1. Stem Cells and Cell Signaling
Cheng-En Lai
BIOE 506: Molecular and Cellular Bioengineering
4/25/2011

2. Overview Background Transforming Growth Factor-β (TGF-β)
Stem Cells
Signaling Pathways
Transforming Growth Factor-β (TGF-β)
SMAD Signaling Pathway
Stem Cell Differentiation Overview
Signaling Examples
TGF-β in various cell types
Other Signaling Pathways
WNT
Notch
Hedgehog
Fibroblast Growth Factor (FGF)
Cross-talk
SMAD/WNT
WNT/FGF/Notch/SMAD/Hedgehog
Conclusions

3. Background: Stem Cells
Self Renewal
Pluripotency
Source for tissue engineering and cell replacement therapies
Similar to cancer cells; stem cells thought to be derived from cancer stem cells
Adapted from System Biosciences (systembio.com)
Understanding stem cells is important for understanding cancer

4. Background: Signaling Pathways
Abnormalities in pathways may give rise to cancer stem cells and tumors
Understanding the signaling pathways and identifying important factors helps to understand cancer transformation as well stem cell differentiation for tissue engineering and regenerative medicine applications

5. Transforming Growth Factor β
TGF-β proteins and TGF-β related bone morphogenetic proteins (BMPs) are important regulators of stem cell differentiation, maintenance, and self-renewal, as well as carcinogenesis suppression.
Comprised of 30 related proteins in the SMAD pathway

6. SMAD Signaling Pathway
The downstream canonical signaling pathway for TGF-β proteins, including activin, nodal, and BMP. Also called the nodal signaling pathway and TGF-beta signaling pathway.
Bind to type 1/type II serine/theonine kinase receptors.
A number of SMAD proteins are activated via phosphorylation
Receptor SMADs complex with common-partner SMAD4 to translocate to the nucleus
Inhibitory SMADs inactivate R-Smads and function as a negative feedback loop.
Signaling cascade resulting in activation and suppression of genes, such as OCT3/4 and Nanog.
OCT3 OCT4 Nanog
Adapted from Blank et al. (2008)

7. Stem Cell Differentiation Overview
BMP-4 is important for proliferation and is mediated by its inhibitory effect on neural differentiation
Shows how changes in signal levels drive differentiation. IE. BMP signals alone are required for formation of the posterior primitive streak, and eventually giving rise to mesodermal tissue
M=maintenance
D=differentiation
Adapted from Watabe et al. (2009)

8. SMAD Signaling Examples
Nodal and activin cooperate with the WNT pathway to maintain ES cells and keep them undifferentiated and pluripotent.
Activin and TGF-β confers mesodermal differentiation depending on amount.
BMP signaling results in mesodermal and ectodermal differentiation in human ES cells.
Nodal signals are important for OCT3/4 expression and maintenance of ES cells.
Activin is important for maintenance of pluripotency, which is possibly done through induction of Nanog and OCT-4
OCT4 and Nanog are important transcription factors in generation of IPS cells
BMP and leukemia-inhibiting factor keep mouse ES cells in the undifferentiated state.[Mishra]

9. TGF-β in Neural Stem Cells
BMP inhibits neural differentiation
TGF-β promotes differentiation in committed progenitors
Inactivation of TGF-β growth-inhibitory functions result in tumor progression
Adapted from Mishra et al. (2005).

10. TGF-β in other Cell Types
Hematopoietic Stem cells
Inhibits early progenitors, while enhances differentiation of committed stem cells
Mesenchymal Stem Cells
Inhibits differentiation and maturation into myoblasts, osteoblasts, and adipocytes, while stimulating MSC proliferation
Basis for efficient wound repair in mesenchymal tissue
Gastrointestinal Epithelial Stem Cells
Inactivation with one TGF-β component (Receptor, SMAD protein) is present in all gastrointestinal cancer
BMP’s and cytokines promote specification, differentiation and proliferation of human ES cells
Deregulation of TGF-β can result in cancer progression as well.
BMPs activate osteoblast differentiation.

11. WNT Signaling Pathway Adapted from Katoh et al. (2007).
Frizzled receptors and LRP coreceptors receive canonical WNT signals, which in turn activate disheveled proteins (DVL).
DVL proteins inhibit the formation of the axin/GSK-3/APC complex, which acts to phosphorylate beta-catenin, tagging it for polyubiquitination and eventual degradation.
Nuclear accumulation of beta-catenin complexes with TCF/LEF transcription factors which activate FGF20, DKK1, WISP1, MYC, and CCND1 genes.
Adapted from Katoh et al. (2007).

12. WNT Signaling Pathway Cell fate determination
Transformation of cancer stem cells due to disregulation
MYC is a transcription factor involved in generation of IPS cells.

13. Notch Signaling Pathway
Delta or jagged ligands bind to Notch receptors.
Gamma-secretase causes the release of the intracellular domain (NICD)
Forms a complex with CSL and MAM and activate genes involved in neural differentiation.
Adapted from Bray et al. (2009).

14. Notch Signaling Pathway
Promotion of neural cell differentiation
Involved in self-renewal of hematopoietic stem cells

15. Hedgehog Signaling Pathway
Shh binds to Patched-1 which stops inhibition of Smoothened.
Smo activates Gli proteins in the nucleus resulting in regulation of genes.
Adapted from Altaba et al. (2002).

16. Hedgehog Signaling Pathway
Induces differentiation of hematopoietic progenitors and neural stem cells
Skin, muscle, and brain cancers develop when pathway is maintained improperly in stem cells

17. FGF Signaling Pathway Adapted from Katoh et al. (2006)
FGF signals are transduced through FGF receptors
Various signaling cascades result in EMT, cell survival, and proliferation/differentiation
EMT is caused by downregulation of E-cadherin
Adapted from Katoh et al. (2006)

18. FGF Signaling Pathway EMT Cell survival Proliferation/differentiation
Cross-talk is seen between WNT and FGF via down-regulation of GSK3β, resulting in tumors with more malignant phenotypes of mammary carcinogenesis

19. TGF-β1 /WNT Pathway Cross-talk
SMAD and TCF/LEF associate to cooperatively regulate genes
Series of experiment by Jian et. al. (2006) show that TGF-β1 addition results in rapid nuclear accumulation of β-catenin in MSCs in a new form of cross-talk.
β-catenin nuclear accumulation is not due to phosphorylation as from canonical WNT pathway
Mediated by SMAD3/GSK3β disruption through TGF-β mediated phosphorylation.
MSCs act differently with TGF-β.
SMAD3 acts as a chaperone for beta-catenin

20. TGF-β1 /WNT Pathway Cross-talk
MSCs act differently with TGF-β.
SMAD3 acts as a chaperone for beta-catenin

21. WNT/FGF/Notch/SMAD/Hedgehog Cross-talk
Balance of all signaling pathways is important for homeostasis and prevention of cancer and congenital diseases
Hedgehog pathway
SMAD pathway
Notch family receptor
Notch family receptor
Hedgehog pathway induced

22. Conclusions
Many signaling pathways with cross talk involved in stem cell proliferation, maintenance, and differentiation
Dependent on differentiation stage, type of cell, local environment, and the identity and amount of particular ligand
Identification of key regulators has potential for generation of iPS cells and cell replacement therapies

23. References
Mishra L, Derynck R, & Mishra B. Transforming growth factor-beta signaling in stem cells and cancer. Science 310, (2005).
Blank U, Karlsson G, & Karlsson S. Signaling pathways governing stem-cell fate. Blood. 111(2), (2008)
Jian H, et al. Smad3-dependent nuclear translocation of beta-catenin is required for TGF-beta1-induced proliferation of bone marrow-derived adult human mesenchymal stem cells. Genes Dev 20, (2006).
Katoh M & Katoh M. WNT Signaling Pathway and Stem Cell Signaling Network. Clin. Cancer Res. 13, 4042 (2007).
Watabe T & Miyazono K. Roles of TGF-beta family signaling in stem cell renewal and differentiation. Cell Research 19, (2009).
Bray S. Notch Signaling: a simple pathway becomes complex. Nature Rev. Mol. Cell Bio. 7, (2006).
Altaba AR, Sanchez P, Dahmane N. Gli and hedgehog in cancer: tumours, embryos and stem cells. Nature Rev. Cancer, 2, (2002).
Katoh M & Katoh M. Cross-talk of WNT and FGF signaling pathways at GSK3-beta to regulate beta-catenin and SNAIL signaling cascades
Katoh M. Networking of WNT, FGF, Notch, BMP, hedgehog signaling apthways during carcinogenesis. Stem Cell Reviews. 3(1), (2007).