Stem Cell Basics 2024Fall PDF

4 Stem Cell Basics Regenerative Biology and Disease Dr. Vijendra Sharma Faculty, Department of Biomedical Sciences, University of Windsor Today’s Objectives Cover common characteristics and differences of stem cells Overview of different kinds of stem cells Details about ESC...

4 Stem Cell Basics Regenerative Biology and Disease Dr. Vijendra Sharma Faculty, Department of Biomedical Sciences, University of Windsor Today’s Objectives Cover common characteristics and differences of stem cells Overview of different kinds of stem cells Details about ESCs – Where they come from – Discovery – How adaptable they are (at which stages) – Culturing ESCs – Introduction of other ways to achieve pluripotency Stem Cell Commonalities Not terminally differentiated Can give rise to specialized cells that make up the tissues and organs of the body. Capable of asymmetric division Each daughter has a choice (fate) – to remain a stem cell or begin on a differentiation course. Expanded lifespan Symmetric and asymmetric stem cell divisions. 29.10.2013, 03:41 (GMT) byLeili ShahriyariNatalia L. Komarova Differences Ways to Asymmetrically Divide Fate can be determined via: Intrinsic (divisional asymmetry) or Extrinsic factors (environmental asymmetry) https://www.youtube.com/watch?v=bej3x2s0K_M ‘General’ Rule of Thumb ** Continue cycling permits symmetric division Asymmetric division/differentiation requires transient exit from the cell cycle. Many differentiated cells/progenitors will re-enter the cell cycle Some cells will become ‘terminally differentiated’ and not re-enter the cell cycle Cell Cycle Phases M = Mitosis S = DNA synthesis G1 = Gap1 G2 = Gap2 G0 = Quiescence Lifespan of Stem Cells Telomerase - a ribonucleoprotein polymerase  Synthesizes telomeric repeats onto the 3' ends of eukaryotic chromosomes  Prevents telomeric shortening and correlates with cell immortality in the germline, stem cells, and certain tumor cells.  Predicted by Olovnikov; Discovered by Blackburn and Greider In culture, some human SCs lose telomerase activity – this is not seen to happen in rodents if cultured properly. Differences Between Different ‘Types’ of Stem Cells Where they reside/originate Growth characteristics vary dramatically depending on the type of stem cell. Differences in their ‘potential’(aka potency) Cues they respond to. Cell surface markers and gene expression signatures Origins of Stem Cells BLASTOCYSTS (parthenote stem cells, embryonic stem cells (ESCs)) – derived from inner cell masses of blastocysts created either by chemical/electrical stimulation (parthenote) OR by normal fertility procedures with sperm and egg. NUCLEAR TRANSPLANT BLASTOCYSTS (process related to animal cloning) – derived from eggs stimulated to divide following complete exchange of genetic material DIFFERENTIATED CELLS –The process of ‘dedifferentiating’ cells. Induced Origins of Stem Cells FETAL TISSUES (heart, muscle, brain, germ cell precursors to sperm and egg, trophoblast cells) – precursor cells more abundant in growing organs; some uncommitted cells. Fetal tissue stem cells ADULT ORGANS (liver, bones, bone marrow, lining of gut, spermatozoa) – reservoirs of cells partially committed to the type of tissue in the organ. Adult stem cells Stem Cell ‘Potency’ Zygote = totipotent Pluripotent - ESCs Multipotent-FTSC & ASC Totipotent stem cell A single stem cell has the ability to become ANY type of cell. Pluripotent stem cell A single pluripotent stem cell has the ability to give rise to the types of cells that develop from any one of the three germ layers (mesoderm, endoderm, and ectoderm) from which all the cells of the body arise. Multipotent stem cell A term that is usually applied to a cell that can differentiate into more than one cell type but not ‘all’ Usually refers to differentiation within a germ layer. Unipotent stem cell A term that is usually applied to a cell in adult organisms means that the cells in question are capable of differentiating along only one lineage. Various Stem Cells Types  Fertilization Cleavage Gastrulation Neurulation Organogenesis Gametogenesis Day 0: Fertilization of the oozyte in the oviduct. 3rd week: Gastrulation Specializations in Mammalian Development Mammalian embryo is protected by the uterus – no need to speed through early development Development of the placenta quickly provides nutrition from the mother, so there is no need for large yolk stores Mammalian eggs are smaller and divide slower than other organisms (difficult to study) Gene transcription begins very early (by the 2-cell stage in the mammal). Formation of Extraembryonic structures (amniotic sac and placenta) begin early (~ 4 days).  Pattern Formation Development relies on an intrinsic understanding of body orientation This is established by cells obtaining a ‘pattern’ where molecular regulators signal differently within the cell Patterning in Different Organisms Mechanisms of pattern formation differ in different organisms. In some species, patterning is defined very early In mammals, patterning is more flexible in the early stages (experimental examples to follow) Patterning has profound implications for controlling cell decisions Cleavage Egg divides into smaller cells No net growth Alternations between DNA synthesis and mitosis. No time for transcription in amphibians - depends on reserves of RNA, protein, membrane, and materials that accumulate during oogenesis. Cells form either a hollow ball (blastula) or a flattened two-layer structure (discoblastula). Production of 16 Cell Morula 8 cell uncompacted stage – fate is not determined Following compaction, the cells are polarized and move to their respective destinations By 16 cell stage, their fate is determined Figure 22-89 Molecular Biology of the Cell (© Garland Science 2008)  Trophectoderm – extraembryonic structures Inner Cell mass – whole embryo proper (totipotent cells, ES cells) Zona pellucida – embryo escapes after a few days to allow it to implant  Morula is still adaptable in mammals Figure 22-90 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008) ESCs Derived from the inner cell mass of the blastocyst. Can be cultured in vitro. Pluripotent Not capable of giving rise to a full embryo on Inner cell Blastocoel Trophectoderm their own mass (ESCs)  16 cell stage 32 cell stage - prim. ecto/endo begin to form 64 cell stage -ICM 13 distinct cells -Supports the tropho 26 ES Cells are a Pluripotent Lineage Signals from gastrulation extraembryonic endoderm layers Gordon Keller Genes Dev. 2005; 19: 1129- 1155 Cells of the inner cell mass (ICM) Primitive Endoderm – amnion, yolk sac and allantois Embryo proper - derived from descendants of the inner cells of the 16-cell stage (epiblast), supplemented by cells dividing from the trophoblast during the transition to the 32-cell stage Hypoblast Epiblast Tanaka et al. Journal of Translational Medicine 2006 4:20 doi:10.1186/1479- 5876-4-20 Blastomeres are Highly Adaptable! Monozygotic – sep. amnion but single chorion and placenta Rare Monozygotic – single amnion, chorion and placenta Monozygotic occurring between 2 cell and morula stage – separate blastocysts (amniotic and chorionic sacs can fuse) Dizygotic - (amniotic and chorionic sacs can fuse) Culturing ESCs from a Single Blastomere Klimanskaya et al. 2007, Nature Protocols Aug. Blastomeres removed from morula at 8-cell stage Cultured in vitro aggregates on feeder layers as with ESCs Avoids ethical issues??? Embryonic Stem Cells in Research Isolated from mouse blastocysts more than 20 years ago (utilized lactational delay). Led to primate than human studies – James Thomson (Univ. Wisconsin) – set up shop across campus Alan Trounson (U. of Melbourne – collab. Singapore and Jerusalem) – pioneer of IVT hot on his tail to isolate human embryonic stem cells Thomson First to Isolate HSCs 1998 NIH started a huge 3-year struggle to fund his research 2001 – Bush – using “existing” hES lines OK, but federal money could not be used to derive more lines 2004 – the death of Reagan – N. Reagan campaign to relax restrictions on embryo stem cell research.  Fifty-eight senators, almost all Democrats, supported the action.  May 2005 – Bill passed to allow gov. funded research on embryonic stem cells extracted from surplus embryos in fertility clinics.  It was later passed by the Senate. President Bush vetoed it -- his first veto of his presidency. (California stands alone under a special proposition) March 2009 – Obama passes bill to allow for new cell lines (created from private funding) to be used even with federally funded $ State law frequently interferes https://www.researchamerica.org/advocacy-action/ issues-researchamerica-advocates/stem-cell-research Canadian hESC Research Laws Must pass stringent review ethics review board. – remain reviewed by ‘oversight committee’ Must have important potential health benefits for Canadians Provided voluntarily with full disclosure of all information relevant to the consent Respect for privacy and confidentiality No direct/indirect payment or financial incentives No creation of embryos for research purposes; Respect individual and community notions of human dignity and physical, spiritual, and cultural integrity Embryonic Stem Cells ICM is plated on a culture dish Crowding (confluence)– replating (subculturing) 6 months – still pluripotent - ES stem cell line Test for surface markers only found on undifferentiated cells Examine chromosomes to ensure they are not damaged Test their ability to proliferate and their ability to form teratoma “Stemness” factors in ESCs: Brace Yourself! A lot of info coming up. We will revisit these points again and again throughout the course. Pathways covered will be REITTERATED with specific examples. You do need to know them and how they cross-talk. Murine Embryonic Stem Cells (MES) Cytokine LIF (leukemia inhibitory factor) BMP4 (bone morphogenic protein 4) Wnt LIF Polyfunctional glycoprotein cytokine–inducible production can occur in almost all tissues. Expressed in the trophectoderm of the developing embryo and in the uterus (required for implantation of the embryo in mice) LIFR (LIF receptor) expressed throughout the ICM. Removing ESC from ICM removes their source of LIF Cytokine superfamily receptors No catalytic domain Interact with nonreceptor protein-tyrosine kinases  Src family  JAK family  Leukemia Inhibitory Factor LIF = cytokine (IL6 family) Attracts non-receptor tyrosine kinases (Jaks) to transduce a signal. Jak/STAT pathway path to gene expression. mESC self- Target genes ↑ self-renewal renewal Figure 1.5 Regeneration Text. Adaptor Molecules Modify the Signal Adaptor molecules can modify the signal in many different ways. Differential pathway activation can have opposite effects on self-renewal mESC self-renewal  PH – pleckstrin homology domains PTB – phosphotyrosine- binding domains SH2 – src homology 2 domains, bind phospho Y SH3 – src homology 3 domains, bind to proline- rich motifs DON’T Freak out! You only need to know the ‘mechanisms’ by which these factors communicate  Multiple ways to get to the same target. The same starting point can result in different endpoints. Regeneration of Bone Leads to BMP 1965 – Dr. Marshall Urist, UCLA – implanted HCl-decalcified bone into the muscle of different animals with normal bone or skeletal system disorders. New cartilage and bone development appeared in or around the donor bone matrix surfaces. ** Substance in the bone matrix - BMP Bone Morphogenic Protein (BMP) Member of the TGF- B family superfamily 30+ proteins; 110-140 amino acids (mature, secreted protein) Four major subfamilies: TGF; Activin / Nodal ; BMP / GDF and miscellaneous Bind to TGFB receptors (Ser/Thr receptors)  Activin subfamily: FSH production erythroid cell differentiation mesoderm induction (frog) TGF- subfamily: cell cycle arrest in epithelial and hematopoietic cells, mesenchymal cell proliferation and differentiation, wound healing, ECM production and modulation, angiogenesis, chemotaxis, immunosuppression, apoptosis, inflammation Transforming Growth Factor-β Subfamilies BMP2 subfamily: Gastrulation, neurogenesis, chondrogenesis, interdigital apoptosis, mesoderm patterning BMP5 subfamily: Development, neurogenesis BMP3 subfamily: Osteogenic differentiation, monocyte chemotaxis GDF5 subfamily: Chondrogenesis in developing limbs BMP-4 - Important for pluripotency but also involved in differentiation and D-V axis formation. The Smad Pathway Three classes: 1. Receptor-Associated Smads TGF – ß: Smad2/3 BMP: Smad1/5 and 8 2. Cofactor Smad (4) 3. Inhibitory Smads (6, 7) Inhibited by Smurfs (1, 2) – Smad ubiquitination regulatory factors Smurf 1 targets BMP Smads Smurf 2 degrades Smad1 and Smad2 Smurf1/2 degrade the TGF-ß receptor with Smad 7 Smad Domains and Their Functions SMAD - combo between Drosophilia MAD and C. elegans sma MH domains – Mad homology domains From Massague, 1998. Ann Rev Biochem. 67:753-  Activin/Nodal Figure 1.5 Regeneration Text. Wnt signalling pathway Wnts are regulators of proliferation and differentiation Wnts are secreted glycoproteins Bind to: – Frizzled cell-surface receptor – Lipoprotein receptor-related proteins 5 and 6 (LRP-5/6) Three different Wnt pathways 1. The –catenin pathway 2. The planar cell polarity pathway 3. The Wnt/Ca2+ pathway Wnt promotes self-renewal of the ESC Wnt induces epiblast to adopt a mesodermal fate. Wnt/β–Catenin Pathway LRP- LDL related protein APC – adenomatous plyposis coli (don’t confuse with anaphase promoting complex) Absence of wnt: β–Catenin either bound to cadherin proteins (transmembrane adhesion proteins) OR degraded. LEF/TCF signaling inhibited by the repressor Groucho in the absence of wnt Wnt/β–Catenin Pathway G-protein R Activation of dishevelled requires signaling proteins (one of which is the ser/thr kinase casein kinase) Wnt signalling = inhibition of GSK-3  = stabilization of  -catenin. –catenin associates with TCF/LEF (multiprotein enhancer complexes) in the nucleus, displacing Groucho and leading to transcription of target genes. Wnt target genes: c-myc; cyclin D; id2; TCF1-4 FoxN1; BMP4; sox-9; c-jun and feedback proteins Pluripotency Regulation in mESCs Transcribe Transcribe new genes new genes PLURIPOTENCY Culturing hES Absence of feeder cells requires matrigel (or lamini-coated plates) in medium with mouse embryonic fibroblast conditioned medium (MEF CM) Important components in medium: LIF/BMP alone - not sufficient to prevent differentiation. (BMP4 INHIBITS renewal!) Activin/Nodal are necessary Fibroblast feeder layers are required in combination with bFGF. Wnt activation can replace MEF CM for short periods of time. Activin/Nodal Necessary to maintain the pluripotent status of hESCs. Not sufficient to sustain long-term hESC growth in a chemically defined medium without serum. Activin/Nodal Figure 1.5 Regeneration Text. Signal Transduction Pathway of FGFRs (RTKR) -FGF2 can maintain long- term expression of pluripotency markers in combination with Activin/Nodal signaling. - Appears to function through the MAPK pathway  Different ligands/receptors cause same effect Same pathways have different C-myc effect HOLY @#&$ WHAT DOES THIS MEAN??? Different pathways activated - Stem cells rely on their ‘home environment’ or “niche. Different pathways = same effect?  End goal is transcription of genes to regulate cell growth conditions (either same genes being activated OR different genes doing same job). Same pathway = different effects  Differences in epigenetics as well as in ‘environment’ (niche), thereby different intracellular communication **Differential GENE expression** the same genes being activated OR different genes doing the Remind me – WHY DO WE CARE???? Making sense of what stem cells need to self- renew between species will deduce the essential themes that characterize stem cell function. Lending insight into normal developmental processes and disease. Manipulation and culture of stem cells – even from different species – represents technology essential for regenerative medicine! Confusing Observations Lead to Important Discoveries Different pathways = same effect mESCs hESCs Lif/BMP4/Wnt Activin/nodal + bFGF Oct3/4, Sox2, + hundreds of Oct4, Sox9 FoxD3, Nanog dissimilar genes FoxN1, Nanog, BMP4, ** The gene families found in common might be important in maintaining pluripotency ** Sox, Nanog, Oct4 Transcription factors that regulate ‘stemness’ and are highly expressed in ESCs. Regulation also controls differentiation Expression is a diagnostic marker of ESCs. Concentration is essential Mutations and deletions is fatal for development Ectopic expression related to cancer and other growth defects **YOU will learn more about these!!! WHY Do We Care??? **This is the basis of iPS (focus of another lecture!) Questions to Ask Yourself What are the different categories of stem cells? How are they the same? How are they different? Where are ESCs derived – what is the process – are there implications in this process that can alter ESCs between organisms? Where during the process are ESCs fate determined? Why would patterning matter for ESCs? How do you culture mouse and human ESCs – what do the differences and similarities tell us? Research update on current advancements in Stem Cell research David L. Stocum Cell Stem Cell STEM CELLS npj Regenerative Medicin e A Brief History of Stem Cell Research