References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Directed differentiation of ES cells into ectoderm

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… What is directed differentiation of ES cells? ES cell Pluripotent 1 Ectodermal cell Mesodermal cell Endodermal cell brain heart pancreas MultipotentDifferentiated 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.

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Why do we care about directed differentiation of ES cells? 2

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Secreted factors keep ES cells pluripotent when cultured 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 ES cellsFeeders Mouse ES cells colonies in culture

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… 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

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… 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

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… 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 6 Implantation

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Distinct signaling pathways specify discrete cell types during development 7 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

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Motor neurons and their diseases Motor neurons – One motor neuron per 10 6 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)

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Stem cell-based approaches to motor neuron diseases Patients iPS cellsES cells Motor neurons Astrocytes… Pathways of degeneration Drug discovery Cell replacement therapy Animal models

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Modeling ALS in a dish Yamanaka method Klf4 Oct4 Sox2 iPS cells induced pluripotent stem cells Skin cells from ALS patients ALS motor neurons Motor neuron nuclei Axons Dimos, JT et al. (2008). Induced Pluripotent Stem Cells Generated from Patients with ALS Can Be Differentiated into Motor Neurons. Science 321:

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… 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

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… How are motor neurons generated during development? Neurons ES cell Ectodermal cell Mesodermal cell PluripotentMultipotent Endodermal cell Neural stem cell Differentiation Lineage restrictions 12

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Specification of motor neuron fate depends on nearby secreted signals Shh Retinoic acid BMPs Wnts Hb9 Hb9::eGFP MNs

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Graded Shh signaling specifies ventral interneurons and motor neurons within the neural tube Motor neuron (HB9 + ) Progenitor Cell Shh Patched/ Smoothened Shh

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Directed differentiation protocol for mouse ES cells into motor neurons 15 Olig2 ES cellsNeurectoderm 2 days 1  M Shh agonist (~3 nM Shh protein) 2 days Motor neuron progenitors Motor neurons GFP Hb9 Witcherle et al., Cell (2002) Hb9-GFP mES cells RA day 2 neurectoderm day 4 motor neuron progenitors day 6 motor neurons RA/Shh

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Dorsoventral patterning of differentiating ES cells 16 Irx3Olig2Nkx2.2 ES cells Neurectoderm 2 days 10 nM Shh agonist 1 µM Shh agonist pMN P0,1,2 2 days p0 p1 p2 pMN p3 Olig2 pMN Irx3 p0,1,2 Nkx2.2 p3

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Timeline for directed differentiation protocol of mouse motor neurons 17 Witcherle and Pelzo (2009)

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Motor neurons from mouse ES cells assayed by injection into chicken neural tube 18 Hb9-GFP mES cells RA/Shh Day 2 neurectoderm Day 4 progenitors Day 6 motor neurons

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Motor neurons from mouse ES cells innervate muscles when injected into chicken neural tube 19 Muscle innervation Mouse motor axons exit chicken spinal cord Injection of mouse motor neurons into the embryonic neural tube of chicken

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

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… 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

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Directed differentiation of ES cells into dopaminergic neurons Dopaminergic 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.

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… 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