Introduction To Stem Cells: Frontiers 2

Week 2

Why use chicken embryos as a model?

They develop outside the mother and are easy to access.

Why use human iPSCs as a model?

They give access to a human developmental system without the ethical concerns of working with human embryos.

What are the learning objectives of this lecture?

Gain an overview of human pluripotent stem cells (PSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and directed differentiation.

What are the key stages of embryo development in order?

Fertilisation, cleavage, blastulation, gastrulation, neurulation, organogenesis.

What is fertilisation?

A sperm fertilises an egg, producing a single diploid cell called the zygote.

What is the zygote?

The single fertilised cell that has two copies of the genome, one from the mother and one from the father.

What does totipotent mean?

The potential to make the totality of cell types — both embryonic cells (germ layers and gametes) and extra-embryonic cells that support development.

What is cleavage?

Rapid cell division without growth, where the zygote subdivides into smaller and smaller daughter cells.

What is a blastula?

The structure resulting from cleavage, where all cells look the same and are called blastomeres.

What is the zona pellucida?

The outer layer surrounding the zygote, analogous to a vitelline membrane in other animals.

What is the polar body?

A small structure found alongside the zygote after fertilisation.

What is the first cell fate decision in development?

At day 3, one group of cells decides to differentiate differently from the others.

What is the first cell lineage to differentiate?

The trophectoderm.

What is the trophectoderm?

A population of extra-embryonic cells that contribute to the formation of the placenta in mammals.

What gene marker identifies trophectoderm?

GATA3.

What morphological feature identifies trophectoderm?

They form an epithelial shell around the outside of the embryo with apical-basal polarity.

What are the cells in the centre of the morula called?

Naive pluripotent stem cells.

What is the morula?

The stage of development at day 3 when the first cell fate decision has occurred, with trophectoderm on the outside and naive pluripotent cells in the centre.

What is the inner cell mass?

A group of pluripotent cells that move to one side inside the embryo at day 3, depicted at the top.

What is the blastocyst?

The stage of development when a cavity appears inside the embryo and the inner cell mass has formed.

What is OCT4?

A transcription factor expressed by all cells in the early embryo layers.

What do naive pluripotent stem cells look like in vitro?

Bright, dome-shaped, rounded colonies with refractive edges, resembling an inner cell mass.

What happens by day 6/7 of development?

The inner cell mass forms a bilaminar disc, a two-layered disc.

What is the epiblast?

The primed pluripotent cell population in the bilaminar disc that will contribute to the adult body.

What is the hypoblast?

A second extra-embryonic tissue that forms beneath the epiblast. In mammals it is often called the visceral endoderm.

What is the yolk sac?

A cavity beneath the hypoblast. In humans it doesn't contain yolk, but in chickens this is where the yolk is situated.

What gene markers identify epiblast cells?

OCT4, SOX2, KLF17.

What gene markers identify hypoblast cells?

SOX17, GATA4, GATA6.

What gene markers identify trophectoderm?

GATA3, KRT18, GATA2.

What is naive pluripotency?

A state of pluripotency found in the early inner cell mass, with less restricted differentiation potential.

What is primed pluripotency?

A more restricted state of pluripotency found in the epiblast, with changed gene expression compared to naive.

What happens by day 11/12 of development?

The trophoblast layer proliferates and differentiates into further trophoblast lineages, and the epiblast cavitates to form the amniotic cavity.

What is the cytotrophoblast?

The innermost layer of the trophoblast, which interacts with the uterus to form the placenta.

What is the syncytiotrophoblast?

The outer layer of the trophoblast, a syncytium formed by cells fusing together to form a larger cell with multiple nuclei.

What is the amniotic cavity?

A cavity that forms within the epiblast by day 11/12.

What is the amnion?

A membrane formed by epiblast cells surrounding the amniotic cavity.

What are amniotes?

Animals whose embryos have an amnion membrane. Humans, reptiles, and birds are amniotes. Fish and amphibians are not.

What is the embryonic disc?

The two-layered bilaminar disc where the epiblast contacts the hypoblast. Only the epiblast layer contributes to the adult body.

What do primed pluripotent stem cells look like in vitro?

Flattened epithelial colonies, like a flat sheet of cells.

What growth factors maintain primed pluripotency in culture?

FGF2 and TGF-β.

What extracellular matrix proteins are used to culture primed PSCs?

Laminin (Matrigel) or vitronectin.

What is Matrigel?

A non-specified mixture of different ECM proteins taken from mouse tumours, with laminin as the major component.

Why do pluripotent cells need stabilising conditions in culture?

Pluripotent and totipotent states are unstable, and cells will spontaneously differentiate if left to their own devices.

What is a teratoma?

A tumour containing lots of different tissue types, which can arise from spontaneously differentiating pluripotent or totipotent cells.

What happens at day 13/14 of development?

Gastrulation begins. Epiblast cells form the primitive streak at the posterior end and start forming the mesoderm and endoderm.

What is the primitive streak?

A strip or streak of cells at the posterior (tail) end of the embryo where gastrulation occurs.

What gene marker indicates mesoderm formation at the primitive streak?

Brachyury.

What is gastrulation?

The process by which pluripotent epiblast cells form the three germ layers of the body: ectoderm, mesoderm, and endoderm.

What is the ectoderm?

The outer germ layer. It forms the epidermis, nervous system, cornea, lens, and other structures.

What is the mesoderm?

The middle germ layer. It forms muscle, skeleton, blood, and other structures.

What is the endoderm?

The inner germ layer. It forms the guts, lungs, liver, pancreas, and other structures.

What are primordial germ cells (PGCs)?

Cells that go on to form the gametes (sperm and eggs). They don't belong to a specific germ layer.

What is the ectoderm's contribution to the body?

Epidermis of skin and its derivatives (sweat glands, hair follicles), epithelial lining of mouth and anus, cornea and lens of eye, nervous system, sensory receptors in epidermis, adrenal medulla, tooth enamel, epithelium of pineal and pituitary glands.

What is the mesoderm's contribution to the body?

Notochord, skeletal system, muscular layer of stomach and intestine, excretory system, circulatory and lymphatic systems, reproductive system (except germ cells), dermis of skin, lining of body cavity, adrenal cortex.

What is the endoderm's contribution to the body?

Epithelial lining of digestive tract, epithelial lining of respiratory system, lining of urethra, urinary bladder and reproductive system, liver, pancreas, thymus, thyroid and parathyroid glands.

What is neurulation?

The process after gastrulation where the embryo begins to establish its nervous system within the ectoderm germ layer.

What is organogenesis?

The formation of organs. The first organ to develop in most animals is the heart.

Why is the heart the first organ to develop?

Without a circulatory system, the embryo would asphyxiate because it relies on passive diffusion for nutrients and gases.

What is multipotent?

The state of stem cells in each germ layer after gastrulation. They can make a multitude of cell types but only from that one germ layer.

What is the Waddington landscape?

A visual metaphor for cell differentiation. Valleys and ridges represent cell differentiation, a cell is a ball rolling downhill, and forked valleys represent binary cell fate decisions.

What does the Waddington landscape show about potency and differentiation?

As differentiation increases, potency decreases. Cells become increasingly specialised as they roll down the landscape.

What did animal cloning show about differentiation?

Differentiation could be reversed. Differentiated cells fused with an enucleated oocyte can revert to a totipotent state.

What did cloning prove about differentiated cells?

Differentiated cells retain the information necessary to form all embryonic and extra-embryonic cell types.

What was the significance of Dolly the sheep?

The first time a mammal had been cloned from adult cells.

How was Dolly the sheep created?

A cell from the breast tissue of an adult sheep was fused with an enucleated oocyte from a donor sheep, and the resulting embryo was implanted into a surrogate mother.

What is induced pluripotency?

The process of reverting a differentiated cell back into a pluripotent state by over-expressing key pluripotency genes.

What are the Yamanaka factors?

KLF4, SOX2, c-Myc, Oct3/4. The minimal set of transcription factors needed to induce pluripotency.

What is the abbreviation for the Yamanaka factors?

OSKM (Oct4, Sox2, KLF4, c-Myc).

What is c-Myc?

A proto-oncogene, one of the Yamanaka factors. When mutated, it can cause cancer.

What are Nanog and LIN28?

Additional transcription factors that can increase the efficiency of induced pluripotency.

What are induced pluripotent stem cells (iPSCs)?

Stem cells generated by over-expressing key pluripotency genes in adult cells, reverting them to a pluripotent state.

What is the advantage of using iPSCs for replacement therapies?

They have the patient's own genome, so the immune system won't reject them.

What is directed differentiation?

Taking stem cells and pushing them to differentiate into a particular tissue or organ by using developmental signalling pathways.

How is directed differentiation guided in vitro?

By using recombinant proteins or small molecule inhibitors to mimic how stem cells differentiate in the embryo.

What is a differentiation protocol?

The particular recipe and sequence of growth factors or drugs used to push a cell into a desired cell type.

What are recombinant proteins used for in directed differentiation?

They are the actual growth factors that bind receptors on the cell surface and activate signalling pathways.

What are small molecule inhibitors used for in directed differentiation?

They competitively inhibit receptors or enzymes in downstream signalling pathways, such as kinase inhibitors.

What is the purpose of differentiation protocols?

To fool cells into thinking they are in the embryo at a particular location, so they differentiate in a certain way.

What is the "14-Day Rule"?

UK law stating that human embryos may be cultured in vitro until 14 days post-fertilisation or the first appearance of the primitive streak, whichever occurs first.

What is the Warnock Committee?

A committee convened in the UK in the 1980s, comprising scientists, religious leaders, and community leaders, to agree rules about human embryo research.

What is the rationale for the 14-day rule?

The primitive streak is the first indication of a single body plan or body axis. Before it appears, the embryo might form two body axes (monozygotic twins), so may not yet be an individual.
The organiser at the anterior end of the primitive streak secretes signals for nervous system development. Stopping before this means the embryo cannot experience pain or suffering.

What is the organiser?

A special group of cells at the anterior end of the primitive streak that secretes signalling molecules to set aside part of the epiblast for the central nervous system.

What is the organiser called in amniotes?

The node. In chickens, it is called Hensen's node. In frogs, it is called the Spemann-Mangold organiser.

What is the neural plate?

A structure that the organiser signals part of the epiblast to differentiate into, forming the central nervous system.

What is epithelial to mesenchymal transition (EMT)?

The process during gastrulation where epithelial cells lose their apical-basal polarity and become mesenchymal cells.

What happens to mesenchymal cells during gastrulation?

They migrate through the embryo to different locations to form the inner mesoderm layer.

What is cardia bifida?

A lethal condition where the mesoderm cells that make the heart fail to join together, resulting in two separate hearts forming.

What is morphogenesis?

Shape creation. The process where cells undergo changes in position, migration, and shape, going from epithelial to mesenchymal and back to epithelial.

What is an organoid?

A miniature, incomplete recreation of an organ grown in vitro from stem cells. It's like an organ but not a full organ.

What is self-organisation?

The process where cells organise themselves and each other to form a body plan, which can be done on a small scale to generate organoids.

What is an assembloid?

A more complex structure made by combining self-organised or directed differentiated cells to make a more sophisticated model.

What is an example of an assembloid?

Combining a retinal organoid with a vascular organoid to make a retina with vasculature.

What are crypt organoids?

Organoids made from intestinal crypts, generated by extracting crypt stem cells from biopsy tissue.

What is the quote by Lewis Wolpert about gastrulation?

"It is not birth, marriage or death, but gastrulation which is truly the most important time in your life."

What is the relationship between potency and differentiation?

They are complementary concepts. As differentiation increases, potency reduces.

What is the direction of time's arrow in development?

As time passes, cells lose their potency and increase their differentiation and specialisation.

What is the difference between totipotent and pluripotent?

Totipotent can make the totality of cell types, both embryonic and extra-embryonic. Pluripotent can make many different cell types but not extra-embryonic tissues.

What is the difference between pluripotent and multipotent?

Pluripotent can make cells from all three germ layers. Multipotent can make a multitude of cell types but only from one germ layer.

What is a blastomere?

A cell in the early embryo during cleavage, before the first cell fate decision.

What is the blastocoel?

The cavity inside the blastocyst.