Dissecting self-renewal in stem cells with RNA interference

1. Dissecting self-renewal in stem cells with RNA interference Natalia Ivanova, RaduDobrin, Rong Lu, Iulia Kotenko, John Levorse, Christina DeCoste, Xenia Schafer, Yi Lun & Ihor R. Lemischka (Nature 442, 533-538 (3 August 2006

2. Stem cells • In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues • In adult organisms, stem cells replenish specialized cells, and maintain the normal turnover of regenerative organs (blood, skin, intestinal tissues etc.) • They can divide indefinitely, in contrast to progenitor cells that also participate in tissue repair

3. Embryonic stem cells • Embryonic stem cells are pluripotent • Most adult stem cells are lineage restricted (multipotent)

4. Therapeutic potential • Therapies are currently in experimental stages (except for bone marrow transplantation) • One possible risk is that transplanted stem cells could form tumors if cell division continues uncontrollably

5. Somatic cell nuclear transfer • Breaking up human embryos offends some people’s moral sensibilities

6. Induced pluripotent stem cells • Yamanaka and Takahashi showed that pluripotency can be induced, first in mouse fibroblasts and then in human facial skin cells Takahashi and Yamanaka, Cell 2006

7. Yamanaka factors • The Yamanaka team used the transcription factors Oct4, Sox2, Myc, and Klf4 • Myc sometimes causes cancer as a side-effects • Yu et al. showed that a similar result can be obtained using the TFS Oct4, Sox2, Nanog and Lin28 • They used human foreskin cells

8. iPSCs replacesomatic cell nuclear transfer • Ian Wilmut announced that he will abandon somatic cell nuclear transfer • The iP technique is more efficient and less problematic to accept socially

9. Control mechanism of pluripotency and differentiation • Different cell types have different gene expression patterns • Gene expression patterns are controlled by the gene regulatory network

10. Selection of genes that are down-regulated during differentiation • Induce differentiation by the addition of retinoic acid and LIF removal • High throughput expression profiles for six days • This results in 901 rapidly downregulated genes • Out of these 65 are known as regulators or are unassigned ESTs

11. Selection of genes whose down-regulated causes differentiation • The 65 candidates are regulators that were down-regulated in the experiment • A causal relationship needs to be established more rigorously • Knockout by shRNA is transferred to daughter cells Knock-down

12. Silencing of genes that cause differentiation Recognizes mRNA non-coding region • Mix the transfected cells with w.t. cells • If the transfected differentiate, their number will decrease because: • Lack of essential differentiation growth factors • Cell cycle slows • Cell adhesions are not formed effectively • Conditions for maintaining pluripotency provided to the culture

13. Results of “differentiation screening” • Two of the hits did not reduce alkaline phosphatase activity or affect cell morphology • Neither did knock-outs of the other genes that were not selected by the screening

14. Activation of the MAPK signaling pathway • ERK was hyper phosphorylated in some knock-downs • The MAPK pathway is activated in trophectoderm Removal of LIF induces differentiation

15. Ruling out non-specific effects • It is still possible that the shRNA experiment induced differentiation but not due to the knock-down • To rule out non-specific effects: • Additional different shRNAs were designed. This provided validation for Nanog, Oct4, Sox2, Esrrb and Tcl1 • A rescue strategy was developed

16. Rescue strategy for TF knock-downs • It is still possible that the shRNA experiment induced differentiation but not due to the knock-down shRNA that targets Non-coding region Only coding region

17. Doxycycline dependent self-renewal • The following knock-downs remained similar to undifferentiated cells: • Sox2 • Esrrb • Tbx3 • Tcl1 • In the other cases the results were not that conclusive, e.g. Oct4 rescue cells grow more slowly but retain ES morphology

18. Doxycycline removal brings up mature cell markers Trophectodermal markers The Trophoblast provides nutrients to the embryo and develop into a large part of the placenta

19. Commitment to specific lineages • What happens if a regulator is constantly turned on in a developing embryo? • Embryoid body: an aggregate of ESCs that “mimics” an embryo • Create an embryoid body and check which lines are missing Markers for all 3 lineages were expressed in NanogR cells Colors= two different clones All 3 markers induced

20. Esrrb blocks mesoderm and neuroectoderm • Activation of Esrrb in the cells of the embryoid body prevented specification into these tissues but not endoderm

21. Expression patterns of silencing-responsive genes • 771 genes are upregulated or downregulated in response to the shRNA treatments • Pattern 1(771): Responsive to most shRNA • Pattern 2(474): Responsive to Nanog,Oct4,Sox2 • Pattern 3(272): Responsive to Esrrb,Tbx3,Tcl1, Dppa4

22. Differentiation inducers controlled by self-renewal regulators • Each of 160 upregulated genes was overexpressed • In 18 cases cell morphology changed, Nanog was down and alkainephosphatase activity was lost

23. Nanog compensate for loss of other self-renewal regulators • It was previously shown that upregulation of Nanog can prevent differentiation even if important substances are removed from the culture • A Nanog expressing vector was inserted to knock-down cells gene shRNA Morphology+ Alkaline phos. activity