Revision cards Flashcards
How has tissue repair and regeneration been present throughout history?
Greek mythology: Prometheus’ liver could regenerate overnight.
275AD: St. Cosme and St. Damian supposedly performed first tissue transplant.
Middle ages: Quest for regeneration through fountain of youth and philosopher’s stone.
1600/1700s: Glanwill made predications that “restoration of grey hairs to juvenility and renewing of exhausted marrow” would be possible.
1868: Ernst Haeckel first used the term ‘stem cells’ coming from ‘stammzelle’ originally used in a phylogenetic context.
1892: Boveri shows SCs are those between fertilised egg and committed germ cells.
1892: Hacker showed that cyclops embryo underwent asymmetric division.
1905: Pappenheim related hematopoiesis to stem cells.
What is a stem cell?
Cells that have the potential to generate different specialized tissue (differentiation) as well as copies of themselves (self-replication).
What are the different ways we can classify stem cells?
- Age of development (embryo or adult)
- Tissue of origin (neural/hematopoietic/umblical cord etc)
- Basis of how many cells they can produce
How can stem cells be classified according to the different cells they can produce?
- Totipotent: all cell types of the human body including thromboblast e.g. zygote
- Pluripotent: derivative from 3 germ layers e.g. embryonic stem cells
- Multipotent: different cell types from a tissue or organ e.g. neural, blood, renal etc
- Unipotent: single cell types e.g. muscle satellite cell
How does differentiation affect the rate of the cell cycle?
Stem cells in the stem cell niche have a slow cell cycle (although this is different in vitro). Once they reach a stage where they are directed to a fate and begin to differentiate (trans-amplifying cells) the speed of cell cycle begins to increase.
How can stem cells be used in a clinical context?
Allogeneic - stem cells derived from a different donor, expanded and stored in the lab before being used to treat other people. e.g. ESC, cord blood cells
Auto-transplant - stem cells to be transplanted are derived from the same patient e.g. from bone marrow, generation of iPS from skin/blood cells by adding Sox2, Oct4, Myc, Klf4
Medicinal interactions with stem cells - recruitment of endogenous stem cells from the same tissue. Use medicines to ‘awaken’ stem cells in the damaged tissues to activate them to repair it.
How can stem cells aid biomedical science?
- Excellent models to screen new drugs
- Models to study genetic conditions (specifically iPS)
- Models to combine the former two (pharmacogenomics) to look for a drug which will compensate a phenotype caused by genes
- Insight into fundamental biological problems
- Direct, potential clinical application for therapeutics
Why are the pathways involved with stem cell differentiation important?
To most, it may seem like you simply flip a switch to cause a stem cell to differentiate into a specific fate. In reality there are many pathways that affect progression.
Loebel et al (2004) said that manipulating stem cell differentiation should be based on what we know of what causes lineage decisions in early embryogenesis.
Is regenerative medicine a new concept?
No! There have been various experiments/transplantations that have been carried out over the years.
- Bone marrow transplantation (1950s)
- Corneal grafts
- Skin grafts for burns victims
- First successful kidney transplant in 1954
- First heart transplant in 1967
What are some of the key issues that need to be addressed before stem cells are used routinely therapeutically?
- Efficacy
- Safety
- Purity and controlled manufacture process
- Identification/maintenance/culture of large no. of stem cells in vitro
- Effective differentiation of stem cells in vitro
- Immunological rejection concerns
- Assessment of functionality
- Identifying and weighing up the risks
- Ethical issues
How are stem cells involved with anti-cancer therapeutics?
Most current anti-cancer treatments work to kill the cancer stem cells themselves which contribute to the rapidly expanding trans-amplifying cells that contribute to the tumour. But they identified that the cancer stem cells were giving rise to tumour progenitors as well which could avoid the anti-cancer treatment. Therefore it is crucial to develop anti-cancer drugs that will target cancer stem cells and progenitor cells.
What recent Nobel prize research has provided advances in stem cell biology?
NP 2007: Capecchi, Evans and Amithies introduced specific gene modifications in mice using embryonic stem cells.
NP 2010: Robert G. Edwards developed in vitro fertilisation.
NP 2012: Gurdon and Yamanaka discovered that mature cells can be reprogrammed to become pluripotent.
Where did the discovery of embryonic stem cells begin?
Stevens et al 1954 found that tetratomas could spontaneously develop in the gonads in stain 129 mice. These tetratomas developed multiple tissue types including hair and teeth, and many structures similar to that seen in embryonic development.
How did the characterisation of embryonic carcinoma cells lead to isolation of embryonic stem cells?
Kleinsmith and Pierce (1964) found that these embryonic carcinoma cells had multipotentcy (capacity to produce all aspects of tumour tissues).
Brinster (1974) put embryonic carcinoma cells from a dark mouse into a light mouse blastocyst to show that the cells became integrated into all tissues to form a chimera.
Evans and Martin simultaneously in 1981 identified that there must be cells in normal development that have similar properties to the embryonic carcinoma cells. They isolated embryonic stem cells from the mouse and grew them on feeder layers.
How were embryonic stem cells isolated?
Martin et al (1981) isolated the emrbyonic stem cells from the blastocyst and placed them on feeder cells. These are embryonic fibroblasts that are treated with chemicals or radiation to stop them from differentiated. They provide the signals needed for the ESCs to stay alive and undifferentiated. This allowed embryoid bodies to form.
The ESCs were tested by injecting them into another mouse blastocyst to create a chimera or implantation into an adult mouse to create a tumour.
What are the properties of embryonic stem cells?
- Derived from the inner cell mass of blastocysts
- Non-transformed, genetically fairly stable
- Indefinite proliferative potential, high amplification capacity
- Stable diploid karyotype (hES less than mES)
- Clonogenic - can grow from a single cell (harder with hES)
- Pluripotent - can generate all fetal and adult cell types in vitro and in vivo and in tetratoma/carcinoma (can’t test all of this for hES cells)
- Incorporation in chimeras (ethical issues with hES although shown in 2016)
- Germline transmission in chimeras - well established in mice (ethical issues with hES)
- Permissive to genetic manipulation
How can you genetically manipulate stem cells in an organism and then visualise gene expression?
Mouse embryonic stem cells injected into a blastocyst will incorporate into all embryonic cell types. You can genetically modify genes to make a reporter line along with a mutation, so a reporter can be used to visualise gene expression. For example, GFP, RFP, or b-galactosidase where you add a substrate to cause it to stain blue.
What on the feeder cell layer causes ESCs to be maintained as undifferentiated?
Leukaemia inhibitory factor is present on feeder layers and controls the proliferation and self-renewal of the stem cells.
LIF interacts with the LIF receptor at the plasma membrane which is associated with gp130. Once bound, LIF initiates the activation of JAK which activates STAT3 to move into the nucleus and contribute to self-renewal.
LIF also activates the Akt pathway which causes the activation of SHP-2 which activates ERK1/2. ERK1/2 inhibits STAT3 and drives proliferation and differentiation.
What happens to the ESCs in the presence or absence of LIF?
In the absence of LIF, ES cells will tend to differentiate but if LIF is present, this will force the cells to preferentially self-renew.
Does LIF work alone to act on the ESCs?
LIF alone cannot drive self-renewal, it must be in the presence of serum too. In serum-free conditions, LIF alone is insufficient to maintain pluripotency and block neural differentiation.
Ying et al 2003 showed that you need LIF and BMP4 (or BMP2) to sustain self-renewal and pluripotency. Without them you will see neural differentiation, turning on of Tuj1 and the turning off of Oct4.
How do LIF and BMP work together to sustain self-renewal?
BMPs induce Id genes via SMADs, these block entry into neural lineages. At the same time LIF via Stat3 inhibits BMPs from inducing mesoderm/endoderm. LIF and BMPs use competing actions to co-operate in combination to sustain self-renewal.
When they first isolated human embryonic stem cells what did they come across?
The first human embryonic stem cells were isolated in 1998. It was technically very difficult to do, the technologies used for mice weren’t easily used for humans. They found that there were some key differences between the nature of mouse and human ESCs as well. Mouse ESCs would grow in a domed column whereas human ESCs would grow in flat columns. Oct4, Sox2 and Nanog are present in both but some of the surface antigens are very different e.g. SSEA+ in mice, SSEA- in humans.
What are the conserved pathways in self-renewal in mice and humans?
Stat3 signalling (although works slightly differently) Nanog, Oct, Sox FGF and TGFb signalling BMPR1a microRNAs Methylation - X inactivation Cell cycle e.g. Rb tumour suppressor Igf2-H19
What are the differences in self-renewal in mice and humans?
LIFR-gp130 Requirement for activin/nodal signalling FGF signalling Cell cycle rates and cell death Rex1 (variable expression in hES) Surface antigens (SSEA and TRA)
What signals are needed to keep mouse and human embryonic stem cells renewal in vitro?
mESCs are grown in 2i (Gsk inhibitor and Erk inhibitor) and LIF.
hESCs are grown in FGF2 and Activin to remain proliferative and undifferentiated.
How are mouse and human epiblasts different?
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.
What are EpiSCs?
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.
What are the two stages of pluripotency in ESCs?
- A naive or ground state (aka inner cell mass-like)
- A primed state (aka epiblast-like)
How can we obtain naive human ESCs?
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.
How can you determine naive and primed ESCs using Oct4?
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.
Why are naive human pluripotent and embryonic stem cells important?
- 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.
Are all human embryonic stem cell lines identical to one another?
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.
What are the characteristics of ICM-like (naïve, ground) cells?
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
What are the characteristics of Epithelial epiblast–like (primed) cells?
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
How do pluripotent stem cells determine the three germ layers?
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.
How do the germ layers form in mouse embryogenesis?
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).
What is the battlefield of pluripotency?
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).
How do the DNA binding domains of the pluripotency transcription factors differ?
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.
How does Oct4 expression influence ES cell fate in vivo and in vitro?
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.
What is produced at different levels of Oct4 expression?
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.
How do Oct4 and Cdx2 interact?
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.
How does Sox2 expression influence ES cell fate in vivo and in vitro?
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.
How was Nanog discovered?
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.
How does Nanog expression influence ES cell fate in vivo and in vitro?
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.
How do Nanog, Sox2 and Oct4 interact with one another?
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.
How was heterogeneity of the stem cell compartment determined?
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.
What is FACS?
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.
Why is Nanog thought to be a rheostat?
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.
What is the landscape of differentiation?
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
Why is heterogeneity important?
Cells from different states may be primed for particular lineages. Different positions in the model will have different propensities.
How can stem cells in culture attain enhanced ability to self-renew?
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.
How can mutations that enhance self-renewal in stem cell culture be identified?
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.
What are embryoid bodies?
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.
What is Derk ten Berge (2008) discover about Wnt signalling in embryoid bodies?
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.
What are the pros and cons of performing differentiation using embryoid bodies?
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
Does the type of embryoid body have an effect on the tissue it generates?
- 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.
How can you improve the reproducibility of the embryoid bodies?
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).
What is direct differentiation?
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.
How to isolate the desired cell types in direct differentiation?
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.
How did Li et al (1998) use lineage selection to generate purify neural precursors?
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.
How has direct differentiation been used in a clinical application?
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.
How did D’Amour et al (2006) produce Pancreatic hormone-expressing endocrine cells from human embryonic stem cells?
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.
What were the problems with D’Amour et al (2006)’s protocol and how did Kroon et al (2008) improve them?
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.
How were in vitro differentiation protocols manipulated to produce pancreatic tissue in vivo?
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.
Explain an experiment that looked at whether we can use transplantation of pancreatic progenitors to treat diabetes?
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.
What is Kelly et al (2011) identify in pancreatic cells?
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).
What are cell surface antigen markers and how can they be detected?
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.
What did Osafune et al (2008) show with regard to production of pancreatic endoderm?
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.
What is the future of 3D differentiation?
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.
What is Waddington’s epigenetic lanscape model?
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.
What were John Gurdon’s experiments in frogs?
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.
How was Dolly the sheep produced?
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.
What is patient-specific stem cell therapy?
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.
Is somatic cell nuclear transfer in humans possible?
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.
How did Takahashi & Yamanaka (2006) induce pluripotent stem cells from mouse embryonic and adult fibroblasts?
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.
How were Yamanaka’s iPS cells tested?
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.
What are the Yamanaka and Thomson factors?
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.
What are the different ways of reprogramming a cell?
- 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)
What are the current methods used to deliver the factors to make iPS cells?
- 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
How have iPS cells been used to treat disease?
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.
How have iPS cells been used to model disease?
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.
What are the issues with the supply of human embryonic stem cells?
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
What are the benefits of iPS cells?
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
What do we need to know about human iPS before therapeutic use?
- Are all iPS the same?
- Develop robust and reliable differentiation protocols
- Relatice efficiency of differentiation methods
- How will future iPS be screened for quality?
What is the major issues with reprogramming and transplantation as seen in mice?
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.
What proof is there of direct differentiation?
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.
How does the ear conduct sound so you can hear?
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.
Why is deafness a problem?
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.
How were auditory stem cells isolated from fetal cochlea used to create hair cells/neurons?
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.
What is the problem with using fetal auditory stem cells?
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.
How does the ear normally develop?
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.
How can you make otic epithelial and neural progenitors from human embryonic stem cells?
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.
How could tranplantation work alongside cochlear implant?
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.
How was tranplantation of otic neural progenitors modelled in gerbils?
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.
How can we take tranplantation of otic neural progenitors to the clinic?
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).
