2013
  • Yamanaka, S. & Blau H.M. (2010) Nuclear reprogramming to a pluripotent state by three approaches. Nature 2010, 465, 704-712.

Fee Band

This project has a Band 2 fee. Details of difference fee bands are available for UK/EU or International applicants.

How to Apply

Find out How to apply for this PhD Project.

Role of integrins and endocytic trafficking in determining cell fate decisions in embryonic stem cells

Principal Investigators: Professor Sue Kimber, Dr Patrick Caswell

Funding

Project available for individuals with self arranged funding.

Description

Human embryonic stem (hES) cells have the capacity to from all tissues within the body, and therefore have huge potential as therapeutic agents in tissue regeneration.  Signals from the microenvironment surrounding the stem cell regulate the decision to maintain self-renewal, or differentiate.  Adhesion to the extracellular matrix (ECM), mediated through integrins, is one important factor within the niche that influences the pluripotency of hES and induced pluripotent stem (iPS) cells, however the mechanisms through which signals from the ECM and other cues are integrated to determine cell fate are not known.

 

Integrins act as receptors for the ECM, providing a mechanical link between the cells exterior and interior.  However, integrins also play an important role in the coordination of signalling pathways downstream of growth-factor and cytokine receptors with cues from the ECM.  Trafficking of integrins through the endocytic system is now recognised as an important regulator of integrin function.  Indeed, endocytosis and recycling of integrins controls the trafficking of co-cargoes, including signalling receptors, and can therefore directly influence cell signalling.  Whilst much progress has been made in understanding the role of integrin trafficking in pathological contexts such as cancer cell migration and invasion, very little is known of the function of integrin trafficking in normal physiology.

 

This project will focus on the role of endocytic trafficking in the decision of stem cells to maintain self-renewal or differentiate, in particular how the trafficking of integrins, and associated cargoes, regulates the signals that determine cell fate.   Techniques will include hESc and iPS cell culture, live cell imaging and other cell biology and biochemical techniques.

 

Related Publications

  • Baxter M, Caramasa M, Bates N, Small F, Murray P, Edgar D & Kimber SJ (2009)
  • Analysis of feeder cell- and serum-free tissue culture conditions for the maintenance of self-renewing human embryonic stem cell lines.
  • Stem Cell Research 3 28-38.
  • Caswell, P.T., Vadrevu, S. & Norman, J.C (2009). Integrins: masters and slaves of endocytic transport.
  • Nature Reviews Molecular Cell Biology, 10(12), 843-53.
  • Soteriou D, Iskender B,  Byron A,  Borg-Bartolo S, Haddock M-C, Baxter M, Humphries, JD, Knight D,  Humphries MJ, and Kimber SJ (2013) Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Proteins for Human Embryonic Stem Cell Maintenance J Biol Chem 288, 18716-18731

Fee Band

This project has a Band 2 fee. Details of difference fee bands are available for UK/EU or International applicants.

How to Apply

Find out How to apply for this PhD Project.

Signaling pathways maintaining human embryonic stem cells

Principal Investigators: Professor Daniel Brison, Professor Sue Kimber

Funding

Project available for individuals with self arranged funding.

Description

The aim of this project is to determine the function of novel genes predicted to have critical roles in preimplantation human development and in disease in later life. Due to the heterogeneity of human embryos and their precious nature, our molecular understanding of the developing embryo is limited. It is known that the period from fertilization through  preimplantation development is a uniquely susceptible window in development, with the embryonic genome being activated as early as the 2cell stage (Vassena et al) and large scale epigenetic remodeling occurring immediately post-fertilization (Santos et al). Therefore, developing our understanding of preimplantation molecular mechanisms has important implications for fundamental understanding of early human development including the origins of disease (Developmental Origins of Health and Disease; DOHaD),  assisted reproductive technology (ART), and therapies using human embryonic stem cells (hESCs) derived from embryos.

 

We have generated global gene expression microarray data and extensively mined the profiles obtained from a wide range of single embryos at developmental stages from oocyte through to blastocyst and its component parts; trophectoderm and inner cell mass (Shaw et al 2013). Our approach suggests that the MTOR/PI3K pathway, which has well documented roles in cancer and disease, also plays a crucial role in early embryo development. In addition, we have identified a number of novel and exciting genes which we believe to be important across and within different developmental stages, including epigenetic regulators. We aim to combine these findings with RNASeq, following recent success using single embryonic cells (Xue et al 2013), to provide information on novel transcripts and alternative pre-mRNA splicing.

 

Our laboratory as part of the North West Embryonic Stem Cell Centre (NWESCC) has generated a number of human embryonic stem cells (hESC) lines and induced pluripotent stem cells,which can form all tissues in the body and so can be used to investigate function in early developmental cell fate decisions, as well as  for tissue regeneration.   The Centre provides a focus for research projects on early human development, pluripotency, generation of mesodermal and endodermal differentiated derivatives and the stem cell niche..

 

Therefore the aims of this project are to:

  1. Perform hESC functional studies on novel genes identified within the microarray analysis.
  2. Perform additional microarray analysis[M1]  and RNASeq on single preimplantation embryos and cells and conduct splicing analysis.

 

Techniques will include pre-implantation embryo culture, hESC culture, immunofluorescence, immunoprecipitation, Western blotting. Real-Time fluorescence and confocal microscopy, flow cytometry, both qualitative and quantitative RT-PCR, lentiviral mediated genetic manipulation, RNASeq and other techniques depending on the results obtained.


 [M1]Put that in in case we want more embryos for array work, which Helen cannot generate herself of course…

 

Related Publications

  • Santos,F. and Dean,W. (2004) Epigenetic reprogramming during early development in mammals. Reproduction., 127, 643-651.
  • Shaw,L. et al. (2013) Global gene expression profiling of individual human oocytes and embryos demonstrates heterogeneity in early development. PLoS. One., 8, e64192.
  • Vassena,R. et al. (2011) Waves of early transcriptional activation and pluripotency program initiation during human preimplantation development. Development, 138, 3699-3709.
  • Xue,Z. et al. (2013) Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing. Nature.

Fee Band

This project has a Band 2 fee. Details of difference fee bands are available for UK/EU or International applicants.

How to Apply

Find out How to apply for this PhD Project.

Signaling pathways regulating differentiation of pluripotent stem cells to chondrocytes

Funding

Project available for individuals with self arranged funding.

Description

HESCs can form all tissues of the body and have excellent potential as agents for tissue regeneration. Our laboratory as part of the North West Embryonic Stem Cell Centre (NWESCC) has generated a number of human embryonic stem cell (hESC) and induced pluripotent cells (iPSCs) lines including those suitable for clinical therapy.  The Centre focusses on research on pluripotency, generation of mesodermal/neural differentiated derivatives and the stem cell niche(2). We are particularly interested in generation of chondrocytes because developing new human models for normal and abnormal chondrocyte function is critical to understanding of chondrodysplasias and osteoarthritis (4), one of the greatest cases of disability in the developed and developing world.  We have previously published an efficient, reproducible 3-step defined protocol for generation of chondrocytes from hESC(1,3) which in due course may be developed to provide  a new cell therapy for OA or cartilage damage in sports injury. Our recent data has shown that highly conserved and homologous member of the bone morphogenetic protein subfamily of TFGβ growth factors (GF): BMP2 and BMP4 have different ability to induce hESC differentiation to chondrocytes. We ask what is the mechanism is this disparate differentiation efficacy? In this project the student will investigate how these two BMPs activate signaling pathways in progenitors and developing chondrocytes from hESCs and IPSCs. You will investigate the interaction of the two BMPs with the extracellular matrix which binds GFs and acts as a reservoir but also presents the GFs to their receptors.  How these GFs interact with the cell adhesion system potentially by cross talk with integrin receptors inducing hESC/iPSC differentiation towards mesodermal intermediates and fully differentiated chondrocytes will be examined. Techniques include hESC culture, immunofluorescence, immunoprecipitation, Western blotting. Real-Time fluorescence and confocal microscopy, flow cytometry, RT-qPCR, pull down assays, lentiviral mediated genetic manipulation and other result-dependent techniques.

Related Publications

  • Oldershaw RA,  Baxter MA, Lowe ET, Bates N, Grady LN,  Brison DR, Hardingham TE & Kimber SJ. (2010). The directed differentiation of human embryonic stem cells towards chondrocytes Nature Biotech 28, 1187-1193
  • Soteriou D, Iskender B,  Byron A,  Borg-Bartolo S, Haddock M-C, Baxter M, Humphries, JD, Knight D,  Humphries MJ, and Kimber SJ. (2013) Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Proteins for Human Embryonic Stem Cell Maintenance J Biol Chem 288, 18716-18731
  • Cheng A,  Kapacee  Z,  Peng J,  Lu S, Lucas R, Hardingham TE,  Kimber SJ. (2014) Cartilage Repair Using Human Embryonic Stem Cell-derived Chondroprogenitors. Stem Cells Translational Medicine in press.
  • Cheng A, Hardingham TE, Kimber SJ  (2014) Generating Cartilage Repair from Pluripotent Stem Cells. Tissue Engineering and Regen Med B 20,257-266.

Fee Band

This project has a Band 2 fee. Details of difference fee bands are available for UK/EU or International applicants.

How to Apply

Find out How to apply for this PhD Project.

The role of circadian clock genes in regulating the chondrogenic potential of human Embryonic Stem Cells

Principal Investigators: Professor Sue Kimber, Professor Qing-Jun Meng

Funding

Project available for individuals with self arranged funding.

Description

Osteoarthritis (OA) is one of the most common joint diseases, affecting ~6 million people in UK, causing severe pain, deformity and a loss of mobility. A common feature of OA is the degeneration and loss of articular cartilage and chondrocytes, the only cell type in this tissue. The current clinical treatment for OA is restricted to symptomatic pain relief. The Meng group has recently identified cell autonomous circadian clocks in cartilage tissue, which controls ~600 rhythmic cartilage target genes, many of these genes have previously been implicated in diseases such as OA (Gossan et al., 2013, Arthritis and Rheumatism). hESC, due to their unlimited capacity for self renewal and pluripotency, provide a potential source of chondrocytes to be used in regenerative medicine towards joint diseases such as OA.  The Kimber group has developed a novel three stage chemically defined protocol to generate chondrogenic cells from a number of hESC lines and induced pluripotent cells (Oldershaw et al 2010 Nature Biotech 28,117 and Cheng et al Tiss Eng 2013). hESC can be induced to generate a 94-97% chondrogenic population expressing SOX9, collagen type IIα1 (COL2A1) and aggrecan in vitro within 14 days.

 

In collaboration with the Kimber group, we have recently identified profound changes of circadian clock genes during the chondrogenic differentiation of hESC.  We are particularly interested in the role of clock genes in chondrogenesis.  Such understanding may help identify key niche factors, which may be harnessed to manipulate differentiation of hESC towards regenerative medicine as well as control clock function. 

 

In this project, we are going to use genetic manipulation of hESC, in vitro chondrogenic differentiation, gene expression studies, real-time clock reporter techniques, lentiviral transduction and RNAseq approaches to address the functional significance of circadian clock genes in hESC differentiation. Exploiting the knowledge of circadian clock control will allow us to generate functioning and native-like cartilage tissue for therapeutic use as well as suggesting new disease models and drug targets for improving in vivo and in vitro matrix formation.

Related Publications

  1. Gossan N, Zeef L, Hensman J, Hughes A, Bateman JF, Rowley L, Little, CB, Piggins HD, Rattray M, Boot-Handford RP, Meng QJ, (2013). The circadian clock in chondrocytes regulates genes controlling key aspects of cartilage homeostasis. Arthritis and Rheumatism; Epub ahead of print.
  2. Meng QJ, Maywood E, Bechtold D, Lu WQ, Li J, Gibbs J, Dupré S, Chesham J, Rajamohan F, Knafels J, Sneed B, Zawadzke L, Ohren J, Walton K, Wager T, Hastings M, Loudon A (2010) Entrainment of disrupted circadian behavior through inhibition of Casein Kinase 1 (CK1) enzymes. Proceedings of the National Academy of Sciences of the United States of America, 107 (34), 15240-15245.
  1. Oldershaw RA,  Baxter MA, Lowe ET, Bates N, Grady LN,  Brison DR, Hardingham TE & Kimber SJ. 2010. The directed differentiation of human embryonic stem cells towards chondrocytes Nature Biotech 28, 1187-1193
  2. Soteriou D, Iskender B,  Byron A,  Borg-Bartolo S, Haddock M-C, Baxter M, Humphries, JD, Knight D,  Humphries MJ, and Kimber SJ. (2013) Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Proteins for Human Embryonic Stem Cell Maintenance J Biol Chem 288, 18716-18731

Fee Band

This project has a Band 2 fee. Details of difference fee bands are available for UK/EU or International applicants.

How to Apply

Find out How to apply for this PhD Project.

Understanding kidney development and disease using human pluripotent stem cells

Principal Investigators: Professor Sue Kimber, Professor Adrian Woolf

Funding

Project available for individuals with self arranged funding.

Description

Pluripotent stem cells (PSP) can form all tissues of the body and have excellent potential as agents for tissue regeneration. Our laboratory as part of the North West Embryonic Stem Cell Centre (NWESCC) has generated a number of human embryonic stem cells (hESC) lines and several induced pluripotent stem cells (iPSCs) (1,2) from human somatic cells using plasmid viral or other reprogramming methods to introduce the conventional ‘Yamanaka factors (3). We have focused on differentiation of PSCs to mesodermal lineages (1).  Recently several new protocols have been developed to generate cells of the major renal lineages (giving glomerular and collecting tubule structures)(4). Since kidney disease is a major problem for health services it may be possible to develop disease specific models from pluripotent stem cells differentiated to kidney progenitors. These could be used to help understand kidney disease and to develop new drugs/treatments for disease. One group of patients are those with ‘renal cysts and diabetes syndrome’ caused by heterozygous HNF1β mutations. Prof Woolf has identified 15 families with proven mutations in this gene(5). The student will derive iPSCS from patient/normal skin fibroblasts or blood cells. Using protocols developed in the lab he/she will differentiate them to ureteric epithelium. Differentiation to kidney progenitors will be compared to wild type iPSCs and hESCs. The mutation will be corrected using gene editing technologies (Crispr-Cas) to prove that the phenotypic differences are a result of the mutation rather than genetic variation. IPSC-ureteric bud organoids will be cultured under conditions which promote cysts development to compare to non-mutant cells. Once the model is established, cells will be manipulated using knockdown/inhibitors to understand more about why the HNF1β mutation causes renal cysts. Techniques will include hESC/iPSC culture/differentiation, immunofluorescence, Western blotting, microarray analysis, confocal microscopy, flow cytometry, quantitative RT-PCR, genetic manipulation and gene editing technologies.

Related Publications

  • Cheng A,  Kapacee  Z,  Peng J,  Lu S, Lucas R, Hardingham TE,  Kimber SJ. (2014) Cartilage Repair Using Human Embryonic Stem Cell-derived Chondroprogenitors. Stem Cells Translational Medicine.
  • McKay TR, Camarasa MV, Bates N, Fitzsimmons J, Brison, DR, Aplin, JD & Kimber SJ  (2011) Generation of continuous immortalised human placental feeders suitable for maintenance of human embryonic stem cells using lentiviral constructs containing BMI and hTert. Stem Cell Research 7,154-162.
  • Yamanaka, S. & Blau H.M. (2010) Nuclear reprogramming to a pluripotent state by three approaches. Nature 2010, 465, 704-712.
  • Takasato M, Er PX, Becroft M, Vanslambrouck JM, Stanley EG, Elefanty AG & Little MH (2013)Directing human embryonic stemcell differentiaon towards arenal lineage generates a sielf orgnaising kidney Nature VCell Biol 16,118-126.
  • Kolatsi-Joannou M Bingham C, Ellard S, Bulman MP, Allen LIS, Hattersley AT , Woolf AS (2001) Hepatocyte nuclear factor-1β: a new kindred with renal cysts and diabetes and gene expressionin normal human development. J Am Soc Nephrol 12, 2175-2180

Fee Band

This project has a Band 2 fee. Details of difference fee bands are available for UK/EU or International applicants.

How to Apply

Find out How to apply for this PhD Project.

Using the CRISPR/Cas9 system to modify gene transcription during hESC differentiation to kidney lineages

Principal Investigators: Professor Sue Kimber

Funding

Project available for individuals with self arranged funding.

Description

The CRISPR/Cas9 system has provided a powerful tool for genetic modification with significantly increased efficiency especially in stem cells: a small guide RNA (sgRNA) complementary to a genomic location of interest acts to bring the Cas9 nuclease to the vicinity. The resultant DNA break is then repaired to generate deletions, insertions or base substitutions at the desired location1. More recently, chimeric proteins between a mutant Cas9 (unable to digest DNA) and activating or repressing domains of transcription factors were created, allowing modification of gene transcription2.

In this project, we aim to utilise this latter technology to drive differentiation of human embryonic stem cells (hESCs) towards kidney cell types by modifying the expression of key genes. We will aim to improve on the efficiency of existing differentiation protocols3 and progressively complement or substitute growth factor-driven differentiation cues with more genetically defined ones. In contrast to the traditional use of cDNA-based overexpression-cassettes, the CRISPR/Cas9-based system has the additional advantages of driving expression from the native genomic locus and – given the very small size of sgRNAs – is more easily deliverable to the cells, with the ability for multiplexing from a single vector.

The student will optimise hESC differentiation to kidney lineages, characterising the resultant (and intermediate) cell types by methods including qPCR and immunostaining. They will design sgRNAs targeting already identified key genes, build the relevant plasmid and/or lentiviral expression vectors and introduce them to the cells at the appropriate time points during differentiation to achieve the desired effect. There will also be an opportunity to identify new key genes via high-throughput expression analysis (microarray/RNA-seq) and target these. Finally, there is scope to develop the technology itself by modifying the activating/repressing domains of the Cas9-based chimeric proteins with a view to optimise the level of expression for each gene.

Related Publications

  • Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013). "Multiplex genome engineering using CRISPR/Cas systems." Science 339(6121): 819-823.
  • Gilbert, L A, Larson M H, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Duodena JA, Lim  WA, Weissman JS, Qi LS (2013). "CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes." Cell 154(2): 442-451.
  • Takasato M, Er PX, Becroft M, Vanslambrouck JM, Stanley EG, Elefanty AG & Little MH (2013)Directing human embryonic stemcell differentiaon towards arenal lineage generates a sielf orgnaising kidney Nature VCell Biol 16,118-126.

Fee Band

This project has a Band 2 fee. Details of difference fee bands are available for UK/EU or International applicants.

How to Apply

Find out How to apply for this PhD Project.