Directed differentiation of ES cells into ectoderm

ReferencesLecture notes (hyperlink)

Activity notes (hyperlink)

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Directed differentiationof ES cells into ectoderm



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What is directed differentiation

of ES cells?

ES cell

Pluripotent

2

Ectodermal cell

Mesodermal cell

Endodermal cell

brain

heart

pancreas

Multipotent Differentiated cells

Directed differentiation of ES cells creates specialized cells in vitro such as neurons, heart muscle cells, endothelial cells from blood vessels and insulin-secreting cells similar to those found in the pancreas, all of which can be used for cellular-based treatment or development of new therapies.



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Why do we care about directed differentiation of

ES cells?

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Secreted factors keep ES cells

pluripotent when culturedSecreted factors (proteins):• Cell feeder layer (fibroblasts) secretes

proteins that interact with receptors in the ES cell membrane to maintain its pluripotency.

• LIF (Leukemia Inhibitory Factor) provided in the media binds LIF receptors in the ES cell membrane to maintain both mouse ES pluripotency and the rate of cell proliferation.

• Serum contains BMPs (bone morphogenetic proteins) that maintain pluripotency of mouse ES cells

• FGF-2 and TGFs maintain human ES cell pluripotency

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ES cellsFeeders

Mouse ES cells colonies in culture



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Directing the differentiation of ES cells

in culture (I)Change growth conditions of ES cells:

• Remove secreted factors that maintain ES cell pluripotency from the media

• Add growth factors to the culture solution that trigger activation (or inactivation) of specific genes in ES cells, in order to promote differentiation into a specific lineage.

Change the surface on which ES cells are growing:• Grow ES cells on non-adherent substrates so that they

aggregate with each other. These aggregates are called “embryoid bodies”.

• ES cells within aggregates will interact with each other. These cell-cell interactions mimic some of the interactions of ES cells in vivo that normally guide their differentiation.

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Directing the differentiation of ES cells

in culture (II)Transfect ES cells with foreign genes:• Adding an active gene or genes to the ES cell genome.

• The gene(s) trigger(s) ES cells to differentiate along a particular pathway.

• This approach is a precise way of regulating ES cell differentiation.

Problems with this technology:• It works ONLY if we know which gene(s) must be active at a

particular stage of differentiation.

• The gene(s) must be activated at the right time, i.e. during the correct stage of differentiation

• The foreign gene(s) are often only required temporarily, but it is difficult to introduce them without permanently changing or “damaging” the genome.

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ES cells form three germ layers during

embryogenesis

Blastocyst

Uterus

Ectoderm

Mesoderm

Yolk sac

Amnion

Endoderm

Epithelial skin cells, inner ear, eye,

mammary glands, nails, teeth,

nervous system (spine and brain)

Blood, muscle, bones, heart,

urinary system, spleen, fat

Stomach, gut, liver, pancreas, lungs,

tonsils, pharynx, thyroid glands

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Implantation



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Distinct signaling pathways specify

discrete cell types during development

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Cell signaling pathways

Motor neuron

Heart muscle cell

(Cardiomyocyte)

Red blood cells

Progenitor cell

Progenitor

cell

Progenitor cell

Shh Patched/

Smoothened

Erythropoietin (EPO)

EPO receptor

Activin/TGF-

BMP-RI



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Motor neurons and their diseases

Motor neurons – One motor neuron per 106 cells in the body– Reside in the ventral horn of the spinal cord– Control movements of muscles – Exist in various subtypes that control different

muscle groups (limbs versus thoracic regions)

Motor neuron diseases– Paralysis from spinal cord trauma– Spinal Muscular Atrophy (SMA)– Amyotrophic Lateral Sclerosis (Lou Gehrig’s

disease or ALS)



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Stem cell-based approaches to motor

neuron diseases



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Modeling ALS in a dish

Yamanaka methodKlf4Oct4Sox2

iPS cellsinduced pluripotent stem cells

Skin cells fromALS patients

ALS motor neurons

Motor neuron nucleiAxons

Dimos, JT et al. (2008). Induced Pluripotent Stem Cells Generated

from Patients with ALS Can Be Differentiated into Motor Neurons.

Science 321: 1218-21.



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Using motor neurons to screen drugs promoting

their survival

Mouse disease models – creating ES cells from existing mouse

model strains– genetic modification of existing ES cell

lines

Human disease models – genetically tested blastocysts from IVF

clinics (SMA)– not applicable to ALS



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How are motor neurons generated during

development?

Neurons

ES cell

Ectodermal cell

Mesodermal cell

Pluripotent Multipotent

Endodermal cell

Neural stem cell

Differentiation

Lineage restrictions

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Specification of motor neuron fate

depends on nearby secreted signals

Shh

Retinoic acid

BMPsWnts

Hb9 Hb9::eGFP

MNs



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Graded Shh signaling specifiesventral interneurons and motor

neuronswithin the neural tube

Motor neuron (HB9+)

Progenitor Cell

Shh

Patched/ Smoothened

Shh



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Directed differentiation protocol for mouse ES cells

into motor neurons

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Olig2

ES cells Neurectoderm

2 days

1 M Shhagonist(~3 nMShh protein)

2 days 2 days

Motor neuron progenitors

Motor neurons

GFP Hb9

Witcherle et al., Cell (2002)

Hb9-GFPmES cells

RA

day 2neurectoderm

day 4motor neuron

progenitors

day 6motor neurons

RA/Shh RA/Shh



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Dorsoventral patterning of differentiating ES cells

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Irx3 Olig2 Nkx2.2

ES cells Neurectoderm

2 days

10 nM Shh

agonist

1 µM ShhagonistpMN

P0,1,2

2 days

p0

p1

p2

pMN

p3Olig2pMN

Irx3p0,1,2

Nkx2.2p3



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Timeline for directed differentiation protocol of

mouse motor neurons

18Witcherle and Pelzo (2009)



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Motor neurons from mouse ES cells assayed by injection into

chicken neural tube

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Hb9-GFPmES cells

RA/Shh

Day 2neurectoderm

Day 4progenitors

Day 6motor neurons



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Motor neurons from mouse ES cells innervate muscles when

injected intochicken neural tube

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Muscle innervationMouse motor axons exit chicken spinal cord

Injection of mouse motor neurons intothe embryonic neural tube of chicken



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Directed differentiation protocol for human ES cells

into motor neurons

Li et al., Nature Neuroscience (2005)

hES cells Earlyrosettes

10 days

1 M Shh agonist + RA

12 days 7 days

Motor neuronprogenitors

Motor neurons

Tubulin Hb9

1 M Shh agonist + RA Late

rosettes

4 days

RA RA

RA

hES cells Day 10primary

neurectoderm(early rosettes)

Day 14secondary

neurectoderm(late rosettes)

Day 33motor neurons

Day 26motor neuron

progenitors

RA/ShhRA



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Dopaminergic neurons and their diseases

Dopaminergic neurons:• Neurons located in the midbrain that secrete dopamine - an important neurotransmitter in the brain

• These neurons degenerate in Parkinson’s disease, a movement disorder.

• Loss of these neurons is associated with muscle rigidity, tremor, posture and gait abnormalities as well as slowing or loss of physical movements.

• These neurons arise during development in response to two signals: Shh and FGF-8.

Dopaminergic neurons



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Directed differentiation of ES cells into dopaminergic

neuronsDopaminergic neurons require Shh and FGF-8 • Mouse EBs are grown in the

absence of serum for 4 days on a non-adherent substrate.

• EBs are transferred to an adherent substrate and grown in a serum-free media that promotes survival of neuronal progenitors.

• After 6-10 days, neural progenitors are exposed to Shh and FGF-8 to induce differentiation into dopaminergic neurons.

• Differentiation of human ES cells into dopaminergic neurons takes a longer time.



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Summary

• Directed differentiation of ES cells into neurons is the production of various neuronal cell types (e.g. motor neurons, dopaminergic neurons) using defined factors.

• The defined factors are crucial for generating these neurons during normal embryonic development.

• Shh is a key signaling molecule that is required for the generation of both motor neurons and dopaminergic neurons.

• However, some factors are uniquely required to produce a particular type of neuron (e.g. RA for motor neurons and FGF8 for dopaminergic neurons).

• Directed differentiation of human ES cells into neurons uses factors similar to those employed for mouse cells.

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Directed differentiation of ES cells into ectoderm