0. Introduction

Question 1

Define CTE

Answer 1

Cell- and Tissue Engineering. The development of biological constructs for implantation

Question 2

Consider the difficulties in cell- and tissue engineering (structure vs. function)

Answer 2

It is easier to replace structure than function, e.g. metabolism and signalling.
Structures may be bone, cartillage or tendon. Function may be liver, pancreas, heart.

Question 3

What kinds of biological material may be transplanted?

Answer 3

Material only (tissue inducing substances)
Cells only
Cells seeded in material

Question 4

What is the aim of transplanting tissue-inducing substances?

Answer 4

T o induce the host cells to secrete signal molecules, such as growth factors

Question 5

What is the aim of transplanting cells only?

Answer 5

The cells may replace the original cells to provide function. Stem cells may induce homeostasis without integration, by signaling molecules. Allows for cell manipulation before transplantation

Question 6

What is the aim of transplanting seeded matrices?

Answer 6

May be either open or closed system. Open system: The cells are seeded in a matrix and transplanted. Closed system: the cells are separated from the body by a membrane, which avoids attack of the cells by antibodies, and allows transport of nutrients and waste

Question 7

What are some observations seen in cell-transplantation?

Answer 7

Isolated cells reform the tissue, due to its effect on the microenvironment. All tissues undergo remodelling, so the entire tissue does not have to be created in vitro. Tissues cannot be transplanted in large volumes due to lack vascularisation

Question 8

Define TEMPs

Answer 8

Tissue-engineered medical products
Newer cell-based therapies that incorpate significant cellular manipulations in vitro

Question 9

Give examples of TEMPs

Answer 9

Chrondrocytes for cartilage repair
Liver and kidney cells grown on extracorporal devices
Encapsulated beta-islets for diabetes type I
Sheets of dermal fibroblasts for ulcers and burns
Genetically modified myocytes for treatment of muscular dystrophy

Question 10

Define CT

Answer 10

Cell-based therapies
Exploits stem-cell capacity, rather than depending on their long-term integration in host tissue

Question 11

Define autologous

Answer 11

Donor and recipient is the same patient

Question 12

Define allogenic

Answer 12

The donor and recipient is from the same species

Question 13

Define syngenic

Answer 13

Donor and recipient are different individuals that are genetically identical, such as homozygous twins

Question 14

Define xenogenic

Answer 14

Donor and recipient are of different species

Question 15

What is the most common origin of cells for transplant (both in the present and historically)?

Answer 15

Traditionally most transplants were allogenic, but it is becoming more common with autologous transplants due to ex vivo modifications that may be performed on the cells

Question 16

What may be an issue with autologous cell transplants?

Answer 16

The autologous cells may have the same susceptibility towards a given disease, since the individual’s cells already once have developed the disease. Therefore it may be necessary to reprogramme or otherwise modify the cells before transplantation

Question 17

What is the main challenge in transplanting tissue engineered constructs?

Answer 17

Rejection of the transplant
RIsk of graft vs. host disease

Question 18

How do extracellular signal molecules leave the secreting cell?

Answer 18

Signal molecules such as proteins, peptides, nucleotides, steroids, carbon monoxide, etc., are released by exocytosis
Some signal molecules diffuse across the cell membrane

Question 19

Explain which types of receptors are activated by lipophilic and hydrophilic signal molecules, and the type of the response they mediate (fast/slow)

Answer 19

Receptors on the cell membrane are stimulated by hydrophilic ligands
Receptors inside the cell are stimulated by lipophilic
Lipophilic molecules are transported in the blood by being bound to carrier proteins
Insoluble (lipophilic) molecules mediate longer-lasting responses, and soluble (hydrophilic) molecules usually mediate shorter responses

Question 20

Define contact-dependent signalling

Answer 20

When the signalling molecules is bound to the surface of the signalling cell.
It is especially important during development and in immune responses (think T- and B-cells)

Question 21

Define paracrine signalling

Answer 21

The secreted molecules only affects the cells in close proximity
Therefore the signal molecules are often rapidly taken up, destroyed by extracellular enzymes or immobilised by the ECM, to avoid them affecting other cells

Question 22

Define synaptic signalling

Answer 22

Neurotransmitters are released in the chemical synapses between one stimulated axon and the post synaptic neuron
It is very fast
Activation of the receptor requires very high concentrations, as the neurotransmitters have low affinity towards their receptors, such that the neurotransmitter fastly dissociated from the receptor and thus is quickly cleared from the synapse

Question 23

Define endocrine signalling

Answer 23

Controls the body-system as a whole
Endocrine cells release hormones into the blood, where they are transported to target cells
It is relatively slo
Activation of the receptor requires low concentration of the signalling molecules, as it greatly diffuses through the blood

Question 24

Define autocrine siganlling

Answer 24

A cell secretes a signal that binds back onto its own receptor
It is very effective when being performed by neighbouring cells of the same cell type
This may, unfortunately, be exploited by cancer cells

Question 25

Define gap-junctions

Answer 25

Specialised cell-to-cell junctions that can form between close plasma membranes and directly connect two cytoplasms via water-filled channels. These channels allow the exchange of intracellular mediators, such as Ca2+ and cAMP, but not macromolecules, such as proteins or nucleic acids

Question 26

How can the same signal molecule have different effects on cells?

Answer 26

The receptors the signal molecules binds to may be different, or the cellular pathways that are initated from the activation of the receptor may be different

Question 27

Give an example of a signal molecule that works differently in different types of tissues

Answer 27

Acetylcholine acts different on skeletal and heart muscle

Question 28

Give examples of how cells may differentiate according to the cells they are next to

Answer 28

Lung bed epithelium cultured alone will experience no differentiation
Lung bed epithelium cultured with intestinal mesenchyme will form villi
Lung bed epithelium cultured with stomach mesenchyme will form gastric glands

Question 29

Define the 4 types of cellular induction

Answer 29

Negative: the collective cells restrict potential of each other
Instructive: cell type of the responder cell changes due to the interaction
Permissive: responder cells contains the potential required to differentiate, and therefore only need the required environment (e.g. ECM)
Reciprocal: tissues signal each other, resulting in differentiation of both tissues: there is no responder or inducer cell, thee two tissues both “give and take” signals

Question 30

What is the nuclear receptor superfamily?

Answer 30

A group of receptors that have a DNA-binding domain and a ligand-binding domain
They may be found intracellularly, either in the cytoplasm or the nucleus
They function as transcriptional regulators to control expression of genes involved in development, homeostasis and metabolism

Question 31

Describe how receptors from the nuclear receptor superfamiliy works

Answer 31

When the ligand binds to the receptor, a conformational change occurs in the receptor, such that the DNA-binding domain binds to a specific DNA sequence in the genome (the receptor-binding element of the DNA). This results in a down- or up-regulation of gene expression

When a ligand is not bound to the receptor, an inhibitory protein is bound to the ligand binding domain.
When the ligand binds to the receptor, the ligand is “encapsulated” by the ligand binding domain and the COOH group. This dissociates the inhibitory protein from the receptor
Then a co-activator protein binds to the whole receptor, and especially binds to the transcription activating domain. This co-activator protein fits right around the receptor when ligand is bound
Then the DNA binding region binds to the specific place on the gene, and transcription is initiated

Question 32

Describe the transcriptional response of hormones binding to its receptor

Answer 32

Early primary response: the steroid hormone binds to the steroid hormone receptors, and this complex activates primary response genes. This induces synthesis of primary-response proteins
Delayed secondary response: A primary-response protein shuts off the primary-response genes (negative feedback), and another primary-response protein turns on secondary-response genes, which induces synthesis of different secondary-response proteins
Hormones are slow acting, so it makes sense that there must be a transition in which proteins are translated before the “actual” proteins are transcribed

Question 33

Name three types of signal transducers

Answer 33

Ions-channel linked receptors
G-protein linked receptors
Enzyme-linked receptors

Question 34

Define ion-channel linked receptors

Answer 34

Involved in rapid signalling between electrically excitable cells. When the signal molecule binds to the receptor, the receptor opens to allow ions to flow through

Question 35

Explain how a G-protein linked receptor functions

Answer 35

Indirectly regulates the activity of a separate plasma-membrane-bound protein, which can either be an enzyme or an ion channel
The interaction between the receptor and the target protein is mediated by a trimeric GTP-binding protein
Consists of a single polypeptide chain that threads back and forth the lipid membrane a total of 7 times
When the ligand binds to the receptor protein, it activates the G proteins (Trimeric GTP-binding proteins). The G protein functions as relay molecules, that couple the receptor to enzymes or ion channels in the membrane. The G proteins have three subunits: alfa, beta and gamma. When inactivated, the alpha subunit has bound GDP. When the G proteins are stimulated by the activated receptor, the GDP dissociates from the alpha subunit, which lets GTP bind instead. The binding of GTP lets the G protein trimer dissociate into two parts: an alpha subunit and a beta-gamma complex. This makes the alpha subunit change shape, so that it may interact with its target proteins. The beta-gamma complex does not change shape, however, the surface of the complex that was previously hidden by the alpha subunit is now free to interact with secondary target proteins. The alpha subunit is a GTPase, so once it hydrolyzes GTP to GDP, it reassociates with the beta-gamma subunit to re-form an inactive G protein

Question 36

Describe the effects of G-protein linked receptor-activation if the target protein is adenylyl cyclase

Answer 36

o If the target protein is adenylyl cyclase, the target protein may activate cyclic AMP from ATP, which binds to inactive protein kinase A (PKA) and activates (PKA). Activated protein kinase A (PKA) translocates to the nucleus, where it phosphorylates inactive CREB. The phophorylated site on the active CREB now functions as binding site for the CREB-binding protein (CBP). The activated CREB then binds to the CREB-binding element on DNA, which mediates transcription of the target gene, which is then translated to the target protein

Question 37

Describe the effects of G-protein linked receptor-activation if the target protein is phospholipase C-beta

Answer 37

If the target protein is phopholipase C-beta, this will activate PI(4,5)P2 (PI 4,5-biphosphate). Activation of PI 4,5-biphosphate will trigger its dissociation into diacylglycerol and inositol 1,4,5-triphosphate. Inositol 1,4,5-triphosphate binds to a IP3-gated Ca2+-release channel in the endoplasmic reticulum, which releases Ca2+ from the lumen of the endoplasmic reticulum. This Ca2+ will bind to protein kinase C together with diacylglycerol (DAG), which activates the protein kinase C.

Question 38

Describe the function of enzyme-linked receptors

Answer 38

Either directly function as an enzyme, or they are directly associated with enzymes that they activate
The transmembrane protein receptor has an extracellular binding site for the ligand and an intracellular site, which is either catalytic or enzyme-binding (it may bind an intracellular enzyme which is then activated, or the activation of the receptor may lead to the activation of an enzymatic response of the intracellular part itself).

Question 39

Give examples of tyrosine kinases

Answer 39

PDGF receptors
FGF receptors
Eph receptors

Question 40

Define tyrosine kinase receptors

Answer 40

The receptor tyrosine kinases may be activated by cell contact signalling, where the receptor ligand is on the cell surface of another cell. E.g. ephrins stimulated Eph receptors
They are often stimulated by growth factors, and the end result of their activation is MAP (mitogen activated kinase, that stimulated mitosis)

Question 41

Describe PDGF receptors

Answer 41

PDGF is a dimer with two receptor binding sites. It cross-links adjacent tyrosine kinase receptors , which initiates intracellular signalling processes

Question 42

Describe FGF receptors

Answer 42

FGF are monomeric ligands that bind in clusters to proteoglycans, that may be in the ECM or on the cell surface
Autocrine FGF-signalling: transmembrane heparan sulphate proteglycan has glycosaminoglycan side chains, which FGF may be bound to. The FGF may activate FGF receptors on the cell itself

Question 43

Describe Eph receptors

Answer 43

Ephrins are membrane-bound signalling proteins that can cross-link their receptors even though they are monomeric, due to clustering on the plasma membrane of the signalling cell
They may either be transmembranous or linked to GPI (phosphatidylinositol) anchors

Question 44

Describe activation of Ras through a receptor tyrosine kinase

Answer 44

A signal molecule activated the receptor tyrosine kinase, which results in phophorylation of the receptor
A Grb-2 (growth-factor receptor bound protein-2) adaptor protein binds to the tyrosine kinase receptor, and binds to Ras GEF (guanine nucleotide exchange factor). Ras GEF stimulates Ras, so it becomes activated by swapping GDP for GTP.
Ras then activates MAP-kinase-kinase-kinase (called Raf). Raf then activates the MAP-kinase-kinase (called Mek). Mek then activates the MAP-kinase (called Erk). Erk phosphorylated downstream proteins, including other kinases and gene regulatory proteins in the nucleus. This results in changes in gene expression and protein activity, causing complex changes in cell behaviour

Question 45

Define MAP

Answer 45

Mitogen activated kinase
Kinase is abbreviated “P” due to phosphorylation