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Overview Mouse embryonic stem cells Human embryonic stem cells Pluripotency genes and network Long-term self-renewal Dir PowerPoint Presentation
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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.

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Presentation Transcript
slide1

Overview

  • Mouse embryonic stem cells
  • Human embryonic stem cells
  • Pluripotency genes and network
  • Long-term self-renewal
  • Directed differentiation
  • Induced pluripotent stem cells
slide2

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

slide4

Generation of embryonic stem cells

Two prominent features of ESCs: long-term self-renewal and pluripotency

isolation of icm cells
Isolation of ICM cells

Mouse embryos

Rabbit Anti-mouse serum

Pipetting

Outer cells are lysed.

derivation of embryonic stem cells from mouse embryos
Derivation of embryonic stem cells from mouse embryos

Martin Evans

2007 Nobel Prize

Karyotype is normal

Evans, M.J. & Kaufman, M.H. Nature292, 154-156, 1981

slide8

Feeders provide factors that maintain embryonic stem cell growth

Day 13 mouse embryos

MEFs: mouse embryonic fibroblasts

Remove heads and internal organs

MEFs

Treat with trypsin and plate cells into a dish

irradiated to stop MEF growth

embryonic stem cells are pluripotent
Embryonic stem cells are pluripotent

Embryoid bodies

Cells of three germ layers

Lowattachment

ESCs

(mixture of differentiated cells)

Teratomas

Mouse injection

slide10

Derivation of embryonic stem cells from human embryos

Jamie Thomson

Univ. of Wisconsin

H9 cell line

ICM-derived

Critical factors:

MEFs, basic FGF

Differentiating cells

Thomson, et al., Science, 1998

slide11

What are the promises?

  • Understand early human development (infertility, birth defects) and control of cell division (cancer)
  • Cell-based therapy
    • 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
slide12

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

slide14

Core ES cell regulatory circuitry

Jaenisch and Young, Cell. 2008

slide15

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;

slide16

Directed ES cell differentiation

Transcription factor landscape

Graf T and Enver T, 2009, Nature

slide18

Conditions for directed differentiation

1. EBs

OP9 co-culture

Expansion

EB medium

EB digestion

Hematopoietic

stem cells

EB

formation

Hematopoietic

stem cells

hESCs

Neutrophils

Progenitor Expansion medium 7d

Terminal differentiation

medium 6-7 d

18 d

2. Co-culture

OP9 mouse stroma cells – hematopoietic differentiation

PA6 or MS5 – neural differentiation

3. Monolayer cultures

slide19

Hypothesis

Differentiated somatic cells can be re-programmed into pluripotent stem (ESC-like) cells with gene(s) important for ESC identity (pluripotency and self-renewal)

Shinya Yamanaka

Kyoto University

These cells would

  • Bypass ethical issues
  • Create patient-specific pluripotent stem cells
24 candidate genes
24 candidate genes

Dppa2 b-catenin Oct4

Dppa3/ Stella Dnmt3l Rex1

Dppa4 Fthl17 Sall4

Dppa5/ Esg1 Grb2 Utf1

Ecat1 Sox2

Ecat3/ Fbx15 Sox15 Klf4

Ecat5/ Eras Tcl1 Myc

Ecat8 Nanog

Ecat9/ Gdf3 Stat3

Gene delivery: Retrovirus allowing gene integration into the host genome

Takahashi and Yamanaka (2006) Cell126, 663-676

putting all 24 genes into mefs reprograms
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) Cell126, 663-676

slide22

Narrowing down the candidates

Oct4 (14)

Sox2 (15)

Klf4 (20)

Myc (22)

Takahashi and Yamanaka (2006) Cell126, 663-676

slide23

iPS cells are pluripotent

Pluripotency markers

EB formation

Teratoma formation

- Saw the same thing with tail-tip fibroblasts

Takahashi and Yamanaka (2006) Cell126, 663-676

slide24

Takahashi K., et al. (2007) “Induction of pluripotent stem cells from adult human fibroblasts by defined factors.” Cell 131, 861-72.

OCT4, SOX2, KLF4, MYC

Yu J., et al. (2007) “Induced pluripotent stem cell lines derived from human somatic cells” Science 318, 1917-1920.

OCT4, SOX2, NANOG, LIN28

Park I.H., et al. (2007) “Reprogramming of human somatic cells to pluripotency with defined factors” Nature 451, 141-146.

OCT4, SOX2, KLF4, MYC

How about human cells?

slide25

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
Pitfalls with iPSCs

<0.1%

  • Low efficiency of derivation
  • Use of C-myc
  • Transgene integration
  • Are they really the same as ESCs?
slide27

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?
slide28

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

,

slide29

- 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

slide30

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

slide31

Human iPSCs

  • 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)

slide32

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

slide33

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.