Exam 1: Tissue Morphology/Cells & Culturing Flashcards
What is embryogenesis?
-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.
What are blastomeres?
New cells arise from early divisions. They are todipotent-capable of forming an entire organism.
What is a blastocyst?
-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.
Blastula
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.
What are the three germ layers?
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
Nucleus
Control center that houses DNA, controls cell division, growth, protein production, & cell death
Ribosomes
produce proteins by assembling ammino acid sequences according to the genetic code
Mitochondria
Mitochondria are the site of respiration and the ‘powerhouses’ of cells, pumping out energy which is then stored in ATP (adenosine triphosphate).
Endoplasmic Reticulum
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.
Golgi Apparatus
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.
Lysosomes
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
Cytoplasm
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
Plasma Membrane
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
Cytoskeleton
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
What happens when cells differentiate?
they attain a
specialized, mature characteristic form
* Different shape & size
* Different molecular content of cytoplasm
Basic Tissue Types:
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
Autonomous Specification
-internally endowed with capacity to achieve fate, doesn’t need outside stimulus.
Conditional Specification
-depends on interactions with other cells or materials
Induction: Methods in which cells influence each other
Diffusion of soluble signals, contact w/ ECM, direct cell-cell receptor contact.
Induction: Types
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)
Cell Adhesion
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
Cell Migration
Requirements
* Generation of mechanical force
* Traction with another surface
Very related to adhesion
Invagination
Cell tissue moves inwards to form a bean shape from a sphere
Involution
Tissue compresses on either side of a sphere to form a bubble
Epiboly
tissue moves in from the side to fill the interior of a sphere
Delamination
tissue separates horozontally
Diffusion of Soluble Signals
Diffusion of signaling molecules, or morphogens, creates spatial
gradients
§ Cells sense their position in the gradient and develop accordingly
Constraints on Morphogenesis
Number of cells that participate
§ Physical dimensions of spontaneous cellular
rearrangements
§ Time scale over which morphogenesis takes
place
Types of Cells
Undifferentiated cells
* Responsible for replenishing old, injured, or dead
cells
§ Differentiated cells
* Specialized cells that perform a unique function in
the body
Properties of Stem Cells
Self renewal and differentation
Stem Cell Self Renewal:
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
todipotent
Generates every cell in the body including the
placenta and extra-embryonic tissue
Can form the entire human being,
from zygotes, cannot self renew
pluripotent
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
Multipotent
broad potential, self renewing
differentiated
non mitotic, functional
Types of Differentiation
Dedifferentiation- undoing dif.
Redifferentiation- undone, then redone
Transdifferentiation- moving from one specialization to another
Epigenetics
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
Types of Stem Cells:
Embryonic, Adult, and induced pluripotent
Embryonic Stem Cells
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
Adult stem cells
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
iPSC(induced pluripotent)
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)
ES Cell markers
OCT-4 (transription factor)
Alkaline phosphatase
Telemerase
Where can we harvest cells?
Autograft, Allograft, Xenograft
Harvesting Methods:
§ 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)
Tissue Disassociation techniques
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.
Cell sorting: Selective adhesion
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
Cell Sorting: Antibody Driven
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
HeLa Cells
first cells successfully cultured
in vitro
* Cervical cancer cells
* Highly proliferative/aggressive/hard to kill off
Cell Culture Media Components
- Water
* Pure water is needed - Balanced Salts (NaCl, CaCl2, KCl, NaHCO3)
* Provide a physiologic ionic environment
* Maintains intra- and extra-cellular osmotic balance
* Physiological osmolality: ~285 mOsm/kg H2O - Energy Molecules (Glucose)
* Glucose most common substance
* Average amount of glucose added is ~5g/L
* Other energy substrates are also often
included (sodium pyruvate) - 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 - pH Change Indicators
* Phenol Red (yellow acidic, purple basic) - 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) - Amino Acids (AA)
* Essential AAs cannot be synthesized by the cells
themselves, so must be added to media - Vitamins
* Most common vitamins in media include Bvitamins (thiamin, folic acid, etc.) and
Vitamin C - 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.
- Conditions the incubator maintains
for mammalian cell growth:
37°C
* 95% Humidity
* 5% CO2
Flask Names
T-Flask (T-#), where # = growth area in cm2
* Petri Dish (P-#), where # = growth area in cm2
cell proliferation is constrained by what factors?
- Cell density
- Nutrient availability
- Waste production
Cell Signaling
: initiated by the generation of a
ligand, which is produced by a sending cell to
alter the physiology of a receiving cell.
Types of Signaling:
Differentiation
Proliferation
Protein Secretion
*and migrate and die
Signaling depends on…
Ligand concentration & other ligand type exposures/environmental factors
Types of ligands
§ ECM molecules
§ Membrane-bound ligands
§ Diffusible molecules
Soluble Signals
§ 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
Receptor
Receptor: transmembrane protein
* Interacts with ligand/signal in extracellular space
* Transduces signal into cell
Autocrine
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.
Paracrine
a form of cell signaling, a type of cellular communication in which a cell produces a signal to induce changes in nearby cells
Endocrine
the signaling molecules (hormones) are secreted by specialized endocrine cells and carried through the circulation to act on target cells at distant body sites
Signal Transduction
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
Adhesion impacts:
Migration
* Cell-cell aggregation
* Cell & molecular transport
Cadherins
Cadherins: transmembrane
proteins that mediate cell-cell
adhesion
* Homophilic binding
* Requires Ca2+
§ Examples:
* E-cadherin
* P-cadherin
* S-cadherin
Ig-Like Receptors
Cell surface receptors mediate
cell-cell adhesion
* Homophilic binding
* Does not require Ca2+
§ Example:
* NCAM (Neural Cell Adhesion
Molecule)
Selectins
Transmembrane proteins that
mediate cell-cell adhesion
* Heterophilic binding
Integrins
Heterodimer - α and β sub-units
§ Extracellular domain binds ECM
protein
§ Cytoplasmic domain connects
cytoskeletal proteins
§ Bind to small peptide fragments
ECM Composition
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.
Collagen
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.
Fibronectin
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
Structural Proteins
Elastin – gives elasticity, found in tissues that
undergo repeated stretching (e.g., blood vessels)
Adhesive Proteins
- 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
Focal Adhesions
Integrin-containing structures that
form mechanical links between intracellular actin
bundles and the extracellular substrate
How does the ECM influence adhesion mediated signaling?
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
Non-reproducible measurement of adhesion
Detaching cells by washing
§ Detaching cells through “flicking”
Cell migration
- Lamellipodia protrude
* Increased number of actin
polymerization - Adhesion to the matrix
through integrins
* Provides a link to actin
cytoskeleton - Contraction of cytoplasm
by myosin-based motors
* Expressed as a traction force
on substrate - Rear release and
displacement
* Enzymatic processes break
the complex or the
membrane rips leaving
integrins behind - Recycling of remaining
integrins
* Integrins transported to
leading edge
Optimal adhesion
Biphasic dependence
§ Weak adhesion
* New attachments can’t form
at leading edge
§ Strong adhesion
* Rear attachments can’t release
Modes of Migration
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
