Transcript MCB 252, Part 2 Genetic inference, lecture 15 The nature of mutations

• Overview
• Mouse embryonic stem cells
• Human embryonic stem cells
• Pluripotency genes and network
• Long-term self-renewal
• Directed differentiation
• Induced pluripotent stem cells
Stem cells, pluripotency and differentiation
Two major types of stem cells
Adult and embryonic stem cells
Self-renewal
The ability to undergo symmetrical divisions without
differentiation
Pluripotency
The ability to give rise to differentiated
cell types derived from all three primary
germ layers of the embryo: endoderm,
mesoderm, and ectoderm
Induced pluripotent stem (iPS) cells
induction of pluripotent stem cells from
differentiated cells
Differentiation of human tissues
Generation of embryonic stem cells
Two prominent features of ESCs: long-term self-renewal and pluripotency
A blastocyst cultured on a petri dish
Day 1
Day 2
•
Day 3
Day 4
Inner cell mass (ICM): a cluster of
cells at the blastocyst stage
Alkaline phosphatase positive
DAPI
Isolation of ICM cells
Mouse embryos
Rabbit Anti-mouse serum
Pipetting
Outer cells are lysed.
Derivation of embryonic stem
cells from mouse embryos
Martin Evans
2007 Nobel Prize
Karyotype is normal
Evans, M.J. & Kaufman, M.H. Nature 292, 154-156, 1981
Feeders provide factors that maintain
embryonic stem cell growth
Day 13 mouse embryos
MEFs: mouse embryonic fibroblasts
Remove heads and
internal organs
Treat with trypsin and
plate cells into a dish
MEFs
irradiated to stop MEF growth
Embryonic stem cells are pluripotent
Embryoid bodies
Low attachment
ESCs
(mixture of differentiated cells)
Teratomas
Mouse injection
Cells of three germ layers
Derivation of embryonic stem
cells from human embryos
Jamie Thomson
Univ. of
Wisconsin
ICM-derived
H9 cell line
Critical factors:
MEFs, basic FGF
Differentiating cells
Thomson, et al., Science, 1998
What are the promises?
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Understand early human development (infertility,
birth defects) and control of cell division (cancer)
Cell-based therapy
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Reduce need for organ and tissue
donors/transplants
Replace mutant or damaged cells for treatment of
diseases such as Parkinson’s disease, spinal
cord injury, muscular dystrophy, heart disease,
liver dysfunction, osteoporosis, vision and hearing
loss
• A short-cut for drug discovery and testing
Transcription factors required for pluripotency
Austin Smith
Oct4 -/- embryo lack inner cell mass
Oct4 -/- cells are not pluripotent
Other important transcription factors:
Sox2 and Nanog
Inner cell mass
Core ES cell regulatory circuitry
Jaenisch and Young, Cell. 2008
Regulation of long-term self renewal
Mouse ESCs
LIF (Smith et al., Nature, 1988)
BMP (or serum) (Ying et al, Cell, 2003)
3i (Ying et al, Nature, 2008)
(Buehr et al, Cell, 2008)
LIF and BMP act on downstream differentiation
signals of MAPK
He S et al. 2009. Annu Rev Cell Dev Biol;
Directed ES cell differentiation
Transcription factor landscape
Graf T and Enver T, 2009, Nature
What would be an ideal method for directed differentiation?
Rapid
Simple
Cheap
Mimic development
Conditions for directed differentiation
1. EBs
EB medium
hESCs
18 d
EB digestion
EB
formation
Hematopoietic
stem cells
2. Co-culture
OP9 mouse stroma cells – hematopoietic differentiation
PA6 or MS5 – neural differentiation
3. Monolayer cultures
OP9 coculture
Expansion
Progenitor
Expansion
medium 7d
Hematopoietic
stem cells
Neutrophils
Terminal
differentiation
medium 6-7 d
Hypothesis
Shinya Yamanaka
Kyoto University
Differentiated somatic cells can be reprogrammed into pluripotent stem (ESClike) cells with gene(s) important for ESC
identity (pluripotency and self-renewal)
These cells would
• Bypass ethical issues
• Create patient-specific pluripotent stem cells
24 candidate genes
Dppa2
Dppa3/ Stella
Dppa4
Dppa5/ Esg1
Ecat1
Ecat3/ Fbx15
Ecat5/ Eras
Ecat8
Ecat9/ Gdf3
b-catenin
Dnmt3l
Fthl17
Grb2
Sox2
Sox15
Tcl1
Oct4
Rex1
Sall4
Utf1
Klf4
Myc
Nanog
Stat3
Gene delivery: Retrovirus allowing gene integration into the host
genome
Takahashi and Yamanaka (2006) Cell 126, 663-676
Putting all 24 genes into MEFs “reprograms”
FBX15: an ESC-specific gene; only expressed in ESCs
bgeo: G418 (an antibiotics that kills the cells) resistance gene
So, cells can survive only when they become ESC-like cells
Viral promoter
Takahashi and Yamanaka (2006) Cell 126, 663-676
Narrowing down the candidates
Oct4 (14)
Sox2 (15)
Klf4 (20)
Myc (22)
Takahashi and Yamanaka (2006) Cell 126, 663-676
iPS cells are pluripotent
Pluripotency markers
EB formation
Teratoma formation
- Saw the same thing with tail-tip fibroblasts
Takahashi and Yamanaka (2006) Cell 126, 663-676
How about human cells?
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Takahashi K., et al. (2007) “Induction of pluripotent stem cells from
adult human fibroblasts by defined factors.” Cell 131, 861-72.
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Yu J., et al. (2007) “Induced pluripotent stem cell lines derived from
human somatic cells” Science 318, 1917-1920.
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OCT4, SOX2, KLF4, MYC
OCT4, SOX2, NANOG, LIN28
Park I.H., et al. (2007) “Reprogramming of human somatic cells to
pluripotency with defined factors” Nature 451, 141-146.
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OCT4, SOX2, KLF4, MYC
Stem cell-based therapy
Regenerative Medicine
Stem Cell Biology
Human somatic cells
Translation
Cellular
therapies
Derivation
iPSCs
•Scale Up
•Quantitative, systematic
approaches
•Quality control
Propagation
Differentiation
Tissue
morphogenesis
“Personalized medicine”
Adapted from: Gepstein. Circ Res 2002 & http://stemcells.nih.gov/info/media/DSC_1187.jpg
Pitfalls with iPSCs
• Low efficiency of derivation
• Use of C-myc
• Transgene integration
• Are they really the same as ESCs?
<0.1%
• Low efficiency of derivation
- Are all four genes expressed in the same cells?
Approach: Using a single retroviral or lentiviral
vector instead of four vectors (2A peptide)
Somers A, et al 2010, Stem Cells (STEMCCA Cre-Excisable lentivector)
Staerk, J et al, 2010, Cell Stem Cell (T cells and myeloid cells)
• Use of C-myc
- Chemical complementation (e.g., with small molecules such as VPA) to
replace C-Myc
Other compounds: Vitamin C, sodium butyrate, ALK5 inhibitor(*, mESC
medium), Apigenin and Luteolin (E-cadherin enhancing)
Reprogramming with small molecules only?
• Transgene integration
- integrating-free vectors
•Episomal vectors followed by selection of integration free cells
•Cre/loxP-recombination system to deliver followed by removal
with Cre- recombinase
•Single-vector reprogramming system combined with a piggyBac transposon
- Protein and mRNA-based
•Delivery of OCT-4, SOX2, Myc and Klf4 mRNA or proteins,
instead of genes, into somatic cells
Protein: polyarginine tag
Mouse, 30 days, the need for VPA.
Human, 50 days, HEK293 cell extracts
Synthetic mRNA:
17 days, 2% efficiency
Are iPSCs as good as ESCs?
Mouse iPSCs:
Can contribute to embryonic development (Takahashi and Yamanaka, Cell, 2006)
Produce adult chimera and are germ-line competent (Okita et al, Nature, 2007)
Are capable of giving rise to every cell in the new born mice (Zhao et al., Nature, 2009)
Journal of Molecular Cell Biology (2010), 2, 171–172
Human iPSCs
1. Global gene expression profiling;
2. Modifications of histone tails;
3. The state of X chromosome inactivation
4. Profiles of DNA methylation
At least for some clones, iPSCs are similar if not indistinguishable
from ESCs (Mikkelsen et al., Nature, 2008)
Stem cell-based therapy
Regenerative Medicine
Stem Cell Biology
Human somatic cells
Translation
Cellular
therapies
Derivation
iPSCs
•Scale Up
•Quantitative, systematic
approaches
•Quality control
Propagation
Differentiation
Tissue
morphogenesis
“Personalized medicine”
Adapted from: Gepstein. Circ Res 2002 & http://stemcells.nih.gov/info/media/DSC_1187.jpg
Disease Modeling using iPSCs
Disease-specific iPSCs
Disease-related differentiated cells
Lee, G., Papapetrou, E.P., Kim, H., Chambers, S.M., Tomishima, M.J., Fasano, C.A., Ganat, Y.M.,
Menon, J., Shimizu, F., Viale, A., Tabar, V., Sadelain, M., and Studer, L. (2009). Modelling
pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature 461,
402-406.
Marchetto, M.C.N., Carromeu, C., Acab, A., Yu, D., Yeo, G. W., Mu, Y., Chen, G., Gage, F.H., and
Muotri, A.R. (2010). A Model for Neural Development and Treatment of Rett Syndrome Using Human
Induced Pluripotent Stem Cells. Cell, 143, 527-539.