Stem Cells and Human Neurogenesis

Question 1

Define stem cells

Answer 1

2 important properties

• Differentiation gives rise to specialised cell types
• Self-renewal means stem cells can give rise to daughter cells equivalent to the parent cell

Question 2

Describe cell potency

Answer 2

• Range of commitment options available to a cell (how many cells can they generate?)
• Totipotent (capacity to form an entire organism eg. zygote)
• Pluripotent (able to form all cell lineages, including germ cells - ectoderm, mesoderm, endoderm)
• Multipotent (can generate multiple cell types that constitute to an entire tissue or tissues - blood cells)

Question 3

Describe embryonic germ layers

Answer 3

• Zygote divides into a blastocyst.
• At blastocyst stage divides
• In gastrulation the inner cell mass forms the endoderm, mesoderm and ectoderm with some germ cells
• Ectoderm: neural and skin
• Mesoderm: Bone muscle blood cells
• Endoderm: liver pancreas lung

Question 4

How are stem cells categorised

Answer 4

• Self renewal requirements
• Differentiation potency
• Where they come from

Types:

• Pluripotent stem cells
• Somatic (adult/tissue specific, also exist in embryois)
• Cancer stem cells (not a physiological stem cell)

Question 5

Describe pluripotent stem cells

Answer 5

• Self renewal is unlimited
• Derived from inner cell mass of embryos (embryonic stem cells - only from culture dish, as can self renew and have pluripotency)
• Somatic human cell types eg. skin (reprogrammed to induce PSCs) - induced

Question 6

Describe regulation of self renewal in PSCs

Answer 6

• Intrinsic factors (Oct4, sox2 and nanog - form transcriptional network and regulate gene expression)
• Extrinsic factors (growth factors, ECM, affect expression of intrinsic factors)

Question 7

How do we assess for pluripotency?

Answer 7

• Embroid body formation should create 3 germ layers (endoderm, mesoderm, ectoderm)
• Teratoma formation (inject cells with cancer to form teratoma. The teratoma should contain tissue from all 3 layers)
• Chimera formation (mouse pluripotent stem cells into a blastocyst, transplant resulting embryos into a mouse the same as the blastocyst donor. If the cell is pluripotent there should be chimeric colour)

Question 8

Describe somatic stem cells

Answer 8

• Neural, skin, blood (bone marrow), intestinal (bottom of crypts)
• Replace injured or damaged cells in our body
• In the brain: subventricular zone and subgranular zone
• Limited self renewal, as they are niche dependent. They are capable of life long self renewal.
• Lower plasticity and will not form teratoma.

Question 9

Describe stem cell niche

Answer 9

• Microenvironment that surrounds and nurtures stem cells and enables them to maintain tissue homeostasis
• Neural: cellular elements (astrocytes, endothelial cells, ependymal) and molecular factors (eg. GF, ECM, CSF)

Question 10

Describe multipotent stem cells

Answer 10

• Can form all the cell types within one system

- Eg. neural stem cells can make neurons, astrocytes and microglia

Question 11

Where do pluripotent cells exist?

Answer 11

• In our body
• All germ cells in our body are pluripotent
• Also in culture

Question 12

Why are PSCs best for neuroscience research?

Answer 12

• Limited accessibility of brain tissues, particularly for studying human brain
• Limitation of plasticity of neural stem cells
• Difficulty in creating stem cell niche for neural stem cells in vitro to maintain their self renewal

Question 13

List uses of PSCs in biological research

Answer 13

• An in vitro model for studying embryonic development (brain development/neurogenesis)
• A valuable cell model to elucidate molecular mechanisms underlying stem cell differentiation/self-renewal and diseases
• A model to study function of specific genes in vitro and in vivo (mouse ES cells) through genetic manipulation

Question 14

Compare embryonic development of mice and humans

Answer 14

• Preimplantation stage human and mice look very similar.
• However, after implantation the mouse has a very different structure to human
• Mouse has a cylindrical shape, and human a disc like shape

Question 15

Compare human and mice brain

Answer 15

• Human brain is 1500g with 86 billion neurons, amnd mouse only 0.4g with 70 million neurons
• Human brain has many sulci and gyri, while mouse brain has much less

Question 16

Describe neural differentiation from PSCs

Answer 16

Must inhibit BMP as this inhibits pluripotency

Question 17

List uses of PSCs in medicine

Answer 17

• Disease modelling (take patients primary cell, make iPSCs and then look for genetic cause, can then test drugs)
• Drug discovery (could also take hESC and manipulate for gene defect then test with drugs)
• Cell therapy

Question 18

Describe cell replacement therapy

Answer 18

• Injecting or implanting live cells into a patient, to replace damaged or died cells for the recover of proper physiological functions

Question 19

List challenges for using PSC for cell replacement therapy

Answer 19

• Differentiation of specific cell types
• Integration and survival (will form teratoma)
• Immune rejection
• Tumorigenesis

Question 20

List possible methods to prevent tumorigenesis

Answer 20

• Generation of iPSCs without changing genomic DNA using small molecules, RNAs and proteins
• Optimal culture conditions to maintain genome stability
• Eliminate undifferentiated cells before transplantation
• Genetic modification of hPSC to prevent tumour formation (toxic ablation - integrate potential toxic gene, administer an inducer and the toxic protein is activated then cells will be killed)

Question 21

Define neural induction

Answer 21

• Induction is a developmental mechanism:
• One tissue secrets signalling factors that instruct an abutting (neighbouring) tissue
  to differentiate to a new cell fate
• Organiser secretes neural inducing signals capable of organising the surrounding cells into the brain and head (eg. hensens node in chicks)

Question 22

Where is the mammalian node?

Answer 22

• At the gastrulation front

- The gastrulation primitive streak formation generates 3 new layers (2nd week in human)

Question 23

Describe the 3 germ layers

Answer 23

Mesoderm:

• PS derived layer of cells migrates adjacent to the epiblast.
• It gives rise to the mesenchyme; and organs: spleen, kidney, gonads, heart, blood vessels and blood. It also gives rise to paraxial mesoderm: somites which are transient structure containing the precursors of muscle and skeleton of the trunk portion of the body

Definitive Endoderm

• PS derived layer forming on top of the mesoderm. There it displaces/replaces the external epithelial layer (extraembryonic epithelium) “visceral endoderm” which surrounds the epiblast prior to gastrulation.
• The endoderm gives rise to the gastrointestinal tract and organs: liver, pancreas, stomach, gut and lungs

Ectoderm:

• Forms by conversion of the epiblast cells that do not ingress through the PS and will give rise to neuroectoderm and epidermis.
• This neuroectoderm will form only the brain, which will also give rise to the craniofacial muscle and bone of the head

Question 24

List tissues that divide form the primitive node

Answer 24

• Anterior endoderm
• Node
• Node derived notochord and prechordial plate
• Loss of node in mouse embryos causes no brain, no notochord but there is a spinal cord

Question 25

Which signals induce brain formation?

Answer 25

• Antagonists of primitive streak signals

Question 26

Summarise brain development

Answer 26

• Brain forms on the “ectoderm” that consists of epiblast cells which do not ingress into the Primitive Streak (PS)
• Brain is induced by signals emanating from tissues derived from the Anterior-PS (mammalian equivalent to Spemann Organiser):
• The Anterior Endoderm (AE) and
• The Node and the mesendoderm (notochord & prechordal plate)
• The AE signals are Antagonists of the signalling pathways: BMP, NODAL and WNT
• The brain grows inside out, from the ventricular zone to the pia surface

Question 27

Describe spinal cord development

Answer 27

• The primitive Streak (PS) generates Mesoderm and Endoderm, but recently it has been shown that the PS mesoderm behind the Node generates neuromesodermal (NMPs) precursors
• Neuromesoderm gives rise to the somites: two rows of condensed structures, which are separated by the node-derived mesendoderm and the neural tissue (neural plate) above the somites that will form the spinal cord neural plate
• WNT and FGF Signals together generate NMPs
• Then FGF alone differentiate NMPs to neuronal precursors; then + Retinoic Acid => spinal cord
• While WNT alone convert the NMPs to somites

Question 28

Where does the PNS develop from?

Answer 28

Neural crest cells

Question 29

List examples of neurocristopathies

Answer 29

• Goldenhar syndrome

- Waardenburg syndrome