Stem Cell Laboratory (Centre for Neuroscience & Department of Pharmacology)
Brief description of project/s
Human embryonic stem cells (hESC)
The discovery of human embryonic stem cells (hESC) opened up many exciting new opportunities to further investigate the basic biology as well as the therapeutical potential of stem cells in general. hESC originate from a pluripotent population of cells, they have a clonagenic potential (i.e. one stem cell can be cloned to give rise to a stem cell line), they are karyotypically normal, they can be propagated indefinitely (self-renewal) and they can differentiate in vitro and in vivo into cells representative of the three embryonic germ layers.
Picture of a hESC colony, maintained on a feeder layer of MEF
1) Signaling pathways involved in the pluripotency of human embryonic stem cells; Involvement of lysophospholipids in stem cell biology:
The signaling pathways involved in hESC self-renewal are still poorly understood. Indeed, while the activation of the Jak/STAT3 signaling pathway by Leukemia Inhibitory Factor (LIF) is able to maintain mouse ESC in the absence of the feeder layer of MEF, hESC cannot be maintained by LIF, suggesting that alternative signaling pathways are required for hESC maintenance of pluripotency. Understanding the signaling networks involved in hESC still remains a challenge.
The culture conditions used to maintain hESC undifferentiated were first based on the presence of a feeder layer of Mouse Embryonic Fibroblasts (MEF) in a fetal calf serum-containing medium. Due to the presence of unknown animal products, hESC grown in these conditions cannot be used for therapeutics. Moreover, these conditions are not optimal for defining the intracellular mechanisms involved in hESC self-renewal and differentiation: use of a feeder layer of fibroblasts from mouse or human origin or their conditioned media, replacement of the feeder layer by Matrigel (an extracellular matrix extracted from the Engelbreth-Holm-Swarm mouse sarcoma), use of serum or Knockout Serum Replacement (KSR, which contains undefined mixture of animal proteins). Therefore, a major goal in hESC research has been the establishment of a culture medium free of animal product. We previously demonstrated that the combination of sphingosine-1-phosphate (S1P) and platelet-derived growth factor (PDGF) is sufficient, in the absence of serum or KSR to maintain hESC undifferentiated and pluripotent (Pébay et al. 2005). This discovery allowed us to work in a chemically defined environment suitable to dissect more precisely the signaling mechanisms involved in hESC maintenance and/or differentiation. We also demonstrated that S1P and PDGF exert an anti-apoptotic effect on hESC as assessed by their ability to rescue hESC from a serum-deprivation induced apoptosis (Wong, 2007). Moreover, the anti-apoptotic effect of S1P and PDGF is abolished when the Erk1/2 or the PI3K pathways are inhibited (Wong 2007).
Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive lysophospholipids that are released by activated platelets and present in serum. These lysophospholipids act on a wide range of cells and regulate numerous cellular functions, including proliferation and differentiation, from the early stages of embryonic development. Most of their effects are mediated by specific G-protein-coupled receptors: S1P1-5, LPA1-3. The downstream signaling pathways modulated by S1P and LPA include the stimulation of PLC, increase of intracellular calcium concentration, phospholipase D activation, arachidonic acid release, inhibition of adenylate cyclase, activation of the small G proteins Rho and Ras, activation of the MAPK Erk pathway. There is now increasing evidence of their role in stem cell maintenance or differentiation. This project focuses on the understanding of the signalling pathways activated by S1P and LPA in hESC and their derivatives.
From Wong and Pébay, Journal of Stem Cells, 2006
2) Differentiation of neural stem cells derived from hESC:
Neural stem cells (NSC) can be easily derived from adult and fetal tissue, as well as directly differentiated from embryonic stem (ES) cells. Studies in mouse, chick and other species have identified several many of the extrinsic and intrinsic key factors that regulate NSC development and differentiation. However in the human, the signaling factors and mechanisms that regulate NSC maintenance and their further differentiation into neural and glial progenitor subtypes still remain largely unknown. In recent years human ES cells (hESC) have been isolated and propagated to generate hESC cell lines. In addition there have been several studies, including those from our laboratory, which directly differentiated hESC to neural derivatives with high efficiency. The use of hESC neural derivatives may provide important breakthroughs in medical research by providing cultured human cells that can be used as in vitro models to study neurodegenerative disorders. If hESC are to be used to study of early human neural development and disease, it is essential to understand the key pathways involved that regulate hESC neuronal differentiation. Thus, we need to explore whether the signaling factors known to be important in mouse and chick neural differentiation are also conserved in the human.
In our laboratory we have established an in vitro model of human NSC differentiation. Treatment of hESC colonies with the BMP antagonist, noggin, results in a highly efficient induction of neural progenitor cells, which can then be expanded as neurospheres and further differentiated to mature neuronal and glial cell types both in vitro and in vivo. Using a range of culture systems, we can morphologically monitor the various stages of neuronal differentiation from hESC to mature neurons and glia, which is very useful for exploring the signals involved in regulating this process.
Some of our main studies are to investigate two different gene families that are both known to play important roles in neuronal differentiation: the canonical Wnt gene family and the SoxB1 gene family of transcription factors. The Wnt proteins are secretable signaling factors that regulate many developmental processes including cell proliferation and differentiation. Wnt proteins bind to the Frizzled and LRP receptor families on the cell surface and transduce their signal either through the release of intracellular calcium or through the beta-catenin pathway, the latter known as canonical Wnt signaling. There have been several in vitro and in vivo studies in mouse demonstrating the role of the canonical Wnt proteins in regulating neural differentiation. The family of SoxB1 transcription factors consists of three members, Sox1, Sox2 and Sox3. Expression and functional studies of the SoxB1 genes suggest they regulate neural development and are commonly used as markers of embryonic, fetal and adult NSC. Our studies in neural differentiation of hESC, together with past studies in mouse, have demonstrated the canonical Wnts and the SoxB1 transcription factors are significant extrinsic and intrinsic signaling factors, respectively, that regulate neural differentiation from ES cells. Our project further explores their biological function in our in vitro model of NSC differentiation from hESC and aims to identify the mechanisms and the co-players involved in this process.
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Key References
Pébay A* Wong R*, Koh K, Nguyen L and Pera M. (2004). Presence of Functional Gap Junctions in Human Embryonic Stem Cells. Stem Cells 22 (6), 883-889. *: equal first authors.
Pébay A* Wong R*,Pitson S, Wolvetang E, Peh G, Filipczyk A, Koh K, Tellis I, Nguyen L and Pera M. (2005). Essential roles of sphingosine-1-phosphate and platelet-derived growth factor in the maintenance of human embryonic stem cells. Stem Cells 23, 1541-1548. *: equal first authors.
Costa M*, Dottori M*, Ng E, Hawes SM, Sourris K, Jamshidi P, Pera MF, Elefanty AG and Stanley EG. (2005). The hESC line Envy expresses high levels of GFP in all differentiated progeny. Nature Methods 2, 259-260. *equal first author
Wong R, Dottori M, Koh K, Nguyen L, Pera M and Pébay A (2006). Gap junctions modulate apoptosis and colony growth of human embryonic stem cells maintained in a serum-free system. Biochemical and Biophysical Research Communications 344, 181-188.
Wong R and Pébay A (2006). Signaling pathways involved in the maintenance of human embryonic stem cells. Journal of Stem Cells 1 (4), 271-282.
Costa M*, Dottori M*, Sourris K, Jamshidi P, Hatzistavrou T, Davis R, Azzola L, Jackson S, Lim SM, Pera MF, Elefanty AG and Stanley EG. (2007). A Method for genetic modification of human embryonic stem cells using electroporation. Nature Protocols 2, 792-796. *equal first author.
Wong R, Jamshidi P, Tellis I, Pera MF and Pébay A (2007). Anti-apoptotic effect of sphingosine-1-phosphate and platelet-derived growth factor in human embryonic stem cells. Stem Cells and Development, in press. Accepted 22.05.07.
Book chapters
Pébay A and Pera M. (2004). Growth factors and the serum-free culture of human pluripotent stem cells. Handbook of stem cells, Volume 1: embryonic stem cells, Part six: Methods, Chapter 51, 529-534. R. Lanza, Editor. Elsevier Academic Press.
Pera M and Dottori M. Stem cells and their development potential. (2005) In: Stem Cells From Bench to Bedside. A. Bongso and EH Lee, Editors. World Scientific Press
Pébay A and Pera M. (2006). Growth factors and the serum-free culture of human pluripotent stem cells. Essentials of Stem Cell Biology, Part four, Chapter 41, 313-316. R. Lanza, Editor. Elsevier Academic Press.
Dottori M and Pera M.F. Neural differentiation of human embryonic stem cells. (In Press) In: Methods in Molecular Biology, Neural Stem Cells, Second Edition. L.P.Weiner, Editor. The Humana Press Inc.
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