2. REGULACE DIFERENCIACE BUNĚK A ZACHOVÁNÍ BUNĚČNÉHO DIFERENCIAČNÍHO STATUSU Tomáš Bárta Image credit: omniprex.com Bi9903 Vývojová biologie živočichů EMVB0311p Základy vývojové biologie 1 Duševní vlastnictví a poskytované studijní materiály Copyright notice ̶ Tato prezentace je autorským dílem vytvořeným zaměstnanci Masarykovy univerzity. ̶ Studenti kurzu/předmětu mají právo pořídit si kopii prezentace pro potřeby vlastního studia. ̶ Jakékoliv další šíření prezentace nebo její části bez svolení Masarykovy univerzity je v rozporu se zákonem. ̶ The presentation is copyrighted work created by employees of Masaryk university. ̶ Students are allowed to make copies for learning purposes only ̶ Any unauthorised reproduction or distribution of the presentation or individual slides is against the law. 2 Embryo How cells acquire different cell fates? Twisting cell fate for good Differentiation! 3 Content of the lecture * Differentiation potential * Spemann-Mangold experiments * Stem cells (basics and classification) * Differentiation mechanisms * Dedifferentiation * How to change cell fate (Reprogramming & Transdifferentiation) * * 4 Differentiation potential Differentiation potential Ectoderm Mesoderm Endoderm 5 Výsledek obrázku pro how to explain epigenetic landscape Totipotent/Pluripotent Multipotent How to change the cell fate? Differentiation Differentiated Differentiation is the process in which a cell changes from one cell type to another - usually, the cell changes to a more specialized type. 6 Cell fate in vivo: Spemann-Mangold Experiments 7 •Sliced (or separated) embryo into two halves leads to the development of smaller, but perfectly normal individual - must contain dorsal organizer region. •Also in two-cell stage embryo. •In humans, identical twins are found in 3 out of 1,000 live births, and arise most frequently by the spontaneous separation of the inner cell mass of the mammalian blastula into two, followed by self-regulation. Cell fate in vivo: Spemann-Mangold Experiments - Explained 8 (A) When the egg is divided along the plane of first cleavage into two blastomeres, each of which gets half of the gray crescent, each experimentally separated cell develops into a normal embryo. (B) When only one of the two blastomeres receives the entire gray crescent, it alone forms a normal embryo. The other blastomere produces a mass of unorganized tissue lacking dorsal structures. Cell fate in vivo: Spemann-Mangold Experiments - Explained 9 (A) When the tissues are transferred between early gastrulae, the presumptive neural tissue develops into epidermis, and only one neural plate is seen. (B) When the same experiment is performed using late-gastrula tissues, the presumptive neural cells form neural tissue, thereby causing two neural plates to form on the host. 10 Cell fate in vivo 11 BONUS: The ultimate example of self-regulation is provided by another mammal, the nine-banded armadillo, in which every blastocyst gives rise to four genetically identical siblings. Polyembryony is the phenomenon of two or more embryos developing from a single fertilized egg. Loughry et al Výsledek obrázku pro how to explain epigenetic landscape Totipotent/Pluripotent Multipotent How to change the cell fate? Differentiation Differentiated Differentiation is the process in which a cell changes from one cell type to another - usually, the cell changes to a more specialized type. 12 Models for explaning the differentiation 13 Stem Cells What are Stem Cells? And how their differ from other cells? A stem cell retains the ability to divide and re-create itself while also having the ability to generate progeny capable of specializing into a more differentiated cell type. Why „stem“ cells? To proliferate and differentiate… 14 Potency defines a stem cell 15 Embryonic vs. Adult stem cells Embryonic stem cells •Derived from inner cell-mass of preimplantation embryo. •The epiblast give rise to embryo, generating all the cell types •The trophectoderm and primitive endoderm give rise to extraembryonic structures, namely the embryonic side of the placenta, chorion, and yolk sac 16 Embryonic stem cells * Derived from inner cell-mass of preimplantation embryo. Ectoderm Mesoderm Endoderm Self-renewal 17 Mechanisms promoting pluripotency of embryonic stem cells Self-renewal Oct4 Sox2 Nanog Differentiation Oct4 Sox2 Nanog •The presence of self-renewal supporting growth factors •The proper extracellular matrix •Cell-to-cell contact •The presence of differentiation-inducing growth factors •Different extracellular matrix •Co-culture with other cell types. 18 Adult stem cells * Sometimes called tissue/organ-specific stem cells * Responsible for tissue homeostasis and maintenance Differentiation does not occur only during embryonic development! 19 Gilbert and Barresi, 2016 Adult stem cells 20 Mesenchymal stem cells can be found in a variety of tissues, including connective tissues, muscle, eye, teeth, bone, and more. They play dual roles as supportive stromal cells as well as being multipotent stem cells. Bone marrow, fat tissue…. Besides tissue-specific stem cells… Cancer stem cells are tumorigenic (tumor-forming) and can be found in tumors, hematological cancers. Possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. Cell progenitor vs precursor * Progenitor = transition stage of a cell that was produced by an asymetric stem cell division – see the formation of blood cells in the previous slide. * Precursor = any ancestral cell type (either stem cell or progenitor cell) of a particular lineage 21 Stem Cell Niche – Stem Cell regulation The stem cell niche supports stem cells! Provides a microenvironment of local and long-range signals that regulate whether the stem cell is in a state of quiescence, division, or differentiation. 22 Gilbert and Barresi, 2016 Stem Cell Niche – Example Drosophila ovarian stem cell niche 23 Gilbert and Barresi, 2016 Stem Cell Niche – Example Ventricular-subventricular zone (V-SVZ) stem cell niche 24 Gilbert and Barresi, 2016 Maintaining the stem cell pool is a critical responsibility of any stem cell niche because too many symmetrical differentiating and progenitor-generating divisions can deplete the stem cell pool. The V-SVZ niche is designed structurally and is equipped with signaling systems to ensure that its B cells are not lost during calls for neurogenic growth or repair in response to injury Stem Cell Niche – Example 25 Gilbert and Barresi, 2016 VCAM1 and adhesion to the niche BONUS: 26 Young blood can rejuvenate an old mouse. (A) Parabiosis fusion of the circulatory systems of two individuals was done using mice of similar (isochronic) or different (heterochronic) ages. When an old mouse was parabiosed to a young mouse, the result was an increase in the amount of vasculature (stained green in the photographs) as well as the amount of proliferative neural progeny in the old mouse. (B) Administering GDF11 into the circulatory system of an old mouse was sufficient to similarly increase both vasculature (green in photographs) and the population of neural progenitors in the V-SVZ (outined red population in photographs and quantified SOX2+ cells in graph). (After Katsimpardi et al. 2014.) BONUS: Models for explaning the differentiation potential 27 Liu, 2021, Nature Yu, 2021, Nature BONUS: Models for explaning the differentiation potential 28 Comparison of early human development in vivo, ex vivo and in blastoid models in vitro. Stem Cells - conclusions * You should be able to: * Define a stem cell and differentiation potential * Know basic classification of stem cells * Distinguish between precursor and progenitor cell * Specify the stem cell niche * 29 Questions? 30 Výsledek obrázku pro how to explain epigenetic landscape Pluripotent Multipotent Differentiation Differentiated 31 32 What is differentiation? Breaking the balance between self-renewal and asymetric division…not only! OK, but how this is achieved? •The presence of growth factors •Cell-to-cell contact •Different cell position •Other aspects of extracellular environment (ECM, stiffness, etc.) Differential gene expression 33 Why cell-to-cell contact is so important? Symetrical cell division Cytoplasmatic determinants distributed evenly to both daughter cells, generating the same cells Asymetrical cell division Cytoplasmatic determinants distributed unevenly, generating two different cells Because of the distribution of cytoplasmatic determinants during cell divisions! Anup Padmanabhan, Mechanobiology Institute, Singapore 34 35 Differentiation is driven by Differential Gene Expression Defining Differential Gene Expression •Differential gene expression is the process by which cells become different from one another based upon the unique combination of genes that are active or “expressed.” •Every cell nucleus of an organism contains the complete genome that was established in fertilized egg. •The unsed genes in differentiated cells are not destroyed – the have the potential to be reactivated •Only a small percentage of the genome is expressed in each cell, and a portion of the RNA synthesized in each cell is specific for that cell type Is the DNA in an organism’s cells that is now expressing different genes truly still the same, however? Does it still possess the same potential to make any cell? Ian Wilmut - 1997 YES! „No genes necessary for development have been lost.“ X A B 36 So how does the same genome give rise to different cell types? 37 Gene expression – Central dogma •Access to genes •CIS-elements •TRANS-elements •Transcription factors •microRNA •DNA methylation •Differential RNA processing • • Regulation of gene expression (+ other mechanisms) maintains cell identity: 38 Gilbert and Barresi, 2016 Modulating Access to Genes Image: yourgenome.org Great, but how cells maintain this „code“ and thus their identity? 39 Access to genes - Maintaining a memory * The modifications of histones can also signal the recruitment of proteins that retain the memory of the transcriptional state from generation to generation as cells go through mitosis. Trithorax family Polycomb family Bind to active genes and keep these genes active Bind to condensed nucleosomes, keep genes inactive (methyltransferase activity) 40 Access to genes - Maintaining a memory The initial domain of Ubx expression is maintained through larval and pupal development by the TrxG proteins. Maintenance of silencing of the Ubx gene anterior to this domain requires both PcG proteins and trimethylation of lysine 27 on histone H3 (H3K27me3). Posterior to its normal domain of expression, Ubx is repressed by the Hox proteins Abd-A and Abd-B, not by the PcG proteins, or by histone H3K27me3. 41 Regulation of gene expression – structure of a gene 42 Regulation of gene expression – CIS elements The promoter, enhancers, and silencers—are necessary for controlling where, when, and how actively a particular gene is transcribed – CIS regulatory elements (on the same DNA molecule) Enhancer/Promoter GFP 43 Gilbert and Barresi, 2016 Regulation of gene expression – CIS elements Enhancers vs. Silencers 44 Gilbert and Barresi, 2016 Regulation of gene expression – TRANS elements - transcription factors Transcription factors are proteins that bind DNA with precise sequence recognition for specific promoters, enhancers, or silencers. Transcription factors that bind enhancers can activate a gene by (1) recruiting enzymes (such as histone acetyltransferases) that break up the nucleosomes in the area or (2) stabilizing the transcription initiation complex. 45 Regulation of gene expression – TRANS elements - transcription factors Gene regulatory network – cell specific transcription factors 46 Regulation of gene expression – TRANS elements - microRNA Image credit: https://spchanlab.files.wordpress.com 47 microRNAs 48 Regulation of gene expression – TRANS elements - microRNA Image credit: Bejarano et al., 2012 49 Regulation of gene expression – TRANS elements - microRNA Image credit: Peskova et al., 2020 miR-183/96/182 miR-183/96/182 PAX6 50 Regulation of gene expression – DNA methylation 51 Gilbert and Barresi, 2016 Two ways to repress…. CpG Regulation of gene expression – DNA methylation Can be the methylation status preserved? YES! 52 Regulation of gene expression – differential RNA processing Differential RNA processing results into different forms of proteins Leading to 38 016 different types of protein 53 Gilbert and Barresi, 2016 Regulation of gene expression – differential RNA processing Another example: •Mutation in intron of myostatin – a negative regulator of muscle growth, creates a new splicing site 54 Regulation of gene expression – differential RNA processing Homo sapiens has around 20,000 genes in each nucleus; so does the nematode C. elegans, a tubular creature with only 959 cells. What’s this worm is doing with approximately the same number of genes as we have? „About 92% of human genes are thought to produce multiple types of mRNA. Therefore, even though the human genome may contain 20,000 genes, its proteome—the number and type of proteins encoded by the genome—is far larger and more complex.“ - Christopher Burge Differential RNA processing – „food for thought“ 55 Differentiation - conclusions * Asymetric cell division * The importance of cell-to-cell contact * Gene expression regulation (CIS vs TRANS), know the basics * 56 Questions? 57 Výsledek obrázku pro how to explain epigenetic landscape Pluripotent Multipotent Dedifferentiation Differentiated Partially or terminally differentiated cell reverts to an earlier developmental stage, usually as part of a regenerative process. 58 Dedifferentiation - regeneration Brockes, 1997 59 Dedifferentiation - regeneration Reversal of the Differentiated State Brockes, 1997 60 Dedifferentiation - regeneration 61 Dedifferentiation – blastema formation The replacement of a large area of tissue can occur through the formation of a blastema comprising undifferentiated cells. •epidermal cap forms that covers the injury •cells from the amputation plane dedifferentiate forming a blastema under the apical epidermal cap •dedifferentiated cells proliferate and undergo re-differentiation to regenerate the various different cell types that make up Regeneration occurs not only via the formation of blastema! •Proliferation of existing cell types •heart regeneration, where pre-existing cardiomyocytes de-differentiate and proliferate •Transdifferentiation •proliferation of biliary epithelial cells, which differentiate into hepatocytes •Differentiation of tissue-resident stem cells/progenitors • 62 Dedifferentiation – blastema formation CREDIT: KAYLEE WELLS / MCCUSKER LAB 63 Dedifferentiation - regeneration What drives it? •inflammatory response •neutrophils and macrophages, which are attracted by signals generated from the dying cells and surrounding tissue. Genetic or chemical ablation of phagocytic macrophages during regeneration results in impaired regeneration. 64 Dedifferentiation - regeneration 65 Dedifferentiation - regeneration 66 Injury induces retinal Müller glia of certain cold-blooded vertebrates, but not those of mammals, to regenerate neurons. Výsledek obrázku pro how to explain epigenetic landscape Pluripotent Multipotent How to change the cell fate? Reprogramming vs transdifferentiation Differentiated 67 Twisting cell fate for good Cell reprogramming into pluripotent state Oct4, Klf4, Sox2, c-myc Somatic cells (fibroblast) D4.jpg Induced pluripotent stem cells Extrinsic conditions (e.g. growth factors, cell culture conditions etc.) Advantages: patient-specific cells Disadvantages: instability of the genome, tumorigenicity, high-costs, safety 68 C:\Users\ntb33\AppData\Local\Microsoft\Windows\Temporary Internet Files\Content.IE5\J58LEY8P\MC900053118[1].wmf C:\Users\ntb33\AppData\Local\Microsoft\Windows\Temporary Internet Files\Content.IE5\J58LEY8P\MC900053118[1].wmf Sir John B. Gurdon – 1958 - 1966 Ian Wilmut - 1997 “This result is of interest since it shows that genetic factors required for the formation of a fertile adult frog are not lost in the course of cell differentiation...” Recipient cell contains factors that are capable to revert somatic cell into embryonic cell. 69 Sir John B. Gurdon – 1958 - 1966 Mozilla Firefox Mozilla Firefox “We describe here some adult frogs which are derived from transplanted intestine nuclei and some of which are fertile.” 70 Oct4 Sox2 Klf4 c-Myc 24 genes, specific for pluripotent stem cells he always took out one gene and followed the efficiency of reprogramming => 10 genes seriously affected the efficiency. 10 genes, specific for pluripotent stem cells he selected 4 genes Mozilla Firefox Shinya Yamanaka - 2006 Mozilla Firefox 71 Shinya Yamanaka – 2006 - 2007 ScienceDirect.com - Cell - Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors - Mozilla Firefox Mozilla Firefox Mozilla Firefox 72 C:\Users\ntb33\AppData\Local\Microsoft\Windows\Temporary Internet Files\Content.IE5\J58LEY8P\MC900053118[1].wmf C:\Users\ntb33\AppData\Local\Microsoft\Windows\Temporary Internet Files\Content.IE5\J58LEY8P\MC900053118[1].wmf Sir John B. Gurdon - 1958 Ian Wilmut - 1997 ? 2pn2pb2.JPG 73 How only 4 genes can reprogram the fate of a cell? 74 F:\photos\export1\D18-10x.jpg D0 D3 D6 D9 D12 D15 D18 D21 I.Shut down of genes maintaining the “identity” of fibroblasts. I.dedifferentiation and upregulation of genes maintaining proliferation II.MET – transition from mesenchymal to epithelial phenotype. III.Establishment of pluripotency gene network. 75 76 77 Graf, 2011 Conversion of one cell type to another cell type. Easily accessible and easy-to-cultivate cell types (fibroblasts, blood cells) are often used. Transdifferentiation Advantages: patient-specific cells Disadvantages: low-efficiency, restricted proliferative capacity, limited cell type diversity, senescence, and do not generally produce progenitor cells 78 adenovirus encoding PDX-1 injected to liver diabetic mice Transdifferentiation of hepatocytes into insulin producing cells. PDX-1 Insulin Activation of insulin expression in human hepatocytes infected with virus encoding PDX-1 Pdx-1 (green) a insulin (red) Activation of insulin promoter (green) 79 Ascl1, Brn2, Myt1 80 actin αMHC Gata4, Mef2c, Tbx5 81 Conclusion – how to manipulate cell fate Výsledek obrázku pro how to explain epigenetic landscape Reprogramming Transdifferentiation Differentiation 82 Conclusion – how to manipulate cell fate 83 * To know what is reprogramming, transdifferentiation and differences between them • Questions? 84 Feedback + contact 85 Mgr. Tomáš Bárta, PhD. tbarta@med.muni.cz