Directed differentiation of ES cells into ectoderm

1. Directed differentiationof ES cells into ectoderm

2. What is directed differentiationof ES cells? Pluripotent 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. Ectodermal cell brain Mesodermal cell ES cell heart Endodermal cell pancreas 1

3. Why do we care about directed differentiation of ES cells? 2

4. Secreted factors keep ES cellspluripotent when cultured Feeders ES cells Mouse ES cells colonies in culture Secreted 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 3

5. 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. 4

6. 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. 5

7. ES cells form three germ layers during embryogenesis Amnion Implantation Uterus Blastocyst Ectoderm Yolk sac Epithelial skin cells, inner ear, eye, mammary glands, nails, teeth, nervous system (spine and brain) Endoderm Stomach, gut, liver, pancreas, lungs, tonsils, pharynx, thyroid glands Mesoderm Blood, muscle, bones, heart, urinary system, spleen, fat 6

8. Distinct signaling pathways specifydiscrete cell types during development Cell signaling pathways Erythropoietin (EPO) Shh Activin/TGF- Patched/ Smoothened BMP-RI EPO receptor Progenitor cell Progenitor cell Progenitor cell Heart muscle cell (Cardiomyocyte) Motor neuron Red blood cells 7

9. 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)

10. Stem cell-based approaches to motor neuron diseases

11. Modeling ALS in a dish Skin cells from ALS patients Dimos, JT et al. (2008). Induced Pluripotent Stem Cells Generated from Patients with ALS Can Be Differentiated into Motor Neurons. Science 321: 1218-21. ALS motor neurons Yamanaka method Klf4 Oct4 Sox2 Motor neuron nuclei Axons iPS cells induced pluripotent stem cells

12. 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

13. How are motor neurons generated during development? Lineage restrictions Pluripotent Multipotent Differentiation Neural stem cell Neurons Ectodermal cell ES cell Mesodermal cell 12 Endodermal cell

14. Specification of motor neuron fatedepends on nearby secreted signals Hb9::eGFP Hb9 BMPs MNs Wnts Retinoic acid Shh

15. Graded Shh signaling specifiesventral interneurons and motor neuronswithin the neural tube Shh Shh Patched/ Smoothened Progenitor Cell Motor neuron (HB9+)

16. Directed differentiation protocol for mouse ES cells into motor neurons RA/Shh RA/Shh RA day 2 neurectoderm day 4 motor neuron progenitors day 6 motor neurons Hb9-GFP mES cells Olig2 GFPHb9 2 days 2 days 2 days 1 M Shh agonist (~3 nM Shh protein) ES cells Neurectoderm Motor neurons Motor neuron progenitors 15 Witcherle et al., Cell (2002)

17. Dorsoventral patterning of differentiating ES cells p0 p1 Irx3 p0,1,2 p2 pMN Olig2 pMN p3 Nkx2.2 p3 Irx3 Olig2 Nkx2.2 10 nM Shh agonist P0,1,2 2 days pMN 1 µM Shh agonist Neurectoderm ES cells 2 days 16

18. Timeline for directed differentiation protocol of mouse motor neurons Witcherle and Pelzo (2009) 17

19. Motor neurons from mouse ES cells assayed by injection intochicken neural tube RA/Shh Day 2 neurectoderm Day 4 progenitors Day 6 motor neurons Hb9-GFP mES cells 18

20. Motor neurons from mouse ES cells innervate muscles when injected intochicken neural tube Injection of mouse motor neurons into the embryonic neural tube of chicken Muscle innervation Mouse motor axons exit chicken spinal cord 19

21. Directed differentiation protocol for human ES cells into motor neurons RA RA RA/Shh Day 10 primary neurectoderm (early rosettes) Day 14 secondary neurectoderm (late rosettes) Day 33 motor neurons Day 26 motor neuron progenitors hES cells Tubulin Hb9 10 days 4 days 12 days 7 days RA RA 1 M Shh agonist + RA 1 M Shh agonist + RA hES cells Early rosettes Late rosettes Motor neuron progenitors Motor neurons Li et al., Nature Neuroscience (2005)

22. 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

23. Directed differentiation of ES cells into dopaminergic neurons • 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. Dopaminergic neurons require Shh and FGF-8

24. 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. 23