Exam 1: Tissue Morphology/Cells & Culturing

Question 1

What is embryogenesis?

Answer 1

-Starts just after fertilization
-Cells go through cleavage (division, but different in that the cells don’t increase in volume, just in number)
-Upon reaching uterus, gastrulation occurs and organs start to develop.

Question 2

What are blastomeres?

Answer 2

New cells arise from early divisions. They are todipotent-capable of forming an entire organism.

Question 3

What is a blastocyst?

Answer 3

-Forms after multiple divisions, pre-implantation
-has a blastocele (fluid filled cavity), trophoblast(surrounds blastocele, forms the placenta) and an internal cell mass (pluripotent cells that will form the entire embryo)
-the internal cell mass from a blastocyst can be isolated; it’s the source of embryonic stem cells.

Question 4

Blastula

Answer 4

Cells migrate to establish 3 germ layers:
Gastrolation is occurring, endoderm moved in. Ectoderm encloses the others. Mesoderm elongated. A new pocket forms (archenteron will be the gut
-Neurulation is also occurring during gastrolation, forms mural tube that will become the spinal cord and brain. Neural crest is created. Technically a fourth germ layer.

Question 5

What are the three germ layers?

Answer 5

Ectoderm-outer skin, parts of eye, pituitary gland
Mesoderm-connective tissue, heart &blood vessels, bone, muscle, kidney, cartilage
Endoderm-liver, pancreas, thyroid, bladder lining, respiratory tract
Neural Crest Cells-neurons, glial cells

Question 6

Nucleus

Answer 6

Control center that houses DNA, controls cell division, growth, protein production, & cell death

Question 7

Ribosomes

Answer 7

produce proteins by assembling ammino acid sequences according to the genetic code

Question 8

Mitochondria

Answer 8

Mitochondria are the site of respiration and the ‘powerhouses’ of cells, pumping out energy which is then stored in ATP (adenosine triphosphate).

Question 9

Endoplasmic Reticulum

Answer 9

The rough ER is studded with ribosomes, giving it a bumpy or ‘rough’ appearance. It folds and tags newly-synthesized proteins before transporting them to wherever they are needed in the body. The smooth ER does not have ribosomes attached to it and is instead involved in hormone and lipid synthesis.

Question 10

Golgi Apparatus

Answer 10

a series of flatted, membrane-bound sacs that packages and distributes substances to the outer cell membrane, where they either become part of the lipid bilayer or leave the cell.

Question 11

Lysosomes

Answer 11

small, spherical organelles that are packed full of digestive enzymes. Their key function is to break down and recycle unwanted material for the cell, such as old cell parts or invading bacteria and viruses. Lysosomes also play an important role in apoptosis

Question 12

Cytoplasm

Answer 12

jelly-like substance that fills the interior space of cells. It is mainly composed of water, but also contains salts, enzymes, and other organic molecules. The cytoplasm surrounds and protects the organelles of the cell and is where many cellular processes (such as protein synthesis and glycolysis) take place

Question 13

Plasma Membrane

Answer 13

semipermeable phospholipid bilayer. The cell membrane controls which substances enter and leave the cell, and also separates the interior of the cell from its external environment

Question 14

Cytoskeleton

Answer 14

a structure that helps cells maintain their shape and internal organization, and it also provides mechanical support that enables cells to carry out essential functions like division and movement. There is no single cytoskeletal component. Rather, several different components work together to form the cytoskeleton
Microtubules are the largest type of filament, with a diameter of about 25 nanometers (nm), and they are composed of a protein called tubulin. Actin filaments are the smallest type, with a diameter of only about 6 nm, and they are made of a protein called actin. Intermediate filaments, as their name suggests, are mid-sized, with a diameter of about 10 nm. Unlike actin filaments and microtubules, intermediate filaments are constructed from a number of different subunit proteins

Question 15

What happens when cells differentiate?

Answer 15

they attain a
specialized, mature characteristic form
* Different shape & size
* Different molecular content of cytoplasm

Question 16

Basic Tissue Types:

Answer 16

Epithelium - continuous 2D sheet of cells, dont move individually, (blood vessel, skin, bladder, kidney lining)
Connective tissue - offer mechanical support, arise from the mesoderm, (bone, ligaments)
Muscle - smooth, cardiac, skeletal
Nervous tissue - CNS- spinal cord & brain, PNS

Question 17

Autonomous Specification

Answer 17

-internally endowed with capacity to achieve fate, doesn’t need outside stimulus.

Question 18

Conditional Specification

Answer 18

-depends on interactions with other cells or materials

Question 19

Induction: Methods in which cells influence each other

Answer 19

Diffusion of soluble signals, contact w/ ECM, direct cell-cell receptor contact.

Question 20

Induction: Types

Answer 20

Negative- collective of cells that restrict potential of each other
Instructive- A responder cell changes due to interaction
Permissive - Responder cells have all potential but require environment
Reciprocal - tissues signal each other
(NIPR)

Question 21

Cell Adhesion

Answer 21

Adhesion molecules enable specific binding
between surface of a cell and neighboring cells
or extracellular matrix
* Integrins – primarily involved in adhesion to
extracellular matrix
* Cadherins – involved in cell-cell adhesion

Question 22

Cell Migration

Answer 22

Requirements
* Generation of mechanical force
* Traction with another surface
Very related to adhesion

Question 23

Invagination

Answer 23

Cell tissue moves inwards to form a bean shape from a sphere

Question 24

Involution

Answer 24

Tissue compresses on either side of a sphere to form a bubble

Question 25

Epiboly

Answer 25

tissue moves in from the side to fill the interior of a sphere

Question 26

Delamination

Answer 26

tissue separates horozontally

Question 27

Diffusion of Soluble Signals

Answer 27

Diffusion of signaling molecules, or morphogens, creates spatial gradients § Cells sense their position in the gradient and develop accordingly

Question 28

Constraints on Morphogenesis

Answer 28

Number of cells that participate § Physical dimensions of spontaneous cellular rearrangements § Time scale over which morphogenesis takes place

Question 29

Types of Cells

Answer 29

Undifferentiated cells * Responsible for replenishing old, injured, or dead cells § Differentiated cells * Specialized cells that perform a unique function in the body

Question 30

Properties of Stem Cells

Answer 30

Self renewal and differentation

Question 31

Stem Cell Self Renewal:

Answer 31

Symmetric Division- daughter cells have identical genetic makeup, can differentiate Asymmetric division- one daughter copy, one new different cell-can be caused by intrinsic mechanisms: localization of cell polarity an desegregation of cell fate determinants, or extrinsic mechanisms: signals from ECM such as spacal and soluble factors

Question 32

todipotent

Answer 32

Generates every cell in the body including the placenta and extra-embryonic tissue Can form the entire human being, from zygotes, cannot self renew

Question 33

pluripotent

Answer 33

Can generate every cell in the body except the placenta and extra-embryonic tissue Cannot form the entire human being embryonic stem cells from blastocyst inner cell mass, self renewing

Question 34

Multipotent

Answer 34

broad potential, self renewing

Question 35

differentiated

Answer 35

non mitotic, functional

Question 36

Types of Differentiation

Answer 36

Dedifferentiation- undoing dif. Redifferentiation- undone, then redone Transdifferentiation- moving from one specialization to another

Question 37

Epigenetics

Answer 37

Stable alterations in gene expression potential that arise during development without altering the DNA sequence * Regulated by: § DNA methylation - cytosines get methyl groups, methylation patterns passed on to daughter cells during division § Histone modifications - histones provide structural support for chromosomes, modification makes chromatin more condensed, less genes available for transcription, also caused by methyl groups

Question 38

Types of Stem Cells:

Answer 38

Embryonic, Adult, and induced pluripotent

Question 39

Embryonic Stem Cells

Answer 39

Embryonic stem cells are derived from inner cell mass blastocyst * Can self-renew indefinitely in culture * Embryonic stem cells used in research are made in a petri dish, not in a woman’s body Needs a feeder layer Advantages * Indefinite proliferation capacity * Pluripotency § Shortcomings * Mouse origin * Human origin § Culture conditions § Immune rejection § Purification of homogenous cell population § Ethical concerns

Question 40

Adult stem cells

Answer 40

undifferentiated cells, which reside in tissue/organs * Hematopoietic stem cells: blood cells, Found in: * Bone marrow * Umbilical cord blood * Peripheral blood § Produces all red and white blood cells, platelets § HSC are identified by the surface marker CD34 * Mesenchymal stem cells: bone and muscle

Question 41

iPSC(induced pluripotent)

Answer 41

isolate tissue, reprogram and wait, the cells become stem cells Advantages: * Cells would be genetically identical to patient or donor of skin cells (no immune rejection!) * Do not need to use an embryo § Shortcomings: * Cells would still have genetic defects * One of the pluripotency genes is a cancer gene * Viruses might insert genes into places where we don’t want them (causing mutations)

Question 42

ES Cell markers

Answer 42

OCT-4 (transription factor) Alkaline phosphatase Telemerase

Question 43

Where can we harvest cells?

Answer 43

Autograft, Allograft, Xenograft

Question 44

Harvesting Methods:

Answer 44

§ Bone Marrow Aspirate * Only need local anesthesia (in upper hip or sternum) * Easily accessible * Use large needle + heparinized syringe (to stop blood clot) typically yielding about 2 mL of marrow * Marrow contains mesenchymal stem cells, hematopoietic cells, adipocytes, endothelial progenitor cells § Uroepithelial Cell Flush-out * No anesthesia required * Method involves repeated filling/emptying with isotonic salt solution § Tissue Biopsies * Skin; take a biopsy punch * Cartilage; arthroscopy to harvest tissue * Heart/Liver/Kidneys (etc.) can use fluoroscopy (to guide to location) and catheter (to retrieve sample)

Question 45

Tissue Disassociation techniques

Answer 45

Cells from tissue biopsies must be dissociated from each other and the ECM for growing in tissue culture plates Mechanical Disruption * Vortex with a digestion buffer * Pipet vigorously * Dice with a scalpel * Enzymatic Disruption * Disrupt the Cell-ECM connections * Trypsin, proteases, collagenases, papain, dispase * Disrupt Cell-Cell connections * Chelating agents (such as EDTA) to disrupt cadherins * Explanted Tissues * May or may not require digestion or disruption. Cells may migrate outward on their own.

Question 46

Cell sorting: Selective adhesion

Answer 46

Isolate desired cell types by manipulating adhesion characteristics § Isolated based on differences based in: * Cell surface receptors * E.g., Coat TC plate with protein that some cells will bind to stronger than others * Adhesion speed * E.g., Remove cell suspension at a given time interval § Pros: Does not require staining or special equipment § Cons: Limited to highly active cells Cell Selection: Antibody Driven

Question 47

Cell Sorting: Antibody Driven

Answer 47

Select a target antigen on cell surface >Magnetic-Activated Cell-Sorting: Label cells with magnetic bead-conjugated antibodies and separate with strong magnetic field >Fluorescence-Activated Cell Sorting (FACS): * Label cells with fluorescently conjugated antibody * Flow through machine to sort fluorescent vs. non-fluorescent

Question 48

HeLa Cells

Answer 48

first cells successfully cultured in vitro * Cervical cancer cells * Highly proliferative/aggressive/hard to kill off

Question 49

Cell Culture Media Components

Answer 49

1. Water * Pure water is needed 2. Balanced Salts (NaCl, CaCl2, KCl, NaHCO3) * Provide a physiologic ionic environment * Maintains intra- and extra-cellular osmotic balance * Physiological osmolality: ~285 mOsm/kg H2O 3. Energy Molecules (Glucose) * Glucose most common substance * Average amount of glucose added is ~5g/L * Other energy substrates are also often included (sodium pyruvate) 4. Buffering Agents * Role is to maintain the pH of cells (typically at 7.2-7.4) * Carbonates, phosphates, and citrates are examples of buffering agents * Most common buffer system is bicarbonate buffer system 5. pH Change Indicators * Phenol Red (yellow acidic, purple basic) 6. Anti-fungal/bacterial substances * Prevents Infections * Ex: Penicillin/Streptomycin; Gentamicin * Not always added since they can influence cells (e.g., altered cell metabolism and increased stress) 7. Amino Acids (AA) * Essential AAs cannot be synthesized by the cells themselves, so must be added to media 8. Vitamins * Most common vitamins in media include Bvitamins (thiamin, folic acid, etc.) and Vitamin C 9. Growth Factors (depends on which type of cells culturing) * Fetal Bovine Serum o ~10% added to cell culture media o Provides a wide variety of macromolecular proteins, nutrients, hormones * Custom Growth Factor Cocktails o For endothelial cells, would include VEGF, bFGF, etc.

Question 50

* Conditions the incubator maintains for mammalian cell growth:

Answer 50

37°C * 95% Humidity * 5% CO2

Question 51

Flask Names

Answer 51

T-Flask (T-#), where # = growth area in cm2 * Petri Dish (P-#), where # = growth area in cm2

Question 52

cell proliferation is constrained by what factors?

Answer 52

* Cell density * Nutrient availability * Waste production

Question 53

Cell Signaling

Answer 53

: initiated by the generation of a ligand, which is produced by a sending cell to alter the physiology of a receiving cell.

Question 54

Types of Signaling:

Answer 54

Differentiation Proliferation Protein Secretion *and migrate and die

Question 55

Signaling depends on...

Answer 55

Ligand concentration & other ligand type exposures/environmental factors

Question 56

Types of ligands

Answer 56

§ ECM molecules § Membrane-bound ligands § Diffusible molecules

Question 57

Soluble Signals

Answer 57

§ Cytokines: Small molecules (5-20 kDa) that influence cell-cell interactions & cell behavior § Chemokines: A subset of small molecular weight cytokines that stimulate migration § Mitogen: Small molecules that promote mitosis § Growth factors: substances capable of stimulating cell proliferation, maturation, and/or differentiation

Question 58

Receptor

Answer 58

Receptor: transmembrane protein * Interacts with ligand/signal in extracellular space * Transduces signal into cell

Question 59

Autocrine

Answer 59

the production and secretion of an extracellular mediator by a cell followed by the binding of that mediator to receptors on the same cell to initiate signal transduction.

Question 60

Paracrine

Answer 60

a form of cell signaling, a type of cellular communication in which a cell produces a signal to induce changes in nearby cells

Question 61

Endocrine

Answer 61

the signaling molecules (hormones) are secreted by specialized endocrine cells and carried through the circulation to act on target cells at distant body sites

Question 62

Signal Transduction

Answer 62

During transduction, the signal is amplified One receptor-ligand interaction leads to the activation of many downstream proteins Binding>enzymatic activity>protein modification>second messenger>proteolysis>protein relocation

Question 63

Adhesion impacts:

Answer 63

Migration * Cell-cell aggregation * Cell & molecular transport

Question 64

Cadherins

Answer 64

Cadherins: transmembrane proteins that mediate cell-cell adhesion * Homophilic binding * Requires Ca2+ § Examples: * E-cadherin * P-cadherin * S-cadherin

Question 65

Ig-Like Receptors

Answer 65

Cell surface receptors mediate cell-cell adhesion * Homophilic binding * Does not require Ca2+ § Example: * NCAM (Neural Cell Adhesion Molecule)

Question 66

Selectins

Answer 66

Transmembrane proteins that mediate cell-cell adhesion * Heterophilic binding

Question 67

Integrins

Answer 67

Heterodimer - α and β sub-units § Extracellular domain binds ECM protein § Cytoplasmic domain connects cytoskeletal proteins § Bind to small peptide fragments

Question 68

ECM Composition

Answer 68

Proteoglycans: protein-sugar complex- Complex that consist of a protein molecule to which chains of GAGs are covalently attached. -Glycosaminoglycans (GAGs): * High molecular weight polysaccharides, which are usually highly sulfated and thus negatively charged § Proteins: extended, fibrous § Other: growth factors, digestive enzymes, etc.

Question 69

Collagen

Answer 69

Constitutes >25% of all protein in humans * High tensile strength * More than 20 different types * Basic structure: alpha helical chains formed by interaction of three polypeptides * Fibrillar collagens – collagen monomers are staggered and crosslinked.

Question 70

Fibronectin

Answer 70

Dimer: two similar polypeptides that are covalently linked together * Subunits linked by disulfide bonds § Each chain contains binding sites for: * Other ECM components * Cell surface receptors

Question 71

Structural Proteins

Answer 71

Elastin – gives elasticity, found in tissues that undergo repeated stretching (e.g., blood vessels)

Question 72

Adhesive Proteins

Answer 72

* Laminin – adhesive peptides RGD, YIGSR, IKVAV * Tenascin – adhesive and anti-adhesive properties * Entactin- found in basement membranes in association with laminin, contains RGD * Thrombospondin – RGD sequence * Vitronectin – present in blood vessels & other tissues, RGD sequence

Question 73

Focal Adhesions

Answer 73

Integrin-containing structures that form mechanical links between intracellular actin bundles and the extracellular substrate

Question 74

How does the ECM influence adhesion mediated signaling?

Answer 74

ECM composition (which proteins are present) dictate integrin binding § ECM concentration § ECM mechanical properties (stiffness/elasticity) § Protein confirmation/variants Extent of cell spreading (i.e., cell area) § Formation of actin filament bundles § Presence of focal adhesions

Question 75

Non-reproducible measurement of adhesion

Answer 75

Detaching cells by washing § Detaching cells through “flicking”

Question 76

Cell migration

Answer 76

1. Lamellipodia protrude * Increased number of actin polymerization 2. Adhesion to the matrix through integrins * Provides a link to actin cytoskeleton 3. Contraction of cytoplasm by myosin-based motors * Expressed as a traction force on substrate 4. Rear release and displacement * Enzymatic processes break the complex or the membrane rips leaving integrins behind 5. Recycling of remaining integrins * Integrins transported to leading edge

Question 77

Optimal adhesion

Answer 77

Biphasic dependence § Weak adhesion * New attachments can’t form at leading edge § Strong adhesion * Rear attachments can’t release

Question 78

Modes of Migration

Answer 78

Chemotaxis: Movement along a soluble chemical concentration gradient § Haptotaxis: An insoluble chemical concentration gradient § Durotaxis: Substrate stiffness gradient Galvanotaxis: Electric current § Contact Guidance: Surface topography § Contact Inhibition: Lamellipodium inhibited by neighboring adjacency