Differentiation of pluripotent cells and transdifferentiation

ReferencesLecture notes (hyperlink)

Activity notes (hyperlink)

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Differentiationof pluripotent cells and

transdifferentiation



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What is differentiationof pluripotent cells?

ES/iPS cell

Pluripotent

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Ectodermal cell

Mesodermal cell

Endodermal cell

brain

heart

pancreas

Multipotent Differentiated cells

Differentiation of ES/iPS 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 (MEFs)

Mouse ES cells colonies in culture



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(I) Directed differentiation of ES/iPS cells

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ES/iPS cell

Pluripotent

Ectodermal cell

brain


Feeders Feeders

✗

✗

✗

✗



More links…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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(I) Directed differentiation of ES/iPS cells



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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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(I) Induced differentiation of ES/iPS cells

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ES/iPS cell

Pluripotent

Ectodermal cell

brain


✗ ✗



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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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(I) Induced differentiation of ES/iPS cells



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ICM 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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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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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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Stem cell-based approaches to motor

neuron diseases



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Modeling ALS in a dish

Yamanaka methodOct4Sox2Klf4C-Myc

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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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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Transdifferentiation

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Transdifferentiation, also known as lineage reprogramming, is a process where one mature somatic cell transforms into another mature somatic cell without undergoing an intermediate pluripotent state or progenitor cell type



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Induced Transdifferentiation

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TRANSCRIPTION FACTORS!



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Induced in vivo Transdifferentiation

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• Cardiac fibroblasts represent 50% of the cells in the mammalian heart• Cardiac fibrobalsts can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT)• Can in vivo over-expression of GMT reprogram in situ the cardiac fibroblats?



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Induced in vivo Transdifferentiation

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Emily Borgelt

Match video file to slide.



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Summary

• Directed differentiation of ES/iPS cells is the production of various mature cell types (e.g. motor neurons, dopaminergic neurons) using defined growth factors or cytokines.

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

• Induced differentiation of ES/iPS cells is the production of various mature cell types (e.g. motor neurons, dopaminergic neurons) using defined transcription factors.

• The defined transcription factors are overexpressed and are sufficient to promote the differentiation of the cells into the appropriate cell type

• Transdifferentiation is the production of various mature cell types (e.g. epithelial cells) from another mature cell type (e.g. blood cell).

• Induced Transdifferentiation is the production of various mature cell types (e.g. motor neurons, dopaminergic neurons) from another mature cell type (e.g. fibroblasts, epatocytes) using defined transcription factors.

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Differentiation of pluripotent cells and transdifferentiation

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