Jenny Nichols
Member of Austin
Smith’s group, Wellcome Trust Centre for Stem Cell Research
University of Cambridge, Tennis Court Road CB2 1QR
E-mail: jn270@cam.ac.uk
Introduction
Unlike most other model organisms, the early mammalian embryo possesses an amazing capacity to regulate its own development. The evolution of the pluripotent compartment that becomes established at the late blastocyst has enabled the in vitro propagation of embryonic cells. Twenty five years ago the first embryonic stem (ES) cells were derived directly from mouse blastocysts in culture using medium supplemented with serum and a ‘feeder layer’ of mitotically inactivated fibroblasts. The process by which ES cells emerge was not understood, but their potential applications were immediately realised to be enormous. The purpose of our research is to try to understand how the pluripotent cells are assigned and maintained in the embryo; how they can be harnessed and propagated in culture as ES cell lines and how the process of ES cell derivation can be controlled and improved.
Current research projects:
- Using defined culture conditions and protocol modifications to monitor embryonic responses and improve the efficiency of ES cell derivation from murine embryos.
- Comparative characterisation of the molecular profiles of embryos from permissive versus recalcitrant strains of mice before and during the process of ES cell derivation
- Investigation of the roles and interactions of the pluripotency-associated transcription factors and signalling pathways during early development.
- Derivation o human ES cells.
Using defined culture conditions and protocol modifications
to monitor embryonic responses and improve the efficiency of ES cell
derivation from murine embryos.
Within a decade of the reports of the first ES cells leukaemia inhibitory
factor was identified as the activity coming from the feeder cells that
inhibits differentiation. More recently we have devised a system to generate
ES cells by replacing the serum in the medium with BMP4 or other factors
that activate the Smad/Id pathway. Thus, a simple model could be proposed
in which LIF blocks differentiation into mesoderm or endoderm and BMP4
blocks the formation of neural tissue.
Addition of selected inhibitors to the culture medium has obviated the requirement for exogenous cytokines for the maintenance and derivation of murine ES cells, apparently by simply removing the option to differentiate. We are currently investigating the effect of these inhibitors on early development and ES cell derivation. Preliminary data suggest that ES cells can be very efficiently derived from mouse embryos cultured from the 8 cell stage in basic medium supplemented with the inhibitors. This efficiency is apparently attributable to the ability of the inhibitors both to ablate primitive endoderm (Gata4+ve), which normally promotes differentiation of the developing epiblast, and to promote expansion of the epiblast (Nanog+ve).
Current collaborators:
Qi-Long Ying, Los Angeles
Jose da Silva (WTCSCR, Cambridge)
Related references:
Nichols, J., Evans, E. P. and Smith, A. G. (1990). Establishment of germ-line competent embryonic stem (ES) cells using differentiation inhibiting activity. Development 110, 1341-1348.
Nichols, J., Chambers, I. and Smith, A. G. (1994). Derivation of germline competent embryonic stem cells with combination of interleukin-6 and soluble interleukin-6 receptor. Exp. Cell. Res. 215, 237-239.
Buehr, M., Nichols, J., Stenhouse, F., Mountford, P., Greenhalgh, C. J., Kantachuvesiri, S., Brooker, G., Mullins, J. and Smith, A.G. (2003). Rapid loss of Oct-4 and pluripotency in cultured rodent blastocysts and derivative cell lines. Biol. Reprod. 68, 222-229.
Ying, Q. L., Nichols, J., Chambers, I. and Smith, A. (2003). BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115, 281-292.
Nichols, J. and Ying, Q-L. (2006). Derivation and Propagation of Embryonic Stem Cells in Serum and Feeder-Free Culture. Methods in Molecular Biology, Embryonic Stem Cell Protocols Volume 1: Isolation and Characterisation, 91-98.
Comparative characterisation of the molecular
profiles of embryos from permissive versus recalcitrant strains of
mice before and during the process of ES cell derivation
We have used serum-free and feeder-free culture conditions
to investigate the differences between the specification and development
of the embryonic and extra-embryonic components in embryos from selected
strains of mice, both before and during the process of ES cell derivation.
Student (joint with Professor Austin Smith):
Laura Batlle-Morera
Investigation of the roles and interactions
of the pluripotency-associated transcription factors and signalling
pathways during early development.
With the aid of transgenic mice and specific antibodies
we are studying the potential requirements and interactions of critical
transcription factors during the establishment of the pluripotent compartment
in the early embryo and during ES cell derivation.
Current collaborators:
Ian Chambers (ISCR, Edinburgh)
Jose Silva (WTCSCR, Cambridge)
Related references:
Nichols, J., Davidson, D., Taga, T., Yoshida, K., Chambers, I. and Smith, A. G. (1996). Complementary tissue-specific expression of LIF and LIF-receptor mRNAs in early mouse embryogenesis. Mech. Dev. 57, 123-131.
Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D., Chambers, I., Scholer, H and Smith, A. G. (1998). Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct-4. Cell 95,379-391.
Nichols, J., Chambers, I., Taga, T. and Smith, A. G. (2001). Physiological rationale for responsiveness of mouse embryonic stem cells to gp130 cytokines. Development 128, 2333-2339.
Chambers, I, Colby, D., Robertson, R., Nichols, J., Lee, S., Tweedie, S. and Smith, A. (2003). Functional Expression Cloning of Nanog, a Pluripotency Sustaining Factor in Embryonic Stem Cells. Cell 113, 643-655.
Derivation
o human ES cells.
We have derived four hES cell
lines from human embryos surplus to requirements of in vitro fertilisation
programmes donated with informed consent using defined medium supplemented
with LIF, BMP4 and basic FGF on human fibroblast feeder cells. These
will presently become available to other researchers from the National
Stem Cell Bank.
Current collaborators:
Outi Hovata (KI, Stockholm)
Alan Handyside (Bridge, London)
Further references since 2002:
Ying, Q. L., Nichols, J., Evans, E. P. and Smith, A. G. (2002). Changing potency by spontaneous fusion. Nature 416, 545-548.
Gabant, P., Forrester, L., Nichols, J., Van Reeth, T., De Mees, C., Pajack, B., Watt, A., Smitz, J., Alexandre, H., Szpirer, C. and Szpirer, J. (2002). Alpha-fetoprotein, the major fetal serum protein, is not essential for embryonic development but is required for female fertility. PNAS 99, 12865-12870.
Jackson, M., Baird, J. W., Nichols, J., Wilkie, R., Ansell, J. D., Graham, G. and Forrester, L. M. (2003). Expression of a novel homeobox gene Ehox in trophoblast stem cells and pharyngeal pouchendoderm. Developmental Dynamics 228, 740-744.
Zhao, S., Nichols, J., Smith, A. G. and Li, M. (2004). SoxB transcription factors specify neuroectodermal lineage choice in ES cells. Mol. Cell Neurosci. 27, 332-342.
Munsie, M., Mountford, P. and Nichols, J. (2005). Transgenic Systems in Nuclear Reprogramming. Methods in Molecular Biology 325, 115-128.
McConnell, J., Petrie, L., Stennard, F., Ryan, K. and Nichols, J. (2005). Eomesodermin is expressed in mouse oocytes and pre-implantation embryos. Mol. Reprod. Dev.71, 399-404.
Kaji, K., Caballero, I. M., MacLeod, R., Nichols, J., Wilson, V. and Hendrich, B. (2006). The NuRD component Mbd3 is required for pluripotency of embryonic stem cells. Nature Cell Biology 8, 285-292.
Kaji, K., Nichols, J. and Hendrich, B. (2007). Mbd3, a component of the NuRD co-repressor complex, is required for development of pluripotent cells. Development; 134, 1123-1132.
