Lecture_Set3 - Embryonic Development In vitro fer9liza9on(concep9on in a dish Stem Cells Shyni Varghese Embryonic Development Embryonic Development ~12

Lecture_Set3 - Embryonic Development In vitro...

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Unformatted text preview: 10/28/15 Embryonic Development •  In vitro fer9liza9on (concep9on in a dish) Stem Cells Shyni Varghese Embryonic Development Embryonic Development ~12 hrs: two genetic material ------ pronuclei 18-20 hrs: fusion of pro-nuclei combining genetic material Two cells Day 1: Fer9liza9on one cell Fertilized egg or zygote 1 10/28/15 Embryonic Development ~ Day 2: Zygote has divided into two cells. These two cells are identical to each other Embryonic Development Embryonic Development Further division into four identical cells Embryonic Development ~ day 3: Eight identical cells 2 10/28/15 Embryonic Development Embryonic Morphogenesis REVIEWS a Surface δS δ E = σδ S First, we present a historical perspective on tissue surface tension during morphogenesis. We then focus on local cell behaviours and the control of intercellular surface tension by adhesion and cortical tension. We also detail how spatial patterns of cell shape that arise in tissues can be Cellsexplained in this context, and how cell shape based sorted and aggregated controls tissue geometry. expression molecules Tissue surface tension and cell sorting Bulk b Cell shape and the geometry of cell aggregates show striking similarities with fluids and soap bubbles, as observed nearly 100 years ago by D’Arcy Thompson in his book On Growth and Form7. The suggested general principles that underlie the control of tissue organization were based on surface tension properties. However, surface tension might refer to different biological entities, Thomas Lecuit in a tissue or an individual for example, a group of cells and Pierre-François Lenne cell8. In this section, we introduce the concept of surface Nat. Cell Biol. 8, 633, 2007 tension as a global tissue property that is controlled by cell–cell interactions. The tissue is simply viewed as a collection of cells with generic properties (adhesion) without consideration of their individuality (cell shape Cell surface mechanics and the and mechanics). Plasticity The ability to undergo a persistent deformation. Embryonic Stem Cells Tissue homeostasis c The property of biological tissues to remain structurally and functionally stable in a physiological environment. Sorting The ability of intermixed, adhesive and mobile cell populations to separate into immiscible adjacent tissues. d control of cell shape, tissue Surface tension The free-energy change when the surface of a medium is increased by a unit area. Strictly speaking, the term ‘interfacial tension’ should be used instead of ‘surface tension’ when the liquid adjoins another liquid or a solid. For simplicity, we use the term ‘surface tension’ in this article. Tissue surface tension The apparent surface tension of a tissue, caused by cohesive interactions (adhesion) between cells. Increasing adhesion in a tissue increases tissue surface tension. Intercellular surface tension The apparent surface tension of two cells that are in contact, caused by the opposite effects of cortical tension and intercellular adhesion. In contrast to the case of tissue surface tension, increased adhesion lowers intercellular surface tension. Figure 1 | Analogy between fluids and tissues. a | Molecular explanation of surface tension in a liquid. In the bulk of a liquid, molecules are, on average, equilibrated by interacting cohesive forces from surrounding molecules (black arrows). At the surface of the liquid, the molecules experience an imbalance of force, with a resultant force (red arrow) that tends to push them away from the surface, thereby introducing a surface tension. The energetic cost δ E for a change δ S of the liquid surface area is simply related to the surface tension σ. b | Cells become sorted and aggregate according to the expression levels of cadherins at their surface. Red cells, which express more cadherins and therefore form stronger cell–cell contacts than grey cells, are surrounded by the grey cells. c | A red cell at the interface between the two cell populations (top) has less favourable interactions with similar cells than a red cell in the tissue interior (bottom). This results in tissue surface tension. By adopting a spherical shape, the red tissue minimizes the interface area with the surrounding grey tissue. d | Sorting behaviour of grey cells that express a given cadherin at their surface and red cells that express a different cadherin. The preferential association of cells that express the same cadherins cause cell sorting according to the differential adhesion hypothesis. Cortical tension The apparent cell surface tension due to the contractile microfilaments of the cell cortex and their interaction with the membrane. Affinity The tendency of cells with similar developmental origins to aggregate. The morphogenetic processes that are described in this Review develop over medium (minute) to long timescales (minutes to hours). It has been shown that, in the case of these medium-to-long timescale processes, the elastic response of the cell — which predominantly occurs over short timescales (seconds) — can be neglected and cells behave like viscous fluids with an equilibrium shape that is dictated by surface tension5,6. The concept ofpatterns and morphogenesis surface tension in groups of cells. A liquid droplet, such as an oil droplet in water, is spherical (FIG. 1a); with this shape, it minimizes its contact area with the surroundings and, therefore, minimizes its surface energy. Increasing the surface area of the drop requires an energy δE, which is proportional to the area increment δS, such that δE=σδS, in which σ is the surface tension of the liquid. The coefficient σ has the dimension of energy per unit surface area and is expressed in Joules m–2. The physical significance of liquid surface tension can be related to the cohesive interactions between molecules in a liquid (Van der Waals forces, hydrogen bonds, ionic interactions9; FIG. 1a, arrows). In the bulk of a liquid, the intermolecular forces that function on a molecule are, on average, balanced. However, these forces are no longer equilibrated at the surface of the liquid — the molecule loses interactions with similar molecules and gains interactions with dissimilar molecules. Surface-tension forces arise from this imbalance of short-distance intermolecular forces, with the resultant force tending to bring surface molecules back to the bulk, thus tensing the surface (FIG. 1a, red arrow). Surface tension causes molecular sorting at the boundary between two different liquids. Cells in a tissue are remarkably similar to molecules in a liquid10 such that, to a good approximation, tissues behave like fluids. First, cells tend to aggregate in clusters in which the surface area of contact with the surrounding environment is minimized. Second, different cell populations can become sorted into two phases like immiscible fluids (FIG. 1b). By analogy to liquids, it is possible to define a tissue surface tension, which can explain cell aggregation and cell sorting4. In the 1940s and 1950s, Holtfreter and colleagues showed that tissues separate by affinity11, a term used by Holtfreter in reference to analogous phase-separation phenomena in chemistry. In a remarkable series of experiments, Stem Cells 634 | AUGUST 2007 | VOLUME 8 This cell Can form the entire human being This cell Cannot form the entire human being Blastocyst To2potent Cells Inner Cell Mass 3. Umbilical cord blood stem cells 4. Amnio2c fluid stem cells Fetus 1. Adult stem cells Primordial Germ Cells 2. Pluripotent stem cells 3 10/28/15 Symmetric cell division Asymmetric cell division 1.  Self-renews 2.  Differentiates Progenitor cell Making more copies of themselves Stem Cells •  Embryonic Stem Cells (ESCs) •  Induced Pluripotent Stem Cells (iPSCs) •  Adult Stem Cells Stem cell Stem cell Pluripotent stem cells •  Under appropriate culture condi9ons will proliferate indefinitely and remain “normal” with an uncompromized karytype •  Maintain their pluripotency. •  They can give rise to all cell lineages 4 10/28/15 Pluripotent Stem Cells Embryonic Stem Cells hESCs colony ESC colony •  Specific Characteris9cs – Markers • Oct 4, Nanog • EBD forma9on •  Differen9a9on •  Teratoma MEF Pluripotent Stem Cells Pluripotent stem cells Embryoid body (EBD) from hESCs ectoderm 200 μm 100 μm 200 μm endoderm mesoderm skin liver nerves lungs eyes muscles blood lining of bones/car9lages gut 5 10/28/15 Pluripotent Stem Cells Pros and Cons of ESC •  Ectoderm •  Mesoderm •  Endoderm Pros •  Unlimited cell source •  Can differentiate into all cell types in human body •  Ectoderm: outermost of the three germ layers Cons •  Allogenic transplantation •  Histocompatible cells for transplantation •  Banks of undifferentiated or differentiated derivatives of ESC •  Teratoma •  Ethical concerns –  Nerve system, epidermis •  Mesoderm: Middle layer between ecto and endo –  Connec9ve 9ssues, car9lage, bone •  Endoderm: Innermost layer –  Lung, pancreas Induced pluripotent stem cells (iPSCs) Induced pluripotent stem cells (iPSCs) Genetically engineering new cells Oct3/4, Sox2, Klf4, and c-Myc (via retroviral system) OCT4, SOX2, NANOG, and LIN28 ((via retroviral system) •  Takahashi K, et al. (2007). "Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors". Cell 131: 861, 2007 Skin cells iPS cells •  Yu J, Vodyanik MA, et al. "Induced Pluripotent Stem Cell Lines Derived from Human Soma9c Cells". Science 318: 1917, 2007 6 10/28/15 Limitations •  •  •  •  Low throughout Efficiency Incomplete reprogramming memory Pros and Cons to iPS cell technology •  Pros: –  Cells would be gene9cally iden9cal to pa9ent or donor of skin cells (no immune rejec9on!) –  Do not need to use an embryo •  Cons: –  One of the pluripotency genes is a cancer gene –  Genomic inser9on: Viruses might insert genes in places we do not want them (causing muta9ons) Technology for iPSCs •  Alternate forms of vectors –  Adenovirus, plasmids, etc •  Delivery of TF’s using chemicals –  Small molecules –  Polymer Adult stem cells •  Mul9potent •  Unipotent PROS ad CONS •  No teratoma •  Could be autologous •  Cant differen9ate into all cell types •  Limited expansion poten9al •  Limited cells source 7 10/28/15 Bone marrow •  Two distinct progenies housed in BM –  Hematopoietic stem cells (HSCs) –  MSCs •  Mesenchymal Stem Cells •  Mesenchymal Progenitor Cells •  BM stromal stem cells •  The International Society for Cellular Therapy, Vancouver, Canada, has recommended the use of the name multipotent mesenchymal stromal cell Mul9potent stem cells •  Ability to differentiate into multiple cell types •  HSC --- haematopoietic stem cells –  can give rise to all blood cell components, including neutrophils, lymphocytes, natural killer cells, dendritic cells, macrophages and monocytes. •  MSC –  can give rise to osteoblasts, chondrocytes, adipocytes, and others •  MSC is a very small population in BM; 0.001 0.01% of the total nucleated cells Mesenchymal Stromal Cells Bone marrow derived Mesenchymal Stem Cells (MSCs) •  Characteristics –  Plastic adherent –  Fibroblast-like cells –  clonal expansion (colony forming unit fibroblast, CFU-F) –  self-renewal –  Differentiation into multiple cell types •  Morphologically, they are, spindle shape like fibroblasts 8 10/28/15 Mesenchymal Stem Cells •  BM derived MSCs show Immuno-modulatory effect –  acute grak vs. host disease (GVHD) –  allogenic heamtopoie9c cell transplanta9on Mesenchymal Stem Cells •  Differen9a9on •  Trophic factors Suppression of T cell prolifera9on MSC-secreted morphogens Cell-cell contact Apoptosis of ac9vated T cells Increase tolerance than immunity 9 ...
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