Revision cards Flashcards

Question

What signals are needed to keep mouse and human embryonic stem cells renewal in vitro?

Answer 1

mESCs are grown in 2i (Gsk inhibitor and Erk inhibitor) and LIF. hESCs are grown in FGF2 and Activin to remain proliferative and undifferentiated.

Answer 2

In humans epiblast is formed very quickly from inner cell mass to create a flat layer of cuboidal epithelium. Mouse is slower and occurs after implantation and forms more of a cylindrical shaped epiblast. This led to the question of whether these differences are due to the species OR whether its the stage at which the embryonic stem cells are isolated. Found out that it was to do with the staging, conventional mESC were naive, conventional hESC were primed. Now have examples of both.

Answer 3

Epiblast stem cells found by Brons et al 2007 with support from Tesar et al 2007 in mice which are far more similar to human embryonic stem cells than mouse embryonic stem cells are. A better equivalent. They are dependent on FGF and Activin, same as hESCs.

Answer 4

- A naive or ground state (aka inner cell mass-like) | - A primed state (aka epiblast-like)

Answer 5

Gafini et al (2013) sought to capture the ground state of human naive pluripotency. Created a reporter line with Oct4-GFP and resistance to antibiotics. He then screened for combinations of exogenous factors that would establish naive cells. Carried out on a background of 2i and LIF. Idenitifed LIF, TGFB1, FGF2, ERK1/2, GSK4, JNK1, p38i to be involved, known as 'NHSM'. Once obtained, he tested the naive cells by adding NHSM to a blastocyst and adding NHSM to primed ESCs, both produced the naive cells.

Answer 6

Genetically, there is a distal and proximal enhancer of Oct4 which determines differential expression between naive and primed. Naive cells use the distal enhancer. Primed cells use the proximal enhancer.

Answer 7

- Fundamental understanding of pluripotency - Easier to modify genetically, more efficient homologous recombination - Gafini et al (2013) showed that you can insert human naive cells into a mouse blastocyst and they will become incorporated and develop into a fetus. Could lead the way for created humanised organs in animals.

Answer 8

The international stem cell initiative looked at the characteristics of 100s of stem cell lines from different labs. Found that there were subtle differences. Osafune et al (2008) showed that there were differences in differential propensity among human embryonic stem cell lines. This affects which one you might use and some will produce a certain tissue more efficiently than another.

Answer 9

``` Mouse embryonic stem cells NHSM-derived human pluripotent stem cells Dome-shaped colonies Reduced doubling time X-chromosomes active Single-cell cloning Use of OCT4 distal enhancer ICM integration LIF-dependent ```

Answer 10

``` Mouse epiblast stem cells Conventional human pluripotent stem cells Flat colonies Increased doubling time X-chromosomes inactivated Poor single-cell cloning Use of OCT4 proximal enhancer Low ICM integration Activin/FGF-dependent ```

Answer 11

Pluripotency implies the ability to self-renewal and generate lineages from the 3 germ layers. This isn't determined by a single molecule but by a set of transcription factors whose expression is carefully balanced.

Answer 12

It begins with the morula, a balloon of cells. It becomes a structure with a cavity known as the early blastocyst. Inner cell mass is present on the inside and trophoectoderm surrounding. In a later blastocyst the inner cell mass begins to look like the epiblast (which will give rise to ectoderm) and a layer is formed on it called the primative endoderm aka hypoblast (which will give rise to visceral and parietal endoderm).

Answer 13

``` Austin Smith (2005) In pluripotent stem cell there is a continual conflict between pluripotency transcription factors that seek to direct ESC differentiation to opposing lineages. Nanog (endoderm), Sox2 (ectoderm) and Oct4 (mesoderm). ```

Answer 14

Nanog has a classic homeodomain (homeobox gene) Oct4 is a Pou gene which means it has2 different DNA binding domains. One of which is a convential homeodomain, and the other which is a Pou-specific domain. Closely positioned together. Sox2 has a high mobility group domain and tends to interact with Oct4 through transactivation domains.

Answer 15

Niwa et al (2000) Oct4 influences ES cell fate. Nichols et al (1998) In vivo: Oct3/4 is essential for pluripotent potential of ICM. Without it, the embryo (inner cells) failed to acquire the potential to produce the different lineages and can only produce extra embryonic tissue or trophoectoderm. In vitro: elimination of Oct3/4 leads to differentiation to trophoectoderm.

Answer 16

1.5 (upregulation): extra-embryonic tissue, mesoderm, endoderm 1.0 (normal): embryonic stem cells 0.5 (downregulation): trophoectoderm The amount of expression of Oct4 determine the fate of the tissue. To keep cells as epiblastic cells, they need intermediate levels of Oct4.

Answer 17

Niwa et al (2005) Interaction between Oct4 and Cdx2 determines trophoectoderm differentiation. There is a reciprocal repression loop between Oct3/4 and Cdx2. As Oct3/4 expression decreases, Cdx2 expression increases. Forced expression of Cdx2 decreases Oct3/4 and leads to differentiation of trophoblast. Oct3/4 and Cdx2 appear to bind in a complex that inhibits their individual transcriptional activity.

Answer 18

Avillon et al (2003) found that multipotent cell lineages depend on Sox2 in early embryogenesis. In vivo: embryos where Sox2 is deleted failed to generate the epiblast. Both Sox2 and Oct4 are required in the lineage leading up to the epiblast formation. In their absense, trophoectoderm is formed. In vitro: Sox2 -/- no formation of inner cell mass, no epiblast, no pluripotent stem cells, no Oct4 expression.

Answer 19

Identified by two independent screens (Mitsui et al AND Chambers et al 2003). - Mitsui: In silico screen. Used digital differential display to find genes which were highly expressed. Northern blot to find location of expression. Some were known, others were unknown. ecat14 (Nanog) was found exclusively in ES cells (now known that it is also found elsewhere). Expressed in plasmids and delivered to cells which were in the presence and absence of LIF. With the addition of Nanog, ESCs can surivive without LIF. - Chambers: Functional screen. Created plasmids with different genes from a library and introduce into cells which are deficient in LIFR, so cannot respond to LIF. Looked for genes that produced a colony without a response to LIF. Identified Nanog cDNA in LIFR-/- cells.

Answer 20

Nango homeodomain protein is transiently expressed in embryo. In vivo: Nanog -/- there is no pluripotent ectoderm, instead forms visceral and parietal endoderm. In vitro: Increased Nanog overcomes the requirement for LIF (and BMP+serum). Nanog -/- ES cells and ICM lose pluripotency and differentiate into extraembryonic endoderm.

Answer 21

They have the internal ability to drive certain differentiated fates. Oct4 (mesoderm), Sox2 (ectoderm), Nanog (definitive endoderm) and Tbx3 (hypoblast). But they can cancel each other out, creating a network of feedback loops that hold the balance together. This is quite fragile, a small change will have a big impact.

Answer 22

Chambers et al (2007) elucidated the heterogeneity using fluorescent reporter lines and FACS. He deisgned a reporter line to have RFP downstream of Oct4 and GFP downstream of Nanog. Yellow shows neither expressing. Then tested it with antibodies to the proteins and used FACS to seprate cells according to colour and fluorescence intensity. Even in a well-maintained stem cell population there is always so GFP pos and GFP neg. He separated these populations to give pure pos and pure neg. After a week, each population was able to regenerate the other, showing that the population was heterogeneous.

Answer 23

A technique used to sort a heterogeneous population of cells. Label the cells using fluorescent reporter line or cell-surface antigens. Produce stream of droplets each containing one cell passing through the laser, charging them. Use deflection plate to separate the cells based on colour and level of expression.

Answer 24

A rheostat produced varying levels of resistance. Nanog is thought to provide variable levels of resistance to differentiation depending on which stage at cell is at.

Answer 25

A model to explain how different cells can be at different levels of differentiation. Based on the idea that cell can move from its stable state to find another stable state. ES cells are at the top of the hill in the crater, but there are different positions in the crater, thereby explaining heterogeneity. It is thought that there are different ridges in the crater, some closer to the edge than others. Cell surface markers may be able to determine a cell's position e.g. centre of crater = SSEA3, further out = TRA-1-60

Answer 26

Cells from different states may be primed for particular lineages. Different positions in the model will have different propensities.

Answer 27

Stem cells have the ability to self-renew, differentiate or die. If there is a LOF or GOF mutation that prevents differentiation or death, cells may have an enhanced ability to self-renew in culture. Mutations that favour self-renewal will be selected for by the culture. Although this can't be used therapeutically, it gives valuable insight into genes that control proliferation and self-renewal. But it is not clear exactly what these mutations do.

Answer 28

Harrison, Baker and Andrews (2007) sought to identify the mutations, what they were like and where on the chromosomes they are commonly positioned. Found that they are mostly gain of function mutations. Led to a larger study to look at LOF and GOF mutations on each chromosome. Found that Ch1, 12, 17, 20, X seemed to have a lot of mutation sites. Ch4 has nothing. This may because it contains genes essential for survival (mutations would cause the cells to die) OR that mutations in Ch4 genes don't give ESCs any growth advantage. Gives indications of areas that contain genes important for growth e.g. gene mutation in ch20 that prevents apoptosis.

Answer 29

Heterogenous aggregates initially obtained from embryonic carcinoma cells. Produced by removing extrinsic conditions for self-renewal and forcing aggregation. They resemble gastrulation and early embryonic development in vivo. Plated on an uncharged plate without ECM so they cells don't anchor and remain floating. Widely studied during 1990s and 2000s. Can be lots of different shapes.

Answer 30

He found that Wnt signalling in embryoid bodies can replicate that found in embryogenesis. Early embryogenesis shows that inner cell mass becomes the epiblast which forms a cyclindrical shape where gastrulation occurs. Cells from the epiblast migrate to form mesoderm. Mesoderm formation is reliant on Wnt signalling, as shown by a Axin-Lacz reporter which shows cells in primative streak activated by Wnt. Berg showed that this was also the case in regions of embryoid bodies. Changing in media can change Wnt expression too. Wnt3a in media leads to faster expression, Dkk1 in media leads to delay in expression.

Answer 31

Pros: - Cheap to produce - Generate 3 separate germ layers - Good for pluripotency assay Cons: - Difficult to control aggregation in a reproducible way (shape/size) - Number of days before they are collected - different degrees of maturity

Answer 32

- Cystic (hollow) EB: best at producing endoderm - Bright cavity (thick cellular surrounding): Good production of the 3 germ layers. Best organised, closer to real embryos. - Dark cavity: more solid, not as organised as bright cavity. Good production of the 3 germ layers.

Answer 33

Many ways seeking to control the shape, number and formation. One way is to make the embryoid bodies from one cell. You can do this by... - Hanging drop method: Put droplets of fluid (10-20ul) on the edge of the plate, each contains one cell. Turn lip upside down and because of the superficial tension, droplet will remain in contact with the dish. - Controlled aggregation: Pyramidal shaped tissue culture wells. You can put a set no of cells in each well depending on application making them form aggregates similar in size and shape (homogenous).

Answer 34

Either using EBs or plating cells as monolayers. Growth factors relevant to specific differentiation will be added. Important variables include the concentration, when they are added and which combination of factors. The substrate that they grow on is also important - some lineages will differentiate well on specific substrates e.g. laminin, collagen, fibronectin etc.

Answer 35

Culture conditions could selectively generate the cell type of interest. However the most common outcome is to have a mixture of cells with the desired cell type contaminated by others. - FACS can be used for specific cell markers to separate intermediate, progenitor cells from final cell types. - Density gradients can be used because different cell types have different buoyancy so will float at different levels [not as effective as markers] - Insert selectable markers using genetic modification, cells will either express GFP or antibiotic resistance gene. Good for research, bad for clinical application.

Answer 36

He replaced part of the Sox2 gene with LacZ or neomyosin antibiotic resistant cassette which doesn't disrupt the gene but allows tracing of gene expression. Neural precursor cells will express sox2, sorted by FACS, given G418 (antibiotic) to purify and enrich (43% to 91%) and then they will differentiate. If you add FGF2 before they differentiate it will encourage the cells to proliferate, increasing the number of neural progenitors. Can adapt these strategies for lots of different applications.

Answer 37

In 2005, successful transplantation of islet cells produced from fetal cells or dead donors into a type 1 diabetic patient occured. Insulin injections were no longer needed. Not widely applicable, very expensive and difficult to carry out as you have to source appropriate cells.

Answer 38

What was already known about pancreatic development: Foxa2 and Sox1.7 mediated the patterning of the foregut, Pax1 expression determined the gut specification to pancreas, Ptf1a expression initiates budding and Ngn3 is expressed when branching occurs. Ins and Glc are expressed during a and b cell development. D’Amour et al (2006) created a protocol whereby they introduced a number of different markers at different time points to replicate pancreatic development and encourage gene expresion. Markers were detected at the level of qPCR (DNA) but a western blot was used to look at protein expression.

Answer 39

In the pancreatic tissue that D'amour et al developed there was the release of C-peptide in repsonse to multiple secretory stimuli, good indication that insulin is being produced. However, there were only a small percentage of insulin-expressing cells obtained. They were not glucose responsive, don't produce proinsulin well, don't maintain expression of key B cell markers, they are more like fetal B cells in character. Kroon et al (2008) improved the protocol, modified components particularly in stages 2, 3, 4 and added other markers to improve quality of progenitors produced in vitro.

Answer 40

Pancreatic endoderm was first produced from human embryonic stem cells using in vitro differentiation. These were then implanted into mice to allow for in vivo maturation. After 3 months, they saw that there was production of 'mini pancreases' which expressed a combination of markers. There was C-peptide produced (showing that insulin was being produced) and there were 3 cell types present: a, B, and Y all containing appropriate granules. These cells were able to respond to glucose and triggered the release of insulin.

Answer 41

Streptozotocin is toxic to the insulin-producing beta cells of the pancreas. STZ can be given to mice to make them diabetic. Pancreatic progenitors were then transplanted to the mice and measured the glucose in comparison to normal animals. Drug and transplant gave the same response to healthy animals showing that the cells can respond to glucose. Removing the explant (cells placed on a mesh so they can be removed later on) causes the mice to become hyperglycemic. HOWEVER, 15% of grafts in mice developed tumours. So obtaining the right molecular profile of the differentiated cells is not enough. There must be evidence for functionality in vitro AND in vivo.

Answer 42

Kelly et al (2011) identified cell surface markers for the isolation of pancreatic cell types derived from human ES cells. They found that there were two key markers identifying the cells, CD142 (pancreatic endoderm) and CD200 (endocrine). Since we need less mature cells for the transplantation, they needed to identify the intrinsic marker (transcription factor) that drive the expression of these cell surface marker. Found that Nkx6.1 labels progenitors and was positive for CD142 cells. Population was separated using CD142 marker and enriched for Nkx6.1 positive. Cells were tested in vitro and in vivo. CD142 cells were able to differentiate and when transplanted into animals they has a similar response to normal glucose response, measured by release of insulin. In the enriched CD142 grafts, none were teratomatous whereas in the unsorted cells, half of the animals developed tumours. However in some cases the CD142 cells didn't engraft/survive but most did (7/12).

Answer 43

They can be very sensitive and selective for the cell type and can give an indication of the function. It allows for analysis of mixtures of cells and manipulation of cell mixtures (cell isolation or elimination). Analysis can be carried out using immunofluorescence, immunohistochemistry, radioimmunoblotting, cytotoxicity. Sorting can be carried out using flow cytometry, magnetic beads (antibody conjugated to a bead), panning, complement-mediated cytotoxity.

Answer 44

Osafune etl al (2008) showed that there are differences in cell lines that affect their efficiency to produce pancreatic endoderm. Screen of 17 different ESC lines showed that although all the cell lines will produce pancreatic tissue HUES 6 and 8 showed enriched pancreatic markers. Therefore it is important to choose the best cell line for the job.

Answer 45

Organoids! Conceptually different from embryoid bodies (less mature). Targeting more complex, late differentiation into organs. Highly organised tissue with more than one functionally relevant lineage. Able to achieve tissue architecture and good differentiation. Performed using combinations of growth factors and ECM. Successful organoids produced for eye, gut and brain tissue.

Answer 46

Differentiation will follow a particular route 'down the hill' seeking a stable state, this will lead to different fates. Pluripotent reprogramming goes against this model, allows the cell to move up the hill instead. Direct conversion is a new idea that allows cells to move from one differentiated state to another differentiated state (transdifferentiation). It has no need for an intermediate pluripotent state.

Answer 47

Investigating the question of whether genes were lost in differentiation or repressed in a complete and irreversible way. Gurdon performed experiments in 60s and 70s. Began with intestinal tissue, from which he isolated highly-differentiated epithelial cells. He nuclearly transferred them into an enucleated recipient egg. This created a complete blastocyst which developed into an adult.This showed that the intestinal cell had all the genetic material to make a whole organism. He then took a graft from the growing embryo and put it into a host embryo, this gave a tadpole which had functional muscle that was derived from original intestine.

Answer 48

In 1996 Reproductive cloning was used to produce Dolly the sheep proving that mammalian cloning is possible. It involved removing the nucleus of a Finn Dorset sheep (white-faced) and fusing it using an electric shock into an enucleated recipient egg from a Scottish Blackface sheep. Fused egg begins dividing normally, it was then placed into the uterus of a foster mother (black-faced) and lamb develops and is born! Dolly lived a healthy life and was able to reproduce -Bonnie! Since then many other animals have been cloned, very important in agriculture.

Answer 49

AKA Somatic cell nuclear programming. Take a somatic cell from a patient biopsy, carry out nuclear transfer to an oocyte to create a clone. Propagate cells in vitro and manipulate them to produce different tissues which can be transplanted back into the patient without any problems with rejection.

Answer 50

Woo Suk Hwang in 2004, fabricated 2 papers that were published in 'Science' that showed that he was able to do somatic cell nuclear transfer with human cells. Most of the findings were fake, not to mention the many ethical issues. In 2013, Masahito Tachibana proved that you can produce human embryonic stem cells derived by somatic cell nuclear transfer.

Answer 51

They predicted that it would be a result of hundreds of factors, research was not well funded. He had already identified a number of genes expressed in ES cells, including Fbx-15. He created a construct using neomyosin resistant cassette and inserted it into Fbx15 loci. Cells with the construct were selected for using 12mg/ml of G418. High expression of Fbx-15 will allow them to survive. Authors selected 24 candidate genes that they thought may play a pivotal role in maintaining ES cell identity. Introduced each candidate separately into Fbx15 Bgeo/Bgeo mouse embryonic fibroblast. None survived showing that a single factor cannot drive pluripotency. Introduced 24 together, generating 22 G418 resistant clones. He selected 12 of these clones, 5 of which looked like ES cells. Analysis of the markers that the clones expressed. Next withdraw individual factors to determine which pool of genes are necessary. Found combination of Oct3/4, Sox2, c-Myc and KlF4 required to induce pluripotent stem cells. Low yield. Nanog was dispensable.

Answer 52

Transcriptome analysis compared the profile of the iPS and ES cells, showed that they were very similar but slightly different. He then injected these cells into a novel mouse and showed that they could produce tetratomas. Derivatives of iPS-MEF10 and iPS-MEF4 can form all 3 germ layers but iPS-MEF3 (Oct3/4, c-Myc, KIF4 no Sox2) only gave undifferentiated cells. Repeated reprogramming from adult fibroblast form tail-tips. Took adult fibroblast from tail of mice and repeated reprogramming. They injected the clones into blastocysts to create chimeras. They obtained 18 embryos at stage 13.5. Two of them had GFP+ cells which contributed to all three germ cells.

Answer 53

Yamanaka: Oct3/4, Sox2, KIF4, c-myc Thomson: Oct4, Sox2, Nanog, Lin28 Thomson tried to avoid using c-myc because myc is an oncogene which encourages proliferation. He was concerned that reprogramming would be more difficult to control. Lin28 inhibits LET7 which inhibits Myc, so Lin28 indirectly activates Myc without it being added itself.

Answer 54

- Somatic cell nuclear transfer: Take nucleus and transplant into enucleated egg. Rapid (<5 hours) - Cell fusion with pluripotent stem cell: fuse a somatic cells with a pluripotent cell to give a cell that expresses both genetic material (tetraploid). Rapid (48 hours) - Transcription factor expression: put specifc TFs into a cell and allow it to take on iPS cell identity. Slow (>10 days) - Small molecule exposure: reprogramming with compounds rather than genetics. Slow (>10 days)

Answer 55

- Integrating virus- deliver a gene which will become integrated into the genome. Random integration is risky because it may be an important region or cause mutations. - Non-integrating vector- will not reach DNA, remains transiently in cytoplasm. No risk of mutations. - Excisable vectors- put genes in and then remove some with a different gene. Very difficult to do. - Protein- strategies using mRNA to increase protein before cellular machinery takes over. - Small molecule replacements - CRISPR-Cas9 can alter specific genes in safeharbour regions

Answer 56

Hanna et al (2007) treated sickle cell anaemia in mice using iPS cells generated from autologous skin. Thalassemia is caused by a mutation that changes the B chain of the haemoglobin making the erythrocyte change in shape. Took a humanised mouse and removed fibroblasts, made iPS cells using Yamanaka factors. They corrected the gene mutation using gene specific targeting. Cells differentiated into embryoid bodies and obtained hematopoietic progenitors. Transplant the corrected hematopoietic progenitors back into the mice. 3 homozygous male mice were irradiated to give them the mutation, have iPS-derived cells transplanted and showed WT allele 6-8 weeks after.

Answer 57

You can study the molecular nature of the disease in vivo and in vitro. Allows for screening of drugs to treat the phenotype (pharmacogenetics). Take a patient cell, make iPS cells and then perform cellular studies in vitro or transplant them to perform drug screens.

Answer 58

Genomic instability Need for a continual supply of high quality embryos Potential for tumour formation Questions regarding functional differentiation Problem of immune rejection Ethically contentious

Answer 59

No requirement for immunosuppressive treatment Opportunity to repair genetic defects by homologous recombination Opportunity to repeatedly differentiate iPS cells into desired cell type for continuous therapy Less ethical issues

Answer 60

- Are all iPS the same? - Develop robust and reliable differentiation protocols - Relatice efficiency of differentiation methods - How will future iPS be screened for quality?

Answer 61

Zhao et al (2011) Cells from same strain of mice usually can be inserted and form teratomas with no immune rejection but when mouse fibroblasts from this same B6 strain were reprogrammed to iPSCs, teratomas formed but injection of these iPSCs were immune-rejected by B6 recipients. There was found to be abnormal gene expression in some cells differentiated from iPSCs that could induce a T-cell-dependent immune response in recipient mice. Immunogenicity of patient derived iPSCs need further evaluation pre-clinic.

Answer 62

Vierbuchen et al (2010) showed that you could differentiate fibroblasts to active neurons that are able to fully function and produce action potentials. This was obtained by a number of factors including Brn2, Myt11, Zic1, Olig2, Asc11. But can now be performed using 2 small molecules. A fradulent paper in 2014 by Obokata et al suggested that you can use an acidic environment to induce direct differentiation.

Answer 63

Sound will travel through the outer ear into the ear canal to reach the inner ear. The sound waves reach the eardrum (tympanic membrane). These vibrations are in turn transmitted to our inner ear by the bones of our middle ear and reach the cochlear. Within the cochlear the organ of corti contains the hair cells (one layer of inner HCs and 3 layers of outer HCs) which will displace against the tectorial membrane. This causes displacement. his motion results in the hair cells sending a signal along the auditory nerve to the brain.

Answer 64

If you have damaged hair cells or neurons you can get deafness. Presbycusis is age-related hearing loss. Becoming more prevalent in an aging population. It is an unmet medical needs because there is no drug treatment. Only therapy is cochlear implant or hearing aids which are not that effective. Idea to replace hair cells/neurons using stem cells is currently being researched. We have about 16,000 HCs and 30-40,000 neurons, but we only need a fraction to communicate.

Answer 65

Chen et al in 2007 and 2009 Obtained fetal material from terminations, dissociated the auditory stem cells and tested how the cells behaved in diferent factors (FGF, IGF, EGF, OSCFM). Combination of these factors plus vitamins and hormones showed they can sustain proliferation. Separated by FACS and looked for markers expressed using qPCR. Showed to be expressing Oct4 and Nestin among others. Under neural and hair cell conditions, hair cells had the molecules and channels (K and Ca) to function but didn't have apical differentiaion, neurons had K channels for action potential firing.

Answer 66

They are ultimately too 'progenitor-like' so they do expand but eventually they have a growth plateau. You need a source of stem cells which you can continually go back to again and again, which won't run out like the fetal cells. So we need to use human embryonic stem cells which can carry on proliferating forever.

Answer 67

Neural plate will start to fall to become the neural tube. Pre-placodal region surrounds it which forms a disc-shaped optic placode (thickening), this is the first primordial of the ear. Neurons and hair cells both come from the optic placode.

Answer 68

It was already known that fgf3 and fgf10 are both needed for otic placode development, fgf3 expressed in the neural tube and fgf10 from the mesenchyme. Expose the cells to fgf3 and fgf10 and measure the intensity of fluorescence of markers Sox2 and Pax8 using a quantative microscope. 20% of cells were positive for markers. Transcriptome analysis identified 40 genes highly expressed in the otic placode (signature). Similar gene markers but morphogenetically different - otic epithelial and neural progenitors. Purify these progenitors and put them under neural and hair cell conditions. Functionally viable with appropriate channels.

Answer 69

Cochlear implant essentially works instead of the hair cells to convert the sound into electrical impulse. But will not function if patient has neuropathy because it requires stimulation of the nerve. Idea is to use a combination: replace hair cells with cochlear implant and neurons using transplanted progenitors.

Answer 70

Gerbils have a lower hearing frequency like us. Insert Ouabain, a Na-K ATPase inhibitor to wipe out neurons in left ear. Take hESC lineand expand populations of ONPs and cause them to differentiate into neurons. Inject cells into the base of cochlear. They form an ectopic ganglion of human neurons (B-tubulin positive). They express glutamate receptors for synpatic junction and reach the brain (anatomically connected). Can measure auditory brain response waves. After transplantation after 10 weeks there is a restoration of at least 1/3 of neurons.

Answer 71

Make sure that the cells are safe and don't cause tumorigenesis. Safe generation and manufacture efficiency (GMP). Good understanding of relationship with host. Combination of technologies (with cochlear implant).

Answer 72

Damage the neurons with Oubain and the hair cells with aminoglycoside antibiotics (Kanamycin or Furosemide). The result is that after one week after treatment there is complete ablation of hair cells and the mice are profoundly deaf. Over 20 weeks you can see a decrease of neurons.

Answer 73

Cochlear implant cannot be carried by the gerbil itself so the animal has a electronic chip system which is fully implantable. The chip is driven by a magnetic field in the cage to generate electricity. You stimulate them with different sounds to establish threshold and monitor their behavourial response. Then you can destroy the nerve, replace with progenitors and measure the brain activity, Shows restoration.

Answer 74

Zygote goes through cleavage and develops into a blastocyst which contains the inner cell mass. From that inner cell mass, embryonic stem cells can be isolated, First done in mice by Martin Evans and Gail Martin in 1981. First done in humans by Jamie Thomson in 1998.

Answer 75

Tumours that are usually benign that typically contains a diversity of tissues including hair, teeth, bone, thyroid, etc. Saccrococcygeal tetratomas described by babylonian historic texts. Dermoid cysts/ovarian tetratomas are much more common. Can be removed by surgery. Similar occurs in testes (Human testicular germ cell tumours) occur when most tumours don't in early age. Contain embryonal carcinoma cells which can differentiate but contain a malignant capacity. Embryoid bodies can develop in tetratomas which look similar to 12 day human embryo.

Answer 76

Ray Stevens in Jackson labs in 1954 discovered that stain 129 mice developed testicular germ cell tumours rapidly. In 1964, Kleinsmith and Pierce showed that you can tranplant an embryonal carcinoma cell into another mouse and it would develop into the whole tumour. Brinster injected embryonal carcinoma cells into a blastocyst which was implanted into a host mother, was able to take part in normal development. Evans and Martin in 1981 took inner cell mass cells and cultured them, they looked very similar to embryonal carcinoma cells.

Answer 77

In 1990 the human fertilisation and embryo act stated that human embryos could be used up to 14 days of development. Must have a license from the HFEA to do research.

Answer 78

These cells undergo genetic changes associated with culture adaptation. Retention of reprogramming factors. Genetic changes associated with reprogramming. Incomplete reprogramming may lead to epigenetic changes. Ethical issues of consent, personal identity, potential to reprogram cells to germ cells.

Answer 79

FGF, BMP, Lif, TGF-B encourage transcription of Oct4, Nanog and Sox2 which can support self-renewal. Culture adaptation is used by the stem cells to select for mutants that will drive self-renewal rather than differentiation. When comparing normal and culture adapted cells, after 4 and 6 days there is greater proliferation of the culture adapted cells.

Answer 80

39 labs contributed 125 hES cell lines, 79 remained normal. Abnormal (culturally adapted) showed that the greater the time spend in culture, the increased probabilities of abnormalities. May have translocation of long arm of chrosome onto chromosome 6. Chromosome 4 doesn't change. 1, 12, 17, 20 are most commonly changed, gene(s) here will give a selective advantage. Human ES cells in testicular tumours shows gains in 17q, 12q, X.

Answer 81

On chromsome 20, there were 'gain' mutations' - extra copy of that region. In this region there were genes that are expressed in embryonic development: BCL2L1, ID1, HM13. Transfect a vector encoding each one of these genes into cells. Overexpression of ID1 and HM13 shows no difference in cells. Overexpression of BCL2L1 does show a selective advantage. It is also expressed in cancer cells.

Answer 82

Hypothalamus is built from novel stem cells in a way that allows stem cells to be maintained over life and change according to needs. Composed of many neurons arranged around the 3rd ventricle. Collectively the neurons mediate homeostasis: temperature, sleep, growth, mood, stress, metabolism, reproduction and electrolyte balance.

Answer 83

- Where does it originate? | - How does it develop?

Answer 84

Hypothalamus can be positioned relative to morphological landmarks. Anteriorly is the optic stalk, posteriorly the cephalic flexure, adjacent to Rathe's pouch and by the mamillary pouch and infundibulum. Identified by the dissection of a chick embryo.

Answer 85

Identifyy a set of molecular markers that weren't dynamic and stayed around for a long time. These markers define different sections of the hypothalamus. -Anterior hypothalamus: Islet1 -Central/tuberal hypothalamus: Fgf10 -Caudal/mamillary hypothalamus: Emx2 Together they mark the whole hypothalamus.

Answer 86

Prechordal mesoderm sits directly underneath the neuroectoderm. Lots of debate surrounding the idea that PM is important for hypothalamus development. Fu et al injected neural epithelial cells above the PM with a dye and fixed the chick plus 150 focal injections. He showed that after allowing developing and analysing the results, he could conclude that basal hypothalamus derives from neuroepithelial cells that lie above the PM.

Answer 87

Cells of the prospective hypothalamus have a distinctive signature. It is thought that PM expresses specific signals to cause it to be a hypothalamic precursor, including BMP7, pSMAD1 and others. Once a precursor, Tuberal cells express fgf10 exclusively in that region. In the case of anterior and mamillary fates there was a question of whether their fates were already specified or whether other signals from adjacent tissues are required. Isolating pHyp by dissection and culturing them ex vivo in a 3D matrix to investigate. Found that cells would grow x20 and differentiate into anterior and mamillary cells of entire basal hypothalamus. Develop with spatial awareness enough to develop small hypothalamus tissue.

Answer 88

Regulatory mechanisms of fgf10 and shh establish progenitors but also feedback to maintain a stem-like cell. Depending on the location of the cell, it will be exposed to different levels. More fgf10 will lead to a stem cell (tanyocytes) being maintained. But if the cells are on the boundary of fgf10 (low exposure) but high exposure to Shh progenitors will be established.

Answer 89

α-tanycytes are self-renewing cells that form the critical component of a hypothalamic stem cell niche. Their proliferation is governed by local fibroblast growth factor signalling. These cells are maintained into adulthood and are both fgf-responsive and fgf-expressive.

Answer 90

A subset of fgf10+ cells express GLAST and Hes5 so CreERT techniques were used to lineage trace these cells. Showed tha adult tanocytes can self-renew, give rise to other tanocyte subsets, neurons which will project to the arcuate nucleus and astrocytes. Involved in stress, energy and reproduction. Lineage-tracing in fish (Her4-CreERT) shows that cells give rise to dopaminergic neurons.

Answer 91

Idea is that due to stem cell location lining the 3rd ventricle, there are lots of fenestration in the capillaries meaning that there is no blood barrier allowing for the stimulation of the stem cells by metabolites/hormones. Stem cells will respond and anticipate need, giving rise to new neurons allowing for adaptation (allostasis).

Answer 92

Freshwater polyp hydra can be cut in half and regenerate the rest of their bodies. Newts/salamanders can perform limb regeneration. We have very limited regenerative capacity, but we can heal after surgery.

Answer 93

There are many different types of cells in the blood which have lots of different functions. Common theme is that they have very poor proliferative capacity and a short lifespan. Every day you lose 10^11 red blood cells yet they are constantly being replenished. Therefore there MUST be a pool of more primative undifferentiated stem cells that can replenish the pool and differentiate.

Answer 94

1950-60s: Effects of ionizing radiation in mice. Bone marrow is one of the most sensitive. X-ray exposure in mice stops production of blood cells and mouse dies. But if mouse is injected with bone marrow from a healthy mouse, mouse survives. Till and McClulloch (1961) found that the spleens of saved mice have colonies/nodules that arise from a single cells (genetic tests) and contain differentiated blood cells and new 'colony forming units'. Second irradiated mouse and inject colony forming units, mice were saved and spleens contains nodules. Colony forming units = Hematopoietic stem cells

Answer 95

Long-term self-renewing hematopoeitic stem cells will become short-term hematopoeitic stem cells and then multipotent progenitors. MPP can become common myeloid progenitors or common lymphoid progenitors. Myeloid progenitors can become megakaryocyte erythrocyte progenitor or granulocyte macrophage progenitors. MEP will eventually lead to erythrocytes or platelets. GMP will eventually become granulocytes or macrophages. Common Lymphoid progenitors can become T or B cells. Cells lost their ability to self-renew and restrict their fates.

Answer 96

In vitro assays and in vivo assays are used to show that hematopoietic stem cells can differentiate but the problem is that this is retroactive classification.

Answer 97

Cell-surface markers can be used to identify HSC. Usually a combination of 5/6 markers. Can look at the presence or absence, high or low expression. Purification methods such as FACS involves labelling the cells with fluorescent markers, passing through laser cell by cell and emission of light. Can identify 4 cell populations: red, green, red+green, negative for both. Thanks to these and other methods, models of hematopoiesis have to be adapted to show that cells are more plastic than we originally thought.

Answer 98

Effector types have very different gene expression according to their function. This means that there must be up and down regulation of different genes. Transcription factors determine gene expression patterns, recruit coactivators/corepressors of transcription, and are often components of multiprotein complexes, providing flexibility.

Answer 99

A transcription factor that is required for development into a specific lineage e.g. GATA1 for Erythroid lineage. Can be tested by KO of TF to see if the lineage is lost. Can change fate of cells they are introduced to. Can antagonise the opposite lineage program (simultaneously upregulating own markers and downregulating opposite markers).

Answer 100

GATA1 is TF for erythroid lineage and PU.1 is TF for myeloid lineage. - GATA1-mediated antagonism of PU.1: Normally PU.1 will bing to its cofactor c-Jun which will upregulate PU.1 target gene. In presence of GATA1 it will outcompete c-Jun and bind to PU.1 preventing transcription. - PU.1-mediated antagonism of GATA-1: GATA1 usually binds to zine finger binding domain to upregulate GATA1 target genes but in PU.1 presences, PU.1 binds to GATA1 and displaces it meaning target genes are not expressed.

Answer 101

-Transcription factors are lineage determinants OR -Transcription factors are executors of externally signalled cues

Answer 102

Initially it was thought that multipotent cells would not express lineage-specific markers however after interrogation of single cells we found that multipotent cells do express lineage-specific genes are a low level. Initially lineage-specific TF cancel each other out, maintaining the cell as undifferentiated but then something happens in the system (random flux of gene transcription or external cue) and one TF is upregulated and cell differentiates.

Answer 103

Many of the mutations that underlie Leukemia are often chromosomal translocations or somatic mutations of key transcription factors in differentiation. e.g. AML1 joining ETO on ch8 from ch21. ETO recruits corepressor complex which wouldn't normally be there. In some cases in chromosomal translocations, chimeric TF are inappropriately activating and repressing genes. Sca/tal-1, LMo2, E2A and runx1 are invovled in chromosomal translocations which create fusion proteins that block the activation of lineage determining factors.

Answer 104

If we know what TF can contribute to which fates there is potential to generate patient-specific cells that are otherwise rare, inaccessible and difficult to expand ex vivo. Could be applied to supplying cells for human disease modelling, therapeutic screening and cell replacement therapy.

Answer 105

It may be the case that extrinsic cues like cytokines direct fate of cells. Cytokines can be instructive and/or selective. Instructive- factor drives fate directly Selective- cytokine eliminates other cell fates by cell death Difficulty with analysis because the outcome for both is the same. Can be tracked using Time-lapse imaging of stem cell fates. Can see every decision made.

Answer 106

M-CSF is a cytokine released during infection and inflammation and leads to increased output of myeloid cells. But is it selective or instructive? PU.1-GFP mouse model where GFP reporter can track PU.1 activity. Use time-lapse microscopy to show that cells could turn from neutral to fluorescent without any cell division/cell death. This shows that M-CSF is instructive and that extrinsic cues can instruct hematopoietc stem cells.

Answer 107

A local tissue microenvironment that hosts and influences the behaviours or characteristics of stem cell. Must have both anatomical and functional dimensions.

Answer 108

Studied irradiated 'saved' mice that had had transplantation of splenic colonies. Sucessful transplantation requires the irradiation of existing HSCs within endogenous bone marrow. This showed that HSCs are competing for a limited source of supportive niches. Schofield (1978) showed that HSCs need to reside in the bone marrow to retain their 'infinite' potential. Stated that the stem cell niche has a defined anatomical location, regulates self-renewal and removal from the niche results in stem cell differentiation.

Answer 109

Needed for clinical treatment - transplantations still require irradiation in bone marrow, exposing patient to unspecific damaging radiation. We cannot maintain adult stem cells for prolonged periods in vitro. Difficult to expand and lose their potential quickly. Comprehending the function of stem cell niches will enable recapitulation of stem cell niche environment and designing better treatments.

Answer 110

First experimental evidence. Group of hub cells at the apex, in contact with both germ line stem cells and cyst stem cells (somatic) via adherens junction. Hub cells release factors to keep it self-renewing. GSC will divide asymmetrically, one cell will remain as a stem cell, the other will become a gonialblast. Eventually after cell division, it will become a spermatocyte. Hub cells produce Upd ligand to activate JAK/STAT. For CSC this is enough but GSC it is not sufficient, it also requires Gbb and Dpp produced by Hub cells to activate BMP, turning off Bam transcription leading to no transcription of differentiation genes.

Answer 111

Contain ovariole which has a germanium at the end. Terminal filament at the very end, followed by Cap cells (hub cells equivalent). Cap cells are in contact with GSCs and may intermingle with Escort stem cells. BMP activation is required and sufficient to maintain GSCs in undifferentiated state.

Answer 112

In males, both GSCs and CSCs require E-cadherin mediated adhesion to the hub for their maintenance. Excess integrin expression in CSCs causes them to outcompete GSCs. Similarly in females, GSCs are maintained by adheren junctions to Cap cells.

Answer 113

Stem cells undergo asymmetric division. Before the cells undergo mitosis, the centrosomes will be duplicated. Daughter centrosome will travel to the opposite side of the cell and mitotic spindle will form pulling the chromosomes from either side. One cell will stay attached to the hub cell and the other will be one-cell diameter away. This means that only the stem cell is in contact with the hub cells, maintaining it as undifferentiated.

Answer 114

Occupancy: where the stem cells are is important, they always need to be in close proximity to niche cells Fate: niche cells will produce local signals which will travel to the stem cell and repress differentiation genes. This prompts asymmetrical division.

Answer 115

There have been several proposed stem cell niches. Hematopoietic stem cell niche is well-recognised to have a high-turnover until skeletal muscle or brain stem cell niches. Most adult stem cells reside in the bone marrow. Surrounded by spongy bone (trabeculae) boundary called endosteium. Then the solid cortical bone and the peristeum boundary. Mammalian stem cell niches provide physical support, soluble factors and cell-mediated interactions to maintain and regulate function of stem cells.

Answer 116

Quiescent HSCs are necessary to prevent stem cell exhaustion. Loss of p21 promotes entrance into the cell cycle. Cycling HSCs: constantlyproducing self-renewing and differentiating daughter cells. Pulse-chase experiment: Feed in vitro cells or mice with labelled food (e.g. BrdU). When cells replicate their DNA they incorporate BrdU instead of tyrosine, labelling the cell. Look for diluted labelling. Time-lapse microscopy: Visualise the cells to see if they do divide or not.

Answer 117

Endosteal vs. Perivascular niche - the environment is not the same. Endosteal niche is full of preosteroblasts, osteoblasts, and osteoclasts whereas in Perivascular niche there are mainly endothelial cells and sinusoids. The structure of the niche determines the junctions, soluble factors and interactions that they will have.

Answer 118

Cell-cell interactions Cell:ECM interactions Cell:Soluble signal interactions

Answer 119

Niches contain both stem cells and stromal support cells. Cells interact with one another through cell-surface receptors, gap junctions and soluble factors.

Answer 120

Macrophages arise from hematopoietic stem cells themselves. But they also regulate stem cells to give signals. For example, cycling HSCs are maintained by CXCL12 and other secreted factors which are secreted by Nestin+ stem cells. Macrophages regulate mesenchymal stem cells to give particular signals. If macrophages are ablated from the bone marrow mesenchymal stem cells are still present but no longer give signals to cycling HSCs.

Answer 121

Autocrine and Paracrine Secreted factors may act locally (within 1-2 cell diameters) or may diffuse throughout the niche. Studies in flies and worms have shown the support cells secrete factors that are required for maintaining stem cell identity. Soluble factors include FGFs, BMPs and Wnts.

Answer 122

Less important for HSCs but other cells are embedded in the ECM. ECM adhesion molecules provide an anchor for stem cells to the niche, a polarity cue for orientation for stem cell mitotic spindle and a platform for signalling of spatial cue and mechanical inputs.

Answer 123

Stem cell niche is a dynamic structure that responds to exogenous signals. It may be the case that we can modify these and use them as a target for therapies. Could use drugs that affect support cell function.

Answer 124

Osteoblastic cells regulate HSCs. Non-random organisation suggests relationship between HSCs and osteoblasts. Osteoblasts are activated by parathyroid hormone through the receptor PPR. Q: Do osteoblasts contribute to the unique microenvironment? Transgenic mouse with a constitutively active PPR receptor shows an increase in trabeculae bone mass and increase in number of HSCs. This could be due to direct effect of PPR on HSCs OR effect of osteoblasts on HSCs. Tested ability of osteroblasts (stromal cells) to support HSCs in vitro. Better in transgenic. In vivo: inject mice with PTH to increase osteoblasts, there is an increased survival advantage after bone marrow transplantation. Therefore enhancing supportive niche function could improve the efficacy of engraftments or influence niche to particular stem cell fates.

Answer 125

Requires a rigid vessel (glass or plastic petri dish) Grown in a monolayer Given certain factors but are isotropic, no gradient. Layered onto matrix, rather than discrete ECm fibrils. Forced apical-basal polarity. Adhesions restricted to x-y plane. Much higher stiffness of material than in vivo (measured in GPa) Often fails to recapitulate the in vivo phenotype, drug response will be very different. Uncontrolled spreading and migration in x-y plane.

Answer 126

Soluble factors can be released in a gradient, similar to in the body. No prescribed polarity. Discrete ECM fibrils are present which the stem cells can attach to. Spreading and migration is sterically hindered. Adhesions distributed in all three dimensions. Low stiffness (kPa range).

Answer 127

Seeking to improve the 2D and 3D models in vitro. Resembles the tissue more but isn't as complex as an actual organ. Increased physiological relevance but decreased experimental tractability due to a lack of appropriate analytical tools. Lancaster and Knoblich (2014) defined it as a 3D structured orginated from stem cells. Contains multiple organ-specific cell types, not spheroids. Capable of recapitulating some specific function of the organ (e.g. excretion, filtration, neural activity, contraction). Cells within organoid are grouped together and spatially organised similar to organ. Organoids have been developed from mammary gland, liver, pancreas, gut, tongue, lung, stomach and prostate.

Answer 128

Cell sorting and Spatially-restricted lineage commitment. e. g. Carrots: place a small piece of carrot under appropriate culture conditions and it will create a mass of undifferentiated cells. If left without external cues will eventually form structures such as roots. e. g. Sponges: dissociated into single cells, can be re-aggregated to reform the main body plant. e. g Amphibian embryos (Townes and Holtfreter 1955) dissociate of epidermal and neural plate cells. Spontaneous re-aggregation leads to cell sorting and segregation of cell types, neural on inside, epidermal on outside.

Answer 129

Cells have differential adhesion properties. When mixed together they are more likely to make bonds with cells of their own type than with cells of the opposite type.

Answer 130

Stem cells will divide and give rise to daughter cells which due to the restraints on the tissue will move away and become far enough away not to be exposed to the signals, allowing for them to become differentiated and for the stem cell to be maintained as undifferentiated. Depending on how far away the daughter cell is, it will be exposed to different signals, leading to different layers forming of different fates.

Answer 131

Velvet-like structure of the gut is due to layer of villi (1mm protrutions) between them are crypts. Highest bodily turnover of cells (4-5 days). Cells include enterocytes (absorbance of nutrients and transportation across gut epithelium), paneth cells (secretion of anti-microbial components), goblet cells (secretion of mucin) and enteroendocrine cells (secretion of various factors).

Answer 132

Located within the crypt. Crypt-base columnar cells (or intestinal stem cells) lie at the bottom of the crypt interspersed with paneath cells. Paneath cells have an important role in maintaining CBC as stem cells, by secreting factors like Wnt, Notch, EGF and BMP. CBC express Leu-rich repeat GPCR 5 aka Lgr5. They divide once every 24 hours and give rise to transit-amplifying cells which proliferate as they move up the crypt and eventually give rise to the other differentiated cell types in the gut.

Answer 133

Adult somatic cells cannot be kept in culture for prolonged periods of time. Prolonged proliferation in vitro may cause the cells to undergo senescence or genomic transformation. And yet in vivo Lgr5+ stem cells can divide every 24 hours fine and dandy.

Answer 134

Aim to design a long-term culture of the intestinal system by incorporating elucidated components of the intestinal epithelium niche. Needs to incorporate signalling molecules AND appropriate matrix support. They used matrigel, a mixture of proteins from mouse sarcoma cells enriched in laminins, not chemically robust although now development of synthetic 'designer gels'. Isolated mouse intestinal crypts and labelled stem cells for GFP (Lgr5-GFP). Crypt preparations were suspended in Matrigel. Titration of key signalling molecules in the medium e.g. EGF, R-spondin etc Result: Single isolated crypt enabled a growth of a crypt villi structure in 5 days, could visualised CBCs and paneath cells.

Answer 135

Active Wnt signalling for crypt proliferation. EGF signalling for enterocyte proliferation. Noggin expression for expansion of crypt numbers. Overcome anoikis of isolated intestinal cells. Anoikis is a form of programmed cell death that occurs when epithelial cells are deattached from ECM.

Answer 136

Dissociating cells from crypt single cells, using FACS for isolation and then putting the cells in appropriate culture conditions allows symmetric crypt-like structures to be built from single cells.

Answer 137

Compared using immunocytochemistry. Contained all the different cell types: enterocytes, goblet cells, paneath cells, enteroendocrine cells. Cell-specific markers were looked for and compared. Paper claims that the two are virtually indistinguishable. Located in the appropriate position (paneth and CBCs are at the bottom). Show polarisation and can be propagated for at least 1.5 years.

Answer 138

Because of the similar physical, cellular and molecular characteristics of organoids with the in vivo tissues the physiological responses are similar too. Could be used... -Experimental tool - humanly relevant -Diagnostic tool - cystic fibrosis -Therapeutic tool - regenerative therapy and drug screens

Answer 139

Dekkers et al (2013) The use of gut organoids will allow testing the response to various cystic fibrosis treatments. They used organoids from WT and CF patients. Used a drug called Forskolin which will activate CFTR channels causing organoids to swell. Results: Strong forskolin-induced swelling was seen in organoids from heathly subjects. Individuals heterozygous for F508del and A445 (mild CF) showed a reduced amound of FIS. Individuals with severe CF showed even less swelling. No swelling seen in E60x4015delATT organoids. Drug screening: used individual/combinations of drugs and compare phenotypes. E60x4015delATT organoids showed no response. Combinations of treatments restored activity of F508 del mutation to 60% of WT level. Therefore organoids display variable responses to CFTR correctors.

Answer 140

Caused by mutations in cystic fibrosis transmembrane conductance regulator (CFTR) which is a channel regulatoing fluid homeostasis. The most common mutation is CFTR F508 del. Each mutation can affect the final phenotype and response to drugs differently depending on the genetic background of the patient.

Answer 141

Cultured organoids can rescue damaged epithelium. Mice in which epithelial damage to the gut bed in the colon is induced with dextran sulfate sodium can be treated with organoids which can heal the damaged bed.

Answer 142

No! Organoids can be derived from both adult stem cells and induced pluripotent stem cells.

Answer 143

hPSCs can be aggregated using embryoid bodies, then grown further with matrigel and appropriate factors. They are then put into bioreactors so that O2 and nutrients can reach the cells. Shows regional specification of forebrain, midbrain and hindbrain structures.

Answer 144

Organoids derived from a patient with microcephaly display smaller neural tissues when compared to a WT, resembling the smaller brain size in the patient. This allows for analysis of the molecular and cellular mechanisms that lead to microcephaly. In 2016, the link between microcephaly and the Zika virus was made. Brain organoids allowed for investigation of the potential mechanisms behind how Zika virus caused microcephaly and other neurological disorders. Dang et al (2016) showed that Zika virus infects neural progenitors and activates TLR3, this triggers apoptosis and attentuates neurogenesis.

Answer 145

- Injury: can be physical (stents), chemical (excess lipids, sugars) or biological (infection or virus) - Repair - Regeneration (introduce new cells or stimulate existing myocytes)

Answer 146

CHD is a common cause of detah and impresses a major economic burden on the health service. Percutaneous coronary intervention is usually used, a non-surgical treatment that sends a metal stent on a wire through to the blocked artery to open it up again. Stents are matched to patient size of artery using X-ray imaging beforehand.

Answer 147

Used to be a major problem in the years following PCI. Drug eluting stents are anti-proliferative and inhibit healing. Namely Taxol and Sirolimus. Growing concerns regarding long term safety, due to a link found in DES causing increased stent thrombosis due to exposure of blood flow to metal of the stent, as a result of delayed or incomplete endothelialisation.

Answer 148

VEGF elution, Oestradilol loaded stents and use of integrin binding peptides have all been researched but with limited success. EPC (endothelial progentior cells) capture stents propose the isolation of EPC from patient's own bone marrow and stimulation of growth over the metal stents to protect from stent thrombosis. However, people with CHD have a declining number of EPC in their bone marrow so is not likely to be successful.

Answer 149

Idea is that stent is pre-coated with human trophoblast derived endovascular cells (HTEC) which express early endothelial markers (PECAM-1) and not late endothelial markers (VE-cadherin) or smooth muscle markers and are anti-inflammatory. Used a pig as experimental model due to similar sized vessels and use of human stents. Stents are placed in a tub with the HTEC and cultured overnight with a rotator. Addition of polymer makes no difference. Stem cells make their own basement membrane and can attach to metal stent. Labeled with III Indium and tracked the label. Electron scanning microscope shows coverage of stent even after an hour. By a week bare stent is covered the same. Result: Although it doesn't make things worse (increase neointima or injury score) it doesn't make things better either (increase lumen). No significant difference.

Answer 150

British Heart Foundation launched 'Mending broken hearts' appeal to drive regenerative medicine research into whether we could use stem cells to regenerate tissue for heart failure. Establishment of dogma has slowed the research field. Potential mechanisms to lead to new cardiomyocytes after injury: -Paracrine stimulation of cardiomyocyte -Progenitor cell proliferation and differentiation -Proliferation of pre-existing cardiomyocytes -Epicardial activation

Answer 151

CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction (CADUCEUS) was a trial carried out in 2009-2010 and enrolled 25 patients following a MI. Cardiosphere-derived cells (CDCs) reduce scarring after myocardial infarction, increase viable myocardium, and boost cardiac function in preclinical models. So CDCs were infused into the infarct-related artery 1·5-3 months after myocardial infarction. The primary end point was death but by 6 months, no patients had died, developed cardiac tumours, or MACE in either group. Compared with controls at 6 months, MRI analysis of patients treated with CDCs showed reductions in scar mass, increases in viable heart mass and regional contractility and regional systolic wall thickening.

Answer 152

- Which is the best approach to use? - Is it best to use ES cells, iPS cells, small molecules? - What is the optimal delivery route? - How can we maintain survival and safety? - What is the mechanism of action?

Answer 153

There are various diseases that are undergoing clinical trials for drugs using stem cells. Namely, age-related macular degeneration, Parkinson's disease, spinal cord injury, diabetes, myocardial infarction. Sensory organs seem to be good targets, lots of clinical trials that are using retinal pigment epithelial cells. When developing a therapy you need to consider the structure of the target, how the disease affects the phenotypes of specific cells and how you can improve that specific cell.

Answer 154

The outmost layer of the eye is the sclera, a white protective fibrous layer. Followed by the choroid,a vasculalture layer containing connective tissues. The innermost layer is the retina, derived from the ectoderm, a light-sensitive layer of tissue that sends nerve impulses to the brain via the optic nerve. The fovea is an area of the retina that has the highest density of photoreceptors, creating visual acuity. The macula is the central functional area of the retina. Retina is made up of many layers. Outer layer is the supporting retinal pigment epithelial cells which are darkly pigmented and prevent light from being deflected. Rods and cons make up the photoreceptor layer. The following layers contain the amacrine, muller, bipolar, horizontal cells which synapse onto one another. Finally the ganglion cells synapse onto the optic nerve which goes to the brain.

Answer 155

Leading cause of blindness in over 55s. Degeneration of retinal pigment epithelial cells and their basement membrane. Can be dry (degeneration due to genetic causes - apoptosis) or wet (neovascularisation causes bleeding and blistering). Polygenic - not one genetic contributor

Answer 156

Macular degeneration in children. Damage to retinal pigment epithelial cells. Genetic cause, in it's typical form STGD1 autosomal recessive Stargardt's disease is caused by mutations in the ABCR (ABCA4) gene on chromosome 1.

Answer 157

In healthy RPE there are established tight junctions, a stable basement membrane (Bruch's membrane) and the rods and cones processes are embedded into the RPEs. In the night the RPEs recycle by apoptosis the outer segments of the photoreceptors to maintain a healthy environment. In degenerated RPEs there is destruction of RPEs meaning there is a lack of support and therefore a degeneration in photoreceptors.

Answer 158

Inherited conditions that affect the photoreceptors, mainly rods. However some genetic variables affect RPE genes and can be targeted with the same cell types for AMD i.e. RPE cells. Can be present alone or alongside profound deafness (Usher's syndrome).

Answer 159

For dry AMD patients are very supplementation with vitamins and antioxidant which can slow progression but doesn't cure it. For wet AMD patients are given intravitreal injection of anti-VEGF which can help in some cases. (VEGF acts to encourage neovascularisation). Since these treatments are generally quite ineffective and limited, stem cell therapy is expected to thrive, often the case when the pre-existing is poor and SCs can offer an attractive alternative.

Answer 160

In macular degeneration you tend to have a blind spotin the centre of your vision because it primarily affects your macula. Retain some level of peripheral vision. In retinitis pigmentosa you tend to lose peripheral vision first.

Answer 161

Can replace... - Photoreceptors: only in purely experimental phase in animal models - Retinal pigment epithelial cells: proof of concept established. Currently in clincal trials.

Answer 162

Can use retinal progenitor cells isolated from different stages of development and integrate them into young and adult mouse retinas (WT). They can engraft and differentiate into photoreceptors, creating synapses. Maximal engraftment and survival occurs when the donor population cam from P3-P7 animals. Progenitors were Nrl-GFP labelled, Nrl is a markers for postmitotic progenitors before they differentiate but after they've stopped dividing. Progenitors from E11.5 survived but failed to differentiate. In pathological circumstances, blind mice were transplanted (peripherin-2-deficient) with retinal progenitors and measured ganglion cell acitvity. Can see that they have integrated and communicating with the pathway - functional integration.

Answer 163

Pearson et al (2012) used Nrl-GFP-treated Gnat1–/– mice to test whether the functional integration of the retinal progenitors had a significant benefit for the vision of the mice. Used a guided water-maze task whereby a mouse is placed into water pool with options of a divided blank screen or stripey screen with a ledge to get out. 50% (by chance) was the threshold. 70% is a pass. Animals with transplantations were able to make behavioural decisions based on the new information given from their sight.

Answer 164

From the previous research performed, mouse progenitors from postnatal days 3-7 have been used. However it would be very difficult to obtain fetal cells from humans at the equivalent stage of development in humans due to the ethical and moral issues. This stage would be equivalent of the second trimester in human pregnancy.

Answer 165

Due to the issues with equivalently aged human fetal cells they sought to produce retinal precursors frome mouse embryonic stem cells using 3D culture system. The ES cells were embedded in matrigel to create organoid exposed to a series of growth factors. Can produce very similar retinal structures and tracked by different markers. Photoreceptors produced in vitro followed a developmental timeline. When grafted into retinas in WT and mutated animals the photoreceptors were able to integrate and differentiate. Photoreceptors from D26 and D29 in vitro were better than those from D34 (like the specific window in NRLP+ cells from the postnatal experiments).

Answer 166

They took a whole retinal sheet from donor embryos that includes both RPEs and neural retina and transplanted it into 10 patients (6RP, 4AMD). 7 (3RP, 4AMD) showed improved visual acuity. However this is a limited tissue source and may be too early (10-15 weeks) so wouldn't express Nrl.). Equivalent stage to the mouse data will be well into the 2nd trimester in humans.

Answer 167

Managed to develop retinal pigment epithelial cells in vitro from primate ES cells. They are easily visualised and can grow spontaneously into colonies. They were transplanted into a model of macular degeneration (royal college of surgeons) rats. Carried out a behavioural test to measure the ability of the rats to track a moving field of stripes to see if they have visual recovering. Transplanted rates shwoed visual recovery very close to WT activity. This established good protocols which can be used to drive differentiation in stem cells to give appropriate phenotype characteristics.

Answer 168

There was a need to show that the retinal pigment epithelial cells developed from human embryonic stem cells will last for a long time and are safe. They showed that the cells could survive for at least 6 months, showed good integration into the retina and no formation of tumours.

Answer 169

Published a report that described an experiment where by 2 patients were transplanted with retinal pigment epithelial cells derived from human embryonic stem cells. 1 with Stargardt's MD and 1 with dry AMD. Human embryonic stem cell line expressed appropriate markers of differentiation (MutF and Pax6). They formed monolayers of epithelial cells. Hestrophin is a molecule which cell can take up and can be used to look for phagocytosis activity of RPEs. Cells untreated or kept at 4 C didn't incorporate PhRodo biomarkers whereas at 37 C they do. This showed that it is an active process because it is dependent on temperature and that cells have phagocytic ability and display pigment = RPE! Two 2 distinct RPE were formed (high and low pigment). Experiment in mice showed that hPRE survived and integrated with no adverse/safety issues. Human patients in a follow-up study in 2014, showed that in 19 patients after an average of 22 months there was engraftment and no evidence of tumours. Monitored for 5/10 years. Small improved in visual acuity.

Answer 170

RPEs produced from iPSCs derived from a 70 year old Japanese woman suffering from wet AMD. In a report produced by Mandei et al (2017) explained the results. iPSCs from two patients were derived but only one was transplanted. The other was stopped due to mutations present in the line. This patient was mildly responsive to anti-VEGF therapy so was given that instead. Cells from first patient survived despite no immunosuppression. No tumours to date, no abnormalities although neovascular membrane has returned. No improvement or deterioration of visual acuity.

Answer 171

Initiative to treat AMD Uses a human embryonic stem cell line such as 'Sheff1' to develop RPEs. Have been able to treat 2 patients with transplant and have shown functional recovery. Produced RPEs were grown on an artificial polymer that creates a well-defined patch of polarised cells that surgeons can easily remove if needs be.

Answer 172

- Manufacture of a reagent to be used in a clinical setting demands the application of strict standards - UK follows the 1968 Medicines Act which inspired the 'Rules and Guidance for Pharmaceutical Manufactures and Distributors' - Manufacture controlled by MHRA (medicines and healthcare products regulatory agency) in UK, FDA in US

Answer 173

GMP requires amongst other things that the donor cells should be obtained ethically and with informed consent. Proven to be pathogen-free (tested for a series of potential contaminants). Individual components used in the manufacturing process should be traceable and ideally xeno-free (without animal-derived material). Should follow clear validated standard operating procedures which usually involves a lot of work adapting conventional research-grade methods.

Answer 174

- Provenance: source of cells, derivation and maintenance - Generation of the right cell type - Purification: Criteria and techniques needed - Characterisation: isolation and expansion, freezing and banking - Quality control: how do you know that each batch works i.e. objective criteria needs to be defined

Answer 175

- Proposed in the mid-19th century by Cohnheim, hypothesised that cells involved in injury repair come from the bone marrow. - 60/70s Friedenstein et al identified adherent (rather than floating), fibroblastic-like colonies from monolayer cultures of bone marrow, thymus and spleen. - Named CFU-F (colony forming units – fibroblast). Able to differentiate into bonee, cartilage and adipose tissue. - Coined Mesenchymal Stem cells in 1991 by Caplan. True identity demonstrated in late 90s when transplants of clonal bone marrow MSCs produced bone in vivo.

Answer 176

Adherent cell populations isolated from bone marrow are highly heterogeneous and may contain several subpopulations. Not all these cells fulfil criteria to be stem cells (unlimited proliferative capacity and multiple lineages). Proposed that non-stem cells should be called mesenchymal stromal cells, but acronym MSCs applies to both!

Answer 177

- Remain plastic-adherent under standard culture (with serum) - Express CD105, CD73, CD90 - Lack expression of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR (label other cells) - Differentiate into osteoblasts, adipocytes and chondrocytes in vitro

Answer 178

MSCs from the neck up are derived from the neural crest, MSCs from the trunk/posterior are derived from the mesoderm. MSCs become mesenchymal progenitor (more restricted) which can become different fates including stromal cells which give rise to osteoblasts, adipoblast, fibroblast-like cells, chondroblast and myoblast.

Answer 179

Initially isolated from the bone marrow. Have been purified from multiple tissues including adipose tissue, placenta, dental pulp, synovial fluid, peripheral blood, periodontal ligament, endometrium, umbilical cord and umbilical cord blood. MSC can be taken when teeth are removed. Evidence suggests that MSCs may be present in any vascularised tissue (niche).

Answer 180

Screened adult and fetal tissue for GFP markers, detecting NG2+ and CD146+ cells in all tissues surrounding small blood vessels. Therefore MSC have a perivascular origin. Cells were isolated using FACS. Selected out cells positive for CD56 (myogenic), CD45 (HSCs), CD34 (endothelial and HSCs). Keep cells positive for CD146. Cell population could attach, grow and expand cell population in vitro for a long time. Maintained expression of CD146 and NG2 for many RT-PCR passages. Cells differentiate into muscle and bone when transplanted into SCID mice. In vitro they produced cartilage, adipose tissue and bone. Cells were positive for CD73, CD90 and CD105 and negative fro CD34 and CD45 confirming their identity. MSCs CD markers are expressed in vivo confirming that the property is not simply acquired in vitro. However, it’s accepted that MSc profile can differ between in vitro and in vivo.

Answer 181

Attractive for autologous transplant potential without concerns of rejection. Traditionally obtained from the bone marrow but fat tissue is becoming a source material. Several clinical trials ongoing, mainly in orthopaedics to replace bone and cartilage. Potential for vertebral cartilage and compression problems or replacing muscle in cardiac infarction.

Answer 182

MSCs have a trophic paracrine effect. They secrete factors that help healing and repair i.e. TGF-B, PGE-2. Have a bystander effects, causing macrophages to become active and exert a repair effect. Produce cytokines which call neutrophils, monocytes, lymphocytes. Help to immunosuppress and control inflammatory environment. But chronically they can be unhelpful in encourage long-term inflammation.

Answer 183

MSCs have an inherent tropism for tumours. Nature of interaction is incompletely understood. Model states that tumour creates a microenvironment, interacting with the ECM, producing metalloproteinase to activate receptor PAR1. Proteinase chops receptor, activating it causing MSC migration.

Answer 184

Gliomas were induced in mice by a human line U87 xenografts in the right frontal lobe. hMScs labelled in red were injected into the carotid artery. Within 7 days they found that cells were in the tumour so had migrated. Wanted to check that the cells weren’t mechanically trapped so injected cells on the contralateral side, still showed that they engrafted and migrated to tumour. Wanted to check that it wasn’t one specific cell line so used U251 which was also targeted by MSCs. This tropism is unique to hMSCs because when other cells were transplanted, did not locate to tumour. To understand how the tumour attracted MSCs, they used transwell culture dishes whereby MSCs are bathed and positioned on the transwell insert with a lower component containing U87 tumour cells conditional media or control separated with a microporous membrane. Test to see if MSCs would migrate in the presence of different factors. EGF and PDGF may be important.

Answer 185

To test if hMSCs could be used to deliver an anti-tumour agent, MSCs were genetically modified with adenovirus to secrete IFN-B. Particularly useful for brain tumours which are very difficult to access locally because of the blood brain barrier. Only the IFN-B secreted by the MSC delivered intracranially increased survival. Promising prospects.

Answer 186

TRAIL = TNF-related apoptosis inducing ligand. Causes apoptosis of target cells through the extrinsic apoptosis pathway. Member of the TNF superfamily which includes the tumour necrosis factor and Fas ligand. Able to selectively induce apoptosis in transformed cells not in normal cells, making it a promising candidate for tumour therapy.

Answer 187

Loebinger et al (2009) transduced hMSCs with a TRAIL-GFP lentivirus that can be induced with doxycycline (switch on or off). Found that tumour cells die when co-cultured with TRAIL-expressing MSCs induced with doxycycline. In vivo: mice engrafted with tumour cells in the lungs showed that doxycycline treatment reduced tumour volume and activity. All untreated animals and MSC treated animals without doxycycline had metastasis. MSCs plus Doxycycline were free from tumours. Now taken to clinical trials.

Answer 188

- Use of embryonic stem cells causes concerns because they require human embryos as their source. - Embryonic stem cells originally derived from embryos that were by-products of IVF, donated to research. - UK now allows embryos to be grown for research for 14 days. - If the couple divorces, widowed or wants to use the embryos with another partner, who has claim over them? - Induced pluripotent stem cells produced from adult cells means there is no need for embryo-derived stem cells, but are they as good?

Answer 189

Moral principles that govern a person’s behaviour or conducting of an activity.

Answer 190

- Aristotle: based on virtues (justice, charity, generosity). Lead to benefit individual and others. - Kant: duty is central to morality. Humans are bound to obey the categorical imperative to respect. - Utilitarianism: guiding principle of conduct should be the greatest happiness or benefit the greatest number.

Answer 191

The duty to prevent or alleviate suffering AND the duty to respect the value of human life. To create stem cells we must destroy an early embryo and therefore a potential human life. But stem cell therapies could be used to treat patients and alleviate suffering for many people.

Answer 192

- The embryo has full moral status from fertilisation onwards - There is a cut-off point at 14 days after fertilisation - The embryo has increasing status as it develops - The embryo has no moral status at all

Answer 193

The human fertilisation and embryology authority regulate by means of a licensing system any research or treatment which involves the creation, keeping and use of human embryos outside the body or the storage or donation of human eggs and sperm.

Answer 194

The human fertilisation and embryology authority determine policies and review treatment and research license applications. Broad range of expertise. Chair, deputy chair and at least half of the HFEA members are not doctors or scientists.

Answer 195

WHO says the world population is rapidly aging, by 2050 22% of the population will be over 60. Increase in age-related degenerative diseases. The challenge is to prolong heath into old-age.

Answer 196

Tissue regeneration declines with age (loss of elasticity, wound healing is slower, bone becomes more brittle) and since tissue regeneration is fuelled by stem cells it makes sense to hypothesise that there is a loss of stem cells or a loss of stem cell function in aging. Proving a decline in stem cell function is very difficult because it is difficult to purify stem cells from most tissues because their markers are similar to progenitors. Lacking adequate models of functionality.

Answer 197

We can easily purify HSCs and assess their function in vitro/in vivio. Peripheral blood is accessible and there are good models for functionality i.e. serial transplantation in irradiated mice.

Answer 198

As a whole, there is decreased immunity, anaemia and increased incidence of bone marrow failure (implications for bone marrow transplantation). Cellularly the number of HSCs actually increases with age however the cells are not very functional, they have a skewed differentiation potential. HSCs from aged mice have an increase in myeloid gene expression (skewed balance – less erythroid and lymphoid). Clonal expansion of HSCs is biased towards myeloid fate.

Answer 199

-Telomere shortening -Energy metabolism and ROS -DNA damage All lead to senescence and decline in function.

Answer 200

Telomeres are repetitive DNA sequences at the ends of chromosomes. Chromsomes form a physical loop at the telomere (t-loop). Every replicative cycle telomeres are shortened and over time this is recognised as DNA damage initiating senescence. Telomerase extends the telomere length but normal human cells have low levels of telomerase (increased in cancer cells). Initially fast, then slow and then speeds up after 70 years.

Answer 201

7 year old patient received a bone marrow transplant from a 60/70 year old individual. Initially showed good engraftment however patient suffered from bone marrow loss within 2 years. Found that the HSCs transplanted had a very short telomere length.

Answer 202

Reactive molecules and free radicals derived from oxygen. Byproducts of oxidative phosphorylation e.g superoxide and hydroxyl radical can damage mitochondrial and nuclear DNA, proteins and lipids. ROS levels increase with age. Antioxidants increase the replicative potential of HSCs upon serial transplantation. FOXO TFs regulate expression of antioxidant enzymes. Conditional deletion of FOXO1, FOXO3 and FOXO4 in mice increases ROS levels and depletes HSCs and neural SCs.

Answer 203

DNA is exposed to spontaneous mutations (in replication/mitosis) and extrinsic mutations (UV light/radiation). DNA has a high capacity for repair but some DNA damage can persist. DNA damage affects stem cell function with age. HSCs from mice with mutations in genes involved in DNA damage response (FANCD1, MSH2, ERCC1). KO mice for DNA repair or telomere metabolism genes leads to premature decline in regenerative function of HSCs.

Answer 204

DNA damage and telomere shortening activate tumour suppressor mechanisms (senescence and apoptosis). ROS can also induce senescence (HSCs lacking FOXO). Senescence markers (b-galactosidase and p16INK4a) increase with aging in many tissues. Senescence requires activation of retinoblastoma and p53 proteins.

Answer 205

Chakkalakal et al (2012) Muscle-specifc stem cells (satellite cells) are located along the myofibribre close to ECM and plasma membrane. They receive signals to keep themselves quiescent (growth factors from fibroblasts). Satellite cells regenerate muscle in response to injury. Aged muscles have reduced regenerative ability and reduced numbers of satellite cells. Exposure of aged satellite cells to serum from young mice rejuvenates the satellite cell response. Therefore there IS a role for the niche in stem cell aging in satellite cells.

Answer 206

Chakkalakal et al. (2012) Transgenic mouse model TetO-H2B-GFP will label all of the chromatin in the cells green. TetO is an inducible promoter meaning that it will only become active and green when the mouse is fed Doxycylin. This enables the pulse-chase method. Cells are labelled with GFP and Doxycyclin is added causing all the cells to fluoresce (pulse). Then you remove Doxycyclin and the cells divide, diluting the fluorescence (chase). Cells that don't divide will retain the signal (label retaining cells) [QUIESCENT], those that do are non-label retaining cells [PROLIFERATIVE]. When separated by FACS they found that aged satellite cells spend less time in quiescent state (more dilution, more proliferation).

Answer 207

In vivo: Transplantation of satellite cells from TetO-H2B-GFP adults into adult or aged mice. There were less aged satellite cells over time in the aged host than in the adult host and they were gradually lost. In vitro: aged cells lost self-renewal and gained differentiation markers showing that they were unable to replenish pool of stem cells.

Answer 208

FGF family of ligands known to affect satellite cell mitogenic activity. RT-qPCR array of FGF ligands in aged and adult purified single muscle fibres. There was a major difference in the level of expression of FGF2 between adult and aged muscle (more mRNA for FGF2 in aged mice). After in situ hybridisation, adult muscles were shown to have almost no FGF2 expression whereas anti-FGF2 was clearly showing FGF2 in the aged tissue. Expressed in muscle but not satellite cells themselves. This showed that FGF2 is a signal from the niche not the satellite cells themselves. Sprouty1 is downstream of FGF2 (neg reg). In young mice: low niche FGF2 and high sprouty1 levels act to maintain the quiescent state. In aged mice: high niche FGF2 downregulates sprouty1 and contributes to sporadic cycling and cell loss. Unknown where FGF2 comes from and why levels change.

Answer 209

- Stochastic model: all tumour cells are equipotent but some will proliferate and increase tumour growth, others will differentiate. It's down to the environment which cells will do what. - Cancer stem cell model: tumours are hierarchically organised like normal tissues. Only certain cells contribute to long term tumour growth and progenitors have limited growth potential. Cancer stem cells are biologically distinct populations which we can isolate and investigate.

Answer 210

Tumours that can show the presence of stem cells. Germ cell tumours containing differentiated cells from all three germ layers and embryonal carcinoma cells. EC cells will recapitulate the whole tumour if injected into a new mouse so are stem cells. Kleinsmith and Pierce (1964)

Answer 211

Can identify stem cells based on their immunophenotype and then purified using FACS, and then their behaviour can be tested using behaviour assays. In AML, there is an increase in the number of myeloid cells and there are only rare cells within the leukemic clone which have the ability to intiate AML growth after transplant into immunodeficient mice (leukemic stem cells).They have a specific immunophenotype CD34+ CD38-. When transplanted expect to see 1 in 40-100 initiating tumour. LSCs have an important role in the high relapse rate of AML patients.

Answer 212

In breast cancer cells they have identified the immunophenotype CD44+ CD24-. Once they had been purified they need to show that they can intiaite the tumour and that the tumour will show similar heterogeneity to the original tumour. Done for a number of other solid tumours: brain cancer, melanoma, lung, gastruc, pancreatic cancer. Controversy for others still exists - difficult to purify and lack of functional assays.

Answer 213

Primary tumour contains a mixture of cells, some may be stem cells, tumour-propagating or more differentiated/committed progenitors. If you take all the populations and place them in a immunodeficient mouse, you can see the tumour-propagating potential of each population. If you are able to graft JUST the tumour-propagating population the frequency of tumours should be much higher. It may be necessary to do serial transplantation because cancer stem cells and progenitor cells may appear similar but serial transplantation will display variation in their TTP. It may be the case that some cells can but don't form a tumour due to lack of stromal cells/niche/permissive environment.

Answer 214

Traditional therapies focus on eliminating highly proliferative cells to reduce the bulk of the tumour. But if stem cells are still present there will be a relapse. Therapies targeting cancer stem cells to eliminate them or differentiate them need to be developed. Combination therapy targeting proliferative AND stem cells could be promising.

Answer 215

- Identifying cancer stem cell markers/obtaining pure population of cancer stem cells - Which/how many cells contribute to tumour growth and progression? - The link between genetic heterogeneity and tumour potential.

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