YR1 Lecture 1H - Stem Cells - Dr Michael O'Connor 2020 PDF

Stem Cells In development, tissue homeostasis, disease and regenerative medicine Associate Professor Michael O’Connor Head of the Regenerative Medicine Laboratory [email protected] 2 John Gurdon Shinya Yamanaka 2012 Nobel Prize in Physiology or Medicine 2012 Nobel prize in physiology or...

Stem Cells In development, tissue homeostasis, disease and regenerative medicine Associate Professor Michael O’Connor Head of the Regenerative Medicine Laboratory [email protected] 2 John Gurdon Shinya Yamanaka 2012 Nobel Prize in Physiology or Medicine 2012 Nobel prize in physiology or medicine John Gurdon (~1962) Shinya Yamanaka (~2007) 3 Overview Stem cell properties - self-renewal - differentiation capacity Stem cell types - pluripotent stem cells - tissue-restricted (‘adult’) stem cells Stem cell functions - embryonic development - tissue homeostasis - disease and treatment Emerging stem cell therapies - clinical trials vs unproven procedures - http://www.stemcellessentials.asscr.org 4 5 Learning Objectives neological & non commicable Ophthalmic ② disease studiee , By the end of this session, you should be able to: Contrast key characteristic features of pluripotent stem cells and tissue specific stem cells and provide at least 2 examples examples of each of these cells Describe the role that tissue-specific stem cells play in tissue homeostasis for at least two organ systems. Explain issues surrounding clinical application of emerging and unproven stem cell procedures 6 What is a stem cell? A stem cell is a cell that can: 1. divide MANY times, making identical copies of itself Stem cell Differentiated cell Symmetric self-renewal cell division 2. make other, specialized cell types (i.e. differentiate) db Asymmetric self-renewal Symmetric differentiation cell division cell division 7 Stem cell types and properties Pluripotent stem cells: - unlimited in vitro proliferative capacity - can make all the cells in the body: - e.g. nerves: treat spinal injury - e.g. retinal pigment epithelium: treat blindness - embryonic stem cells (from donated surplus IVF embryos) - induced pluripotent stem cells (‘reprogrammed’ somatic cells) Tissue-restricted (‘adult’) stem cells: - restricted in vitro proliferative capacity - can make only one or a few specialized cell types - blood system - intestines - ocular lens - tissue homeostasis: stem cell… transit amplifying cell… effector cell zygote (totipotent) Fertilisation ↑ = formation It all starts here… Normal stem cell biology plantation ( -- - Ectoderm (a inner all t I - Gastrulation - form man) Placenta appropriate body plan, Fertilization = zygote formation organs and (totipotent) placenta Mesoderm Endoderm - organism grow - repair injuries from T insults that occur W - throughout organism's. Lifetime - postnatal growth 8 9 Human development It is not birth, marriage, or death, but gastrulation, which is truly the most important time in your life." Lewis Wolpert (1986) Implantation and gastrulation Blastocyst (~14 days) Blastocyst (~5 days) Cell divisions (i.e., proliferation) Zygote Fertilization Oocyte 10 Stem cells and gastrulation Tissue-specific (‘adult’) stem cells Totipotent cell (will produce >10 trillion cells in the adult body) Pluripotent cell (e.g. cells of the blastocyst inner cell mass) Tissue-specific (‘adult’) stem cells 11 Normal stem cell biology Stem cells are involved in development, homeostasis, wound healing: Totipotent cells: can make any cell in the body or placenta - e.g. zygote Pluripotent cells: can make any cell in the body - in vivo: blastocyst inner cell mass cells and epiblast cells - in vitro: human embryonic stem cells and ‘reprogrammed’ somatic cells - cells derived from hESCs are currently in clinical trial - cells derived from hiPSCs are planned for clinical trial (2014?) Multipotent stem cells: can make a range of cells in an organ - e.g. haematopoietic and intestinal stem cells G - HSCs have been used clinically for >30 years Unipotent stem cells: can only make one type of cell - e.g. lens epithelial cells (eyel Pluripotent stem cells: human embryonic stem cells (hESCs) 12 Donate to other couples Discard colony IVF - Excess embryos Embryo bank (PLURIPOTENT) ↓ of see e Derive hESCs - normal - disease-specific from PGD embryos - Call types) Pros: Cons: - can differentiate to any cell type - ethical concerns (e.g. destruction of an embryo) - can produce disease-specific cells for study - clinical use may require immunosuppression to avoid - economically viable for clinical applications transplant rejection 13 Pluripotent stem cells: induced pluripotent stem cells (hiPSCs) Pros: Cons: - possibility of patient-specific stem cells - known differences to hESCs (critical???) - alternate source of disease-specific stem cells - not economically viable clinically with current - does not require embryo destruction reprogramming methods Reprogrammed cells (i.e., hiPSCs) for clinical therapy? 14 - obtain nucleated somatic cells (e.g. skin/blood/other cell) - culture to expand cell numbers ~ 2 to 4 weeks - introduce cDNA for reprogramming factors and a ‘selection’ method - e.g. transcription factors that regulate the pluripotent state - viral methods: exogenous factors randomly inserted into the genome - non-viral methods: factors inserted into genome then excised; factors not inserted ~1 day to 1 week - culture cells while ‘selecting’ for pluripotent cells (e.g. via antibiotic resistance) ~6 to 8 weeks Timeframe: - characterize resulting cells to confirm pluripotency >>3 months with no problems ~1 week (very limited characterization) - unsuitable for acute injury? (e.g. spinal injury) - generate and purify differentiated cell types of interest ~1 to 3 months (e.g. retinal pigment epithelium) 15 Comparison of stem cell properties Pluripotent stem cells: - unlimited in vitro proliferative capacity - can make all the cells in the body: - e.g. nerves: treat spinal injury - e.g. retinal pigment epithelium: treat blindness - embryonic stem cells (from donated surplus IVF embryos) - induced pluripotent stem cells (‘reprogrammed cells’) Tissue-restricted (‘adult’) stem cells: - restricted in vitro proliferative capacity (can be difficult to obtain and/or maintain) - can make only one or a few specialized cell types - blood - intestines - ocular lens - tissue homeostasis: stem cell… transit amplifying cell… effector cell - Multipotent haematopoietic stem cells (HSCs) 16 - HSCs found in: bone marrow; peripheral blood; umbilical cord blood ~1 in 100,000 bone marrow cells are HSCs: at any one time many are non-dividing - BILLIONS of new blood cells are made DAILY - the stem cell’s environment (i.e. niche) regulates the stem cell behaviour Shiozawa et al. 2008. Leukemia. 22:941-50. Multipotent haematopoietic stem cells (HSCs) ① Multipotent stem cell: (hematopoietic stem cell) Common lymphoid progenitor ② Common myeloid progenitor Transit amplifying cells: (to increase cell numbers) Effector cells: (red and white blood cells) 17 18 Multipotent haematopoietic stem cells (HSCs) # To understand molecular events to biological * Cell control processes or correct when required. tramplation may not required if a alraybe the biology welt enough. Understand molecular events to control the process when required e.g., GCSF is used to mobilise patient’s HSCs from bone marrow to peripheral blood before harvesting for bone marrow transplants Multipotent haematopoietic stem cells (HSCs) Nature Immunology 2002 Jul;3(7):687-94. G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating Abstract G-CSF induced HSC mobilization is widely used for clinical transplantation; however, the mechanism is poorly understood. We report here that G-CSF induced a reduction of the chemokine stromal cell derived factor 1 (SDF-1) and an increase in its receptor CXCR4 in the bone marrow (BM), whereas their protein expression in the blood was less affected. The gradual decrease of BM SDF-1, due mostly to its degradation by neutrophil elastase, correlated with stem cell mobilization. Elastase inhibition reduced both activities. Human and murine stem cell mobilization was inhibited by neutralizing CXCR4 or SDF-1 antibodies, demonstrating SDF-1 CXCR4 signaling in cell egress. We suggest that manipulation of SDF-1 CXCR4 interactions may be a means with which to control the navigation of progenitors between the BM and blood to improve the outcome of clinical stem cell transplantation. 19 20 Circulation Research 2013 Mar 1;112(5):816-25. An open-label dose escalation study to evaluate the safety of administration of nonviral stromal cellderived factor-1 plasmid to treat symptomatic ischemic heart failure. Abstract Rationale: Preclinical studies indicate that adult stem cells induce tissue repair by activating endogenous stem cells through the sdf-1:chemokine receptor type 4 axis. JVS-100 is a DNA plasmid encoding human stromal cell-derived factor-1. Objective: We tested in a phase 1, open-label, dose-escalation study with 12 months of follow-up in subjects with ischemic cardiomyopathy to see if JVS-100 improves clinical parameters. Methods and Results: Seventeen subjects with ischemic cardiomyopathy, New York Heart Association class III heart failure, with an ejection fraction ≤40% on stable medical therapy, were enrolled to receive 5, 15, or 30 mg of JVS-100 via endomyocardial injection. The primary end points for safety and efficacy were at 1 and 4 months, respectively. The primary safety end point was a major adverse cardiac event. Efficacy end points were change in quality of life, New York Heart Association class, 6-minute walk distance, single photon emission computed tomography, N-terminal pro-brain natruretic peptide, and echocardiography at 4 and 12 months. The primary safety end point was met. At 4 months, all of the cohorts demonstrated improvements in 6-minute walk distance, quality of life, and New York Heart Association class. Subjects in the 15- and 30-mg dose groups exhibited improvements in 6-minute walk distance (15 mg: median [range]: 41 minutes [3-61 minutes]; 30 mg: 31 minutes [22-74 minutes]) and quality of life (15 mg: -16 points [+1 to -32 points]; 30 mg: -24 points [+17 to -38 points]) over baseline. At 12 months, improvements in symptoms were maintained. Conclusions: These data highlight the importance of defining the molecular mechanisms of stem cell-based tissue repair and suggest that overexpression of sdf-1 via gene therapy is a strategy for improving heart failure symptoms in patients with ischemic cardiomyopathy. 21 Understand molecular events to control or correct biological processes when required. Cell transplantation may not always be required if you understand the biology well enough. 22 Multipotent intestinal stem cells The intestinal villus and crypt: - absorptive cells: enterocytes - secretory cells: enteroendocrine, Paneth and goblet cells - BILLIONS of new intestinal cells are made DAILY 23 Multipotent intestinal stem cells Effector cells: (absorptive and secretory cells) Transit amplifying cells: (to increase cell numbers) Multipotent stem cells: (intestinal stem cell) Stem cells in development, homeostasis and healing 24 Stem cells provide: - a source of functional cells when required - regularly dividing (e.g. gut stem cells) - irregularly dividing (e.g. some haematopoietic stem cells) Transit amplifying (progenitor) cells: - usually the first cell ‘type’ produced by stem cells - provide large bursts of cell division (over a short time-frame) to expand cell numbers - the cells produced by progenitor cells are the functional cells needed (e.g. red/white blood cells, etc) - this hierarchical arrangement may protect against cancer - fewer stem cell divisions minimizes the chance of genetic mutations during DNA replication Controlled delivery: - stem cells act in response to biological cues in the local environment (i.e. niche) - developmental cues during embryogenesis - homeostatic cues during every day life - injury cues during wound healing 25 Cancer stem cells hypothesis Cancer: a disrupted balance in tissue homeostasis (especially proliferation) Cancer stem cell hypothesis - a ‘low frequency’ (somewhat controversial) cancer stem cell is responsible for cancer growth - if true, need to target/destroy these cancer stem cells or will get cancer recurrence Quiescent and Proliferating cycling stem cells progenitor cells Cancer stem cell model Progenitor cell model Effector cells - no reocurance 26 Applications for stem cell research Help better understand development of human tissues: - what genes or proteins are involved in normal tissue development? - what genes or proteins are involved in congenital defects, disease or injury? - provide leads for finding new cures Aid new drug development: - make specialized cell types for: - drug screening for new candidate drugs - laboratory based toxicity testing to minimize toxicity testing in animals Use in regenerative medicine: - stimulate cells and tissues in the patient to re-grow or repair - grow replacement cells or tissue in the lab for transplantation (‘tissue engineering’) 27 / Novel drug discovery maintain pluripotent stem onl e differentiate them ↳ the to effective are of in the lab produce cells that interest to study your. Initiate repair mechanisms Fix or kill diseased/injured cells - high-throughput chemical screening: identify drug candidates for further testing - test toxicity of candidate new drugs: more accurate than animal testing 28 Regenerative medicine therapies ↳ Phamacetical patient & tissue Injured or diseased organ supplied that san be then that would to the stimulate. resident stem Biologics/Pills differentiate move expand Tissue-specific stem cell Applications: spinal injury, macular degeneration, stroke, diabetes, cartilage injury, cataract, Alzheimers, Parkinsons, etc, etc the 29 Tissue engineering therapies d Take a human pluripoint Transplant d Human pluripotent stem cell - stee cell neurom- - Expand Differentiate in vitro into: in vitro - neurons - retinal pigment epithelium - etc, etc Applications: spinal injury, macular degeneration, stroke, diabetes, cartilage injury, cataract, Alzheimers, Parkinsons, etc, etc 30 WSU Regenerative Medicine Human pluripotent stem cells Generate/purify LECs (can make 106-108) Use these to make >100,000 functional human micro-lenses in vitro… ABC News 19 March 2018 Investigating cataract formation, anti-cataract drugs, and lens regeneration as an improved treatment for child cataract Challenges for developing new stem cell therapies 1. Research-scale technical challenges - make/purify/proliferate effector cells ↳ - investigate molecular mechanisms of diseases 2. Moving from lab-scale to production-scale - do small-scale techniques work at large-scale? - quality assessment and quality control of cells to transplant? 3. Overcome tissue rejection - are a patient’s own tissue-restricted stem cells suitable? (e.g. leukemia) - need a wide range of human embryonic stem cell lines? - are patient-specific ‘reprogrammed cells’ feasible - current high cost of generating reprogrammed cells - time required (>3 months): suitability for acute injuries? (e.g. spinal damage) 4. Develop appropriate clinical transplantation protocols - what cell type is needed (e.g. mature cell or a progenitor cell)? - when to transplant (e.g. early- or late-stage disease)? - how many cells needed? - delivery method: cell suspension or adhered to a matrix? (what matrix composition?) - avoid tumour formation 31 32 Current stem cell-based therapies Current most common diseases treated with stem cells: - leukemias (treated with hematopoietic stem cells) ~ - - lymphomas (treated with hematopoietic stem cells) - - [corneal burns (limbal stem cells)] > harvest - umbilical blood-variablity Bore manm * I harvest Example of hematopoietic stem cell therapy: - harvest hematopoietic stem cells (HSCs) to use in treatment - HSCs used to be taken from bone marrow - HSCs now ‘mobilized’ into the blood stream (via growth factors) & harvested intravenously - HSCs from umbilical cord blood also being used - kill malignant cells via chemotherapy and/or radiotherapy - transplant (inject) new hematopoietic stem cells - autologous transplant (patient’s own cells used) - allogenic transplant (donor cells used) - syngeneic transplant (cells donated by identical twin) - provide immunosuppression when necessary to protect against rejection 33 Other stem cell ‘treatments’ ~$100 per 60 mL www.stemcellfacecream.com New Scientist, 3 August 2009 Stem cell tourism – patients paying for treatment at illegal "guerrilla" clinics – continues to be a lucrative racket. Police in Hungary last week arrested individuals they suspect of running an illegal stem cell treatment clinic in Budapest. Reuters reported the police saying that the treatments were unproven, based on stem cells taken from embryos or aborted fetuses, and cost as much as $25,000 per person... 34 Stem cell tourism BBC News, 18 February 2009 A boy treated with foetal stem cells for a rare genetic disease has developed benign tumours, raising questions about the therapy's safety. The boy, now 17, received the stem cells in 2001 at a Moscow hospital and four years later scans showed brain and spinal tumours, PLoS Medicine reports. Israeli doctors removed the abnormal growth from his spine and tests suggest it sprouted from the stem cells... Donor-Derived Brain Tumor Following Neural Stem Cell Transplantation in an Ataxia Telangiectasia Patient. Amariglio et al. 2009. PLoS Medicine. 6(2):e1000029 Conclusions: This is the first report of a human brain tumor complicating neural stem cell therapy. The findings here suggest that neuronal stem/progenitor cells may be involved in gliomagenesis and provide the first example of a donor derived brain tumor. Further work is urgently needed to assess the safety of these therapies. Stem cell clinical trials vs unproven treatments 35 How do clinical trials differ from the unproven stem cell ‘treatments’: - publically available data that underwent peer-review - multiple in vitro and in vivo pre-clinical studies - had to obtain approval from health regulatory body (FDA in USA) - clinical trials will evaluate the safety and then the efficacy of the therapy - as a patient/physician you should always CHECK THE CLAIMS and be aware of vested interests Phase I studies assess the safety of a drug or device Phase II studies test the efficacy of a drug or device Phase III randomized & blind testing; hundreds to thousands of patients Phase IV (Post Marketing Surveillance): after a drug/device approved for consumer sale - compare against other treatments - monitor long-term effectiveness - determine cost-effectiveness - outcomes can see the drug/device restricted or taken off market http://www.youtube.com/watch?v=ZjYfQUm1_6g&list=UU1DIKSs_IYh5xpBoygj_V3w&index=12 36 1052 novel stem cell clinical trials (2017: Fung et al. Stem Cell Reports) - only 46% of 393 completed trials had published results 37 38 - 63.7% (mainly early-stage) trials reported +ve outcomes - 48 trials registered by known stem cell tourism clinics … none had reported results - reporting guidelines needed for early-phase clinical trials Resources on stem cell tourism 39 - desperate patients - unscrupulous operators with unproven ‘treatments’ - need better patient education for better patient protection http://www.stemcellessentials.asscr.org https://www.youtube.com/watch?v=947F1Mgaji8 International Society for Stem Cell Research http://www.closerlookatstemcells.org/Home.htm Stem Cell Therapies: Now and in the Future The Australian Stem Cell Centre Patient Handbook What you should know about experimental therapies overseas and what is happening on your own doorstep. December 2009 Find out what's possible. Know what to ask. We have all heard about the extraordinary promise that stem cell research holds for the treatment of human diseases. Clinics all over the world claim to offer stem cell treatments for a wide variety of conditions. But are all of these treatments likely to be safe and effective? The ISSCR provides information to help you evaluate these claims. 40 Summary Pluripotent stem cells: can generate ANY cell type of the body; can be maintained in definitely in vitro; are usually karyotypically normal unless generated from disease-specific cells; examples =… ? Tissue-specific stem cells: typically only produce the cells of the tissue in which they are found; have extensive proliferative potential (life-long in some cases) but usually generate transit amplifying cells from which effector cells arise; are difficult to find in vivo so are typically difficult to keep as stem cells in vitro; are usually karyoptypically normal; examples =… ? Clinical and patient issues relating to experimental stem cell therapies vs commercial provision of unproven stem cell therapies 41 Further Information Managing the potential and pitfalls during clinical translation of emerging stem cell therapies. Main, Munsie and O’Connor. Clinical & Translational Medicine. 2014. 3:10. The 3R principle: advancing clinical application of human pluripotent stem cells. O’Connor. Stem Cell Research & Therapy. 2013. 4(2):21. Novel ocular therapies from new cell sources. O’Connor. Australian Optometry Pharma. 2012. March issue. ABC News 19 March 2018 WSU stem cell discovery offers hope for adult and childhood cataract 42 Thank you