Introduction to stem cells
Lecture 1: Introduction to Stem Cells
Extra reading: Julia Polak
Stem cells renew through mitotic division and differentiate
Totipotent- differentiate into all tissues (e.g. fertilised egg)
There is no indication into what they will differentiate into
Pluripotent= differentiate into three germ layers
Multipotent= self renew to only closely related family of cells
Oligopotent= only closely related cell types e.g. myeloid and lymphoid cells
Unipotent= differentiate into 1 cell type
Drug development with- non human cell models, tumour derived cell models (genetically different), cadaveric cells
Therapy and regenerative medicine- not as far as reality
Divide stem cells- most potent to least potent, embryonic (derived from the inner cell mass of the blastocyst), fetal, perinatal, adult
They give medication to hyper ovulate to harvest remaining embryonic cells because of pluripotency (infinite self renewal), very controversial (religion
Fetal stem cells- not used much as its from terminated pregnancies, limited potency (multipoint), they are grouped with adult stem cells, was used fro Batten disease (neuronal ceroid lipofusion), missing lips enzymes
Stem Cells company in Palo Alto, attempted, very limited Stem cells, first ever FDA approved trials
ReNeuron first patient trials in the UK for stroke patients
In China, worked on Neuro-Regeneration, repair and functional, recovery
Perinatal Stem Cells= amniotic fluid and fetal epithelial cells (that flaked off felt skin)- multipoint stem cells in amniotic fluid, characteristics of mesenchymal stem cells
Amniocentesis, not common as there is a 1% of miscarriage
Placenta (amnion, villi, blood) usually used for eye injuries as a source of stem cells
Villi- 5-7 week old placenta, more potency the younger it is
Pluristem, MSC like cells, termed adherent stroll (ASC) cells, derived from placenta and used allogeneically
ASCs differ from MSCs in unique and proprietary way Pluristem expands these cells
Blood- CD34+ cells, isolated from umbilical cord
Umbilical cord (blood and Wharton Jelly)
How much does this cost (usually 1-4k)
However there are insufficient nucleated cells, 60% collected, could help a person 40-50kg
CHORI company> sickle cell anaemia and thalassemia
They do sibling cord blood banking preserved at no cost for a child that may need transfusion
Wharton Jelly- fibroblast and macrophages, within the umbilical cord, easy to get them and lots of cells for MSCs
Adult stem cells have limited potency, oligopotent/unipotent, however the are autologous so no immunorejection, cannot divide indefinitely
Lecture 2: MSCs
MSCs, not really stem cells but stromal cells
understand MSC and what they can do immunomodulatory characteristics, principles of clinical applications
Stem cells can divide and renew and differentiate
Multipotent can differentiate into some cell types
Plastic adherent cellular fraction of many organs contains stratal progenitor cells that can give rise to colonies of fibroblastic morphology
Stromal cells are not dormant but move around and divide
This cellular subset CFU-F totally or partially corresponds to a proposed multipoint progenitor cell population, able to differentiate into adipocytes, chondrocytes and ostocyte
Most commonly source of MSC: Whartons jelly umbilical cord, fat adipose tissue, bone marrow
Bone marrow also hematopoetic stem cells, aspirate bone marrow you have both and then separate in culture, puncture over iliac crest (very painful, not easy to get volunteers)
Older you get the slower the MSC renewal and differentiation, not as many good cells, best from children
Can take from adipose tissue, aspirate fat, isolate the cells from fat and some have MSC characteristics
Can take from Wharton jelly
They look like fibroblasts but no specific marker to identify the two they have certain surface markers but not specific
Pre clinical studies find potential with MSC, ameliorate some disease
Cell-replacements therapy, e.g. cartilage replacement for arthritis, cell empowerment (secrete growth factors and help to ameliorate the disease)
Many studies have attempted to exploit potential of MSCs, cannot replace any tissue, rate of MSC engraftment, will never become functional liver cells for examples but may have some markers
Inject the MSC and notice improvement but when they look where the MSCs are they couldn’t find it, difficult to correlate the difference of MSC
MSC work the best when you use them for treatment of inflammatory disease, stimulate the MSC to secrete cytokines to modulate immune response
Therapeutic effects depend on their ability to regulate inflammation, not tissue replacement
As part of cell empowerment promote tissue repair (secrete growth factors and help neighbouring cells, trophic cells, chemokine and regulate inflammatory response
They can promote tissue regeneration but not by engrafting but by secreting different factors
Modulate immune response, prevent over production of inflammatory t cells, promote normal t cell production
We know about MSC for 50 years, first reported in 1970s, immunosuppressive characteristics
MSCs affects both innate and adaptive immunity, inflammatory cytokines will induce them for anti inflammatory growth factors
The amounts and kinds of inflammatory cytokines cary during the initiation and progression of the disease
What is the effective dose, half million and 5 million per kg of the body, not yet been optimised
New approaches; iPSC-derived MSC, extracellular vesicles
Cynata doing it, huge source of MSC and employ in different therapies, harder to harvest from umbilical cord and chasing donors use for indefinite time
Extracellular vesicles: not cells but what they secrete, with pro inflammatory cytokines, collect what they secrete and inject in the body instead of the MSC, used will exosomes (CSF, blood, urine) used as cargo, exosome based vaccines and anti-tumour
Get from adipose tissue, isolate ADSC (MSC)> cell culture> stimulate cell and use exosomes, drug delivery
Biogenesis of each subtype of EV, microvesicles, exosomes, apoptotic bodies (result of disintegrate of apoptotic cells)
Potential mechanisms, growth factors, used in inflammation, angiogenesis, cell proliferation, ECM/scar tissue remodelling, in vitro you can titrate and get better response with exposures than MSC injected in the bodies
Number of trials using MSCs is rapidly increasing, 7x more in 10 years, clinical trials=humans, pre-clinical=animals
Most of clinical trials are not designed to prove hard-core scientific evidence of clear benefits for the patients, just about injecting cells in body
Knee arthritis x-ray, MSC differentiated into chon,ost, adipocytes, can we inject cell in and repair cartilage, may do a little something but will never repair cartilage
Decrease symptoms but never rebuild the cartilage, hard to measure evidence/not clear
Does not prevent clinic trying to sell this to patients, many clinics offering this treatment, 1000-12000 USD, marketed for profit
Limited potential of differentiation, only the three types of cells, not replacement of tissue but can secrete factors to reduce inflammation, new tech to work better and make them more available but it is only the beginning
Lecture 3: hESCs- Human Embryonic Stem Cells
Type of Pluripotent Stem cell
understand the origin of hESC, their derivation and potential use
Able to discuss advantages and disadvantages for use in clinic
And compare them with other SC types
Early stages of development:
fertilisation>single cell into zygote> divide by 2 to blastocyst by day 5: 32 cells
Only one sperm cell makes it through zona pellucida
Travel through the fallopian tube
When they reach uterus> start to hatch and expand
Day 7 implantation occurs cells secrete proteins
Part of problem- baby embryo in the uterine wall
Can be compared to tumour invasion, similar mechanism and very quick
Placenta connects to blood vessel
In vitro, give hormones to stimulate ovulation, collect egg and mix with sperm and then development and culture blastocyst in incubator, some embryo transferred and frozen
Fertilise 8 eggs, you get 6 perfect blastocysts, not transfer all 6 and transfer 1 and freeze the rest
Either discard spare embryos or donate them for research, supernumerary embryos, NHS covers storage cost up to 10 years
Some ethical arguments, they would be discarded anyways, better donated for research so they can be used instead of discarded
How they are derived:
hESC derived from inner cell mass, clump of the cells, give rise to body and ball around is placenta
Use laser to take pipettes with vacuum and fold in between under microscope and snap it
What is left is the inner cell mass
Then transfer into cell culture dish
Not sure for best conditions, giving the hESCs feeder cells (fibroblast are mitotically activated but won’t divide, secrete growth factors to help the SC survive to continue to grow)
Day four is initial outbreak, trophoblast cells around them, carpet of the feeder cells
Secrete proteases to attach to uterine wall, kills the cells around
Day 6 colony getting bigger and less big cells around, remove them
No more trophectic cells secreting proteases
2 weeks embryonic stem cell extensive line
Derived from blastocyst, 5-7 days development and inner cell mass
how do we know they are hESC, they are markers for pluripotent SC, OCT3/4,NANO, phosphatase activity present during development stage
Few things to check, pluripotent, differentiate into three germ layers, mesoderm and ectoderm, few weeks later we check the markers
Check in vivo, can divide infinitely in the right conditions
All three germ layers, put in immunocompromised mice, start to grow and differentiate into germ layers, teratomas, remove and check the clear distinction between mouse tissue and tumour
Proof of three germ layers to prove potency
Next problem; stability, check resolution of karyotyping, check how deep there is a mutation, e.g. chromosome number 13 about 100 megabits (100km)
Conventional karyotyping, notice is chromosome bases are missing, not notice
Go even further, one base pair is the size of 1mm, not expenisive but so much data so not easily analysable
Sequencing and different approaches to genome sequencing can give rise to different resolutions
isolate inner cell mass, destroy the egg, ethical issue
Can we bypass al this? Yes its possible not to destroy egg but how?
Blastomere extraction from the human embryo, has to be monogenic, linked to sickle cell, Huntingtons
Certain stage you remove one cell> multiply genome, amplify gene and using PCR to determine which mutation is present
Check which embryo is viable to transfer to the woman
What if you remove one cell and let embryo grow and use it to make embryonic stem cells
Technology to bypass ethical issues and was done, derived from single cell
Why are they used in clinical uses:
to use them you have to differentiate, have to know which factors to use, improving over time
To use cells in clinical setting, cannot derive from standard lab, cleanliness/contamination
All clinical grade cell lines derived from specific conditions, not much special equipment, easy but need specific environment
Once you have clinical grade line that is safe, differentiate into cell types to cure certain diseases
First test in animal (pre-clinical) and then if they work you go to humans
1998 first, clinical trials in 2010
2015 more trials
Where they are happening: UK, Europe, France, Israel, Brazil, Japan, US so not everywhere
first clinical trial, Geron’s phase 1 (California) for spinal cord injury, first you have to differentiate them into specific cell type, neuro-progenitor cells
Maybe get spinal cord to regenerate
Derived motorneurons, worked in mice (immunocompromised rat)
Published data in 2005
Had to get approval from regulatory body (FDA), to make sure patients are safe and not harmed, very detailed and long process, lot of money and time to collect data (21000 pages)
Took four years to get approved, and 2009 they got approved
Was then put on hold for another year because of problem in one study
First patient treated in Atlanta in 2010
Has to happen between 7-14 days after treatment
Look for safety and efficacy
Treat 1 patient and see what happening and if after a month nothing happens another patient can be treated
Within a year 2011 they presented data with no side effects, 1 month later they stopped the study> because of money
They didn’t get it to work, doubt the approach and value of companies stock nosedive, they had to close the trial and fired the entire team to focus on something else
The fired team gathered together they bought all the patents and started to re-trial and were more careful this time
Problem to prove safety not enough cells were injected, proved no tumour growth so they increase the injection of cells
1 million-20 million cells injected> ended with positive results, have to find the right balance for dosage, does not work immediately, difficult for investors to understand it
Second clinical trial called ACT
Published in 2004 that ESCs they can differentiate to retinal pigment epithelium, outmost layer of the eye
Age related diseases, Startgardt disease and we have no therapy for them to prevent blindness
Macular degeneration, blind over next 10 years
Repeated with 18 different Embryonic stem cell lines, tried to cure the rat, kind of positive rat 70% of vision back, within one year they got approval, accessibility to therapy side made it easier
2011 they treated the first person, 77 year old woman in LA and it kind of worked, started clinical trial in London on 2012 first time in Europe
Cell line was derived from a single cell and yes it worked, 2019 there was 32 clinical trials, 24/47 were for eye diseases
Don't know when availability will be for everyone, reduce costs, NHS not covering
They started slowly declining and increase with induce pluripotent stem cell trials
Lecture 4: iPSCs- Induced Pluripotent Stem Cells
Understand reprogramming
Able to discuss advantages and disadvantages for use in clinic
Understand the different of iPSC and other stem cells
1988: ESC lines derived from human Blastocysts
2006: iPSC lines derived from human somatic cells, Shinya Yamanaka, Nobel prize in Physiology or Medicine after 6 years of discovery
Dolly the sheep- Clone using techniques from ESC development, using 4 genes and the use of retroviruses from somatic cells
Konrad Hochadliger, Rudolf Janeich and Shinya Yamanaka
Cells have to be reprogrammed, iPSC revolution has started
Cellular preprogramming: ESC chromatin is globally decondensed (open). Open chromatin is transcriptionally active. Upon differentiation chromatin become more condensed and transcriptional inactive
Reprogramming re-opens chromatin, making it more active
Chromatin re-opening during reprogramming may not always be complete and thus leaves an epigenetic memory of the original cell type
Open-leaves transcription of all gene potential
Oct3/4, Sox2, Klf4,Myc, iPS reprogramming factors from fibroblast
Different ways to reprogram, retrovirus, lentivirus (Integrating methods), micro RNA, Proteins (non-integrating methods), using today combinations of mRNA and micro RNA, has highest efficiency
Timeline of different reprogramming methods, lentivirus from day 60 to expand cells, with mRNA it is now in 2 weeks with no chromosomal damage
Over time the technology is advancing, mRNA has zero footprint, low efficiency are validated for only one cell type, now it is a lot more easy
Fibroblast add the microRNA and mRNA and starts to change, visible by day 14 and day 20 it is finished
Day 14 normal fibroblasts, day 16 cell becomes smaller and group together by day 18
iPSC Line validation, with the same markers, three germ layers
Characteristics compared to embryonic stem cells, high proliferation, availability, spontaneous differentiation, capacity to produce diverse cell types, same as embryonic but different to MSC or adult stem cells
Applications of iPSC cells, use them to model disease and test different drugs, Neural disease you can take skin biopsy, reprogram into iPSC, differentiate to neuron’s and understand the pathology and also what you can actually test new compounds to get rid of traits and toxicity tests for new drugs
E.g. iPSC in Modelling Cardiac disease e.g. arrhythmia, use of propanol to reduce arrhythmia
e.g. iPSC in modeling neural disease, similar to autism related (RTT, IGF1
Cell therapy using iPSCs, 2007 used to cure sickle cell anaemia in mouse model, generated from autologous skin, restore the expression of the mutated gene it can be cured
Took cells from mouse> iPSC> corrected mutation using differentiation and put it back into the same mouse and it works, normal cells replaced diseased cells
Directed differentiation of pluripotent stem cells, same issue like embryonic hard to know how to get there to correct differentiation
As time passed more and more technology availability, CDI reprogram to make iPSC and sell different cell types, iPSC from different diseases, typical human biology, and representing specific disease states cells,
iPSC-based therapy, autologous iPSC cell-based therapy, they have potential and they are free of ethical issues attached to hESC, and no immunorejection as you take from the person itself
However- after few years they discovered it is expensive, takes a long time to establish iPSC lines, validate and differentiate into target tissue, after time cell divide and gather numerous mutation e.g. skin, starting from not pristine model with a number of mutations
Personalised therapy would not work, they discovered now that it is not going to work
Solution: allogeneic transplantation from HLA matching donors, human leukocyte antigen, e.g. blood type but with HLA, many more genes, not as simple to find combination
HLA homozygous donor, 2 genes, transfer to more people
Clinical iPSC bank in Japan, the government decided to invest 1.1 billion dollars to push regenerative therapies in Japan, try to find HLA homozygous to give to more and more people, enough lines to treat 32% of the population
How can you find the people that have the homozygous HLA, selection of healthy super-donors
Very homogenous population, 170 different lines covers 90/80% of the Japanese population, 75 lines
However in UK or USA is different, not homogenous and the strategy would not work
Screening for HLA Haplotypes in cord blood banks, database and select proper genotype, otherwise would need to screen 100,000 to just get top 10 lines
How to reduce expenses, homozygosity, and at least 3 HLA loci, A,B,DR, blood type O, Clean medical history and malignancies
They went to cord blood banking , avoiding accumulation of somatic mutation over the lifespan, younger the cleaner the genome
Is this strategy introduce a bias- socioeconomic status, privileged population, current likelihood of an American or Canadian patient finding an HLA match ranges from 32 to 88 percent depending on the persons ethnic background with ethnic minorities at the low end of the range
Japan has the second lowest gap between rich and poor among developed countries, homogeneity helps to develop iPSC
Additional quality control, exam sequencing to prevent dangerous mutations, it is expensive, done with QC Workflow, there are also other tests needed to validate the quality and safety of the lines before clinical uses
How do we bypass the costs of this? Then therapy will not be available for everyone
Genome editing and universal donor done with CRISPR, we can engineer cells using unedited pluripotent cell, remove HLA molecules from cell surface and becomes pluripotent universal donor cell, was discovered a few years ago
HLA Class 1 and HLA class 2, remove micro globulin, HLA-e most common, already in clinical trials and is the future
Clinical trials was with macular degeneration because worked with Embryonic stem cells, Masayo Takahashi in 2010
iPSC-derived AMD treatment (Acute Macular Degeneration)
Autologous iPSC-derived RPE cells> cells can mutate so start to get away from autologous, 2014, everything else was allogeneic
Number of companies started to use iPSC cells, differentiation into mesenchymal/stromal stem cells, uniform population and the same source, using those in therapy> Cynata
Fate therapeutics, differentiating into INK and using in immoncologist to treat different tumors
Manufacturing iPSC cells, has to be in a very controlled environment and high skills, 2015 was first clinical trials and then they started to grow, similar to hESC, mostly in eye diseases
iPSC based therapy standardisation, how to characterise cells and make it cheaper to make available for everyone else, will take many years
Dont know how long those cells will stay there, macular degeneration is genetic, cause the cells to die, put new cells but you didn’t change the environment> lots of unanswered questions
Lecture 5: Ethical aspects of stem cell research and cellular therapies
identify and assess the most relevant ethical and political issues with stem cell research
Understand the science is not devoid of political and ethical values
Assess the public policy approaches to regulate hESC research
Assess media reports and websites on cellular therapy
Ethics Hype Vs. Real, speculation, exaggerated and not a priority
e.g. designer babies, IVF- more embryos to ensure fertilisation, not regulated by government, impossible and exaggerated, not one gene that determines traits and instead combinations, not possible in UK because of regulations, e.g. 1 embryo
But is it real? The controversial embryo tests the promise a healthier baby, some polymorphisms can be associated with some diseases, sold to general public, performance of polygenic risk cores in screening, prediction and risk stratification…
Some people and scientific agree but may not be important
Embryonic stem cell research and ethics, regulations and selling cells to consumers, genetic mutations
hESC first derived in 1988 beginning of new field, a lot of debates around bioethics, it is the mark of an educated mind to be able to entertain thought without accepting it- Aristotle
hESC results embryo destruction or one opposes embryonic stem cell research and accepts the benefits will be foregone; moral pluralism in liberal democracies, co-existing view, cultures, political beliefs and religious beliefs
Reasons to support, freedom of research, therapeutics benefits, improving IVF technology
Opposing hESC; not destroy embryos, principles of subsidiarity use alternate, slippery slope concerns (small thing will be a chain reaction to a bigger event, usually negative effect)
Moral status of the embryo: competing views, sanctity of life (full moral status), potentiality view (gradualist view, time and event relevant moral status), no moral status view (not a problem but an obstacle)
Who is right? Proposed solution is a compromise, can we force one moral view on those who do not share it, not impose their opinion, hard to find view to make everyone happy
Middle ground approaches: discarded-created distinction, impermissible to derive and use stem cells from embryos solely for the purpose of stem cell research, critique is the inconsistency of surplus embryos that won’t be used> no significant moral difference
The use-derivation distinction- cannot use embryos to derive embryonic stem cell line, use what is readily available
No matter the middle ground there will always be problem with consistency and we did not solve the problem, but public policy view accepts
Countries have different regulations and opinions, no one trying to change their opinions, UK, Spain, France and Sweden, regulated but possible to do research, but Germany and Italy it is banned, can import stem cell lines, Lithuania is all prohibited
How can the differences of opinions be explained, religions Islam, Bible and Talmud
Pathos (Passion), Globus (Logic) and Ethos (Trust) - Aristotle
Pathos- the battle of suffering using snowflake babies by President Bush
Ronald Reagan- wanted to use embryonic stem cells, Parkinson’s
Search for ethical nation (Germany), completely banned but import from other countries, guilt from the past
Logos- refers to reason, general teachings in christianity vs jewish, cannot “play god”, positive attitude for technology and science
How collective identities can influence regulatory approaches e.g. UK, science= common sense, positive, Japan= risk of the collective body so science and technology are embraced, Germany= made mistakes so should not rush and impose regulations
How to cell stem cell therapies to consumers, lot of regulations but why? Blind to be cured with stem cells vs breast enlargements, people who do not know the facts they fall for it
Media hype mislead general public e.g. Germany Cell Center, claimed to cure macular degeneration for 10,545 euros, we still do not have the answer, autologous stem cell therapy so government cannot tell what to do with body, 1 person died so closed company
Moved to Jordan and renamed to MEDRA, 2010, recruiting in the US but do injections in the Dominican Republic and then banned them, changed name to Stem Cell of America, claimed to cure heart, kidney, liver and lung disease
Now can do Parkinson’s, ALS and Autism
William C Rader, lost license so could not practice, charging 25,000 dollars for one treatment
Hotspot cities in the US are in West of US (LA)
Stem cells adipose and bone marrow for orthopaedic treatments like arthritis
2021, 5x more clinics for stem cell research
Advertisement for fake stem cell therapy, new policy on ads for speculative and experimental medical treatments, be careful of wording
Ethics of genetic manipulation of embryos in SC research- prize using CRISPR for gene editing, make immune against HIV, discovered by chance, mutation of HIV receptor in 2018
WHO said no more gene edited babies but years later the countries re-butted for example in Russia
Lecture 6: Regulatory Science, the challenge for advanced therapies
advanced therapies: gene therapies, somatic cell therapy, tissue engineered components e.g. macular degradation, epithelium on Brooke’s membrane
Difficulties in regulation of therapies, less successful, things that shouldnt be happening are happening, e.g. blindness from stem cell therapy, died as consequence of therapy, broke out in tumours and died a month after, came into ER with stroke> stem cells in skull for baldness
What’s in the tube? How’s it working? Unlicensed therapies The ‘equipoise problem’. Conflict of Interest and informed consent
What are the therapies that is being injected, problems in manufacturing cell therapies, arises from the way cells are > problems
Work in animals vs work in human, cells are not clinical grade in the lab in a very small scale, work out expansion program (enough cells for clinical trial) and cells change
Stem cells change, don’s sit still, how do you know if its the same cell that was useful, cells pick up mutations in culture
Asterias: spinal cord injury therapy, cells trying to produce were oligodendrocyte, were indeed these cells, analysed expanded culture, cytometry were positive for nesting and NG2 (only 50%), weren’t carrying the marker that was needed
e.g. Human pluripotent stem cell mutations that arise, analyse cell lines, whole series developed mutations in p53 protein, Onco gene, mutations are dangerous ad puzzling
Don't know what to do, do we inject? Didn’t know what the mutations did, never gave them the cells
How’s it actually working, pathway establishes; mode of action (don’t understand when the potency was being lost)> potency assay (how potent it is per unit, allows to compare with different materials)> release of assays
E.g. neural stem cell therapy to treat stroke, animals with stroke and cells showed major improvement, clinical grade human line, but… based off stem cell, replacing lost tissue, worked in animals, but when brain sectioned there weren’t any stem cells after 4 weeks, no idea what mode of action is, immunomodulation, angiogenesis, neurotrophic mechanisms…
The science gap, stem cell can do much more than what they actually do, very responsive cells by doing something, doing other things than making other cells, don’t know how to develop functional assays
Unlicensed therapies; therapies that went wrong
The way stem cell therapies are marketed, in UK largely MHRA, protected because of socialised medicine, route in isn’t through medical practice but through the internet> leads to strange places
A lot of clinics, applied online and got cell therapy, not approved by the FDA
Enormous market for these therapies
Why isn’t the FDA doing anything about it? Very political, always in public eye, Scott Gottleib, dishonest and exploitation, a lot of opposition, suppress innovation, only have right to deal with federal business not in one state not go through borders, loophole, what constitutes a medicinal product and practice of medicine
Direct to consumer marketing in why the therapies are proliferating
e.g. child with leukaemia, are those cells a therapeutic product are they a medicine… NO, autologous graft, vs fat cells and changed into sc into brain> practice of medicine
Ability to practice medicine doesn’t always go through medicinal products, e.g. take out gall bladder
Autologous therapy, has to be homologues, same job as before e.g. blood cells from bone marrow vs fat tissue cells
Minimally manipulated, blood cells not manipulated vs the fat cells, when is manipulation minimal?
Everyone is different, active arm of trial vs personalised medicine
What should happen? Randomised control trial of technology (CarT cells trial for leukemia, gene therapies for cancer, find mutants in the gene causing cancer, everyone different, personalised gene therapy vector to recognise the tumour, how do we regulate that
The ‘hospital exemption’- terminally ill patient but ideas proposed, special license for the unlicensed medicine, one-off
Randomised Trial problem; the equipoise problem, is placebo controlled trial ethical? Deliberately withheld therapeutic medicine, lot of resistance, duty as doctor is to treat individual, how to get over this> if medicine in question is in equipoise if the medicine works or not is genuinely uncertain, don’t know if it will harm or treat
Who decides if its in equipoise? Ask doctors if its ethical, almost 100% vs patients 47% and 17% for Parkinson’s wouldn’t join trial
Not in equipoise in case of patients, lot of hype around stem cells, why people going through unlicensed clinics
To be ethical; benefit analysis, informed consent, trial should be conclusive or informative, avoid conflict of interest
Informed consent and conflict of interest; companies making money from product have little incentive to perform clinical trial (90% fail)
A clinical trial does not remove the conflict of interest; treatment of autism using stem cells, unethical… clinical trials failed, despite the failure duke is still selling the therapy
Stem cells might have failed in clinical trial but she needs to promote the company, very real problem and the regulation is extremely challenging, needed to resolve
Jack Price- the future of brain repair
Lecture 7: Stem Cell Niche
Describe the concept of the stem cell niche
Discuss specific examples of signals exchanged between stem cells and their niche
Describe how niche can be manipulated in vitro and in vivo
Cancer progression is modulated in the niche
How targeting the niche could benefit therapeutically
Niche: all of the physical, chemical and biological factors a species needs to survive, stay healthy and reproduce in an ecosystem> stem cell biology
History and the concept of stem cell niche: microenvironment
e.g. differences between intestinal villi, lung, aorta…
Work of James Till and Ernest McCulloch in 1960s experimentally confirmed the
development of blood cells from a single precursor hematopoietic stem cell (HSC). Raymond Schofield in 1978 has proposed the concept of a “niche” as a specialized
microenvironment housing stem cells.
He hypothesized that the ability of stem cells to self-renew and retain their identity
depends on the environment provided by neighboring (non-HSC) cells.
He also proposed that the progeny of a stem cell will undergo differentiation unless they
can occupy a similar 'niche’
Experimental evidence for stem cell niche was first provided by invertebrate models (Drosophila germ line stem cells)
Common features of SC niche: intrinsic state of the niche, microenvironmental stimuli, remodelling of the niche by stem cells by secreting different factors
What Factors: cells getting in touch with other cells, junctions in epithelia or neurons, signallng, chemokine, cytokines, different growth factors secreted and they talk to each other, influence each other, e.g. inflammatory microenvironment, stimulate mesenchymal SC to secrete factors to calm inflammation die, if you know you can design therapies, inflammation and scarring, macrophages, t-cells, secrete factors
Extracellular matrix, group of molecules secreted by cells, network secreted by cells e.g. collagen network, elastin… assemble into intricate network they keep growth factors and release at different times, different from niche to niche
Physical factors such as sheer forces, sheer stress, string of the blood and endure that, bone and cartilage and pressure when walking
Cartilage is just ECM and chondrocytes, cannot engineer only ear, because of physical force, same cartilage in knees, first time exposed to physical pressure so they will just crush
Important how the physical factors influencing all cells
e.g. hypoxia and metabolism, different blood of oxygen in different parts of the body, cells exposed to less oxygen than what you are breathing, if you put them in different environment, different genes will be transcribed will be something else
e.g. metabolism, there are different pathways, cancer metabolism, cells exposed to different environments they react differently
Niche interactions: haematopoetic stem cells, e.g. bone marrow, other factors influencing how they behave, secreted factors chemokine, Wnt
Intestinal stem cell niche, different cell types in the intestinal membrane and enzymes, secreted different factor
Epidermal stem cell niche: Secreted factors Wnt, EGF, cell-cell contact is differentiation, ECM is polarity, physical factors is elasticity and topography
Manipulation of stem cell niche: can we manipulate the niche in vitro/vivo and learn it in stem cell biology and use to treat diseases, number of different ways to approach this: topography, nature of matrix… number of ways
e.g. patterning ligands on substrates
Cancer stem cell niche: healthy tissue provides signals for proliferation, healthy balance but when cancer starts to form, cells that change are secreting different factors and signals to keep under control lessens, at the beginning niche can keep in control and after it cannot
Two different hypotheses; hierarchical model of carcinogenesis, stochastic model of carcinogenesis, read to understand how the expansion of the cancer can happen
Cancer stem cells can self-renew and independently divide and go somewhere else, their plasticity and dormancy correlates with their therapeutics resistance, stronger than the niche and modulate all the factors present in the niche
Stem cell niche as therapeutic target, blood, skin, muscle, bone
During transplant treat patient to mobilise the stem cells , move from bone marrow to blood and isolate them, modulating the niche will allow higher amount of stem cell in the blood
Skin: trying to make wound healing and support there are different strategies, silicon and collagen sponge so blood vessels can bind and restore the tissue, artificially made, be able to support wound healing, e.g. necrotic tissue in the arm
Muscle: fibrous tissue, again ECM, the network surrounding the matrix, they can guide the blood vessels to help heal the wounds, growth factors, elasticity, lateral force transmission, alignment of cells
Satelite cells are unipotent and need some guidance that retain the ability to regenerate muscle
How can this be done, providing ECM from urinary bladder and submucosa, no cells taken and usually from pig and then all thats left is ECM, looks like net
Put it to heal the muscle and get mobility to the patient
Bones too, bone tissue scaffolds, immobilise to help bone to heal, making different scaffolds, add cells and molecules in between the broken part of the bone to promote healing
e.g. Physiomechanical strategies, collagen, growth factors, hydrogels etc, bone grafts in dentistry
Stem cells receive signals from the niche and communicate with their surrounding, they modulate niches and niche modular the cells so understanding how it works allows you to play with the niche and stem cell differentiation and used for therapeutic purposes, use an example and be able to go into depth
Lecture 7: Genetic Modifications in Stem Cell Research
what is genetic modification?-is the direct manipulation of a cell/organism’s genetic material, adding genes, correcting genes, mutating genes
Stem cells used for genetic modification, ESC, adult SC, iPSC, Universal donor and cancer SC, self renewal property distinguishes from most other terminally differentiated stem cells
Self renew- pluripotent and proliferative capacity
Sc can be propagated by asymmetric differentiation allowing a stem cell to generate one stem cell and one differentiated progenitor cell
ESC- pluripotent to circulatory system, nervous system and immune system, ectoderm, endoderm and mesoderm
NANOG, OCT4 and SOX2 which ensure the maintenance of pluripotency and suppression of genes that lead to differentiation, factors
Adult Stem cells, in the blood give rise to different progenitors> T cells, beta cells
Tiny population of undifferentiated reserve
Possess different tissue markers, organogenesis, tissue homeostasis, carcinogenesis
iPSC= Yamanaka factors, OCT3/4, CMYS, SOX2, and KLF4, reprogramming factors
High proliferations, unlimited source of specific cells
Universal donor iPSC= address the allogeneic rejection problem by manipulating the HLA expression in stem cells
Single cell line differentiated into multiple therapeutic cell products
How can we do this, editing the genes HLA factors, b2m, rfxank genes and add suicide systems
Cancer stem cells= asymmetric differentiation, self-renewal, origin is not clear, genetic alterations, mutations in progenitor cells
Surface molecule targeting therapy
Challenges for CSC, need to learn more of how to modify them
Homologous recombination, new genetic material needs to be integrated, Homologous recombination, nucleotides sequences are exchanged between two similar or identical molecular of DNA< crossing over during meiosis, double strand break repair
How to apply it, deriving sequences that are homolog, host DNA harbouring a marker gene at a specific locus> low efficiency
Generation of knockout mice> take part of gene if you want to interact, induce a mutation, stop codon use to prevent from making the protein, look for desired phenotype
Generation of marker for lineage tracing, allows us to follow cell fate decisions of stem/progenitor cells, populations and their descendants within a living organism, based on visualisation of the cells lineage via a time and spatially controlled reporter gene expression in stem/progenitor cells and passed on to cell progeny
Lineage tracing of hair follicle bulge stem cells, are gene under the control of a promoter specific for the cell population of interest, GFP expression occurs in hair follicle bulge stem cells as soon as promote becomes activated during the cell migration, permanent genetic modification is passed on to the cell progeny, independent for are expression, and facilitates tracing of the cell lineage, in cells that are not in the bulge cell lineage, the are gene will not be expressed and therefore the reporter gene will not be expressed
Integrating viruses, virus integrated into DNA of host cell, then DNA replicates as the cell genome after cell division, integrated viral DNA is duplicated and usually distributed equally to the two cells that result
Life cycle of retroviridae
Which one is more efficient, problems access, non-diving, ex vivo procedure only, ideal cell target: pluripotent, self- regenerating stem cells
Bone marrow, skin, skeletal muscle, adipose
Viral vectors are integrating into the host genome, retrovirus, lentivirus, remove viral structures and replace with trans gene or other gene of interest
How can this be improved; genome editing; how does it work, molecular scissors, homing devices, identify where to cut guided RNS, template, correction
Using site directed nucleases- SDNs
Diverse application of CRSPR, double stranded DNA break, HDR and NHEJ
Cas9 scissors, after double stranded break
NHEJ and HDR occurs after the DSB in genomic DNA, homology directed repair
Gene editing using base editors, doesn’t require double stranded break so higher efficiency
ABE and ADE, cytosine and adenine
Can also manipulate the epigenome
What is gene therapy, gene therapy is define as a set of strategies that modify the expression of an individuals genes or that correct abnormal genes, for example gene addition/ augmentation, introduction of an additional functioning copy of the defective gene. Used to treat loss of function conditions
Recessive dystrophic epidermolysis bullosa, collagen 7 deficiency
Autologous epidermal grafts
Keratinocytes were used using the viruses
12 months showed remarkable effect
How can we improve the long lasting effect cos after 3 months nearly no cells found
Scalable tissue stem cell manufacturing through CRSPR-edited autologous IPS cell therapy- using iPS cells
No animal- derive components in the cutter system, not matrigel (xenon-free)
Gene editing using plasmid and protein based methods
58% biallelic correction of homozygous mutation
Keratinocytes under feeder free conditions
After 2 months, retain the properties and will longer be expressed and detected
iPSC cells have better regenerative potential
Functional restoration of collagen 7 after base editing
Lecture 8: Haematopoiesis
production of blood cells, in bone marrow
Different types of blood cells
I healthy adult accelerates upon certain physiological stressors, infection
Decrease with age
Unchecked growth of HSCs and or immature blood cells result in leukaemia
In utero, production of blood cells begins in embryonic sac, 14-19
Second trimester in the liver 3-7 months
Bone marrow is primary site of blood making from 7 months gestational age
Bone marrow, prom birth- primary site
Wishing medullary cavity of long and axial bones, in children bone marrow present in all bones
Replaced by fatty blob as we get older, proportion to age, 70 years old, 30% cells
Periosteum outer surface of bone, cortical bone outer layer of compact bone, trabecular bone-inner layer of spongy bone, endosteal layer is at interface of bone and BM
Bone marrow aspiration- unexplained low blood cell count, abnormal cell populations, degree of cellularity, trephine needle to posterior superior iliac spine, cytogenetic, flow cytometry, trephine biopsy, Perl’s stain- iron less common
HSC- rare 1 per million nucleated cells, highly proliferative, ability to self-renew, multi potency, mostly dormant protects from genomic damage, capable of 50 cell divisions, lymphoid and myeloid cells decreases, less myeloid SC
Origins of HSC, McCullough and Till, experiments in mice, irradiated mice, small nodules grew on the spleens of mice, differentiated into the 3 primary blood lineages, red, white, platelets
Confirmation of clonality, sublethal irradiation, definitive evidence that the spleen colony forming unit were derived from single clonogenic SC
HSC hierarchy, common lymphoid progenitor and myeloid, demonstrates HSC have greater proliferation but lowest frequency
Balance between self renewal, symmetric cell division and asymmetric division
Once they become active because of cytokines, get expansion, asymmetric division is a maintenance source, differentiation
Markers:CD34, transmembrane phosphoglycoprotein, used to identify, deletion and enrichment for HSC bone marrow transplant, CD38- population, however expressed by multiple other non HP cel type, potential to misidentify
Stem cell niche: specialised environment, will lose steaminess, Schofield, unable to function in spleen compared to marrow, histological studies, very closely related to endosteum, impotence of osteoblasts importance in regulating bone marrow HSC, marker for true stem cell niche
Ability of cytokines to mobilise HSC into circulation, closely related to blood vessels, sinusoidal blood vessels
SLAM family, signalling lymphocyte activation molecule, CD150, CD48 and CD244 more accurate markers to identify HSC
Growth Factors, cytokines for differentiation of HSC, IL3, IL4 and GMCSF, GCSF and IL5
5% myeloblasts in bone marrow, common myeloid progenitor> promyelocytes, nuclei no longer visible and then neutrophils
Lymphoid differentiation, common lymphoid progenitor> b and t cells, natural killer cells and plasma cells
Constituents of blood; plasma (yellow), red blood cells, erythrocytes (white blood cells and platelets)
Erythrpoeisis, formation of red blood cells, sacs of haemoglobin, 10^12 new blood cells each days, 120 day life span, 50% of the blood volume (hematocrit)
Hemoglobin synthesis, mitochondria, iron from transferrin, and global chains by ribosomes
Red blood cells progenitors, GATA2, important transcription factor, FOG1, BFU and CFU, differentiation for haemoglobin
Pro, Bas, Early pol, Late Ort, Reit and RBC, 16 divisions in total to Retic
EPO- regulated by erythropoietin, hypoxia detected by kidneys, formation of EPO which signals differentiation, EPO useful in high levels of high blood cells, reduced is cancer bone marrow and increased renal cell carcinoma
Granulocytes- most frequent white blood cell, phagocytes innate immune system, polymorphs because of multi lobed nucleus, 6-24 hours life span, neutropenia- severe risk of infection and sepsis
Granulopoeisis- no further division from myelocytes
Granulocytes- basophils and Eosinophils, basic vs acidic dyes, infrequent B, Histamine release B> allergy, E defence against parasites, and fibrin clots E
Acute vs chronic myeloid leukemia, example of aberrant proliferation of HSC, undifferentiated vs differentiated, driver cells
Lymphopoeisis, 20-30 per cent of leukocyte population, very dense nucleus, cannot differentiate between t and b cells, 80% t cells, cell mediated immunity, B cels natural killer cells, ALL and CLL for malignant transformation
Monocytes- phagocytic cells, convoluted nucleus , migrate into connective tissue, differentiate into macrophage and possess lysosomes, skin langerhans cells
Thrombopoeisis- formation of platelets, form platelet plug for vessel damage, tissue repair, inflammation and immunity, lifespan 7-10 days, Anucleate cells
Megakaryocytic, present in bone marrow, precursor to platelets, terminal maturation processes, endomitosis, direct result of cytoplasmic restructuring
Regulated by TPO, produced in the liver, binds to receptor cMPL, platelets reduced TPO increased and opposites
Recent studies megakaryocyte biased HSM, directly into it
Aplastic anaemia- autoimmune condition, low formation of blood cells, HSC transplantation, Eltrombopag and romiplostim, activators of megakaryocytic, established in TPP
HSC transplantation- curative therapy replacement of SC from donor, conditioning from chemo and radio, cells infused 24 hours after conditioning, engraftment 1-3 weeks post infusion, systemic immunosuppression, GvL vs GvHD, control potential leukaemia
Sources of HSC, peripheral blood SC, administration of GCSF, disrupts adhesion to bone marrow storm, autologous SC transplant, Myeloma and Lymphoma, higher dose of chemotherapy, Allogeneic
Alternative SC source, HLA matched donor-sibling,HLA matched unrelated donor
Cord blood transplantation, high proliferative SC and progenitor, fewer activated T cells, delayed or failed engraftment
Lecture 9: Cord-Blood Banking
Anthony Nolan- cord blood collection
What is umbilical cord blood, 3 vessels, vein and 2 arterioles
Allogenic use of HLA matched to donor
First 1988 Paris
Used to treat malignant and non malignant, Leukemias
8-16% of all transplant per year
Anthony Nolan, equity access, 42 million donors worldwide, 830000 cord blood units globally, CBB diverse inventories, better matched grafts and improving overall prognosis, 82% caucasian, new HLA or ethnic combinations can be collected on day one
Filling the gaps: 4000 procedures per annum, crd blood important resource for those who cannot find adult donor for urgent cases, ethnicities who dont have a match is where cord blood comes in
Aphorises increase and Marrow decreases
Advantages; HLA mismatches are better tolerated, less stringent match requirements, less chronic graft vs host disease, better quality of life and relapse control, inventory frozen and ready to go
Disadvantages; limited cell dose may cause longer neutrophil engraftments, acute GvHD rates are high, second donation from same donor is not possible, CBU selection os more complicated, concerns around quality and potency of product, cost if double transplant is required
As a result each potential transplant must be assessed in order to weigh out the particular graft type in order to determine if the use of cord blood may be appropriate or not
Built in 2007 in Nottingham Trent Uni campus
2008 obtained HTA license, 2019 more involved in cell and gene therapies
Anthony Nolan 5 fixed collection sites at major UL maternity hospitals, KCL hospital
Model of collection; public cord blood bank, maternity staff, collections Sunday to Thursday
Private Banking? Not guaranteed that it will be used, 50% chance of being partial match, 25% not match at all
Model of collection: Consent, Collection (cannulate cord itself to push blood through, placenta is elevated and cord is clean to minimise bacterial and fungal contamination), Transported daily, Reception (received in the cord blood bank, check paper integrity age of unit, temperature report retrieved from data loggers), Daily workflow (placed in incubation to check for bacteria, quarantine)
Initial QC, lab is responsible for determining the quality of unit received, less than 30 hours, determined by a number of factory, cell content, sc content, flow cytometer> viability of unit
Flow cytometry, antibodies to stain the cells, to detect and plot anything that comes off
Processins, AN uses Sepal 2 system, good MNC and TNC yield, good stem recovery, wash, cooling system, 40 minutes process
Adding DMSO- added using syringe over 15 minutes, mixed, temperature cooling, temperature trace of the whole process is produced> final bag has smaller segments for further testing
Controlled rate freezing- slowly and steadily to minimise damage
Cryogenic storage, -190 degrees celsius minimise TWE after CRF freezing, quarantine and clinical/ searchable
Post process quality criteria- safety, identity, purity and potency
Viability of CD45, CD34, negative for IDM tests and microbiological tests, 2 or 3-4 attached segments
Full validation process
AN perform CB searches on request from Transplant centres in the UK, for international via bone marrow donors worldwide
Once a TC expresses interest in a unit either preliminary or final report will be issues depending whether cord blood is reserved for patient
CBU shipment, shipped in dry shipper at -196 degrees, extra tests like DNA for chimerism analysis or plasma for further testing
Unit is temperature monitored and transferred to storage tanks once at TC
Ensures no transient warming events occurred during the transit
Shipped a week prior incase of any transport issues, allows patient to undergo condition once the graft has safely arrived
First international transplant 2012 to Australia, mixed race aboriginal Philippine patient
Has remained at high rate during the start of pandemic, cord blood supplies material for cell therapy, provides starting material for advanced therapies, more sustainable business model
Quality not quantity
Driving quality; HTA licensing, inspections every 2 years, Net Cord FACT accreditation, inspect cord blood banks against more rigorous standard in the world
How does quality affect the patient, best possible chance for patient
UR single CBT Vs Double CBT
Wide spectrum of confidence level for use of CBU in UK, internationally imported at high cost, 20% are selected by UK TCs
Advise on CBU is marbling or stratification indicating poor mixing during DMSO addition, possible leaks, thawing and infusion to aid cord infusion
Gravity feed manifolds attached to giving sets, pall wash bags can be provided too if required, remote trouble shooting advice assistance for CBU with bag defects
Also used for fresh and frozen cord blood, fresh cord tissue, fresh amnion, direct dispatch from collection sites, IDM screening, research tissue banks
Lecture 10: Hippocampal stem cell neurogenesis
Niche
What is it
Molecular control and regulation
Functionality
Modulate
Production of new neurons by SC
Differs from pre-natal neurogenesis
NSC exist along the entire Neuroaxis
Oligodendrocyte, Astrocyte, Neuron, but does not replenish after injury in adults
Does adult neurogenesis occur?
1965> evidence of new neurons in hippocampus of adult rats
Sub ventricular zone (SVZ) of lateral ventricles, sub granular zone of dentate gyrae (DG)
SVZ, come from, this niche> olfactory bulb in rodents
Don’t migrate far, SC proliferate, migrate and differentiate to stem cells, integrate circuits
Rodents; 4-6 weeks from NSC to neurons
GFAP expressed by NSC, Sox2> NSC marker
Neuroblast> granule neuron
Human brain DG> Eunice Friesen, Carbon dating on neurons C14 levels postmortem
If person have the neurons they were born with> flat line> showing neurogenesis throughout life
Not the olfactory bulb, SVZ> striadon, DG, not clear what for at the moment
Slow neurogenesis, 700 new neurons in adult humans in hippocampus per day
Don’t need that many to be functional, not there to replenish dead cells
Why is hippocampus- privilege, why is DG and SVG and not spinal cord
transplantation> non-neurogenic SC (spinal cord) extract, half into DG or SVZ and half back in spinal cord
SC nothing happened> no neurons
DG/SVZ we can make neurons> environment is very important
No neurons in spinal cord as not the niche
What’s in the niche= hippocampus (very vascularised), NSC sheeting the blood vessels, Astrocytes, feeder cells…
Astrocytes modulating the Stem cell niche, co-culture them neonatal and adult hippocampus astrocytes
Neuronal marker MAP2 and GFAP for Astrocytes
BrdU> drug picked up by dividing cells
Astrocyte surrounding neural stem cells
Molecular control neurogenesis; FGF2> fibroblast Growth F, EGF> epidermal GF, CCg> co-factors of FGF
mRNA, transcription factor, epigenetic factor
Hundreds of factors that modulate neurogenesis
Wnt signalling> astrocyte secreting Wnt, expressed sub granular layer, neuronal development, DAPI> strain the cells, DCX double coating? Wnt pushing to become neurons, In-Vitro> used of lentivirus express GFP, put into D6, dominant- D6> blocks Wnt signalling, over-express Wnt> less increase than in Vivo, sore point in 1 place
Why are they important> number not that impressive/ can newborn neurons have speciality?> learning and memory/ spatial learning, how to block neurogenesis> block cell proliferating, any cancer drug stops neurogenesis> brain fog/ depression (read paper on Wnt)
Functions- pattern separation differentiate between memory, better pattern separation younger
Too much neurogenesis> fine balance> disturb established context memory, don’t remember when you were young
Neurogenesis importat for mood and depression, decrease neurogenesis, prozac neurogenesis
Neurodegenerative disorders, mood cognitive decline e.g. Parkinson’s, Alzheimer’s, depend what stage
Change in neurogenesis occurs at very early stage
After progression stimulating neural stem cells could prevent of slow down Alzheimer’s disease
Modulate neurogenesis, more stem cells producing neurons rather than more
Measure neurogenesis using bio-marker> stable never convert to Alzheimer’s disease, predicting up to 3.5 years before diagnosed
How to modulate neurogenesis= take into account functionality
learning> increase neurogenesis, spatial learning
stress> decrease neurogenesis, chronic
Sleep deprivation> decrease neurogenesis, chronicity
intercourse> increase neurogenesis
Exercise if enjoyed too, e.g. rats on treadmill
older> decrease
Occurring throughout life but the rate differs
BrdU injection, marker for cell proliferation
Morris veterans task, milky white water, swim to platform> spatial awareness after 6 days
Barbaric experiment> young-young, young-old, circulatory system
Diet for neurogenesis> intermittent fasting,5/2 diet, not fasting but re-feeding increases neurogenesis
Omega-3, Curcumin, vitamin deficiency, food texture, soft diet 20-30% decrease
Diet modulates cognition and memory, caffeine exception, coffee metabolites> not good for neurogenesis
Lecture 11: Stem cells in Bone repair
basic bone biology: bone cells, skeletal development, how bone is formed, 206 bones
Bone remodelling throughout life, physiology, mechanical forces, bone quality
Why regenerate bone?
Endogenous/exogenous stem cells in maintenance and repair of bone
Regenerative/ therapeutic strategies
Developmental origins, cranial neural crest (head bones), mesoderm (rest of bones)- right type of lineage
Osteoclasts, blasts and chondrocytes, cells and molecules, cell to cell molecules, disease
Bone- living tissue, continuously turning over, support, muscle attachment, houses haematopoeisis, reservoir for calcium, phosphate, homeostasis
Bone cells keep the niche alive for haematpoesis
Osteoblasts- cells that form bone
Bone is type 1 collagen, cartilage is type 2 collagen
They are polarised cells (nucleus stuck on one end of cell, unmineralised matrix and mineralised left to right
As they deposit mineral they become engulfed in the bone matrix
One they get engulfed they called osteocytes
Osteoclasts- bone eating/resorption, multinucleate, mediated by integrins, dissolves through acid demineralise- how it functions, complicated cell
Where do they come from- MSCs/SSC- makes all menechymal cells, bones, muscle, tissue, for bone factors is Runx2 and Osx, Sox9,5,6 for cartilage, then marker genes to express lineage, Dmp1 and Sost
Osteoclast made from blood- HSCs, macrophage lineage (phagocytic cells), colony stimulating factors (come from stroma, CSFs) M-CSF and RANKL
Ossification (bone formation), Intramembranous and endochodral
Intramembranous- all bones in our head, bone formed in membrane, direct formation of OB precursors, formation of periosteum, differentiation to osteoblasts, matrix deposition, vascularisation, continued growth and remodelling
Endochondral- vertebrae, from mesenchyme and blood vessel, hypertrophic chondrocytes, osteoblasts, blood vessel, proliferating chondrocytes, epiphyseal cartilage (end of bone), secondary ossification
^ bone remodelling- osteoblasts and osteoclasts
Why remodel- calcium homeostasis, maintaining blood calcium hypo (too little)
Skeletal homeostasis/ bone mass, too much bone osteosclerosis
Adaptation/mechanical forces
How does it happen, cycle format- quiescence, osteoclasts reabsorb bone (differentiate, activate, recruitment), reversal phase with osteoblast (recruitment and proliferation) 10% per year
Basic multicellular unit (BMU)- 6 months, skeleton is constantly remodelling, what you eat is what you gain
Osteoblasts and clasts, make factors, bidirectional regulation, RANKL, soluble mediators and growth factors
Regulated at systemic level (hormones, vitamins…) growth factors (TGF, Osteoprotegerin)
Local factors (Prostaglandins) and transcription factors (Runx2)
Osteoporosis, estrogen deficiency (menopause)
Therapy: antiresorpative
Other regulators of bone mass/remodelling- body builders have higher bone mass, mechanical loading, 30% more bone in serving arm in tennis
Different bones- differential regulation by loads, compressions, tension, torsion
Julius Wolff- functional adaptation 1892, bones can adapt to mechanical loads, implications for tissue regeneration
Bone laid down along lines of maximal compressive or tensile stress, external forces determine remodelling, osteocytes respond to mechanical loads, the mechanosensors of bone
High bone mass- sclerosteosis, mutation in sclerostin (loss of function mutation) sclerotin expression in osteocytes
Sclerositn- an osteocyte specific gene, relation to mechanical loading, secretion decreases with mechanical loading, increased bone formation Wnt signalling, anti sclerostin antibody used as therapy to regenerate bone
Why regenerate- what kind of bone, stem cell base, fractures, ow do you heal fractures, endochondral ossification recapitulated, therapy- recruitment/ activation of correct precursor
Maxillofacial injury, development intramembranous ossification recapitulated
Distraction osteogenesis, happens often with tumour, make a cut and pull bone apart to form, it is intramembranous
Cartilage regeneration- growth plate responsible for bone growth, dwarfism (growth plate mutation)
Osteoarthritis, cartilage repair
Stem cell sources, ESCs and iPSCs, Pluripotent stem cells
Coax the intrisic stem cells, endogenous adult SC (+growth factors)
Autologous therapy, culture in lab
Markers of MSCs- not good yet in therapies, not very good in vivo markers
MSC: bone marrow, osteogenic, chondrogenic, adipogenic
Skeletal Stem cell (discovered 5 years ago)
Adipose derived SC in calvarial repair
Which stem cells pluripotent stem cells (ESC and iPSC)
Tissue regeneration, PSC cell differentiate, models embryonic development, well-defined, controlled, efficient isolation of specific progenitors, unlimited source of undifferentiated/ differentiated cell types
Gastrulation: 3 germ layes, in vitro differentiation, inject extra-uterine site, teratocarcinoma
Manipulation of ES cells, ES cell differentiation in vitro recapitulates development, gastrulation in a dish
ectoderm> brachyury+ primitive streak> mesoderm > osteoclasts, chondrocytes and osteoblasts
Chondrocyte lineages- how does the embryo make different types of cartilage, can lessons from development be applies to in vitro PSC differentiation approaches
BMPs drive chondrogenesis and endochondral ossification, extracted from demineralised bone matrix, unique ability to induce entire cascade of bone formation via endochondral ossification
GDF5 drives joint development and articular chondrocytes
PSCs can be directed to derive two separate chondrocyte lineages, important implications for cell-based therapies
Lecture 12: Skin stem cells in skin renewal and ageing
know how to describe skin, structure and function, how niche determines skin stem cells, role in skin homeostasis, skin ageing, biological consequences of stem cell dysfunction
Largest organ, every 27 days new skin slower as you age, top 18-23 layers made of dead cells
Epidermis (thin, few hundred micrometres), dermis and hypodermis (adipose tissue)
An organ with a divers array of cell types, sweat glands, blood vessels
Function: to protect (UV. Radiation, dehydration, bacteria, traumas), regulate body temperature (excrete water, ammonia, vitamin D), sensation (feel heat, pain, pressure, touch, nerve cells important physical and emotional)
Epidermis, corner, granulosum, spinosum, basale, dermis, protection (corneocytes)
Integrity of SC is essential for survival of terrestrial life
SC as a brick and mortar model, structural proteins of corneocytes, structural lipids
Functional lipid barrier require lipid synthesis, secretion and post-secretory processing
Atopic dermatitis or eczema allergens can enter, without brick and mortar model
Homeostasis: different locations of stem cells in skin, not only in epidermis, each of them have a different role, wound healing, neural crest processors
Stem cell niche, refers to microenvironment specific anatomic location, topographical cues, nutrient, complex dynamic structure
Epidermal homeostasis- going through renewal, basal layer cannot divide any more, constant turnover, speed depends on signals from dermis
Two models of epidermal regeneration: symmetric division model, asymmetric division models, 3-steps involving a TA cels, 2 steps no TA cell
Circadian rhythm in epidermal regeneration, cues from outside, UV- differentiation and proliferation (night)
Role of bone marrow in skin repair- skin distress signal, then mobilisation and recruitment of a sub-population of bone marrow cells to keep skin graft
Movement and growth- elastic recoil properties on collagens, main part of dermis
Major ECM components of dermis collagens, elastic, protogylcans and GACs
70% of skin is made from collagen, high tensile strength, not flexible, flexibility come from elastin and the ratio between the two is very important
Most abundant is collagen 1 but there are many different collagens, maintains skin structure and integrity, collagen 3 is 15%, both of them are 95%, ratio changes after wound healing
Elastin is only 4%, responsibility for ability to stretch, skin loses ability to recoil, if skin is overstretched striae (stretch marks) occur
Cleavage (tension) lines: elastin and collagen fibres orientated in some direction more than others, important in surgery, if it is parallel, less scarring and healing is faster
In foetus there are no scars, they can heal without visible trace
Pathology of the scars, changes with collagen ratios, hypertrophic or keloid scars
Proteoglycans and GAGs- name used for six different types of long linear polysaccharide chains composed of specific disaccharide cells, complex network in our skin
HA synthesis, proposed method for HA breakdown
How skin is ageing is slowly degrading
How epidermis connected to dermis, collagen 7
Network, slowly gets less abundant with age, less and we can see how it changes, wrinkles, cannot be changed easily, many factors influence, intrinsic ageing and photoaging
Both factors affect stem cells in the niche, homeostasis of the skin is changing, flattening if the epidermis and the junction, permeability becomes thinner (dry), immune cells decrease, melanocytes irregular pigmentation, Collagen not produced as much as before, elastic fibres disintegrate
Loss of mature collagen secretion of signalling enzymes, MMP expression
How can we prevent skin ageing
Billycan changes expression in different parts of the body, lower as people age, not only focus on collagen
Genome, epigenome, proteome- cells acquire mutations that change characteristics
Ageing- p16 present, controversy if it slows down stem cells in older people, ageing due to imbalance between symmetric vs asymmetric division> losing lineage specificity, progeny may not be functional, tissue intergrity and physiological function of the skin
Wnt, Notch signalling
Genotoxic stress> DNA damage
Local and systemic envrionemt
Cells escape from stringent homeostasis and arise to cancer stem cells
Are skin anti-ageing products worth of buying- NO, even simple moisturiser will the the same as the most expensive, repairing skin barrier is just moisture and lipids, the only true one is sunscreen will protect photo damage
Lecture 13: Mechanotransduction and stem cells
describe concepts of mechanotransduction and elastic modulus
Appreciate how cells sense the stiffness of their environment
Explain scientific evidence behind the theory that cells behave as tensegrity structures
Understand how stiffness affects stems cell differentiation and its implications for tissue engineering
Explain how cytoskeletal shape/spreading/tension affect stem cell lineage commitment
Describe emerging evidence that intestinal organdie maintenance and differentiation is mechanosensitive
Cells are subjected to mechanical forces
Cells apply mechanical forces
These forces play a role in cell differentiation
The cell is not a bag of water
Cell cytoskeleton: microtubules, actin filaments and intermediate filaments
Mechanotransduction: link between the sensing of mechanical cues and the subsequent cell response, cells sense mechanical forces applied to them and they generate mechanical forces to probe their environment
Bone adapts to its loading environment to maximise the load it can carry using the minimum about of material
Intrinsic versus extrinsic cues, cell differentiation
Intrinsic- cell density and shape, ECM elasticity and topography, soft vs stiff
Extrinsic- fluid flow, hydrostatic pressure, compression, tension
Feedback loop between nucleus and mechanical cues: mechanical cues> alterations in cytoskeletal tension> nucleus> cell fate, motility and behaviour
Stiffness and modulus obeys Hooke’s law, assumptions- continuum, isotropy, small deformations, linear elasticity, E= elastic modulus size-independent measure of stiffness
Elastic (Young’s) modulus of tissues; fluid blood or mucus to least stiff to bone and glass most stiff
Cells apply mechanical forces: cells generate internal forces via their cytoskeleton, feel stiffness, how do cells apply/sense force?
Integrins- integrin heterodimer
ECM- nucleus communication
Tensegrity structures, connected by continuous tensile elements, isolated components in compression
Cell attachment
Is the cell a tensegrity structure
Cytoskeleton- integrated system, stiffness increased with increasing applied stress, similar to intact tensegrity models, but different from isolated elements, speed of mechanical responses exceed soluble messenger responses by orders of magnitude
Stem cell control- speed of mechanical responses exceed soluble messenger responses by orders of magnitude, biochemical diffusion, mechanical physical displacement
Mechanotransduction and stem cells; what are the implications of these effects on stem cells
Density dependent MSC differentiation; plating density of MSC affects osteo vs adipogenic differentiation, control for cell-cell interactions
Stem cell shape- cell spreading directs hMSC lineage commitment between osteoblasts and adipocytes
Cytoskeletal effects- cytoskeletal tensile forces play a role in hMSC lineage commitment
Rho/ROCK= Rho>ROCK>tension, cell shape, soluble factors, osteoblasts, adipocyte
Stem cell commitment- mechanical cues, changes in cell shape influence lineage commitment, cell shape itself is likely a driving factor in development, in hMSC these effects are mediated by the generation of cytoskeletal tension through the RhoA/ROCK pathway
Polyacrylamide hydrogels
Focal adhesion formation: larger, more stable focal adhesions on stiff substrates, more defined stress fibres on stiff substrates
Muscle cells> differentiation, lower cell, upper cell
Matrix stiffness effects
MSC differentiation, expression of markers for neurogenic, myogenic and osteogenic differentiation correlated with tissue stiffness
Blebbistatin: small molecule inhibitor of non-muscle myosin
Alters the ability of the cell to generate contractile forces
hMSC gene expression is influenced by substate stiffness, this effect is blocked when cells are treated with blebbistatin
Cardiomyocytes maintain normal beating frequency when cultured on matrices with similar stiffness to heart tissue
Myocardial infarction
Mechanoregulation in development: hippo pathways-organ growth control
Yap/Taz in mSC mechanotransduction, YAP/TAZ loacalisation is regulated by cell shape
YAP/TAZ is regulated by ECM stiffness and requires tension of the actin cytoskeleton
Cell spreading, Stiff ECM and high contractile forces> activation of YAP and TAZ
Confined cell adhesion, soft ECM and low contractile forces> inactive YAP and TAZ
2D versus 3D culture of stem cells
Intestinal organoids
Intestinal stem cells in synthetic hydrogels, Lgr5+ stem cells proliferated poorly in soft (300Pa) hydrogels, but were well supported in stiffer hydrogels (1.3kPA)
Synthetic hydrogels for gut organoids- degradable softening hydrogels are required for organdie formation
Synthetic hydrogels for gut organoids
Local softening drives crypt formation
Mechanical cures drive crypt formation- Lgr5+ stem cells localise to curved ends of defined shapes
Epithelial patterning: tissue geometry controls organdie patterning through cell shape-mediated regulation of YAP and Notch signalling