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White blood cells
have different morphology based on function; tell which type it is and what it does
macrophages phagocytize--> fuse with lysozymes (proteins digested, etc)
no waste going on
large bone marrow cell with a lobulated nucleus responsible for the production of blood thrombocytes (platelets), which are necessary for normal blood clotting.
What is the migration of white blood cells out of the bloodstream?
the response is initated by signal molecules produced by cells in the neighborhood or by complement activation. Some mediators act on capillary endothelial cells, causing loosening of their attachments to their neighbors so that the capillaries become more permeable. Endothelial cells are also stimuated to express selectins, cell-surface molecules that hrecognize specific carbs that are present on the surface of WBCs in the blood and cause sticking to the endothelium. The inflamed tissues and local endothelial cells secrete other mediators called chemokines and the chemokines act as chemoattractants, causing the bound WBCs to crawl between the capillary endothelial cells in into the tissue
- WBC need to get out of the circulatory system and to the damaged area of tissue to 1) get rid of debris and 2) protect you from foreign substances
- As a result of damage, signaling molecules diffuse--> changes take place
- In response to damaged tissue, changes take place
- Endothelial cells need to slow the cells down so they can escape and proteins are produced
- --> one protein is selectin: has a lectin binding domain and is membrane bound; it is going to slow WBCs down
- After WBCs are slowed down, they need to be grabbed.
- Integribs with an EC domain and can grab the ECm are used to grab the WBCS
Function of integrin and what must be done to active integrin.
fx: to prevent unnecessary activation , we have inactive integrins
activator protein--> development of binding site compatible with cell surface protein on WBC--> held in place--> moves through endothelial cells and migrates through the tissues to do this--> secretes proteases to get through basal lamina and ECM, cytoskeleton, etc
Fibrobasts and fat cells are __.
- are large, membrane-spanning, heterodimeric proteins.
- All members of the integrin family adopt a shape that resembles a large “head” on two “legs,” with the head containing the sites for ligand binding and subunit association. Most of the receptor dimer is extracellular, but both subunits traverse the plasma membrane and terminate in short cytoplasmic domains.
What are stromal cells?
- supporting role for bone marrow
- 1) store energy for various blood cell types
- 2) fibrblasts secrete ECM and also help maintain stem cell population in bone marrow
- ==> pockets in bone marrow for stem cells to exist
What was the study done to test whether single stem cells are responsible for all blood cells?
They exposed an animal to high doses of x-rays, destroying most of the hematoopoietic cells, causing the animal to die within a few days. If, however, the animal receives a bone marrow transplant, it allows recovery and repopulation of bone marrow.
Cells are then sorted and the different fractions are transfused back into irradiated mice. If a fraction rescues an irradiated host mouse, it must contain hemapoietic stem cells. Cells are then labeled to figure out stem cells
If a fraction rescues an iradiated host mouse, it has hemopoietic stem cells.
What did the irradiated mouse study show?
it showed that stem cells are characterized by a specific combo of cell surface proteins; it is about 1 in 10000 cells
How do you label the hemotopietic cells in the irradiated cells for example?
- you can use a retrovirus, which will insert cell randomly
- --> all cells have retrovirus in different place--> spleen (cells lodge into these organs) for example
If the retrovirus is in the exact same spot, one stem cell gave rise to all of them because it is inserted into one spot
What do hemopoietic stem cells depend on?
contact with stromal cells;
the contact-dependent interaction between Kit and its ligand is one of several signaling mechansm thought to be involved in hemopoietic stem-cell maintenance
the multipotent stem cell divides infrequently to generate more multipotent stem cells or committed progenitor cells; as they go through their divisions, the progenitors become more specialized in the range of cell types that they can give rise to.
committed progenitor cells
limited in the number of times that they can divide before differentiating to form mature blood cells
Explain Kit and Kit ligand (SCF).
- SCF can be transmembrane or soluble; cleavage of the extracellular domain makes SCF soluble
- the transmembrane form is formed by alternative splicing that excludes exon 6
s a cytokine that binds to the c-KIT receptor that plays an important role in hematopoiesis
SCF plays a role in...
the regulation of HSCs in the stem cell niche in the bone marrow. In the bone marrow, HSCs and hematopoietic progenitor cells are adjacent to stromal cells, such as fibroblasts and osteoblasts (Figure 2). These HSCs remain in the niche by adhering to ECM proteins and to the stromal cells themselves.
SCF binds to the c-KIT receptor (CD 117), a receptor tyrosine kinase
with the exception of mast cells, expression decreases as these hematopoietic cells mature and c-KIT is not present when these cells are fully differentiated
Why is serum used for growth of cells?
Serum used because it has growth factor. People look for specific molecules telling cells to grow
eventual differentiation of cells
with a specific CSF, you can grow stem cells and control differentiation
- partially differentiated erythrocyte; divides rapidly
- at some point, a different signaling molecule binds--> differentiation takes place--> no more nucleus since it extrudes its nucleus to become an immature erythrocyte (reticulocyte)--> the reticulocyte loses its mito and ribosomes to become a mature erythrocyte--> no new cell division
-blast: undergo certain number of cell division in reponse to one signaling molecule
Explain the IL3 receptor.
promotes the survival and proliferation of the earlier erythroid progenitor cells; in its presecne, much larger erythroid colonies develop from cultured bone marrow cells in a process requiring a week or so
Explain the sharing of subunits among CSF receptors.
human IL3 receptors and GMCSF receptors have different alpha subunits and a common beta subunit; their ligands bind to the free alpha subunit with low affinity, and this triggers the assembly of the heterodimer that binds the ligand with high affinity
What allows differentiation to occur?
signals from the environment allow differentiation to occur; when conditions wear out, a new signal leads to more differentiation
Marrow Stromal Cells- history
Cohnheim injected dye:
inected dye insoluble aniline dye into bone marrow--> precipitated into cells--> animal sacrified and sliced--> searched for labeled cells
Found RBC and WBC and fibroblast like cells
suggested: fibroblasts could migrate through bloodstream and help populate other tissues
Marrow Stromal Cells- history
- working on bone marrow extracts; when grown in culture= non-adherent cells
- --> adherent are fibroblasts and adipocytes
Freidenstein took culture from bone marrow and cultured adherent cells
- --> cell division--> spindle like--> continued growth--> little foci developing--> some cells grew faster than others
- adherent cells seemed to differentiate into different cell types
Example of Freidenstein experiment
- Collagen type II found in cartilage; create ECM--> become chondrocytes (pre-cartilage cells)
- --> others were calcified= bone cells/ osteocytes
- --> others were turned into adipocytes--> supply energy
- --> others were myoblasts
Marrow Stromal Cells- history
Tweak culture conditions (CSFs)
CSFs (in serum were individual molecules that acted as ligands)
gained control over differentiation of bone marrow cells
Does it make sense that thy can differentiate into different things?
Yes, because adipocytes have energy, chondrocytes (cartilage before bone), and myoblasts are in bone
Do they actually get out of bone marrow and populate other areas/ tissues?
experiment: transgenic mouse= replaced a collagen gene with another collagen gene from humans
- designed a specific set of PCR primers that had human collagen genes in cells
- irradiated another mouse to wipe out bone marrow and replace with marrow of transgenic mouse
- wipe out bone marrow= the human populates it
- --> will there cells start showing up tissues in which human gene were showing up was measured; found in various places
Applications for Marrow stromal cells
degenerative arthritis: culture their myostromal cells and allow chondrocyte developent--> or osteocytes for poorly healing bone
poorly healing bone
gene therapy: osteogenesis imperfecta
skeletal muscle development
- develops from mesodermal tissue due to signals-->leads to production of TF, which
- 1) turn off proliferation
- 2) activate mature muscle genes that produce mature proteins
They reinforce their own activation-->reactivates themselves
What is the role of myogenic regulator proteins in muscle development?
signals result in teh synthesis of the four closely related myogenic gene regulatory proteins, MyoD, Myf5, MyoG, and Mrf4. These gene regulatory proteins activate their own as well as each other's synthesis in a complex series of feedback loops. These proteins in turn directly activate transcription of muscle structural genes as well as the Mef2gene, whcih encodes an additional gene regulatory protein. Mef2 acts in combination with the myogenic proteins in a feed-forward loop to further active the transcription of muscle structural genes, as well as forming an additional positive feedback loop that helps to maintain transcription of the myogenic genes
template protein in sarcomeres that positions thick filaments midway between the Z discs; it acts as a molecular spring, with a long series of immunoglobulin-like domains that can unfold one by one as stress is applied tot he protein; part of each titin molecule is elastic and chagnes length as the sarcomere contracts and relaxes; the other part is associated with a myosin thick filament
What does titin do?
it gives elasticity to muscle; after differentiation, its hard to undo that stuff
requires its own stem like cell--> myoblasts: proliferating undifferentiated muscle stem cells that, under proper conditions, fuse and become muscle cells
What is further specialization of muscle cells/
- slow-twitching cells and fast twitching cells
- Slow twitching: more mitochondria and more iron--> red
- Fast twitching: less mito and less iron--> not red
What is a muscle stem cell type?
Explain regulation of satellite cells.
they are regulated by myostatin and are the stem cells of adult skeletal muscle, normally held in reserve in a quiescent stae but available when needed as a self-renewing source of terminally differentiated cells; when themuscle is damaged or stimualted to grow, these cells are cativated to proliferate
can reproduce indefinitely unless you have muscular dystrophy
structural protein that connects the cytoskeleton of muscle cells to the surrounding extracellular matrix through the cell membrane
What are the connections of the cytoskeleton to the ECM important for?
- they are critical to maintaining cell volume
- a dystrophin mutation can lead to a conection not being intact nor optimal, thus causing gaps and influxes of ions and therefore damage to/ complete destruction of a muscle cell
What experiment was done to ask the question, do stromal cells get out of the bone marrow and migrate?
- A gene coding for fusion protein with a reporter gene was created (B-gal)
- Under control of the myosin light chain promoter (Myosin LC-3F), they viewed only hte places that these cells were activated
- All cells in the mouse had this gene
- Control: satellite cells
- Mouse and scalpel--> wound in calf muscle
- mouse was injected with satellite and bone marrow cells
The bone marrow contributed in a similar way to satellite cells; myostromal cells can conribute and grow into muscle cells