Differentiation of pluripotent cells and transdifferentiation

1. Differentiationof pluripotent cells and transdifferentiation

2. What is differentiationof pluripotent cells? Pluripotent 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. Ectodermal cell brain Mesodermal cell ES/iPS 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 (MEFs) 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. (I) Directed differentiation of ES/iPS cells Pluripotent Multipotent Differentiated cells ES/iPS cell Ectodermal cell brain ✗ ✗ ✗ ✗ Feeders Feeders 4

6. (I) Directed differentiation of ES/iPS cells 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. 5

7. 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 6

8. (I) Induced differentiation of ES/iPS cells Pluripotent Multipotent Differentiated cells ES/iPS cell Ectodermal cell brain ✗ ✗ 7

9. (I) Induced differentiation of ES/iPS cells 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. 8

10. ICM 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 9

11. 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)

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

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

14. Stem cell-based approaches to motor neuron diseases

15. 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 Oct4 Sox2 Klf4 C-Myc Motor neuron nuclei Axons iPS cells induced pluripotent stem cells

16. 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

17. 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)

18. 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

19. 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

20. Transdifferentiation 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 19

21. Induced Transdifferentiation TRANSCRIPTION FACTORS! 20

22. Induced in vivo Transdifferentiation • 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? 21

23. Induced in vivo Transdifferentiation 22

24. 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. 23