Embryonic Stem Cell Differentiation Laboratory

The directed differentiation of embryonic stem cells (ESCs) has been put forward as an avenue for the generation of mature functional cell types that could potentially find clinical application in the treatment of conditions where specific cell types are missing or diseased. For example, HESC derived insulin producing beta cells could potentially replace scarce cadaveric islets currently being used as a treatment for type 1 diabetes. Similarly, the possibility of generating large volumes of red blood cells by a manufacturing process could alleviate the growing shortage of donor derived blood for transfusions now being experienced worldwide. However, hampering progress toward these outcomes is an inability to efficiently direct the differentiation of ESCs to any desired outcome. The Embryonic Stem Cell Differentiation Laboratory has a number of areas of interest including the generation of therapeutically important cell types from ESCs. The projects listed below are a sample of those available for honours, PhD or post doctoral research.

Eligibility: Students applying for honours should have an aggregate second and third year average of approximately 75% and should be interested in pursuing a career in science. Students interested in PhD positions should have the same plus an honours score above 80% (H1 or equivalent) and therefore be eligible for either an APA or Monash University scholarship. Exceptions will be considered on a case-by-case basis. If you are unsure about your suitability please forward a copy of your CV to either Ed Stanley (ed.stanley@med.monash.edu.au) or Andrew Elefanty
(andrew.elefanty@med.monash.edu.au) along with your expression of interest. Direct enquiries to either Andrew (+61 03 9905 0650) or Ed (+61 03 9905 0651) are also welcome.

1. Transcriptional Profiling of Pancreatic Progenitors

Background: Pancreatic b cells generated from differentiated embryonic stem cells (ESCs) represent a potential alternative to cadaver-derived islets for the treatment of type 1 diabetes. We have previously developed an embryonic stem cell line in which cells expressing a gene critical to the early development and function of the pancreascan be identified by the co-expression of a fluorescent marker (Micallef et al, 2005 (pdf)). Using this cell-line we are able to direct the differentiation of embryonic stem cells toward an endodermal and pancreatic fate. We have also generated a mouse line from these cells allowing a direct comparison between the cells made in the laboratory and those formed during embryogenesis (Holland et al, 2006 (pdf)). We have previously employed gene-profiling experiments to map the course of ESC differentiation and find new genes important to the differentiation process (Hirst et al, 2006 (pdf)). We now wish to apply similar strategies to dissect the process of pancreatic differentiation from ESCs.

Developmental expression of GFP in Pdx1^GFP/w mice mirrors that reported for Pdx1.

Fluorescent images of E9.5 Pdx1GFP/W and wild-type littermates demonstrated expression of GFP in the emerging dorsal (db) and ventral (vb) pancreatic buds.
Overlay of bright field and fluorescent images (Merge) of the Pdx1^GFP/W embryo shown in A.
Overlay of bright field and fluorescent images of an explant culture of E12.5 pancreas. D,E. GFP and Pdx1 (detected with an anti-Pdx1 antibody) expression in the same explant. The insets in the lower panels show the discrete nuclear localisation of Pdx1 in comparison with pan-cellular GFP.

This project will involve the molecular phenotyping of different populations derived from Pdx1GFP/w ESCs and Pdx1GFP/w mice in order to match the developmental stage of cells from in vivo and in vitro sources. This analysis will also be used to identify novel markers of ESC differentiation.

Techniques to be employed will potentially include fluorescence activated cell sorting (FACS), RNA isolation, ESC differentiation, whole genome microarray analysis and in silico interrogation of gene expression (microarray analysis), tissue culture and microdissection.

2. Gene targeting vectors for genetic modification of Human Embryonic Stem Cells

Background: The ability to genetically modify human embryonic stem cells is critical to utilising these cells to study their commitment to specific cell lineages. We have previously used gene targeting in mouse ESC cells tag genes whose expression marks specific stages of embryogenesis (Micallef et al 2005 (pdf), Ng et al 2005 (pdf)). We have recently begun applying this same technology to generate HESC lines with genes encoding fluorescent proteins inserted into specific loci whose expression marks a variety of interesting cell types (Costa et al, 2005 (pdf)).

This picture is taken from Costa et al (2005) and shows that the HESC line, Envy, expresses green fluorescent protein in all kinds of differentiated cell types. Panels A to I show neural cell types expressing GFP, J to K shows GFP+ embryoid bodies, M to O demonstrates that GFP expression is maintained in blood cells and S to U shows GFP expressing liver like cells. Envy cells will be very useful for tracking HESCs and their derivatives following transplantation and in cultures containing mixtures of different cell types.

This project will involve the design and building of gene targeting vectors using a variety of different cloning techniques. Students will be instructed in aspects of vector design and assembly. Preliminary experiments will involve testing of vectors in appropriate cell lines.

Techniques will potentially include polymerase chain reaction (PCR), Real time PCR, restriction digestion and ligation, HESC culture, Flow cytometry, Vector design, Southern Blot analysis, Cell electroporation and transfection.

3. Lineage specification during ES differentiation

Background: The specification of particular cell lineages during ESC differentiation is dependent on the growth factor milieu and media composition the ESCs are exposed to. In order to efficiently produce cell types with therapeutic value it will be necessary to guide ESC differentiation along specific pathways. Our laboratory has established protocols for the directed differentiation of mouse and human ESCs to a variety of different cell types including heart, blood, endothelium, endoderm and neurons using a serum free media (Ng et al 2005a, Ng et al 2005b (pdf), Micallef et al 2005 (pdf), Costa et al 2005 (pdf))(see below picture).

This project will involve further analysis of the culture conditions promoting the formation and patterning of ectoderm, mesoderm and endoderm from human embryonic stem cells.

Techniques will potentially include HESC culture and differentiation, Flow cytometry, Real time PCR, cell transplantation into immunodeficient mice, Statistical data analysis

4. Red Blood cell development from Human embryonic stem cells

Background: The eligibility criteria for safe blood donation are continually being restricted in an attempt to limit the transmission of pathogens (both known and unknown) to the recipients of blood products (http://www.arcbs.redcross.org.au/). Human embryonic stem cell derived Red Blood Cells (RBCs) represent a potential alternative to donor-derived blood, provided methods for the production of this cell type can be optimised. During development, red blood cell formation (erythropoiesis) occurs at a number of embryonic sites and transits through a series of sequential developmental switches culminating in the generation of mature enucleated erythrocytes (adult RBCs). A key focus of the Embryonic Stem Cell Differentiation Laboratory is to understand and control the formation of red blood cells from HESCs in an efficient and scalable manner.

This picture shows a multi-focal colony of human red blood cells differentiated from human embryonic stem cells in a serum free medium. The colonies form in methylcellulose (a semi-solid media) and each colony, which contains hundreds of cells, is derived from a single red blood cell progenitor. Red cells made in this way could eventually replace blood collected from donors and provide a safe and reliable source of blood cells for use in blood transfusions. See Ng et al 2005b

This project will involve dissection of the developmental stages of erythropoiesis using genetically tagged HESCs as tool to track and identify each stage.

Techniques will potentially include HESC culture and differentiation, Flow cytometry, Real time PCR, cell transplantation into immunodeficient mice, gene targeting, Southern blot analysis.

5. Analysis of the function of Slain, a novel stem cell gene

Background: We have previously used gene-profiling experiments to identify a new family of stem cell genes expressed in mouse and human embryonic stem cells (Hirst et al, 2006 (pdf)). Slain1, the founding member of this family, is highly conserved throughout vertebrate species yet shares no sequence similarity with any previously identified proteins or protein subdomains. We have generated a mouse line in which the bacterial beta galactosidase gene has been inserted into the slain locus and are using this line to track expression of Slain1 in the mouse. Other experiments aimed at placing Slain into the context of existing biochemical or signalling pathways are also underway. Understanding the function of Slain1 is likely to provide a novel insight into factors regulating the growth and differentiation of embryonic stem cells.

This figure shows expression of the Slain1 gene in the brain of an adult mouse in which the Slain1 locus has been tagged with the bacterial beta galactosidase gene.

This project will involve analysis of Slain1 function using in vivo and in vitro models, including Slain1 knockout mice, ESC differentiation and protein-protein interaction studies

Techniques will potentially include ESC culture and differentiation, Flow cytometry, Real time PCR, mouse genotype and phenotype analysis, histological analysis, immunohistochemistry, gene targeting, southern blot analysis, fluorescence microscopy.