Organoids and stem cells

Question 1

What were the first 3D cultures generated from?

Answer 1

They were generated from mouse mammary glands

Question 2

How were mouse mammary glands made?

Answer 2

Used primary mammary epithelial cells

When cultured in 2D these cells did not reproduce the physiology of mammary cells, cells were not producing milk as they wanted

They then cultured the primary mammalian epithelial cells on a reconstituted extracellular matrix (now called matrigel). This changed the way the cells were organizing in their cultures.

Generated cysts with functional properties that mimicked the physiology of the mammary gland

Question 3

What are organoids?

Answer 3

Multicellular, self-organized, functional in vitro “replicas” of organs

Complex 3D cultures that reproduce the development of a specific organ

Question 4

What are organoids derived from?

Answer 4

Derived from cells that differentiate to give rise to several different cellular types

Question 5

What do organoids acquire?

Answer 5

Acquire the structure/tissue organization of a complex organ within an organism

Brain tissue, gut cells, auditory organoids

Question 6

What is the source of organoids?

What do these stems cells produce?

Answer 6

Stem cells

More stems cells and differentiated cells

Question 7

What are the 2 main sources of stem cells?

Answer 7

1. ESCs derived from the ICM in a blastocyst (pluripotent) and give rise to a new organism.
2. Adult stem cells can also be found in many organs and tissues in an adult, needed to maintain homeostasis. They are mutlipotent so can give rise to a discrete range of cell types, generally with one particular organ.

Question 8

Intestinal stem cells in action

Answer 8

• Stem cell descendent tracing in the mouse intestine using the CreERT method
• The promoter of the gene lgr5 (expressed in intestinal stem cells) (expressed by stem cells) (transgenic) drives CreERT expression
• Crossing this transgenic with the Cre-activatable Rosa26-LacZ reporter leads to LacZ irreversible expression in Lgr5+ cells (+Tam)
• We can see the early cells that express LacZ that generate whole rows of cells of the interest

Question 9

What are iPSCs

Answer 9

Induced pluripotent stem cell

Question 10

How can we induce iPSCs from somatic cells?

Answer 10

Somatic factors can be taken and expose them to the expression of of Oct3/4, Sox2, Klf4 and cMyc which transforms somatic cells into pluripotent stem cells - YAMANAKA FACTORS

Cells can be taken from tissues of specific individuals to generate iPSCs

Question 11

How do Yamanaka factors influence the ICM?

Answer 11

They reproduce the ICM state

Question 12

What does the Zika virus cause?

Answer 12

Newborns with microcephaly (smaller head)

Question 13

When it came to modelling the zika virus why was this difficult when done in mouse?

Answer 13

When it came to modelling the Zika virus is was very complicated because mice brains and human brains are very different, specifically the cortex. The way it differentiates is different enough whereby modelling the disease was not so easy

Around the time of the outbreak the first brain organoids were being grown, they became an attractive model for the mice model and might be appropriate to model the disease

Question 14

How was a brain organoid developed to investigate Zika virus in development?

Answer 14

• One of these studies designed a mini-bioreactor that was more efficient to grow brain organoids in a normal laboratory setting
• The mini-bioreactors spin cells in culture inside so they are better oxygenated so they can grow and give rise to bigger organoids that are more reproducible because of their small cell death and no up-doses in the centre of the organoid – a problem of big organoids
• Another thing that they optimised was the conditions of growth factors necessary to grow brain organoids, so they could grow region specific brain organoids
• Once generated they could infect the organoids with the Zika virus and track how they were developing, analyse the phenotypes and understand the mechanisms that were leading to these effects

Question 15

What was the problem with normal brain organoids?

Answer 15

• The first problem that they encountered was the fact that brain organoids were very heterogenous, difficult to grow and use for systematic analysis because of the heterogeneity.

Question 16

What was the findings from modelling zika virus in organoids?

Answer 16

• They compared a reference that looks at the organisation of the forebrain compared with an infected forebrain
• They found that when they were infecting the forebrain was the virus was preferentially infecting progenitor cells
• The virus would slow down the proliferation of the progenitor cells and would eventually die by apoptosis
• The cortex was slowly being depleted of neural progenitor cells
• Apoptosis would also spread to the neuronal layer
• This slowing of the progenitors, promotion of apoptosis was that the infected organoids were reduced in size, thin small ventricles, thin cortical layer
• This gave a good model of the microcephaly seen in new-borns as a result of the Zika virus
• They also found that infecting organoids when they were still young, they were more susceptible to infection than older organoids – same in humans in pregnancies

Question 17

What next steps need to be taken in zika research?

Answer 17

• Molecular mechanisms underlying microcephalic phenotypes in infected conditions, molecular pathways affected by virus
• Finding therapeutic approaches
o Validation of antibodies for vaccine development?
o Identification of therapeutic targets and drug effectiveness?

Question 18

What is cystic fibrosis?

Answer 18

• Cystic fibrosis: a genetic disease due to mutations in CFTR (encodes for a channel that is found in the epithelial cells of many of our organs)

Cystic fibrosis is caused by mutations in the gene that produces the cystic fibrosis transmembrane conductance regulator (CFTR) protein. In people with CF, mutations in the CFTR gene can disrupt the normal production or functioning of the CFTR protein found in the cells of the lungs and other parts of the body …

Question 19

What are gut organoids composed of?

Why would gut organoids be useful?

Answer 19

They reproduced a good model of the gut epithelium, formed from lots of cells that formed this type of epithelium

Gut organoids could help us to determine whether a patient will be receptive to the current available treatments and also to find new treatments, personalised treatments for patients

Question 20

How were organoids grown for CF patients?

Answer 20

• Biopsy from CF patients were taken, and the organoids are grown from the adult stem cells that are taken from the biopsy. These organoids cultures can be expanded and test drugs to find the correct drugs for that particular patient.

Question 21

How can CTFR be recovered in patients with CF?

Answer 21

• There is a drug called Forkskolin that will activate CFTR, so when you expose a healthy organoid to this drug CFTR becomes activated, there is ion movement that leads to the swelling of this organoid
• CF organoids will have defective CFTR and when the drug is given the organoids do not swell, or that efficiently.
• However if we give a drug that can partially cover the function of CFTR then when exposed at the same time to forkstalin will lead to swelling
• This led to the identification of a number of compounds that were useful to recover the activity of CFTR in the patients that the organoids were derived.
• This led to new treatments for patients and people with same allelic variants

Question 22

How was a complete optic cup grown using stem cells?

What factors were essential to promote optic cup formation?

Answer 22

• It has been shown that a complete optic cup can be grown as an organoid model
• They started with embryonic stem cells (mice cells), the stem cells aggregated to form the neural epithelium, a part of this neuroepithelium would then express Rx and would start evaginating to form the optic vesicle and then an optic cup
• They found there were two essential factors that were important to promote transformation and growth of the optic cups: Activin and Laminin-rich extracellular matrix

Question 23

What cellular changes permit invagination of the optic cup?

What areas changes shape?

How was the proximal region changing?

What did these events happen in the absence off?

Answer 23

• They showed how the evagination of the optic vesicle is giving rise to a neuroepithelium that is initially quite rigid, because along the apical surface of the epithelium there is an accumulation of acto-myosin cables which is evident when they label phosphomyosin.
• At the onset of optic cup folding the acto-myosin was lost in the most distal part of the optic vesicle. This led to a more flexible epithelium in the distal region, this allows it to fold inwards, as optic cup folding progresses. It was also helped because the distal region of the tissue started to proliferate much faster than the rest of the optic vesicle.
• They also identified changes in the shape of the cells at the edge between the distal region and the proximal part of the optic vesicle
• They also realised the proximal region was getting thinner and more rigid to form the retinal pigment epithelium
• These structures were able to differentiate to give rise to the retina with all the different neuronal types
• All of the morphogenetic processes occurred in the absence of the lens and extraocular mesenchyme. These are essential sources of signals needed for transformations.

Question 24

Now that scientists were able to produce an eye organoid showing optic cup invagination, they compared mouse to human eye development

What were the differences?

Answer 24

• Differentiation timescale is different:
Mouse organoids: full evagination at day 7; cup formation by day 9
Human organoids: full evagination at day 16; cup formation by day 26
• They show different sizes! The sized matched with the mice and human.
• The neural retina is thicker in human organoids as occurs in the normal embryo
• The complement of photoreceptors is different: both rods and cones in the human retina – mouse retina has very few cones
• Photoreceptors maturation is fast in mouse organoids, slow in human organoids – mimicking the situation in vivo

Question 25

What signal is needed for eye cup folding?

Answer 25

Wnt

Question 26

How does Wnt cause optic cup folding?

Answer 26

• Wnt are expressed by the extraocular mesenchyme that is driving RP specification and also contributing to the mechanisms that drive the folding of the optic cup
• In the case of the organoids the Wnt signals seem to be coming from the adjacent non-eye tissue in these organoids instead of the extraocular mesenchyme tissue

Question 27

How does the optic cup acquire dorsal ventral patterning?

Answer 27

Tbx5: dorsal retina

Vax2: ventral retina

Question 28

How can organoids be useful in regenerative medicine?

Answer 28

• As a source of tissue/cells for transplantation approaches
• As a model to search for mechanisms driving regeneration:
To identify potential molecular targets
To search for small compounds promoting regenerative cellular responses

Question 29

How can organoids potentially be a source of tissue for retinal degeneration?

Answer 29

• This summarises the pipeline that could be used to regenerate damaged retinal tissue
• This idea exploits obtaining somatic cells from patients, generating iPSCs in culture and generating retinal organoids
• We can then dissociate and isolate particular cell types to be used in transplantation, they can also transplant differentiated tissue rather than just singular cell types
• Masayo Takahashi and her team went further and transplanted retinal tissue grown from iPSCs in two patients with age-related macular degeneration

Question 30

How can organoids been used to mechanisms driving regeneration

Answer 30

• Identifying molecular targets:
p27Kip1, Retinoblastoma (promote cell cycle exit)
Wnt/ßcatenin (promote proliferation)
• Screening for regenerative compounds:
Small molecule screens
• Informed by the knowledge of mechanisms driving regeneration in non-mammalian vertebrates