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6 Directed Differentiation Regenerative Biology and Disease Dr. Vijendra Sharma Faculty, Department of Biomedical Sciences, University of Windsor Key Concepts Definition of fate Directing differentiation – basic principles of change (differentiation) and memory *m...

6 Directed Differentiation Regenerative Biology and Disease Dr. Vijendra Sharma Faculty, Department of Biomedical Sciences, University of Windsor Key Concepts Definition of fate Directing differentiation – basic principles of change (differentiation) and memory *more pathways Methods for monitoring directed differentiation of ESCs Adult stem cells – overview of basic principles Plasticity in ASCs General role of the niche – importance in ASCs Comparisons of ESCs and ASCs Cell fate “Cell fate” is what cells should become. Fate vs. choice Normal cells (blue) entering senescence (green to yellow) or apoptosis (purple to red) after treatment with chemotherapy. Image source Fate Decisions Dictate Development Throughout Life Early fate decisions regulate the growth of the early embryo Fate decisions also occur throughout life within an organism (adult stem cells) Regulation of fate dictates variability between organisms and WITHIN organisms. *These utilize the same principles* WHAT REGULATES CELL CHANGE?? WHAT REGULATES CELL MEMORY?? Change = Differentiation A. The process by which embryonic cells differ from one another with distinct identities and functions. B. Involves the emergence of cell types such as muscle, nerve, skin, and fat cells. C. Achieving a stable terminal state (not just transitory differences). D. Change in gene expression to produce "luxury" proteins. E. Is Characterized by the protein profile present in the cell. The earliest stage of cell differentiation is cell determination – the commitment to a subset of cell fates. Cell fate SOME specified cells keep their fate even when isolated (determined). This is tested by transplantation (some cells change their fate – not determined). Figure 22–7 Molecular Biology of the Cell, 5th Edition  Physical Diversity Between Cell Types within an Organism Same genome – yet different cell types differ dramatically in their structure and function. During differentiation cells selectively become one type of cell or another HOW? Physical Diversity Throughout the Life of an Organism Cells differ in: Number of divisions Repair capacity When they fully mature Number/type of changes through life (morphology, functional changes) Experiments to Demonstrate that DNA is not “Changed or Lost” During Developmental Decisions Differentiated donor did not lose any DNA  Gene activity can be changed by fusion of differentiated cells Fusion of chicken red blood cells (inactive nucleus) with human cancer cells leads to reactivation of chicken genes. Human cells have cytoplasmic factors that can activate chicken genes. Fusion of human (non-muscle) cells with rat muscle cells induces human muscle gene activity. Gene expression of cells that are differentiated is controlled by cytoplasmic factors (i.e., TFs), which can be altered. History Lessons Fate is sensitive to the ‘environment’ DNA is not changed Fate = either change (differentiation) or memory … what is the molecular process regulating these events? Differences in Proteins by Two Human Tissues 2-D electrophoresis Differences outweigh their similarities For similar proteins, even their abundance differs between tissue types. Selective Gene Expression Position Influences Fate One group of cells changes the fate of another group of cells. Info passes via: 1. Secreted diffusible molecule (inducer molecule) 2. Surface molecule receptor 3. Gap junction (channel) ** Generally, the signal is limited in time and space Figure 22–10 Molecular Biology of the Cell, 5th Edition Common Inducer Molecules Pathway Ligand Receptor Inhibitors/Mod. RTK EGF EGFR Argos FGF FGFR ephrins EphR S/TKR TGF  TGR R Chordin (Sog), BMP (Dpp) BMPR noggin Nodal Wnt Wnt (wingless) Frizzled Dkf, Cerberus Hedgehog hedgehog Ptc, smth Notch delta notch Fringe “Morphogen” is a Long-Range Inducers that Exert Graded Effects Vary in concentration Threshold concentrations for different fates. Negatively regulated by inhibitors. *wnt is one of these* Figure 22–14 Molecular Biology of the Cell, 5th Edition Lateral Inhibition Example of Lateral Inhibition and Cell Diversification **Notch pathway is known for mediating these interactions Figure 22–60 Molecular Biology of the Cell, 5th Edition Ligands in mammals (drosophila): Delta-like (Delta) and Jagged (Serrate) Cells also use selective gene expression to REMEMBER what type of cell they are 1. Positive feedback loops (supports Master gene regulation) 2. Combinatorial gene control 3. Cycles of Repressors (i.e. Circadian Rhythms) 4. Epigenetics (also directs change)  Positive Feedback Creating Memory Figure 7–68 Molecular Biology of the Cell, 5th Edition An example of one Gene Controlling the Production of a Whole Organ Figure 7–77 Molecular Biology of the Cell, 5th Edition Master Regulatory Genes Toy (twin of eyeless) ey (eyeless) sine oculis (SO) eyes absent (eya) dachshund gene (dac) Ey binds directly to target genes encoding the lens crystallins, rhodopsins, and photoreceptor proteins Figure 7–77-78 Molecular Biology of the Cell, 5th Edition Combinatorial Gene Control During Development Combining a few regulatory proteins can generate many cell types during development. The production of each regulatory protein is self- perpetuating once it has been initiated (through cell memory). Figure 7–76 Molecular Biology of the Cell, 5th Edition Repressive Cycles (Circadian Rhythms) Circadian rhythms allow living organisms to live in phase by altering day and night... Dr. Phillip Karpowicz | Our Researchers at the UWindsor Circadian Clock Functions Through Series of Repressors Figure 7–72 Molecular Biology of the Cell, 5th Edition What makes memory/change HERITABLE? EPIGENETICS: Heritable change that is not because of a change in sequence Rely heavily on modifications to histone and DNA and regulation of RNA. Epigenetics Regulate Cell Specific Traits Globin genes expressed exclusively in erythroid cells 5 globin genes in total - expressed at different stages in development and in different organs ( embryonic yolk sac;  yolk sac and fetal liver;  &  adult bone marrow). LCR Control of Globin Expression Individually regulated, yet also regulated by locus control region (LCR). LCR controls chromatin condensation – in erythroid cells (but no other), the globin locus is decondensed. ** The LCR is deleted or mutated in patients with a severe inherited form of anemia, thalassemia. Histone Acetylation DNA Methylation Typically turns OFF genes **Note methylation at other regulatory regions can result in genes turning ON (recall the imprinting of the Igf2 gene in the parthenogenesis lecture!) Types of Methylation 1. De Novo methylation. **The initial imprint** Some evidence that repeats may alter chromatin structure signals de novo methylation. 2. Maintenance Methylation. Copies DNA so that methylation pattern on newly replicated DNA strand is identical to previous. 3. Demethylation of DNA. The existence of active demethylation is controversial. 4. Passive demethylation. Occurs due to the lack of maintenance methylation. Inheritance of DNA Methylation Imprinting Example in the Mouse Example of somatic cell showing which gene is expressed - this case is paternal imprinting Allows the offspring to mark which gene came from which parent and express genes accordingly (methylated gene usually silenced). Histone Methylation Histone is also methylated at specific residues (lys and arg primarily); it can also be ubiquitinated. Remodelling of nucleosome alters protein recruitment to DNA Most often repressive - - can even recruit DNMTs Examples of histone methyl. which is activating. Activation of ‘stemness’ genes (Oct4, Sox2, Nanog) Differentiation genes hypermethylated - Methylation of stemness genes - Activation of differentiation genes (demethylation) Figure 9.11 Stocum text  Bivalent Chromatin Regulation Example in H3 (bivalent domains): http://focus.hms.harvard.edu/2006/050506/epigenetics.shtml 40 Directing Differentiation Is Differentiation Permanent??? Full differentiation is ‘normally’ stable. Cells can be altered in regenerating tissues. Dedifferentiation is when differentiated somatic cells are restored to a more undifferentiated, multipotent condition. Transdifferentiation is when a differentiated somatic cell can change into a differentiated cell of a separate lineage. Transcription factor cross-antagonisms in a cascading landscape of unstable and stable cell states. Thomas Graf & Tariq Enver Nature 462, 587-594 (2009) doi:10.1038/nature08533 Germ lineage destinations ENDODERM: Stomach, colon, liver, pancreas, urinary bladder, lining of the urethra, epithelial parts of trachea, lungs, pharynx, thyroid, parathyroid, intestines. MESODERM: Skeletal muscle, skeleton, dermis of skin, connective tissue, urogenital system, heart, blood and lymph cells, spleen. ECTODERM: Central nervous system, lens of the eye, cranial and sensory ganglia and nerves, pigment cells, head connective tissues, epidermis, hair, and mammary glands. Directing diff in vitro: Start: Prep pure ESC populations Techniques used for Directed differentiation Ebs: 3D pros and cons Stromal: pros and cons of subculturing with another cell type – factors secreted; separating cell types ECM: simplest – lacks pros from above methods - expensive Gordon Keller Genes Dev. 2005; 19: 1129-1155 Spontaneous Differentiation of Human Embryonic Stem Cells Free radicals and reactive oxygen species (ROS)  intracellular messengers  direct cardiomyogenic differentiation of stem cells Physical stimuli  Electrical pulses, mechanical forces, and heat treatment  Reports of Stress and “stemness” Retracted https://www.scientificamerican.com/article/stress-induced-stem-cell-findings-are-re tracted/ Directed Differentiation of Human Embryonic Stem Cells Get rid of uncontrolled spontaneous differentiation and induce the desired paths Defined culture milieu: Defined serum: known growth and differentiation-promoting factors customized for the desired cell. Using select TFs/ligands Gene therapy Addition of Hormones or Growth factors Specific ligands/TFs can be used to direct Fate Can also be valuable ‘markers’ for differentiation Functional Analysis Cohen and Melton Nature Reviews 2011 Highly Dependent on the Timing and Order of Treatments Thomas Graf & Tariq Enver Nature 462, 587-594 (2009) doi:10.1038/nature08533 Video for Culturing ESCs into Cardiomyocytes Use this URL to watch a video of culturing mESCs into cardiomyocytes using the embryoid body technique https://www.jove.co m/video/825/in-vitro -differentiation-mou se-embryonic-stem- mes-cells-using-han ging Screening Methods for Mapping Differentiation Differentiating ES cells can be identified: Changes in the morphology Down-regulation of expression of stem cell- specific markers Up-regulation of markers for differentiated cell types Immunohistochemical staining Morphology ‘Picture’ overexpressed genes can be mapped Stained with primary antibody directed against different antigens Fluorescent conjugated secondary antibody to detect expression Measuring Gene Expression Immunohistochemistry or Flow cytometry Introduction of reporter gene  expression depending on a gene that is specifically expressed in proliferating, undifferentiated cells Microarray  analyze of the transcription of the entire genome of a cell in a specific cell phase  compared with other cell types or stem cells in a special phase can be identified Real Time (q) Reverse-transcriptase (RT) - PCR (qRT-PCR or RT2-PCR)  Sensitive and quantitative method for determination of the differentiated state Molecular markers for Pluripotency will be lost during differentiation Growth factors ex. FGF-4 (human ES cells) Transcription factors ex. Oct-4 Extra cellular matrix proteins Cell surface antigens ex. SSEA-3 (human), SSEA-4 (human), SSEA-1 (mouse), human tumor rejection antigen (TRA-1-60, TRA-1-81) Enzyme ex. alkaline phosphatase ES Genes Downregulated During Differentiation (human) Transcription factors such as Oct-3 and PEA-3 Growth factors i.e. FGF-4 Cell cycle regulators i.e., cyclins D1 and E Site-specific histone methylations:  methylation or acetylation H3-Lys4  methylation of H3-Lys9 Germ Line Differentiation Markers Markers that indicate different germ layers  Alpha fetoprotein (AFP) (endoderm)  VEGFR2 (KDR) (mesoderm)  neural cell adhesion molecule (NCAM1) (ectoderm) Flow Cytometry Frequently Used in Stem Cell Biology I like this video for the effects – note however that most often we use cell surface antibodies to identify stem cells – or differentiating populations. https://www.youtube.com/watch?v=B2zr eF2dnWk ESCs (pluripotent) can Become ALL of the Cell Types in Your Body  Embryonic Stem Cells Along the way, they make other populations of stem cells Adult Stem Cells Adult Stem Cells – Brief History 1940s The research of adult stem cells begins. 1950s Infusion of bone marrow cells could reconstitute the blood system and save lethally irradiated mice. 1960s Dividing cells in the brain can give rise to nerve cells. Solution to ethical and medical problems? Adult stem cells Multipotent adult progenitor cells (MAPC) Tissue-specific, unspecialized cells Plasticity - The ability to differentiate into cell types of other tissues. Primary roles: maintain and repair the tissue of which they are found. Source Slow dividing in vivo until activated by disease or tissue injury. Cell Cycle Checkpoints Protect Cells: They Also Protect Adult Stem Cells Errors in Inappropriate cytokinesis environmental M G1 conditions Errors in DNA fidelity G2 S Errors in synthesis Checkpoints Aid in Stem/Progenitor Decisions M G1 Quiescent ASC G2 S Differentiation Cells Can Adapt to Changing Conditions ↓nutrients ↓oxygen M G1 G2 S Cell cycle re-entry Checkpoint Adaption is Required to Expand of Stem/Progenitor Cells - Development - Injury - Maintenance ASC or - in vitro expansion M Progenitor G1 G2 S - Adult Stem Cell self-renewal - Progenitor expansion - Inhibit differentiation Proliferative Potential in vitro Adult Stem Cells Holoclones – greatest reproductive capacity; correspond to the slow-cycling cells in vivo (i.e. the putative stem cells) Paraclones – short replicative lifespan; flat morphology; terminally differentiated Meroclones – transition stage between holoclones and paraclones; cells have differing reproductive potential. First Major Hurdle! ASC purification – isolation of the SCs without harming surrounding tissue Difficult to expand in culture.  Part of solution – find the genes involved in asymmetric cell kinetics. Genes Involved in Directing Differentiation of Adult Stem Cells Cell cycle regulators In general – competency = arrest in the cell cycle Viral infection methods have allowed us to successfully manipulate these cells and utilize them in xenograph and transplant in vivo experiments and in vitro culturing techniques. Competency = Arrest in the cell cycle Example – p53 - Up-regulation – induce asymmetric division. Competency signal Family members p63 and p73 also involved in differentia tion Stem Cell Niches Stable custom environment for stem cells. Subsets of tissues that can indefinitely house one or more stem cells and control their self-renewal and progeny production in vivo. Many niches have one or more specialized cell groups (skin, GI tract). Extracellular matrix and adhesion molecules (basement membrane)  Help secure the niche spatially and may modulate the concentration of adhesion and other signaling molecules. Niche Structure Junctions Between Cells at Niche Junctions provide physical attachment, but they also regulate signaling to play direct roles in regulating differentiatio n decisions. Quiescence and the SC Niche Stem cells preserve their lifespan by tightly controlling the number of times they divide. Adult stem cells do this by staying in G0 in the niche (held there largely by inhibitors of the cell cycle – like p53) Differentiation requires 2 steps – 1. stimulation to exit from G0 to commit the SC to divide AND 2. competency (needs to exit the cell cycle at least temporarily). Plasticity Ability of an ASC from one tissue to generate the specialized cell type of another tissue Example: Adult stem cells from bone marrow generated cells that resemble neurons Intrinsic cues: “Master switch” genes – produce transcription factors whose function is to regulate the next level of genes in the hierarchy, eventually leading to individual tissue types. Extrinsic dues: Hormones. Apparent Plasticity; Lineage Conversion Pluripotent stem cell Lineage committed stem cell Stem Cell Recap SIMILARITIES DIFFERENCES Expanded lifespan Different sources Self-replicate Different potency Differentiate Proliferative Proliferation is held ‘in capacity check’ in vivo  Checkpoint Adaption is a Hallmark of Cancer Errors in cytokinesis Inappropriate M environmental G1 conditions Errors in DNA fidelity G2 S Errors in synthesis Historical Views on Cancer 1) All tumor cells can form new tumors and are, therefore, equally tumorigenic. 2) Unregulated growth is due to the serial acquisition of genetic events leading to the expression of genes that promote cell proliferation with concomitant silencing of growth- inhibitory genes and blunting of cell death. 3) Cancer is a proliferative disease. A tumor removed by surgery i n 1689. Cancer Stem Cell Theory 1) Tumors arise from cells termed cancer stem cells that have properties of normal stem cells, particularly self-renewal and multipotentiality (a minority) of tumor cells. 2) Unregulated cell growth is due to a disruption in the regulatory mechanism in stem cell renewal. 3) Cancer is a stem cell disorder and not strictly a disruption in cell proliferation/cell death. Discovery of “Cancer Stem Cells” 1994 Discovery that only a select population of cells within cancer can re-constitute the tumor. These cells were more ‘immature’ than the bulk tumor mass. Dr. John Dick, U. of Toronto Cancer Stem Cells: Identifying Properties Capable of reforming a heterogenous tumor Self renew High proliferation potential Long-lived cells Give rise to short-lived, differentiated cells Highly influenced by signals from their microenvironment Highly drug-resistant Cancer Stem Cells: Identifying Properties Debatable points: Are the minority subpopulations in a given tissue Mainly appear to be in a quiescent cell-cycle state Characterized by specific surface markers  Origin of Cancer Stem Cells Modified from Bjerkvig R et al. Nat Rev Cancer. 2005;5:899- 904 Higher Lineage CSC Correlate with Prognosis Visvader G&D 2009 Therapeutic Implications Resistance to treatment → absence of the targeted biological property (imatinib/Gleevec in CML) → quiescent state → expression of efflux proteins protecting vs xenobiotic toxins Relapse Metastasis Strategies to Target Cancer Stem Cells Immunotherapy against stem-cell-specific markers Combination of treatment vs tumor burden and treatment vs cancer stem cells Therapies promoting differentiation of cancer stem cells Targeting Cancer Stem Cells for Better Treatments Tumour Regression Cancer Stem Cell Complete Specific Therapy Remission Conventional Therapy Relapse Acute Promyelocytic Leukemia: Cancer Stem Cell Properties Regenerative Principles Guide Therapeutic Decisions Acute promyelocytic leukemia: from highly fatal to highly curable Zhen-Yi Wang1, and Zhu Chen1,2 Blood. 2008 Mar 1;111(5):2505-15.