CELL PROGRAMMING
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Rathjen Laboratory - CMGD Adelaide
Telephone: +61 8 8303 5354 or + 61 8 8303 4671
Facsimile: +61 8 8303 5338
Email: peter.rathjen@adelaide.edu.au
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Research Focus
 Stable
pluripotent embryonic stem (ES) cell lines were isolated from the early mouse
embryo over 20 years ago. The ability to contribute to all the tissues of the
embryo and adult after reintroduction into the embryo, coupled with the ability
to differentiate extensively in vitro, has led to the widespread use of these
cells as vectors for transmission of genetic alterations into the mouse germline,
for modelling embryonic development and as a source of cell populations for
experimental analysis. With the isolation of embryonic stem cells from human
embryos in 1998, interest in the scientific and commercial application of ES
cell technology has increased dramatically. The potential to exploit human ES
cells as a source of cell populations with therapeutic applications has led
to enormous interest in processes that regulate and control the differentiation
of ES cells in vitro. We have been working with mouse ES cells for more than
15 years, developing in vitro differentiation technologies that promote the
differentiation of ES cells to specific cell lineages, recapitulating the developmental
processes of gastrulation in the early mammalian embryo. These models provide
a unique opportunity to identify and characterise differentiation and developmental
processes at a cellular and molecular level. Moreover, these differentiation
methodologies and the understanding at a molecular level of the regulatory mechanisms
will have application to the production of therapeutic agents from human ES
cells.
Research Projects
Recent publications
Australian Collaborators
- Australian Stem Cell Centre
(ASCC) Biotechnology Centre of Excellence, Melbourne, VIC
- Professor David Gilbert, SUNY, Upstate Medical University, Syracuse, NY,
USA
- Professor Shinya Yamanaka, Department of Stem Cell Biology, Institute for
Frontier Medical Sciences, Kyoto University, Japan
- Professor Anna Wobus, Institut für Pflanzengenetik und Kulturpflanzenforschung,
Gatersleben, Germany
- Professor John Hopwood, Women's and Children's Hospital, Adelaide, South
Australia, Australia
- Dr Charles Claudianos CMGD, ANU, Canberra, Australia
- ARC/NHMRC Research Network in
Genes and Environment in Development (NGED)
- Healthy Development Cluster, University of Adelaide, Adelaide, South Australia,
Australia
- Healthy Aging Cluster, University of Adelaide, Adelaide, South Australia,
Australia
Pluripotence in mammalian embryonic
stem cells
Pluripotence in mammalian embryonic
stem cells: What makes these cells tick?
 Pluripotence
describes the developmental competence of cells that display a specialised differentiation
capability - the ability to give rise, through differentiation, to all cell
types of the embryo and adult. Within the mammalian embryo the pluripotent lineage
is first apparent as a small population of cells massed to one pole of the expanding
blastocyst, the inner cell mass (ICM). Pluripotence persists in cells of the
primitive ectoderm, or epiblast, of the pregastrulation embryo, and is lost
as somatic cell populations are formed by differentiation. In culture, pluripotence
is maintained in embryonic stem (ES) cells, cell lines derived from the ICM
of many mammalian species, including mouse or human blastocysts.
Using a conditioned medium, MEDII, and ES cells we have recapitulated the formation
of primitive ectoderm in culture. ES cell-derived early primitive ectoderm-like
(EPL) cells are pluripotent but differ from ES cells in their gene expression,
differentiation potential and response to cytokines. Understanding the signalling
molecules required for the formation of EPL cells, and the ability to compare
two ontogenetically related pluripotent cell populations has given us a unique
window into the processes and pathways that underlie the maintenance and loss
of pluripotence. This model has been developed using mouse ES cells and we are
currently transferring this knowledge to a human ES cell model.
Signalling pathways required for the formation
of EPL cells from ES cells
Characterisation of MEDII has identified L-proline dependent signalling
pathway(s) as responsible for the alterations in gene expression and differentiation
potential associated with the formation of EPL cells. Inhibition of the transport
of proline into the cell, through antagonism of an amino acid transporter, can
abrogate the effect of L-proline, suggesting that entry of L-proline into the
cell is required for the formation of EPL cells. We are currently screening
for downstream effectors of L-proline to gain a complete understanding of the
intracellular pathways involved in the formation of EPL cells from ES cells
in culture.
Identifying intracellular pathways required for
maintaining the transcription of pluripotent regulators in ES cells
Remarkably little is known about links connecting the extracellular environment
and the regulation of pluripotence at the level of transcription, the extracellular
factors and intracellular signalling pathways that directly regulate the activity
of the pluripotent cell transcriptome. The POU-domain transcription factor Oct4
has been recognised for some time as a key regulator of pluripotence: deletion
of Oct4 results in a loss of the pluripotent cells within the developing blastocyst.
Using a comparative approach based on ES and EPL cells in culture we have initiated
projects to understand the signalling pathways that regulate Oct4 within a pluripotent
cell.
Epigenetic control of pluripotence and cell commitment
Chromatin structure and composition plays an important role in regulating
and maintaining gene expression and cell state. Changes in chromatin are absolutely
required for differentiation of ES cells, and also to 'reprogram' differentiated
cells to a pluripotent or less differentiated state. Work from our collaborators,
the Gilbert laboratory (SUNY), has demonstrated that timing of DNA replication
at specific gene loci can change at particular stages of ES cell differentiation,
and that this correlates with gene expression changes. This suggests a correlation
between changes in gene expression, replication timing and changes in active
and inactive chromosomal domains upon loss of pluripotency and commitment to
differentiated lineages. However, little is known about how active and silent
chromosomal domains are set up early in development and differentiation.
Our work aims to identify changes in chromatin structure (histone modifications,
DNA methylation, binding of chromatin modifiers etc), determine the timing of
these changes at key points in the differentiation pathway (such as, loss of
pluripotency and commitment to a differentiated lineage) and their temporal
correlation with gene expression changes. The use of our ES cell differentiation
system can provide important temporal information; for example, (i) are new
chromatin domains becoming established post-EPL cell stage or during EPL 'maturation',
and (ii) are pluripotence genes silenced by sequestration into heterochromatin
domains and when does this occur? This approach will also identify co-regulated
genes and aid in the elucidation of common regulatory mechanisms that underlie
the epigenetic changes.
Understanding the role of CRTR1 in ES cells
CRTR1 is a novel member of the CP2 family of transcription factors with
the ability to repress transcription from CP2-responsive genes. CRTR1 was initially
identified as differentially expressed between ES and EPL cells in culture:
in the embryo CRTR1 is similarly expressed, with transcripts seen in the pluripotent
cells of the ICM and down regulated on formation of the primitive ectoderm.
CRTR1 is expressed later during development, predominantly in the tubules of
the developing kidney where it becomes restricted to the distal convoluted tubules
and persists into adulthood. The phenotype of a recently reported CRTR1-/- mouse
suggests that the gene product is not essential for the maintenance and differentiation
of the pluripotent cells of the early embryo, but is required for normal kidney
function.
We are employing two approaches to understanding the role of CRTR1 in pluripotence,
molecular characterisation of the protein and identifying target genes. The
repression domain of CRTR1 has been mapped to a region encompassing amino acids
48-200. This is consistent with the identified repression domain in LBP9, the
human homologue of CRTR1. In heteromeric complexes CRTR1 acts to reduce the
activation capability of CP2, thereby repressing the expression of CP2-responsive
genes. We are continuing experiments to elucidate the mechanism by which CRTR1
exerts repression. The identification of target genes in ES cells is being undertaken
using microarray comparison of cell lines over-expressing either CRTR1 or modified
version of the protein capable of transcriptional activation. Potential target
genes will be validated by their presence in CRTR1-chromatin fractions.
Lineage-specific differentiation
of pluripotent ES cells
Lineage-specific differentiation of pluripotent ES
cells: Understanding the molecular cues that regulate differentiation to the
primary germ lineages during mammalian gastrulation.
Manipulation of the differentiation environment during EPL cell differentiation
results in lineage specific differentiation to either the mesendodermal lineages
or ectodermal lineages. In both cases differentiation occurs without the formation
of the extraembryonic endodermal lineages.
The formation and differentiation of EPL cells in cell aggregates (suspension
culture) in the presence of MEDII (EBMs) results differentiation to the ectodermal
lineage and results in a population of neurectoderm. Differentiation within
EBMs is homogeneous, synchronous and proceeds via the formation of a number
of transient, temporally distinct intermediate populations, providing a unique
system to capture and analyse intermediate populations such as pluripotent primitive
ectoderm and bipotential definitive ectoderm.
When differentiated as embryoid bodies (EPLEBs) EPL cells form a mixed population
of mesoderm and definitive endoderm, analogous with the cell populations that
are formed within the primitive streak. In contrast, the differentiated cell
products that form during ES cell differentiation within embryoid bodies (EBs)
comprise a more diverse array of cell types that include derivatives of the
ectodermal lineage and the extraembryonic endoderm lineages. The formation of
a more defined set of differentiation products within EPLEBs, coupled with the
lack of endogenous signalling that emanates from the extraembryonic endoderm,
suggests these bodies provide a superior system for understanding the molecular
mechanisms underlying lineage formation and for the formation of cell populations
with clinical applications.
Analysis of EPLEBs suggests that differentiation proceeds via a series of defined
events that recapitulate the events of the primitive streak in the gastrulating
embryo:
- The patterning of EPL cells to posterior epiblast.
- An epithelial to mesenchymal transition (EMT) that correlates with cell
differentiation and formation of mesendoderm.
- The differentiation of mesendoderm to mesoderm or endoderm.
By analysing differentiation within EPLEBs, and comparing it to differentiation
in EBMs, we are identifying and characterising the signalling molecules and
pathways that regulate the developmental outcome of differentiating EPL cells,
the decision to form either mesendoderm or ectoderm.
Deciphering the body's language
Deciphering the body's language: The signalling pathways that lead to positional
specification in the neural tube.
Cells within the developing neural tube become specified, or patterned, with
respect to the dorsal-ventral and anterior-posterior axes. Patterning of the
neural tube and subdivision of the neural progenitors is proposed to underlie
the complexity of the mature nervous system. By manipulating the differentiation
of EPL cells we can form unpatterned, or naive, neural tube-like cell populations,
or neurectoderm, within EBMs, that can be exploited as a model to understand
the molecular mechanisms of neural tube patterning.
To recapitulate the processes of cell patterning, ES cell-derived neurectoderm
has been exposed to known patterning signals. In response to Sonic hedgehog
(SHH) and retinoic acid EPL cell-derived neurectoderm expresses genes specific
to the ventral and posterior neural tube, respectively, providing proof-of-concept
that these cells can be patterned. The response to these factors appears uniform,
with over 90% of the cell population up regulating appropriate gene expression.
We are using this system to understand two aspects of positional specification
in the neural tube:
Specification of the dorsal/ventral boundary
The interaction of the SHH and BMP4 signalling pathways is proposed to determine
the boundary between the dorsal and ventral neural tube, although the mechanism
is unclear. By adding varying concentrations of both SHH and BMP4 to unpatterned
neurectoderm we can show that BMP4 acts to prevent signalling by SHH and the
up regulation of ventrally expressed genes. This has led us to propose a model
that predicts the formation of distinct dorsal and ventral domains, whereby
BMP4 emanating from the overlying ectoderm prevents ventralisation of the dorsal
neural tube by SHH. In contrast, the ventral domain of the neural tube is determined
by BMP4 signalling antagonists, emanating from the notochord, which prevent
BMP4 antagonism of SHH. o
Identification of novel signalling pathways required
for anteriorisation of the neural tube and formation of the forebrain
The signalling pathways that establish anterior identity and underlie
the formation of the forebrain are poorly understood. The forebrain is induced
from the neural tube by signals emanating from an adjacent organiser, the anterior
visceral endoderm (AVE). The AVE expresses Hesx1, a homeobox transcription factor,
knock-out of which leads to truncation of the forebrain. We have generated an
in vitro 'AVE' by introducing Hesx1 into HepG2 cells (Hesx1:HepG2 cells) and
have exposed unpatterned neurectoderm to the resulting conditioned medium. These
EBMs demonstrate an anteriorised gene expression, indicated by an induction
of forebrain specific marker genes. Microarray analysis of Hesx1:HepG2 has identified
a number of candidate signalling molecules that could regulate anteriorisation.
Publications (since 2000)
Johnson, B.V., Rathjen, J., and Rathjen, P.D. Transcriptional control of pluripotency:
decisions in early development. Current Opinion in Genetics & Development
(2006), 16:447-454
Kavanagh, S.J., Schulz, T.C., Davey, Pl, Claudianos, C., Russel, C, Rathjen,
P.D. (2005) A family of RS domain proteins with novel subcellular localisation
and trafficking. Nucleic Acid Research 33: 1309-22
Morris, M.B., Rathjen, J., Keough, R.A., Rathjen, P.D. (2005) Biology of embryonic
stem cells. In 'Human Embryonic Stem Cells'. Eds Odorico, J.S., Pederson, R.A.,
Zhang, S.C. BIOS Scientific Publishers, pp 1-28.
Pralong, D., Lim, M.L., Vassiliev, I., Mrozik, K., Wijesundara, N., Rathjen,
P., Verma, P.J. (2005) Tetraploid embryonikc stem cells contribute to the inner
cell mass of mouse blastocysts. Cloning and stem cells 7: 272-8
Lang, K.J., Rathjen, J., Vassilieva, S., Rathjen, P.D. (2004) Differentiation
of embryonic stem cells to a neural fate: a route to rebuilding the nervous
system. Journal of Neuroscience Research 76: 184-92
Rathjen, J.R., Rathjen, P.D. (2004) Embryonic Stem Cells. Isolation and application
of pluripotent cells from pregastrulation mammalian embryo in Stem Cell Handbook
p.33-43, edited by S. Sell, Humana Press Inc., Totowa, N.J., USA.
Rathjen, J., Washington, J.M., Bettess, M.D., Rathjen, P.D. (2003) Identification
of a biological activity that supports maintenance and proliferation of pluripotent
cells from the primitive ectoderm of the mouse. Biology of Reproduction 69:
1863-71
Rathjen, J., Rathjen, P.D. (2003) Lineage specific differentiation of mouse
ES cells: formation and differentiation of early primitive ectoderm-like (EPL)
cells. In Methods in Enzymology, Vol 365: Differentiation of embryonic stem
cells pages 3-25, edited by J.N. Abelson and M.I. Simon, Elsevier Academic Press
Pelton, T.A., Sharma, S., Schultz, T.C., Rathjen, J., Rathjen, P.D. (2002) Transient
pluripotent cell populations during primitive ectoderm formation: Correlation
of in vivo and in vitro pluripotent cell development. Journal of Cell Science
115:329-39
Rathjen, J., Haines, B.P., Hudson, K., Nesci, A., Dunn, S., Rathjen, P.D., (2002)
Directed differentiation of pluripotent cells to neural lineages: homogenous
formation and differentiation of a neurectoderm population. Development 129:2649-61
Rodda, S.J., Kavanagh, S.J., Rathjen J, Rathjen P.D. (2002) Embryonic stem cell
differentiation and the analysis of mammalian development. International Journal
of Developmental Biology 46:449-58
Stead, E., White, J., Faast, R., Conn, S., Goldstone, S., Rathjen, J., Dhingra,
U., Rathjen, P., Walker D., Dalton, S. (2002) Pluripotent cell division cycles
are driven by ectopic Cdk2, cyclin A/E and activities. Oncogene 21: 8320-33
Cui, S., Hope, R.M., Rathjen, J., Voyle, R.B., Rathjen, P.D. (2001). Structure,
sequence and function of a marsupial LIF gene: Conservation of IL-6 cytokines.
Cytogenet Cell Genet 92: 271-278
Faast, R., Thonglairoam, V., Schulz, T.C., Beall, J., Wells, J.R. E. W., Rathjen,
P.D., Tremethick, D.J., Lyons, I. (2001) Histone variant H2AZ is required for
early mammalian development. Current Biology 11: 1183-1187
Rathjen, J., Dunn, S., Bettess, M.D., Rathjen, P.D. (2001) Lineage specific
differentiation of pluripotent cells in vitro: A role for extraembryonic cell
types. Reproduction, Fertility and Development 13: 15-22
Rathjen, J., Haines, B.P., Hudson, K., Nesci, A., Dunn, S., Rathjen, P.D., Directed
differentiation of pluripotent cells to neural lineages: homogenous formation
and differentiation of a neurectoderm population. Development (in press)
Rathjen, J., Rathjen, P.D. (2001). Mouse ES cells: Experimental exploitation
of pluripotent differentiation potential. Current Opinion in Genetics &
Development 11: 589-595
Rathjen, J., Rathjen, P.D. (2001) Formation of neural lineages from embryonic
stem (ES) cells. The Scientific World (in press)
Rathjen, P.D., Rathjen, J. (2001). Directed differentiation of embryonic stem
(ES) cells for therapeutic application. J. Tumor Marker Oncology 16: 112
Rodda, S., Sharma, S., Scherer, M., Chapman, G., Rathjen P. (2001) CRTR-1: A
developmentally regulated transcriptional repressor related to the CP2 family
of transcription factors. Journal of Biological Chemistry 276: 3324-3332
Whyatt, L.M., Rathjen, P.D. (2001) IFN-inducible mammalian expression systems.
Methods in Molecular Biology 2001, 158:301-18
Haines, B.P., Voyle, R.B., Rathjen, P.D. (2000). Intracellular and extracellular
Leukaemia Inhibitory Factor proteins have different cellular activities, which
are mediated by distinct protein motifs. Molecular Biology Cell 11, 1369 - 1383.
Lake, J., Rathjen, J., Remiszewski, J., Rathjen, P.D. (2000). Reversible reprogramming
of pluripotent cell differentiation in vitro. Journal of Cell Science. 113,
555-566.
Rathjen, P.D., Verma, P. (2000). Precision genetic modification of the mammalian
genome. In White, D.H., and Walcott, J.J. (eds) (2000). Emerging technologies
in Agriculture: From ideas to adoption. Proceedings. Pp 49-58. Bureau of Rural
Sciences, Canberra.
Voyle, R.B., Rathjen, P.D. (2000). Regulated expression of alternate transcripts
from the mouse Oncostatin M gene: Implications for Interleukin-6 family cytokines.
Cytokine 12, 134-141.
Whyatt, L.M., Rathjen, P.D. (2000). IFN-inducible mammalian expression systems.
Meth. Mol. Biol. Ed. Tymms, M. and Kola, I. Humana Press.
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