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Cross-section through layers of cells

· Yellow = accumulation of proteins, forming a filter/barrier between epithelium and underlying connective tissue

· Above the barrier, epithelial cells are densely packed as a protective layer. If the pink are nuclei and dark are membranes to make a cell, connective tissue is mostly not cells. There is a lot of space that makes up the ECM. Molecules need to fill this space. Glycoaminoglycans (GAGs): refers to hyaluronan, proteoglycans, and glycoproteins.

· All tissues have fibroblasts; main function is to supply molecules to ECM. Elastic fiber is a fibrous protein that has some elasticity. Collagen is a structural rope-like protein to help hold things together.

· Function of ECM is to hold things together by making connections between cells and other components, and gives structure and stability to ECM (support between cells). This is what the GAGs do

Glycoaminoglycans (GAGs)

· Unbranched polysaccharide chains of repeating disaccharide units. Do not need to know specific names of sugars

· Repeated on average 70-200 sugars long. Simple to make, do not take too much energy. Long straight chains, easily folded, can fit into many spaces

· Characteristics: large, lots of negative charges (carboxyl, sulfate groups) which are important to their function in ECM

· Function: Take up space in ECM, attract cations (usually Na). Sodium accumulates in ECM, which then attracts water. This attracted water gives the organism turgor pressure (tissue can be compressed while still maintaining its structure).

· Also help regulate secreted proteins from cells. GAGs create channels that the proteins can travel through. Can be signaling proteins trying to get into bloodstream

· Create caverns of various sizes, can direct movement of proteins. Can also bind proteins as they move through ECM. Can be accumulated in an area that can enhance their activity, but also reduce their activity somewhere. GAGs can store them for future use

Hyaluronan

· GAG composed of repeats of up to 25,000. it is not sulfated (lacking some negative charges). Also not linked to any proteins

· Its role is to resist compressive forces in joints and other areas under high stress. Also space filling molecule during development and wound healing

· Composed of repeating disaccharide unit without sulfates

Proteoglycans

· Other kind of GAGs. Attached to a core protein before being secreted. Covalent linkage between an amino acid in protein (usually a serine) and repeat disaccharides attach using a linker tetrasaccharide. Come in many sizes and shapes

· Come in many shapes and sizes, but functionally doing the same thing (taking up space, providing turgor pressure)

(42 genes) fibrillar or fibrillar-associated, network, anchoring

· Proteins found in ECM. Most abundant is collagen. Rope-like structures that go in all different directions to hold ECM together. Collagen is extremely dense, relatively inflexible molecule. Can bend but not stretch

Collagen

· Exons = 54 nucleotides

· (6 Gly-X-Y repeats)

· It is composed of multiple strands made up of 3 subunits. Sequence glycine X and Y (which can both be proline). Proline forms kinks in backbone of polypeptide to direct orientating of next amino acid

· Collagen forms helical structures, but not a typical alpha helix. Glycine is needed because it's small enough to fit between the 3 strands. 3-stranded structure is wrapped around other 3-stranded structures

· Collagen is triple helical strands held together by covalent linkages

· Resists stretching and compression and holds things together

Elastin

· Opposite characteristics of collagen. Found around blood vessels. Very irregular compared to collagen.

· Irregular in structure because it is associated with tissues that are frequently stretching and relaxing, such as the lungs

· Still need to provide some structural integrity. Do not have secondary or tertiary structure, called random coils. Held together by red cross-links (covalent bonds) that hold structure together

Adhesive Proteins in the ECM

· Have to hold everything to each other and hold cells together in ECM

· Need adhesive proteins (2 types)

1. Fibronectin

2. Laminin

Laminin

· Yellow from first slide is a protein called laminin. Makes up basal lamina. Adhesive proteins secreted into ECM to form a barrier and surround things. Small molecules can still get through, but it still surrounds things (like a fence)

· Can be found around muscle fibers to separate it from connective tissue, barrier between epithelium and connective tissue. Between blood and kidney barrier. Works as a filter to allow certain things through

· Multi-subunit protein with many binding domains. Mostly self-associates to form sheets of basal lamina (binds with itself)

Crystallins

· Embryo developing has a region of cells that are exactly the same

· Called optic cell which is where eyes will grow. Ectoderm surrounds embryonic cell vesicle (hollow ball of cells that forms where eyes will develop). These are all cuboidal cells; one side of these cells will elongate. These are the cells that are closer to the retina, and will develop into the lens. Lens is disc shaped structure that filters light that comes in and focuses it on photoreceptor cells of retina.

· After a while, these elongated cells start making crystallin proteins (water soluble), that will completely fill primary lens fibers. Will fill up so much that other cellular functioning will stop. Are still alive, but all other cellular functions are inhibited.

· When it becomes an adult lens, there is a front (right) that is still cuboidal, capable of proliferation, and can still divide (proven using BrdU experiments)

· Stacked cells that developed during embryonic development, filled with proteins that are still present in adulthood and are used to focus the light

Photoreceptors of the eye

· Series of neurons that send electrical signals to brain to identify what is being looked at

· The photoreceptors receive the light coming into the eye after passing through several layers of neurons

· Retina has 120 million rods (yellow), responsible for seeing low light

· 6-7 million cones involved in seeing color

Rhodopsin-transducin

· Activating an electrical system using a protein called rhodopsin (G-coupled receptor); membrane-bound protein. G-coupled receptors are called 7 transmembrane domain proteins (have 7 alpha helices)

· Bound to a co-factor called retinal

· When a photon of light hits it, undergoes a conformational change that activates G-coupled protein (transducin)

· Begins a cascade of events that starts with hydrolysis of GTP in response to photon of light at rhodopsin that triggers depolarization of neuron which transmits information through interneurons to ganglion cells and to photoreceptors of the brain to indicate what is being viewed

Photoreceptor cells are receiving light all the time and can take damage

· Even though the cells are permanent, are the proteins at the top replaced?

· Pulse-chase experiment was done where triated leucine was fed to animals, taken up by their cells, and cells produced would be radioactively labeled with a leucine (red). Proteins produced are being sent into layer of membranes (lines) which is where G coupled receptors are embedded.

· Experiment showed that leucine is incorporated into proteins, and then proteins slowly migrate to the end. Are absorbed and recycled by pigmented epithelial cells at the top

· This means that photoreceptor cells, despite being permanent, experience turnover to keep them healthy and functioning

· Turnover occurs in form of proteins

Simple duplication

· Renewal by duplication

· Cells that have differentiated (mature cell type), but have maintained ability to divide. Examples are liver and endothelial cells

· Cells divide, but where is the cell in its path toward differentiation?

· Liver filters toxins, digests fats using bile (secretes into digestive system), stores glycogen (energy storage), pumps proteins into bloodstream (protein production), regulates blood glucose levels

· Mature liver cells are called hepatocytes (red), green = bile duct. Every liver cell is in contact with a capillary

Hepatocytes

· Blood supply (red), endothelial cells (blue) line circulatory system

· Hepatocytes (gray), bile ducts (yellow)

· Kupffer cells are a form of macrophage (immune cell) in liver; break down RBC. Also responsible for ethanol induced liver injury

· Greenish looking diamond shaped cells are fibroblasts; are present in balance between hepatocytes. Help in ECM production and overall liver function

· Liver can regenerate (2/3 of rat liver can be removed and will grow back). Once it gets back to original size, it will stop growing (will not over or undergrow). Can happen as many as 12 times in 1 rat, but can happen up to 50 times based on growth capabilities of cells grown in culture. Liver transplants are therefore one of the most successful transplants

Liver regeneration

· Small red dots in blue are called fenestrations (gaps/holes in endothelial cells that allow molecules to get through)

· When regrowth of liver is happening, something has to trigger hepatocytes (mature cells) to re-enter the cell cycle and divide

· Once we remove some of the liver, is the signal for liver growth coming from internal signal in remaining liver cells, or is there an external signal telling cells to grow back?

· Experiment: Using 2 rats, are connecting their blood flow to each other. Go into one rat, remove part of its liver. Expecting that missing liver to grow back since it's being exposed to a rat's blood supply that does have a complete liver

· Rat that did not have liver removed responded from rat that did have liver removed. Rat missing liver grew it back. This proves that this is controlled by an external signal.

Liver regeneration pathway

· Liver cell under normal conditions has membrane receptors implanted. Wishbone shaped receptor is a uPA receptor that binds a protease called urokinase, which becomes activated when liver damage occurs. Protease is an external protein in ECM, binds to uPA receptor. Active protease will start proteolytically attack proteins in ECM. One protein in ECM is called Pro-HGF (pro = inactive, precursor form; hepatocyte growth factor (HGF)).

· Cells preemptively position pro-HGF in ECM nearby. When liver takes damage, cell will increase number of uPA receptors in its membrane. These attract more protease that will digest ECM around cells (white balls). Protease cleaves Pro-HGF and activates it (black ball into sunglass shape)

· pro-HGF binds to c-Met (HGF receptor) present in hepatocyte membrane as a monomer. When HGF is activated, it binds c-Met as a dimer, triggering a cascade of signals that reach the nucleus and get hepatocyte to enter cell cycle (G0 -> G1/S). Still perform their functions as duplication occurs

· Protein called TGF-beta (transforming growth factor beta) that is rarely produced in normal hepatocytes. Before damage occurs, no TGF-beta is made. But when cell re-enters cell cycle, TGF-beta levels increase. It works as a stop signal and tells cells to exit cell cycle. Level of TGF-beta is proportional to number of cells growing back

· When damage to liver occurs, fibroblasts also need to be regenerated. Fibroblasts can grow back faster than the hepatocytes if liver damage is occurring over a long period of time. Fibroblasts continue to produce more and more collagen because they grow back faster than the hepatocytes. This leads to cirrhosis of the liver, and it can no longer function.

Endothelial cells represented in blue lining blood vessels

· Basal lamina (yellow) made of adhesive protein lamina. Separates blood supply from surrounding tissue. Is semi-permeable. Usually a size-dependent barrier. Small molecules (vitamins, minerals, nutrients) can get through, but there are also situations where whole cells need to get through. WBC need to get out of blood supply and into tissues if they're needed. Need to be able to digest through basal lamina

· Directed by endothelial cells and other cells called pericytes lead to development of other layers (elastin, flexible layer that allows blood vessels to expand and contract while maintaining shape). Expanding/contraction is controlled by smooth muscle to propel blood. If blood vessel gets big enough, loose connective tissue dense with collagen (light green) surrounds largest blood vessels

· Capillaries only have endothelial cells and basal lamina. Cells cannot be farther than 50 microns from a blood vessel in order to diffuse

· Lumen of capillary contains thousands of RBC

· Capillary has pores (holes) called fenestrations; gaps that control size of molecule getting through basal lamina into tissue

Pericytes

· Associated with capillaries, cells on outside are called pericytes. Wrap around capillary, some will penetrate through basement membrane and contact endothelial cells to communicate with them and regulate their function. Can also do this in a paracrine way by releasing signaling molecules that pass through a short distance. Pericytes release them and diffuse through to bind endothelial cells

Help maintain homeostatic and hemostatic (conditions in blood supply) function within capillaries. Abundance of them at blood-brain barrier to stop things from getting into CNS. Also clean up debris via phagocytosis around capillaries

Angiogenesis

· Growth of new blood vessels. Occurs after wound healing/tissue damage

· Cells can fuse and form capillary-like structures with openings that could allow for blood flow. Somewhere adjacent to existing blood flow, there is cell proliferation (can be good or bad). Additional cells need oxygen, way to get rid of waste, and nutrients, so they also need a blood supply. Group of cells too far from blood supply need to signal to existing blood supply to grow towards them. Signal is sent from cells in low oxygen situation. Signal binds to a receptor on endothelial cell surface. Mature cell types (endothelial) can undergo simple duplication after receiving this signal

· Endothelial cells start growing in direction of signal, releasing something called a pseudopodial process (extension of cell). Cell will divide sooner than normal to make multiple cells. As those cells divide, a lumen starts opening that is continuous with existing blood supply. Continues happening until growing cells bump into and fuse with an existing capillary to form a circuit

Cells are under low oxygen conditions (low HIF)

· Release a signal called hypoxia induced factor (HIF)

· HIF: Helix-loop-helix family member; transcription factor. Made of 2 subunits, alpha and beta. Beta subunit is always expressed, alpha subunit is produced but under normal oxygen conditions getting to tissue, there will be a prolylhydroxyalase (hydroxylates proline in degron of alpha subunit) that is active. Alpha subunit is eaten up because its degron is activated. Ub-ligase activates and adds ubiquitin to it and is chewed up.

· Under low oxygen conditions, prolylhydroxyalase is inhibited by increase in succinate (increased by citric acid cycle due to low oxygen conditions). Alpha subunit is still present. Alpha and beta subunits can bind and work together as a transcription factor

· Alpha-beta bind to promoter of a gene called VEGF (vascular endothelial growth factor), a signaling molecule (red dots). Are produced and secreted by cells that need a blood supply. Diffuse through ECM until they come in contact with an endothelial cell in nearby capillary. Stimulate binding of VEGF to the receptor on surface of endothelial cells. Cells re-enter cell cycle via cell cycle and produce proteases

· Make proteases to break through basal lamina so that endothelial cells and start growing in new direction, chew through ECM as new cells are made. Grow in direction of signal until fuse with another capillary

Stem cells

· Relatively undifferentiated cell that can continue dividing indefinitely and undergo terminal differentiation

· Stem cells need to be in a protected environment that promotes growth and is connected to a place where the products of those stem cells are needed

· One daughter cell needs to stay in region maintaining capability of indefinite division

Other daughter cell travels towards terminal differentiation. Assuming that differentiated cell will replace something that has been lost or needs to be repaired

Cross-section of epidermis (skin)

· Pink (keratinocytes) are filled with keratin (long fibrous monomer with sticky ends)

· Keratin falls into acidic or basic categories based on amino acid make-up

· Wrap around each other in alpha helical coiled helix (dimers). Basic pairs with acidic. Relative pairs change depending on where keratin is being used or its function

· Dimer wraps around other dimers and sticky ends will bind together to make densely packed, strong, somewhat flexible keratin proteins

Loose connective and dense connective tissues of the skin

· Loose connective tissue has more cells and fewer fibers compared to dense connective tissue

o Green stripes = collagen

· Dense connective has fewer cells and more structural proteins in ECM (collagen)

· Epidermis

o Layer above loose connective tissue; has a basal lamina below

o Has keratinocytes attached to it and multiple layers of keratinocytes with different cell types mixed in (immune cells, melanocytes)

o As keratinocytes move further away from the loose connective layer, they change in shape and size and have a different kind of keratin filling them up

o At the top of the epidermal layer, the cells will be so densely packed with structural protein that all cellular functions cease. These cells die once they reach the top

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Fibronectin

Multi-subunit protein made up of repeated domains (mostly antiparallel beta sheet).

· Depending on domains linked together, can get different binding sites in domains to attach to other components in ECM

· Disulfide bonds at cysteine residues hold 2 subunits together. Fibronectin can be incredibly soluble (during developmental stages and wound healing). In normal differentiated adult tissue, more insoluble form of fibronectin is produced

Loose connective tissue is dermis

· Loose connective tissue is bottom layer

· Keratinocytes are contained in middle pink layer. Size and shape changes as they undergo differentiation in epidermis

Cell proliferation

· Basal lamina (yellow): Filter between epidermis and dermis

o Small molecules can pass through, cells cannot unless they use proteases to chew through the layer

· There are 4 different layers of cells, but cell division is only happening in the bottom layer attached to the basal lamina

· Layer 1: Proliferation only occurs in basal layer. Stem cells begin as unipotent stem cells (only become keratinocytes). Given that keratinocytes are not observed in other 3 layers, environment in lower layer promotes proliferation there and not in the other layers

· Layer 2: As we move up, keratin begins building up. End up with prickle cell layer because they look like they have appendages (appear prickly). Produce proteins called cadherins that they imbed in their membranes. Adjacent cells attach to these membranes and give them appendages that make them look prickly

o No cell division occurs here

· Layer 3: Granular layer. Filled with keratin that look like granules. Cells are under stress and begin dying

· Layer 4 (top pink): 10-20 layers of keratinized squamous cells; cells are dying off, protective layer where cells are connected to each other in earlier layer. Impervious to external substances because the cells are connected to each other

o Lots of division needs to be happening in basement membrane to replace this top layer

Hair follicles

· Hair is made of keratin, contained in hair follicles. Based on BrdU experiments, halfway down a hair follicle, there are stem cells present

· Once produced, will migrate away from bulge region and can go in 2 directions. If they go up, they become part of epidermis (skin). If they migrate down, they move down to hair follicle and fill it with keratin to become part of growing hair.

2 hypotheses as to regulation of stem cell differentiation (nature versus nurture)

· Environment in which cells are growing (nurture) and internal components of cells (nature)

· Environmental asymmetry: stem cells are maintained because of their associated with factors in the external environment

· Once cells divide, one of 2 daughter cells one of them stays associated with environmental factor and remains as a stem cell. The one that moves away and loses the signal begins differentiating. Location determines whether cell remains a stem cell or not

· Divisional asymmetry (nature): cell is full of molecules, copies DNA, goes through division, and cytoplasm is divided in half. Cytoplasm prior to pinching off may not be homogeneous, so some factors within the cell are unequally given to one cell more than the other. Internally, cells will inherit different set of molecules that regulate different molecules. One cell may inherit molecules that keeps it a stem cell, and one may not inherit those molecules and begin differentiating

Growing skin cells

· Rolling basal lamina with basal layer with cell division. As moving away from this layer, division is not happening. This is in a location with no hair growing. BrdU experiments were used to try to identify stem cells

· Can vary how long BrdU is given to animal (pulse) and how long you stop giving it to animal (chase). Options are short pulse short chase, long pulse long chase, short pulse long chase, long pulse short chase.

Stem cell division

One stem cell divides, 1 of the daughter cells remains a stem cell and one differentiates. As a committed transit amplify cell, it eventually becomes a differentiated cell

Founder stem-cell population

· Developmentally, cells need to differentiate from clumps of cells by communicating with each other. Establish pattern within growing embryo. Become a founder stem-cell population

· As tissue grows, transit amplifying cells help make tissue that needs to regenerated

Stem cells in the lumen of gut

Lining of gut, single layer epithelium to promote absorption of food

Has many requirements as skin but is not very thick

Arranged in crypts into underlying dermal tissue. Villi (protrusions) go up into lumen of gut. This affords some of the same protection and microenvironments of epidermis (skin)

Cells at top (blue) are damaged because they're exposed to digestion of food. Digestive juices digest things associated with cells. Cells last 3-7 days and require constant turnover. Need a stem cell population that is not subjected to same digestive juices. Stem cells are hidden in crypt away from digestion at the bottom (red). True stem cells because they proliferate forever and do it relatively slowly (once every 24 hours). Once they divide, movement of non-stem cells is upward until they reach the villi. Yellow regions are cells proliferating more rapidly (transit amplifying); they had moved up from the crypt and received new signals to divide faster. Need to decide what cell type they will commit to

Figure 22-16. Renewal of the gut lining. (A) The pattern of cell turnover and the proliferation of stem cells in the epithelium that forms the lining of the small intestine. The nondividing differentiated cells at the base of the crypts also have a finite lifetime, terminated by programmed cell death, and are continually replaced by progeny of the stem cells. (B) Photograph of a section of part of the lining of the small intestine, showing the villi and crypts. Note how mucus-secreting goblet cells (stained red) are interspersed among the absorptive brush-border cells in the epithelium of the villi.

Most abundant cell types in the gut lumen are absorptive cells

· Absorb nutrients and transfer to bloodstream. Need things to flow easily, so goblet cells are made to secrete mucus and keep things fluid

· Paneth cells are used as defense cells; secrete proteins called defensins in case bacteria enter the crypt. Prevent bacteria from causing infection. Located in bottom of crypt.

· Enteroendocrine cells release signaling molecules to allow for regulation of cells. Do not need as many since they release signals

· Went from single stem cell to pluripotent stem cell (stem cell that can differentiate into more than 1 kind of cell)

Differentiation of stem cells in lumen of gut

· Cells are stained to see relative abundance (left)

· Starting to differentiate as they move up through crypt

· Right is gut cancer. Something caused disproportionate growth of one cell type, resulting in tumor growth.

Signals for differentiation

· Wnt (far right) protein is binding to a transmembrane receptor called frizzled. Wnt signal is transmitted across membrane into cell and moves through proteins. Ends up at a protein called beta-catenin, which is a gene regulatory protein. Genes it regulates are associated with cell proliferation in the crypts of the gut.

Beta-catenin

· Beta-catenin gets destroyed (left)

· Destruction complex is disassembled by Wnt signal (right)

o Beta-catenin is still present, so it can bind with LEF and transcribe genes for cell proliferation

Stem cells in the lumen of gut have to develop into 4 different types of cells and into the right proportions.

Example takes into account both mechanisms (nurture and nature)

· To decide whether to develop into absorptive or secretory cell:

o Stem cells divide and have unequal inheritance of certain cellular components, leading to uneven gene expression in each

· Protein called notch imbeds into membrane and binds to receptor on another protein. Notch is activated because of inheritance on one cell but not on the other. Environmental factor causes Notch on one cell and receptor on other cell to interact. Cells communicate as to which cell becomes what.

· Happens within the crypt

· Combination of internal/external signals ensures proportions of cell types is right

EphB/ephrinB

· As cell is leaving crypt, how does it know to stop dividing in the villi?

· Pair of proteins EphB and EphrinB (receptor and signal)

· While in crypt, cells are producing EphB. When they move into the villus, they start producing EphrinB (proliferative --> non-proliferative)

· When something goes wrong with EphB and EphrinB

EphB mutant does not give cells signal to shut off, so they continue dividing when they're not supposed to

Ephrin-Eph signaling controls cell segregation between crypts and villi

· When something goes wrong with EphB and EphrinB

EphB mutant does not give cells signal to shut off, so they continue dividing when they're not supposed to

(A) Proliferative cells (including the stem cells) and Paneth cells express EphB proteins, while the differentiated, nondividing cells that cover the villi express ephrinB proteins. The repulsive cell-cell interaction mediated by encounters between these two types of cell-surface molecules keeps the two classes of cells segregated.

(B) In a normal gut, as a result, Paneth cells (brown stain) and dividing cells remain confined to the bottoms of the crypts.

Signals defining the intestinal stem-cell niche

Maintaining non-proliferative state of cells in villi

Wnt signaling causes proliferation of cells in crypt, but gets transferred to another signal in the villus. Signals move up through core region of villus (yellow). Start producing BMP (bone morphogenic proteins) in core of villi. Reinforce signals not to divide anymore.

A megakaryocyte among other cells in the bone marrow

· Bone marrow in blue has megakaryocyte. Has processes (limbs) that extend through basal lamina, through bloodstream. Has pockets of cytoplasm with a membrane enclosing them containing proteins involved in blood clotting

· Its enormous size results from its having a highly polyploid nucleus. One megakaryocyte produces about 10,000 platelets, which split off from long processes that extend through holes in the walls of an adjacent blood sinus.

The migration of white blood cells out of the bloodstream during an inflammatory response

· WBC need to break out of bloodstream. If there is a wound in tissue away from bloodstream, how do blood cells know to get there? Similar signals make way to endothelial cells once injury has occurred

· Endothelial cells loosen up and provide gaps for blood cells to move through. The cells are moving rapidly through the bloodstream, so how do they hold on to the membrane

Selectin

Cells upregulate a transmembrane protein called selectin, similar to Velcro. As cell goes by, sugar groups at end of protein on the cell surface bind to a binding domain on selectin and slows flow of cells past damaged area.

Integrins

· Other adhesive proteins called integrins make a stronger connection between cell surface and endothelial surface. Cells are bound to the region after being slowed down.

Cells can then start migrating through endothelial cells

· Need to digest them to make holes big enough to pass using proteases

The response is initiated by signal molecules produced by cells in the neighborhood (mainly in the connective tissue) or by complement activation. Some of these mediators act on capillary endothelial cells, causing them to loosen their attachments to their neighbors so that the capillaries become more permeable.

· Endothelial cells are also stimulated to express selectins, cell-surface molecules that recognize specific carbohydrates that are present on the surface of leucocytes in the blood and cause them to stick to the endothelium.

· The inflamed tissues and local endothelial cells secrete other mediators called chemokines, and the chemokines act as chemoattractants, causing the bound leucocytes to crawl between the capillary endothelial cells into the tissue.

Bone marrow showing different cell types

· White spots look like gaps, but they appear cell-shaped. They are filled with lipids and are similar to adipocytes.

· In bone marrow, referred to as marrow stromal cells. As we age, these become more abundant compared to other cell types and can lead to osteoporosis.

Marrow stromal cells

· During development of blood cell types, marrow stromal cells have connections with earlier stages of development

· Can form a connection between stem cell that forms the other blood cells

Spleen colony assay experiment

Rescue of an irradiated mouse by a transfusion of bone marrow cells.

· Spleen is a storage site for certain cells in body (circulating cells in blood system)

· Irradiate rat number 1 in an X ray machine until all bone marrow cells are killed. Rat will die unless it is given bone marrow from a healthy rat (rat number 2). Injected into circulatory system of rat 1. Cells circulate and reconstitute bone marrow cells that were lost. Some of the cells don't make it into the bone marrow and get stuck in the spleen. Cells that are stuck there will continue to grow and develop. Should be able to remove the spleen and see colonies of cells due to implanted founder cell

· Bone marrow from donor has different cells at different stages of development.

· Rat 1 is recovered. How can we use this experiment to determine whether 1 type of stem cell is responsible for development for all the different blood cells? Before injecting bone marrow from donor into irradiated mouse, cells were grown in culture and exposed to a virus. Virus infected donor cells, got inside, and integrated itself into DNA of donor cells. Viruses insert themselves randomly in the genome in every donor cell. Want to go into spleen and selecting colonies of cells to find every type of blood cell. Analyze the DNA from those cells to see where virus inserted itself using Southern blotting. Take genome from cells isolated, run on gel, and use a probe out of the viral sequence and use it on the blot. Looking to see one of every cell type with virus in the same spot. If this is identified, then that means the cells originated from a pluripotent stem cell which had the virus inserted into its genome. As the cell divides, the new cells will have the viral DNA in the same location

Hemopoiesis

· Proved that all the blood cells originated from one pluripotent stem cell. Stem cell divides, one cell stays as a stem cell and one has to decide what to differentiate to

· Cell makes choices as it commits to becoming a certain type of cell. Based on signals coming from bone marrow and what the body needs in that moment

Dependence of hemopoietic stem cells on contact with stromal cells

· Where will the stem cells be kept in the bone marrow?

· Marrow stromal cells in orange form attachments with stem cells in bone marrow. Stem cell makes a receptor called Kit.

· Stromal cell produces a molecule called Kit ligand, also known as SCF (stem cell factor). If this connection is made between receptor and ligand to stromal cell, then the cell is a stem cell. Red signal indicates that stem cell will continue to proliferate at a slow rate to maintain stem cell population

Receptor and SCF

· SCF is a transmembrane protein. Has an amino terminal that dangles in the membrane

· Has a cleavage site in its amino terminal end that can become a soluble region. SCF can be anchored in the membrane or can be a soluble thing in the membrane.

Colony-stimulating factors (CSFs)

· Cell differentiates based on signal being sent from bone marrow

· Known as colony stimulating factors that stimulate different cell types. If supplied with correct factor, factors bind to receptors and trigger gene expression to make stem cell to develop into a specific cell type

IL3 and GMCSF

· Examples of colony stimulating factors

· Depending on what signal they hear, they will activate certain genes and move down a certain pathway

· Sharing of subunits among CSF receptors.

· Human IL3 receptors and GMCSF receptors have different a subunits and a common b subunit. Their ligands are thought to bind to the free a subunit with low affinity, and this triggers the assembly of the heterodimer that binds the ligand with high affinity.

A developing red blood cell (erythroblast)

· Developing RBC. Top is a progenitor to a RBC (committed to a RBC but not fully developed). Erythroblast (-blast: cells that will become a certain type and will rapidly divide a certain number of times)

· During development of RBC, nucleus is kicked out (dark red) and it is filled with hemoglobin

Blast cell (BFC-E)

goes through a certain number of divisions. Receives signal to stop dividing and forms several erythrocytes at the end

Some of the parameters through which the production of blood cells of a specific type might be regulated

· Pushing a stem cell in a direction

· Still do not fully understand how to make a stem cell differentiate into one type of cell, but we can control certain conditions (keep it alive, control division of progenitor and transit amplifying cells)

Marrow Stromal Cells (MSCs)

• 1867: Cohnheim injected dye

• 1970's: Freidenstein, non-adherent cells

• 1980's: Tweak culture conditions (CSFs)

1867: Cohnheim injected dye

· Cohnheim injected a dye into a bone marrow of an animal. This labeled many of the bone marrow cells and let the cells develop and leave bone marrow to circulate bloodstream.

· Found that some of the MSCs left bone marrow to populate other tissues. Suggested that MSCs weren't stuck in the marrow all the time and could do other things

1970's: Freidenstein, non-adherent cells

· Cells can be grown outside of the body in petri dishes. When going into bone marrow, some cells have to be grown in specific conditions.

· Took bone marrow, let them grow in petri dish, and poured of blood cells to keep the MSCs. Adipocyte looking cells eventually differentiated into different things, such as bone, muscle, or cartilage cells.

· Cells initially in fibroblasts but look like adipocytes can turn into many different cell types in culture. Did not know how or why

1980's: Tweak culture conditions (CSFs)

· Changing culture conditions could change what cells differentiated into; changed colony stimulating factors to turn MSCs into what researchers wanted.

· Understood that pathways responded to signals to trigger differentiation

Osteogenesis imperfecta

· Working with condition called osteogenesis imperfecta (brittle bone disease during fetal development). Mutant form of collagen that is much weaker grows in the bones. Collagen is a major structural component in bones.

· Could researchers take MSCs and see if they are contributing to other things such as bone marrow? Could they modify gene expression to cure brittle bone disease?

· PCR bands at the top from a collagen gene. M = mouse (smaller), H = human (bigger). Took human form of gene and replaced the gene in the mouse. + = mouse had human gene implanted. - = controls that had mouse gene

· Let mice grow and sampled tissues around mouse body to see if the genes had showed up after 1 month and after 5 months. Mice with human gene inserted still had the human gene present after 5 months. Human gene also ended up showing in lungs and cartilage. Cartilage makes sense since it is bone adjacent, but why did it show up in the lungs? Suggests that bone marrow cells, once implanted, can support grow of blood cell types but can also get out and migrate

Are there stem cells in muscles?

Yes, this is how muscle growth occurs

Muscle cells fuse together to form multi-nucleated fibers. Within fibers, striping is showing mature muscle proteins with sarcomeres that allow for muscle contraction

Helix-loop-helix factors

· Have patterning that allow cells to develop into different muscle components. Have a variety of transcription factors. Signal activates them, they activate another transcription factor (Mef2), they all reinforce themselves (sign of terminal differentiation) to activate muscle structural genes (actin, myosin, etc.)

· Once these genes are expressed, they remain expressed since this process leads to terminal differentiation

Myoblast fusion in culture

· In culture, pre-muscle cells (left) look like fibroblasts

· Once signals are heard by cells, gene expression is altered and some cells start fusing (light green on left), called myoblast

· Proliferate a certain number of times and start fusing into multinucleated cells

· Form muscle fibers with multiple nuclei. Nuclei migrate to periphery because there is no room for nucleus in the muscle fiber

Satellite cells

· With proper training, can add to existing muscle fibers and make them bigger. Requires additional cells

· White spots are nuclei associated with muscle fiber, are on outside doing job

· Second type of nucleus associated with muscle fibers called satellite cell (muscle stem cell). Surrounded by ECM different than the other cells. It is a unipotent stem cell, will only turn into muscle. Similar to a myoblast

· Satellite cells drift into direction of transit amplifying cells, possibly because they have a limited number of divisions they can make

Muscular dystrophy

· Continuous damage to muscle cells; that after some time cannot be repaired anymore. Due to mutations in a protein called dystrophin

o Dystrophin: Structural protein associated with muscle cells

o Provides a unit between contractile proteins in muscle by connecting to proteins in the membrane, which are in turn attached to ECM components

· Muscle cell in muscle fiber membrane (gray) separating muscle fiber (bottom) from ECM (top)

· Myosin walks along cytoskeleton actin filaments (yellow)

· Contraction of muscles occurs with actin, but also requires an "anchor" in the form of dystrophin that is bound to proteins in the membrane of the muscle fiber, which are then connected to ECM components on outside of muscle fiber, in order for movement/displacement of one muscle cell to the other

o When muscle fibers are contracted, they are pulling against dystrophin, which is attached to membrane proteins, which are attached to the ECM

· Muscular dystrophy causes a loss of binding between dystrophin and membrane. Need to be replaced and repaired

· When muscles contract, muscle cells are not pulling on or against anything, which causes them to prematurely rupture due to excess stress on the muscle

o Ruptured cells then need to be repaired/replaced

Do MSCs escape bone marrow, travel through circulatory system, and repair damaged muscle tissue?

Did an experiment to answer this question

Used gene-modifying technique, equivalent of spleen colony assay and then used a reporter gene (beta-gal), which turns blue if it makes the desired protein

Myosin protein is a globular domain with a long alpha helical tail with a neck region that needs to be reinforced. Called light chains

Put light chain-3F gene in front of reporter gene. Incorporated it into genome of a fertilized mouse egg. Everywhere that muscle proteins are made, light chain and beta-gal will be produced. Mouse's bone marrow will have this gene, called a genetically modified mouse (GMO)

In D, did damage to muscle in front of its shin. Took muscle from donor mouse (genetically modified) and injected MSCs it into wound site of first mouse. C shows muscle fibers being made and labeled by beta-gal from the donor mouse. In response to damage, nuclei are making beta-gal. MSCs are made to differentiate into muscle cells.

Cause damage to front of shin again and see if cells can get there on their own from bone marrow

Damaged region (C and D) has labelled cells appearing in repair region. E shows a blood vessel with a labeled cell moving through it, suggesting that MSCs can get out of bone marrow, get into bloodstream, migrate through, and get out of bloodstream to reach site of injury and repair it

The family of connectivetissue cells

· Fibroblast is a type of stem cell that can turn into different cell types if given the correct signals and if not too far along the differentiation process

· Cartilage cells can go back to fibroblast cells via de-differentiation

Cartilage cell differentiation

· When cartilage cells are grown in culture and differentiate into mature chondrocytes, secrete collagen type 2 into ECM. This collagen indicates that they are cartilage. Fibroblasts secrete more of collagen type 1

· If chondrocytes are grown in culture, and are put in densely enough, will stay chondrocytes. If the cells are spread out too much, they would revert back to a fibroblast-like phenotype by changing the collagen they are releasing. Evidence from growing them in culture

· Adipocytes, neutrophil (WBC)

· Fat cushions around organs, stores energy, provides insulation

CCAAT/enhancer binding protein

· Peroxisome proliferator: Activated receptor

· Pluripotent stem cell in purple. Purple into green is when cell decides to turn into a pre-adipocyte.

· Genes for other cell types are shut off and packaged away in the DNA, and pre-adipocyte genes are expressed. Pref-1(pre-adipocyte factor 1) is one of the genes expressed, but it is no longer expressed once the cell is no longer a pre-adipocyte

Pref-1

· A transmembrane protein, also called Notch. Outside of the cell, it has EGF (epidermal growth factor repeats). Used to communicate to other molecules. Extracellular domain can be cleaved to make it a soluble signaling molecular. Has a binding domain to bind another EGF domain called Delta

· Alteration of gene expression occurs as a result of activation of Notch

Fibroblast-like cell precursor

· Fat cells are not lost, but shrunken down

A fibroblast-like precursor cell is converted into a mature fat cell by the accumulation and coalescence of lipid droplets.

· The process is at least partly reversible, as indicated by the arrows; the dashed arrow indicates uncertainty as to whether a differentiated fat cell can ever revert to the state of a pluripotent fibroblast.

· The cells in the early and intermediate stages can divide, but the mature fat cell cannot.

Leptin

· Using 2 mice

· Right is the wild-type mouse, left is the mutant

· Mutant mouse has a mutation in the Obese gene, which codes for leptin.

· Leptin is put out by white fat cells and sends signal to brain indicating organism is full.

. The leptin-deficient mutant fails to limit its eating and becomes fat (3x the weight of a normal mouse).

· Region where fat accumulates on mouse is called fat pad

· Graph shows wet weight, DNA content, and triglyceride content of fat pad

· Region where fat accumulates on mouse is called fat pad

· Graph shows wet weight, DNA content, and triglyceride content of fat pad

· Left represents before leptin injections, right represents after leptin injections

· Did the mouse lose weight in response to leptin injections? Leptin injections in Ob (obese) mouse led to loss of weight in fat pad. Is it losing triglycerides or cells? Statistically speaking, same amount of DNA = same amount of cells, so the amount of fat in those cells dropped

Beta-actin

· Beta-actin is a housekeeping gene so same band shows in all 3 instances. Same amount of cDNA is put in the cells, so differences in other bands are significant

· A and B are involved with oxidizing fats. Genes in C are involved with packaging away fat. Their expression dropped indicating that fat is being metabolized

Ob mice response to leptin injections

· Pref-1 expression normally occurs in adipose tissue and is no longer expressed once the cells have differentiated into adipocytes (terminal differentiation)

· On day 1 in the Ob mice, the mice are not expressing Pref-1 as expected because fat pad is mature

· In response to leptin injections, Pref-1 starts getting expressed again in the fat pad, suggesting that some of those cells have de-differentiated and are starting to become more fibroblast-like

· Suggests that adipocytes could possibly be de-differentiated

Process of Wound Healing

1. Hemostasis - Clot

a. Release of epinephrine by cells around wounded tissue

b. Initially constricts blood vessels and reduces bleeding

c. Platelets arriving at wound site will secrete PDGF (platelet derived growth factor), a signaling molecule that will start remainder of clotting process

2. Inflammatory - Vasodilation, phagocytosis

a. Blood vessels dilate so that blood flow isn't as great and there is less pressure/stress on weakened tissue

b. WBC arrive at wound site and digest wounded tissue, destroyed cells, foreign material that may have entered characterized by swelling, redness, pain. Happens during first 2-5 days. Inflammation occurs after clot forms

3. Proliferative - granulation, contraction, epithelialization

a. Replacing missing tissue. Cover wound. Tissue looks granular in nature due to proteins and collagen being secreted by cells repairing the wound.

b. Fibroblasts fill in defects, capillaries form. Wound is contracted to reduce area that needs to be fixed.

c. Epithelium (keratocytes) starts proliferating

4. Remodeling

a. Tissue needs to be reconstituted as best as it can, but it won't be the same as it used to be

Organisms capable of regeneration

· Regeneration can occur unidirectionally (lobster can grow back claw, but claw cannot grow back lobster)

· Bidirectional: Worms can grow back in 2 directions (cutting a worm in half makes 2 worms)

· Planarians are the ultimate regenerators. Can grow head, tail, body back etc. Cells know where they are and can grow back missing parts using patterning

o As long as cells know where they are relative to where they're supposed to be, the rest of the organism can grow back

· Only one vertebrate (9) in the diagram can successfully undergo regeneration

Regeneration

Restoration or new growth by an organism of organs, tissues, etc. that have been lost, removed or injured. Usually involves multiple tissue types and does not involve scarring. Tissues are functional; same strength, function, resistance to stress, neuronal connections, ability to move

Urodele (Ambystoma mexicanum)

· Urodele: Vertebrates (amphibians) that can undergo regeneration

· Can lose a body part (i.e., tail) if being chased by a predator to escape

Emperor newt

· Emperor newt (A). Top of head (1), limbs, eye (retina, lens), lower jaw can grow back. Once these body parts grow back, they look like original, function like original, and involve multiple tissue types

Regeneration in emperor newts

· Arm of newt was cut on either side of elbow, and it was followed on series of days. Arm grew back no matter where it was cut. As arm grows back, it appears wider and more bulb-shaped. This is area of regenerating cells is called a blastema characterized by rapid cell proliferation and de-differentiation. As regeneration completes, everything differentiates into cell type it needs to be. Skin grows back faster during regeneration than in normal wound healing. Keratocytes more rapidly enter cell cycle, have higher proliferative rate (more cells made), and are always expressing integrins (cell surface proteins that allow cells to drag themselves across wound area). Integrins are always upregulated in these amphibians, but need to be signaled to upregulate during wound healing

Blastema

· Bulb depicts blastema

· Cells that are growing are either undifferentiated or de-differentiated stem cells. Remodeling is a constant feature of blastema

· Epithelium growing over the top (A); epithelium has under it is basal lamina. Blastema does not have a basal lamina, instead is called an apical epithelium

· Forming macrophages to digest things

· Proteases produced for remodeling as well as protease activators. Also need protease inhibitors to ensure proteases do not digest too much. Produced at much higher levels in blastema than in wound healing

Regeneration of lens in newt eyes

· Cross-section through eye of newt. Circular structure is the lens. Experiment surgically removed the lens, which grew back. In humans, once the lens is lost, it cannot be repaired or regenerated.

· Pigmented epithelium cells (PECs) will re-enter cell cycle (de-differentiate) in response to absence of lens. Will lose their pigment and start to form circular structure that becomes the lens. Cells in front can proliferate, cells in back pack together more tightly, begin filling up with crystallins. After many days, lens grows back and is fully functional.

· Trans-differentiation: Cells that were not producing crystallins and were not lens cells were able to proliferate via de-differentiation and become a different type of cell (PECs --> Lens cells)

· Researchers have also removed PECs from chickens and humans and grown them in culture. After messing with colony stimulating factors and media, some of the PECs can start producing crystallins. Cells in general can do this, but in the organism (human and chicken), they cannot

Myotubes

· Experiment where researchers were growing in culture cells that would develop into myotubes

· Myotubes: complex, highly differentiated cell type.

· These cells were grown in culture, labeled for detection, and were injected into blastema of regenerating newt arm. Some of the myotubes after arm grew back were incorporated into the muscle. Some of the myotubes de-differentiated and then re-differentiated into other cell types. Label was found in cartilage and bone. Something about blastema environment makes cells de-differentiate

Retinoblastoma

· Different serums were used to grow the cells. Protein concentrations were affected. One protein (retinoblastoma) was phosphorylated

· Within blastema, myotubes that were put in were getting affected by microenvironment.

· Their cell proliferation proteins were phosphorylated and they could re-enter the cell cycle (de-differentiate)

Retinoblastoma protein (Rb)

· Active form on left (red). In active form, it prevents cell proliferation/growth. Referred to as a tumor repressor gene. Rb binds to gene regulatory proteins that would otherwise activate proliferation genes.

· Rb is deactivated when the protein is phosphorylated. It can no longer bind to gene regulatory proteins, and they can bind to promoters and trigger cell proliferation

How do body parts removed from the newt know where to grow back?

· Take a proximal dissection (near) shoulder and distal (near) wrist cut. Cut off the blastema (D) and attach it to proximal region of second newt.

· The rest of the arm grew back, but the second blastema caused another hand to grow in the place where the hand goes. Indicates that blastema knew where to grew.

· There are signals being sent by cells in body of animal. As signals move down to arm, they get weaker and cells growing back hear the signals at the volume that it makes it to them. (darker region = less signal received). Cells gauging relative location based on strength of signals. Happened during original developmental stages, but is also happening in adulthood. This is unusual considering this process is shut down after the animal grows

Retinoic acid

· One of the signals is called retinoic acid (signals are strong near secretion point but dissipate).

Newt arms were cut off at different places. · Take ceramic bead, coat with retinoic acid, and plant it into different parts of arm. Caused extra body parts to grow (elbows, forearms) due to extra retinoic acid signals

Assuming that regeneration in MRL mice is similar to newts

· Have multiple tissue types, no scarring, regain of function

· Poked hole in control mouse's (C57BI/6) ear and MRL mouse's ear

· Hole shrunk in control mouse's ear indicating scar tissue covering the wound

· 2 Different strains of MRL mice had the hole close completely

Pictures during regeneration/regrowth process

· B and C are 2 different versions of MRL. Cuts were made down the middle. Anything new is on the left, old tissue is on the right

· 1-2 days after hole was made. Scabs formed (clotting process) happened in both. Control did not have growth of cells (re-epithelializing) like in MRL

Day 5

· MRL had complete re-epithelialization (multiple layers), separation of cartilage and appears bulbous. Cells are proliferating. Looks like blastema of salamander

· Control has scab, epithelium beginning to grow back slowly

Day 10

· Control: Epithelium has covered back over wound and some cells grew back

· MRL mice had blood vessels, epithelium, hair follicles grow back. Look like undifferentiated cells

Day 20

· Control: No longer growing; skin covered, collagen underneath, a few cells grew back. some remodeling might occur

· MRL: 2 sides of hole have grown back and met in the middle. Epithelium needs to fuse and form normal epithelium

Day 81

· Top is control. Gap in the middle indicates hole in ear that did not grow back

· Bottom is MRL mouse. Epithelium has fused, underlying tissue is fused and continuous. Muscle, cartilage, skin, hair follicles have all grown back

Staining for laminin (bright yellow/green)

· Left is control, right is MRL

· Control: Thicker tissue is epithelium that's growing. Bright yellow line is basal lamina that formed under epithelium. Starts early and persists as time goes on

· MRL: Basal lamina looks like it was deposited, then after some time, it was digested as blastema grew. Associated with regeneration in urodele amphibians (apical epithelium). Underlying cells can communicate with epithelium via signaling to tell them to keep growing

· Shows wound healing versus regeneration

Microarray

· Can go into a blastema, dissect newly produced cells from it.

· Extract RNA from those cells and use it to probe a microarray to see which genes are being expressed

· Used radioactively labeled cDNA to probe. 2 dark bands indicated a hit (gene identified)

· Pref1 expression increased around day 20 when tissue began growing back. Gene is being expressed during regeneration

· MRL mice had higher levels of Pref1

o Pref1 works by keeping stem cells in the proliferative stage

· Laser capture microdissection also showed high levels of Pref1 in MRL mice

· Immunostaining with an antibody against Pref1 showed that cells in the blastema of MRL mice were producing Pref1

· Gene associated with stem cell proliferation is maintained/expressed

Basal lamina in MRL mice during wound healing/regeneration

· Needs to be digested in order for damaged epithelium to grow back

· Basal lamina prevents communication (i.e., signaling molecules) from reaching other cells beneath the basal lamina

· To remove the basal lamina in the MRL, it had to be digested

· Basal lamina in MRL was digested using MMPs (matrix metalloproteases)

· More remodeling was occurring in the MRL mice than the control because more proteases were digesting the ECM so that newly formed cells had room to grow and could excrete the ECM they wanted as they matured

Proteases used during regeneration

· Showing MMPs in MRL mice tissue

· Proteases are produced by black dots in remodeling tissue

Heart regeneration in MRL mice - figure 1

· Incision made under ribcage, took cold probe (blunt end metal instrument) after placing in liquid nitrogen. Cold traveled through diaphragm and into heart of mouse (right ventricle)

· MRL and control (C57BL/6) mice hearts were injured at the right ventricle (RV) using a cold probe

· Could have shocked mouse or used an SA node inhibitor drug

o SA node helps to regulate heartbeat, so inhibiting the SA node can stimulate a heart attack in the mouse. Not practical for this experiment

· Looking for reproduceable techniques and reducing collateral damage to other parts of body

Heart regeneration in MRL mice - figure 2

· HE stained mouse heart tissue (control versus MRL) after injury to right ventricle

· Pictures were taken between days 15 and 16 during healing process

· MRL mouse had more rapid heart generation and myocardial tissue seems to be regenerating

· A and B: Pink is undamaged tissue, purple is damaged tissue

o B, MRL: Muscle fibers are lined up in an orderly fashion as if they are trying to replicate undamaged tissue

§ More myoblast-like, pre-muscle cells

o A, control: Newly formed tissue is disorderly and not mimicking healthy tissue; only attempting to fill in the gap

§ Proliferate, secrete collagen, fix damage

· C, control: No hole in the heart, so ventricle is still partially functional. Looks more like scar tissue

· D, MRL: Still have organized cells, but underneath it looks like muscle tissue

· E, control: At day 60, there is more scar tissue holding the ventricle together

· F, MRL: Cardiac tissue has grown back around right ventricle

Heart regeneration in MRL mice - figure 3

· Pulse chase with BrdU, BrdU stained showed up as dark circles (labeled nuclei). Those cells during pulse experiment took up BrdU and incorporated it into DNA

· Control: E and F; BrdU shows up in scar tissue but not in cells that look like myocardial cells

o Cell proliferation is taking place in response to injury

· C and D: Showing BrdU taken up in cells that look like muscle fibers

· B: Blue one was stained twice with DAPI (fluorescent stain) that labels nuclei (DNA)

o Allows for accurate cell counts; how many nuclei are there versus how many took up BrdU? This ratio was used in the next figure

Heart regeneration in MRL mice - figure 4

· Using same slides of heart dissection to create a mitotic index: Counting relative to cell division

o Indicated percentage of control and MRL mice cells dividing

o Comparing number of BrdU-stained cells with DAPI-stained nuclei

· Control has lower mitotic index than MRL mice, indicating that the MRL mice have more cell division

· Compared amount of cells to amount of cells in BrdU

· Control mouse had low levels of nuclei with BrdU, where MRL mice had up to 20% of their cells expressing this

· Each bar represents an individual mouse; all of the MRL mice represented regeneration given the amount of cell division that is occurring. Mouse 6 (bar 6) had a low number that does not fit with the rest

o This could be due to more severe damage that was done to the mouse, or maybe the mouse wasn't injured enough and cell division is not occurring as much as expected

Heart regeneration in MRL mice - figure 5

· Verifying that the cells that are growing back are cardiomyocytes - used same slide of cells in all 3 images

· A: Used an antibody against alpha-actinin (muscle-related protein) to stain for muscle cells

o Identifies muscle cells

· B: Used secondary antibody for BrdU which produced green color

o Stained BrdU to indicate which cells were recently divided

· C: Superimposed A and B images to give yellow color. Indicates that BrdU labeled nuclei are cardiomyocytes

o Proved that cells that are growing back are cardiomyocytes

o Stained for both proved that muscle cells were growing, which is expected in the case of regeneration

o Proof that regeneration is growing back the damaged cells

Heart regeneration in MRL mice - figure 6

· EEG (echocardiograph) of the mice's hearts proving that the regenerated mouse hearts were also functional after growing back

· Testing contraction of ventricular chambers

· A: Showing series of heartbeats after hooking up electrodes. Bars represent volume of blood in different heart chambers, how they contract, and how they expand to draw in new blood

o Showing volume of blood in each chamber which can be used to calculate how much blood is being moved by the heart

o Known as a functional assay: Is heart functioning properly?

· B: Shows averages of blood volume moved by the right ventricle

o To compensate for damage done to the heart and being unable to contract as much, more blood was being pumped in

o 1 month: Hearts of mice developed a bigger right ventricle to compensate

o 3 months: Right ventricle returns to normal size during regeneration in MRL mice since it has regained its functionality

§ Functions like it did pre-injury

Heart regeneration in MRL mice - figure 7

· Focusing on scar development (collagen) and the amino acid mainly found in collagen (hydroxyproline) since it has kinky prolines that can be wrapped around to close wounds in tissue

· A, left: Measuring how much collagen is in heart

o Cutting heart, grinding it up to measure collagen levels

o Increase in collagen levels as expected, but remodeling occurred in later days

· A, right: MRL mice produced collagen and then dropped drastically until day 60. Indicates that remodeling is occurring for longer time period than control

· B, left: Collagen gene expression raised, then dropped

· B, right: Collagen gene expression raised, then dropped

o Where did the collagen that was expressed in the MRL mice go? Proteases chewed it up during the remodeling process in order to fix the wound more effectively over time

Conclusions from the evidence provided

· Can we call this regeneration? Yes, MRL mice regained function after injury and the cells that were produced were muscle cells to replace the ones lost

· This paper focused on one tissue type; regeneration usually comprises growing back multiple tissue types

o This is a drawback, but the heart is mainly composed of heart muscle cells and nothing else

· Also did not see a blastema indicative of regeneration

· Could do follow-up experiments on whether the cells are myoblasts, whether de-differentiation occurred