Lecture 2 - Tissue microenvironments and organoid systems

embryonic niche

embryonic niche

contains cell communication, the ECM, soluble factors and hypoxia/metabolism

extraembryonic endoderm

receives and returns signals to the epiblast and trophoblast cells

extracellular matrix

dynamic and complex protein network with unique biophysical, mechanical and biochemical properties specific to each tissue

what is the ECM structure in connective tissue like?

more elastic

what is the ECM structure is bone marrow like?

stiff

supportive cells

supports the stem cell niche in close contact with the stem cell and secreted factors for stem cells to respond to

neural inputs

provided in adult tissues and fetal tissues where circulatory and nervous systems begin

acellular components

matrix, soluble factors, chemicals, proteins, polysaccharides and lipids

blood vessels

bring nutrients to the stem cell niche and carry away waste

villus

terminally differentiated cells with specific function absorbing nutrients, barrier for regulation and secretes proteins and molecules important for gut function

lamina propria

mixture of blood vessels, immune cells and mesenchymal cells like fibroblasts

transit amplifying zone

differentiated progenitor cells that go through a limited number of cell divisions migrating out of the crypt to mature functions on the villus

crypt base

niche for intestinal stem cells (rapid turnover)

how often does differentiation and migration occur in the intestine?

every 1 to 2 days

enterocyte cells

create a barrier through which dietary molecules are absorbed

enteroendocrine cells

hormone producing cells that influence nearby cells

goblet cells

secrete gel forming proteins to create a mucous layer

paneth cells

secrete protein factors that control intestinal stem cells

active intestinal stem cells (aISC)

the classical intestinal stem cell that is always undergoing renewal at the base of the crypt adjacent to the Paneth cells

reserve intestinal stem cells (+4 rISC)

dormant stem cells that are only activated in light of chemical exposure or when the cell can be potentially damaged

LGR5

intestinal stem cell marker found at the crypt base of the stem cell niche

b-galactosidase

encoded in the reading frame of the LGR5 gene and produces a blue metabolic product so that we know where the LGR5 is located in the intestine

what is the goal of lineage tracing experiments?

limiting the labelling of cells and tissues

why is low cell density at the start important for good lineage tracing?

because if the density is too high it’ll be hard to pick out where the stem cell is

BrdU

analogue of thiamine that becomes incorporates during DNA synthesis and labels cells who have undergone cell division

when stem cell is labelled…

all of its progeny will also carry that label because it is distributed to daughter cells over a few or many divisions

when a non stem cell is labelled…

the label stays within the cell

why shouldn’t we use a reporter gene in lineage tracing experiments?

because it can be turned on or off so we’ll lose track of the progeny

what is the role of LGR5 in Wnt signaling?

supports the signaling pathway by blocking the endocytosis and degradation of the frizzled/LRP receptor complex

what happens to the signaling pathway in the presence of R spondin?

the frizzled/LRP stays at the cell membrane and disrupts the b catenin destruction complex allowing the accumulation of b catenin and high LGR5

what happens when b catenin is ubiquinated?

it gets degraded and the LGR5 receptor is low

peri cryptal fibroblast

secretes Wnt towards the bottom of the crypt which activates canonical signalling supporting the maintenance of intestinal stem cells

BMP

promotes differentiation of intestinal stem cells

noggin

BMP antagonist

when are BMP levels high?

at the villus

when are BMP levels low

at the base of the crypt when there is high wnt signalling

mesenchyme

supports the villus and crypt architecture as well as high rate of nutrient absorption by increasing surface area of the gut

what does compartmentalization of villi and crypts do?

• increases absorption rate by increasing intestinal surface area

• provides continuous cell turnover by storing lots of transit amplifying cells

• protects intestinal stem cells in the crypt base from pro-differentiation signals

cell culture

the removal of organs, tissues, or cells from an animal/plant and their subsequent placement into an artificial and controlled environment that is conducive to growth

organ culture

a whole part of an organ is maintained/grown, which allows for differentiation and preservation of architecture

explant culture

small pieces of tissue are attached to a vessel and bathed in culture medium; the individual cells slowly migrate into the medium where they can divide and grow

slice culture

thin slices of an organ are maintained in culture medium

dissociated culture

tissue fragments are isolated and digested with proteolytic enzymes which creates a suspension of single cells

in vitro cell growth conditions

• medium: has to have salts minerals, glucose (ex: DMEM)

• nutrients: amino acids, vitamins, hormones, animal serums

• growth factors: EGF, FGF, LIF

• temperature: 37

• gasses: O2 and CO2

• antibiotics: penicillin, streptomycin

• ph: 7 - 7.5

adherent culture

monolayer, anchorage dependent

what do cells need to grow in adherent culture?

either can grow on the plastic of the culture dishes/flasks or they require an ECM component to grow (ex: collagen, fibronectin, laminin)

non adherent cell culture

anchorage independent, suspension, free floating cells

holoclar

graft of corneal epithelial cells grown on a fibrin scaffold from limbal stem cells for patients with eye burns (autologous cell therapy)

autologous cell therapy

uses cells from the patients own body to replace damaged tissue

scaffold

3D environment that promotes cell attachment, differentiation and function that is important for cells who normally rely on some physical support to carry out their function

limbal stem cells

a type of eye cell

hydrogels (synthetic matrices)

highly absorbent, interconnected network of polymer chains often used as 3D scaffolds for tissue engineering

• can absorb and maintain fluid

basal lamina extracts

naturally occurring scaffold

Matrigel

a naturally occurring scaffold made from basement membrane extract from a cancer cell line but can’t control for physical properties (hydrogel derived product)

2D cell culture

soluble gradients are absent, forced apical basal polarity, continuous layer of matrix, high stiffness, adhesion restricted to one plane and unconstrained spreading and migration in one plane

3D cell culture

soluble gradients are present, no polarity , discrete matrix fibrils, variable stiffness, adhesion in all three dimensions and spreading and migration are hindered

what tissues are good for matrigel?

breast tissue, neural, endothelial, blood or mucus, lung

what tissue is good for plastics?

bone

bioreactor

a manufactured instrument intended to biologically support cells/tissues might agitate the cell aggregates

gel embedding

at the end of the 3D liquid culture process it exchanges the growth medium that lies on top and helps diffuse the cells into the gel to set up a concentration gradient (upgraded suspension culture)

2D cell monolayer advantages

simple and efficient, low cost, high reproducibility

2D monolayer disadvantages

cells lose their phenotype , lack cell - cell and cell - matrix interactions, fail to mimic the cellular function and signaling pathways as seen in vivo

3D cell aggregates advantages

better mimic cell-cell and cell-matrix interactions compared to 2D models

3D cell aggregates disadvantages

transiently resemble cell organization and interactions, lack capacity to recapitulate tissue organization, lack potency for self renewal and differentiation, difficult to maintain long term cultures

animal models advantages

closest to recapitulating body functions and cellular interactions in human tissues, can predict how a disease may develop or how a treatment may be responded to

animal models disadvantages

differences in human and animal biology, limited usability in imaging and high throughput studies, high cost maintenance

why is extrapolating results from model systems to humans difficult?

• human physiology is very different (lifespan , metabolism of ibuprofen and warfarin)

• biological processes are specific to humans and cannot be modelled in other animals

• humans are not inbred

organoids

miniature, self organizing 3D structures formed in vitro created by pluripotent or multipotent stem cells that are lower in complexity compared to in vivo tissues

how are intestinal organoids created and maintained?

they are created from digested intestinal tissues or a single Lgr5 intestinal stem cell and it is maintained liquid suspension or in Matrigel

ISC self renewal

factors are usually added to nurture spheroids which then self organize and expand into mini gut structures

when do we know when an organoid begins to form?

when the cell stats to self pattern and the radial symmetry of the spheroid breaks

FACS (fluorescence activated cell sorting)

uses light lasers and detectors to separate a mixture of cells based on the fluorescence of the individual cells

stem cell purification

purification can occur physically by antibody labelling or genetically by reporter gene labelling and coupling that with the antigen of interest so that it is recognized by the flow cytometer

antigen

any substance that causes your body to produce antibodies

RSPO1

soluble protein activator of the Wnt signaling pathway that increase the size and number of intestinal organoids from digested mouse small intestine

polymers of ECM proteins that support intestinal organoid transformation

3D PEG hydrogel, fibronectin, laminin - 111, collagen type 1

microfluidics

technologies that precisely control the movement of liquids using micro scale geometries and channels

how does microfluidics work?

the dead cells shed into the lumen and clogs up the channels but if there is movement due to right concentration and mixture of the media there will be no build up

application of organs on a chip

intestinal parasite infection and study life cycle within the mini gut tissue and test drug efficacy to kill parasite while leaving the normal cell type intact

how does spontaneous differentiation occur?

by removing cytokines/factors to destabilize transcriptional network that supports the undifferentiated state in culture

embryoid bodies

large 3D cell aggregates that are grown in suspension culture but can also be plated onto a matrix permitting adhesion migration and further differentiation into the 3 germ layers

what allows the observation of radially organized germ layer progenitor cells in the embryoid bodies?

comprehensive antibody panels or multiple fluorescent gene reporter systems

Chi

chemical activator of Wnt signaling promoting tissue patterning

aggregated mouse embryonic stem cells (gastruloids)

grown in suspension and shaking cultures showing differentiation and body axis formation reminiscent of a spinal cord and tail but no true head

human gastruloids

• no evidence of anterior neural development

• pluripotent human embryonic stem cells cant give rise to certain extraembryonic stem cells

enteric nervous system formation in gastruloids

primitive spinal neurons innervating the gut tubes in 20 - 40 days can be observed (PNS development)

human embryo research laws

in the case that human embryos are created for reproductive purposes but are no longer needed research is allowed only if:

• no genetic alteration of the human gametes or embryos

• no embryo transfer for continuing pregnancy if manipulation is not directed to ongoing development

• research only takes place during the first 14 days after fertilization