IPSCs Advantages and Disadvantages Essay Plan PDF

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Discuss, using appropriate examples, the advantages and disadvantages of using induced pluripotent stem cells for disease modelling and cell therapy. Introduction: What are induced IPSCs? Adult cells can be artificially induced to become unspecialised cells, like embryonic stem cells –’Induced pluripotent stem cells’. Refutes previous dogma that cell differentiation is irreversible. Ø Since human iPSC lines have now been generated from a variety of patients with genetic diseases it is now possible to develop in vitro disease models by differentiating the pluripotent cells to cells of the affected tissue Ø BUT 1st we need reliable reproducible and efficient differentiation protocols to drive production of just desired cell type

Main Body: iPSCs made to date not identical to ES and may contain genetic mutations of host origin or acquired BUT could be used in same patient (no immune rejection) Macula degeneration the Japanese story Autologous repair- using patient’s own reprogrammed iPS cells • 2014 Kobe- IPSC-RPE (made from the patient’s own fibroblasts) put into the back of the eye of a Japanese woman with macula degeneration She recovered and her sight has not degenerated further (as untreated eye has) • HOWEVER in a second patient- cells had genetic abnormalities and trial stopped. Repair from donor iPSCs • In 2017 first (Japanese) patient treated with RPE differentiated from iPSCs derived from a donor i.e. allogeneic Reprogramming is possible with many differentiated cells of all three major cell types in the body (germ layers) iPSCs to model many different particualrly monogenic diseases and use these models to understand development, determine aberrant pathways in disease and identify new drugs through high through put drug screens. PBMCs Periferal blood mononuclear cells. KLF-4 Kruppel like factor 4 tf expressed in ES cells and knock out dies perinatilly from dehydration: - Lin 28 can replace Klf-4 - John Gurdon and Shinya Yamanaka jointly won the Nobel Prize in Physiology and Medicine in 2012 for showing the adult somatic cells can be reprogrammed to become pluripotent. - Take a cocktail of factors and use retroviral transfection to transfect cells and analyse cells after this – widdled down to 24 factors then 4 factors. Retroviral transfection of a combination of only 4 factors shown to induce mouse embryonic fibroblasts to become pluripotent cells, named induced pluripotent cells (iPS cells). Success rate of less than 0.1%. - 4 factors necessary for this were: Oct4 and Sox2, cMyc and Klf-4. Monitor colony numbers – large number of colonies when 4 factors used, few colonies when 3

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factors used (ie: lacking cMyc or Sox2), no colonies when lacking Oct4 or Klf-4, no colonies with 2 factors. These factors important in generating pluripotent stem cells.

BUT IPS cells SOMETIMES have had problems: Ø Reactivation of c-myc in differentiated tissues of resulting chimeras lead to tumours (original study)… so may need to reprogram without myc (if using integrating virus) Ø Epigenetic pattern not identical to ES (>DNA –Me in iPSCs); abnormal transcriptional inactivation of certain chromosome regions (Chrom 12 in mice) Ø Virally re-programmed cells gain mutations Ø Promoter occupancy by ‘4 factors’ not identical to ES cells Ø Telomere lengthening varies Ø Telomeres shortened in somatic cells : ES cells have mechanisms to maintain telomeres centred on telomerase reverse transcriptase complex. In vitro different iPSCs differ in amount of telomere lengthening (Wang e t al 2012) Telomerase plays a critical role in reprogramming. Developments in IPS technology: - Avoid use of c-myc - Reprogramming of many other cell types (neural, epithelial, pancreatic, myeloid etc) - Use of poly cistronic vectors (one vector carrying all the factors needed) - Use of 2 or 1 factor in cells already expressing other factors (generally already expressing sox-2 and/or klf4) - Use of non integrating adenovirus (instead of retro or lenti-virus) as vector : Sendai most efficient - Use of plasmids (ie no virus and non-integrating) - Use of removable, transposon flanked, genes (piggy Bac) - Transfection of synthetic mRNA or protein for the factors - Kinase inhibitors to improve efficiency - Use of small molecules: direct reprogramming - microRNA - Successfully transfected hESCs with a plasmid contain these factors which provided a non integrating source. Although expression transient and success rate now much lower (1:1,000), they got cells expressing ES markers and able to contribute to all three germ lineages (Yamanaka then many others). - Non-integrating adenovirus is good because when factors are put in, no integration occurs in active gene to produce mutation. Sendai-virus is most efficient. - Plasmids can also be used. Applications of Ips cells: - IPS cells useful for studying human developmental events in vitro but also more translational applications. - Somatic cells can be taken (most common are dermal fibroblasts and blood) and cultured with pluripotency inducing factors, before selecting and expanding IPSCs to

produce tissues for patient-specific cell therapy, drug screening and human disease models. Use of Pluripotent Cells for Disease modeling: Overall Strategy: Take somatic cells from a person (eg: a patient with a mutation in a gene leading to disease) to make IPSCs with the mutation to make cell of interest (eg: neuron, cardiomyocytes etc). Can look at other tissue to analyse unnoticed changes. Important to have a control – eg: a sibling, will have a genome similar to the patient, sibling cell can be differentiated and compared to mutant cells. Sibling genome is not identical to patient. CRISPR-cas9 can correct mutation in cells and produce differentiated cells from gene-corrected IPSCs. Can use normal IPSCs and induce a mutation to produce differentiated cells to analyse disease. Functional analysis of all of these models can show what mutant gene does. Functional analysis compares markers produced in mutant and non-mutant cells as well as function (eg: APs in neurons, cell behaviour in vivo). Models can also be used for drug screening – can you correct differences caused by mutation using drugs? Potential use of IPSCs to allow correction of genetic defects and provide therapy: murine proof of principle • It was a powerful ‘proof-of-principle’ that iPSCs could one day fulfil their potential in fighting human disease. • Humanized sickle cell anemia mouse model. Mice rescued by transplantation with hematopoietic progenitors obtained from autologous iPSCs in vitro. Human mutant Betaglobin allele corrected by gene specific targeting. • Can you use these cells themselves in therapy? Patient cells will be self-cells – if mutation can be corrected and cells are differentiated into affected cell, eg: cardiac myocyte in a heart condition, these corrected cells can be implanted in host tissue to use them for therapy. May be possible, still inconclusive. • Proof of principle from mice – fibroblast taken from skin of a transgenic mouse carrying human sickle cell anaemia gene, reprogram them, correct genetic defect using Oct4, Sox2, Klf-4 and Cmyc using a viral method, sickle cell anaemia gene mutation corrected via homologous gene recombination through a viral method (now achieved with CRISPR-cas9) and differentiate them to haematopoetic progenitors via embryoid body method, inject them into the mice. IPSCs began to produce normal blood cells and no longer suffered from anaemia. First IPSCs from patient with genetic disease: Disease; Amyotrophic lateral sclerosis (ALS): motor neuron cell death in spinal cord and motor cortex. Skin biopsy taken from an 82-year old woman with ALS; fibroblasts reprogrammed IPS cells then induced to develop to form motor neurons AlS is the disease Stephen Hawkins had. Manycases sporadic but More than 20 genes have been associated with familial ALS, of which four account for the majority of familial cases:[45] C9orf72 (40%), SOD1 (20%), FUS (1– 5%), and TARDBP (1–5%).[19 Proof of principle: IPSCs taken from patient with genetic disease. Patient was elderly, patient suffered from Amyotrophic lateral sclerosis  leads to death of motor neurons. Skin biopsy

taken from an 82-year old woman with ALS; fibroblasts reprogrammed. IPS cells then induced to develop to form motor neurons. Individual with genetic disease – take cells to be reprogrammed into IPSCs and differentiate them specifically into cells/tissue that are either normal or diseased. Transcript-ohmics or proteomics analysis used to look at transcripts and proteins made then this can be used for bioinformatics to tell you what pathways are involved/effected. Cells can be used to develop new therapeutics. NO study has shown definitively that hESCs/hiPSCs can be uniformly (100%) targeted to just one specific cell type. However protocols that highly enrich for specific cell types have been reported mostly using a combination of exogenous factors (e.g. growth factors) and sometimes combined with cell selection IPS derived neurons keep their genetically programmed defect Ø Skin fibroblasts from a patient with Spinal Muscular Atrophy: mutations in Survival Motor Neuron 1 gene leading to reduced protein and loss of α motor neurons (death by 2 yrs) Ø Reprogrammed to IPS Ø Differentiated IPS cells to neurons Ø Neurons retained disease phenotype Ø Much reduced incidence of neuron formation compared to wild type (healthy) neurons Ø 4 weeks after differentiation, there is a non-significant decrease in choline acetyl transferase in the SMA mutant neurons but after 6 weeks there is a significant decrease. Fewer neurons in the mutant IPSCs that have differentiated neurons than in the wildtype IPSCs. Potential for studying disease in vivo. Ø iPSCs as disease models works best for monogenic diseases. Isogenic lines will be screened to see if mutation can be seen with a drug. hESCs and hiPSCs produce functional glucose responsive pancreatic β cells in vitro and in vivo in mice - Cells only produce insulin not other pancreatic hormones - Cells respond differently to different doses of glucose as well - PSC differntiated beta cells secrete insulin in response to multiple sequencial high glucose challenges - A schema for directed dif new modified from old method - B hues 8 beta cells; - C PH cells ;polyhormonal cells resembling fetal Beta cells - D primary pancreatic beta cells - All challenged sequentially with glucose ( conc indicated and depolarised to reset with KCl in between). - KGF=FGF7 - SANT1 sonic hedgehog antagonist - LDN inib of BMP R1 - XX1 gamma secretase inhibitor

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Cells that were produced from previous study were poly-hormonal, ie: made more than proinsulin and insulin, not as useful. In studies this was refined and they found they made cells that only produced insulin and not other pancreatic hormones, cells responded differently to different levels of glucose – glucose increases, more insulin released in these cells. These cells were much closer to adult beta cells compared to the old protocol cells.

iPSC generated 3D vascularised liver buds in vitro: - hiPSC→HNF4a+ hepatic endoderm - HNFa+ endoderm cultured with HUVECs and hMSCs - Self organisation to form ‘liver buds’ with integration of ‘vasculature’ - After in vivo engraftment: - showed vascular perfusion + albumen and α1-AT secretion - Improved survival of (immunodeficient) mice after gangcyclovir induced liver failure - Specified hepatic cells (immature endodermal cells destined for hepatic cell fate) selforganized into three-dimensional iPSC-liver buds. - by interacting with endothelial and mesenchymal cell. - HUVEC and MSC stroma provides support (FGF + BMP pathways). - 43% of cell in liver buds After in vivo transplantation formation of functional vasculature in vivo stimulated maturation of iPSC-liver buds - into tissue resembling the adult liver. - Dextran perfusion though liver showed functional blood system though engrafted blood vessels after transplant. - Generate HNF4a and hepatic endoderm + HUVECs + hMSCs to hepatic endoderm precursors  self organisation to form liver buds. Liver buds could be implanted into an immunocompromised mouse under the kidney capsule, liver buds grafted well and amalgamation of vessels within the buds with the external mouse vessels showed blood flow through organoids. After in vivo engraftment, liver buds were put into mice that had been treated to induce liver failure – partial rescue of liver was seen using the liver buds. - Liver buds: SIP3A7 and albumin markers shows relative expression of HNF4alpha. Showed a fully human blood-brain barrier chip that accurately predicts drug permeability and can be perfused with whole blood. Utilizing patient-derived tissue, they recapitulate disease-specific defects. They establish a platform to advance drug screening and disease modeling. And showed PoP of drug sensitivity and permeability. iBMECs (iBMECs: iPSC derived Brain microvascular endothelial cells) Cultured under Laminar Flow Express Tissue-Specific Markers of the Human Brain Microvascular Capillary Wall. To test the functionality of the human BBB-Chip, assessed whether proteins could be selectively filtered. Indeed, IgG and albumin remained confined to the blood side, while transferrin accumulated on the ‘brain’ side. Using a BBB-Chip, with endothelial cells from Huntingdons disease iPSCs permeability was increased across all dextran sizes examined, suggesting significant disruption to BBB function. Huntington’s disease: BBB becomes permeable. Take pluripotent stem cells  differentiate them to brain microvascular endothelial cells and take primary human pericytes and primary

human astrocytes put into model > brain and blood side developed in model, permeability across this interface observed using RNA sequencing  lots of functions was seen in model barrier similar to that of the BBB. Same scenario used but IPS cells made into microvascular epithelial cells as well as IPS-derived neural cells and put them together to form a brain side and a blood side  trans-epithelial resistance looked at (high resistance should be seen if interface is selectively permeable)  because this was done on a chip, you could put blood into blood side with labelled molecules to see if permeability barrier is similar to that of BBB. Looked at the crossing of immunoglobulin, albumin and transferrin. Transferrin crossed the BBB, whilst immunoglobulin and albumin do not – this was seen in the model, suggesting it reflected the BBB well. This was repeated in IPS-derived neural cells which had the Huntington’s mutation and trans-epithelial resistance was reduced in this model and selective permeability was compromised – good disease model for Huntington’s. IPS cells may have ‘Epigenetic memory’ IPSCs ‘remember’ the differentiated cell type they came from- having a bias to give progenitors that differentiate into that or related cell types Nt ESCs from neural transfer into oocyte develop to blastocyst and make ESCs. IPS cells may have epigenetic memory – ie: remembering the differentiate cell type they came from, so chromatin marks are not as they should be for ES cells but show similarity to their cell origin, so progenitors produced in bias of their prior cell type. ES and IPS cells made from different cell types of a B6Cba mouse with a mutation, generated blastocysts to make ES cells, also generated fibroblasts to generate IPSCs, also took fibroblast nuclei and injected into a nucleated oocyte  oocyte activated, blastocyst formed, ES from blastocyst produced. IPSCs also produced from blood. All of these cell lines used to produce 2 cell lineages – either bone or blood. Colony number shows that if nuclei transfer was done, there was a good number of colonies for each blood cell type produced. Fibroblast IPSCs differentiated to blood cells did not produce many colonies. Blood IPSCs could be reprogrammed to blood lineages to produce different blood cells, indicating that these IPSCs retained memory of being a blood cell, making it easier for them to differentiate into a blood cell. Bone cells derived from a fibroblastic-like cell type, fibroblast IPSCs shows high expression of bone cell genes – cells have a memory to make bone. Methylation pattern across entire iPSC genome does not faithfully mimic that found in ESC. Comparison of iPSC methylation at specific sites across the chromosomes with ES and adult cells: - Reprogramming errors common - Large megabase sections of DNA, often at telomeres and centromeres, incorrectly methylated - Many differences common to all IPS cells and differences inherited with division - DNA methylation in pluripotent cells unstable and prone to greater errors compared to differentiated cells Reprogramming may cause mutation in iPSCs even without viral vectors. Even non integrating techniques can generate iPSCs with new mutations, some in genes involved in cell proliferation, cancer and development.

BUT growing the cells in culture seems to select against some genetic abnormalities, e.g. de novo gene copy number variation goes down with culture. -The iPSCs become more like ES with time in culture! • Some effects improved by culture others exacerbated; • New mutations arise during extended iPS culture and accumulate with more division cycles. Hussein et al 2011 Hussein et al 2011 Nature 471, Zhang Nagy • But de novo gene copy variation goes down with culture

NDUCED PLURIPOTENT STEM CELLS

Induced Pluripotent Stem cells (iPSCs) are adult dierenated cells which have been genecally reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important in maintenance of the de9ning proper es of embryonic stem cells (selfrenewing, nondierenated, pluripotency). Discovery It was previously thought that cell dierenaon was irreversible, and once a cell had dierenated, it cannot return to a less dierenated state to recapitulate development. However, several studies have proven this theory wrong. In 1996, Keith Campbell’s lab successfully reprogrammed dieren ated mammary epithelial cells in an oocyte via somac cell nuclear transfer (SCNT), resulng in the genera on of an embryo and cloning of Dolly the Sheep. However, this process was very ine@cient, with only 1 in every 350 fusions actually producing a pluripotent embryo which would develop into a lamb. This prompted the search for an alternave way to reprogram dieren ated cells to generate pluripotent cells. In 2006, Takahashi & Yamanaka successfully reprogrammed mouse embryonic Abroblasts to become pluripotent cells, named induced pluripotent stem cells (iPSCs). Yamanaka won the 2012 Nobel Prize in Physiology and Medicine for this breakthrough. Derivaon/Generaon (Takahashi & Yamanaka 2006  MURINE iPSCs) They achieved this through retroviral transfecon of a combinaon of 4 factors; Oct-4, Sox-2, cMyc and Klf-4. Ini ally they tested 24 factors, and through serial passaging to select cells capable of self-renewal and colony formaon, narrowed this down to 4. Oct-4 is absolutely required for pluripotency, regula ng expression of dierenaon genes (for example through acvaon of polycomb genes – di erenaon repressors). Sox-2 is a cofactor of Oct-4 which facilitates repression of dierena on genes. CMyc is associated with cell cycling, and promotes proliferaon and metabolism, whilst Klf-4 inhibits dierenaon and upregulates Sox-2 and Nanog. The combinaon of these 4 factors together could produce cloned cells which when analysed, displayed the characteriscs of embryonic stem cells such as selfrenewal, pluripotency, expression of surface markers SSEA-1 and transcrip on factors Oct-4, Sox-2, Nanog. In 2007, the Arst HUMAN iPSCs were generated from adult Abroblasts via lenviral transfecon of Oct-4, Sox-2, Nanog and Lin28. Lin28 and Nanog were used to replace cMyc and Klf-4

of the original “Yamanaka factors”. Today, reprogramming of many types of dierenated cells is well established, such as ligaments, tendons, carlage, epidermal cells and neurons. Characterisaon IPSCs demonstrate an ES-like gene expression paEern and are maintained as self-renewing, pluripotent cells in medium with LIF and BMP (murine) or Acvin and FGF-2 (human). They have a...