Midterm 3 Lec 25-36 Flashcards
What are the cardiac muscle cells called? How are they connected? Why is this important to the function?
- cardiomyocytes
- They are connected with each other via gap junctions (pores)
- Electrical signals are spread form cell to cell causing coordinated contractions of the heart
Where are cardiac muscle found and what is their functions?
- They are found in the heart
- provide the force needed to pump blood throughout the body
Do humans make new heart cells throughout their life?
- Evidence that humans make new cardiomyocytes at an extremely low rate through 14C dating
Background
- Molecules in the body contain carbon, normally 12C which is more common in the air → In the 1950s/1960s nuclear bomb testing produced 14C which was incorporated into CO2 and therefore plants which where eaten by animals and humans
- 14C was therefore in our DNA during cell division therefore new cells would have it
Experiment
- researchers were able to look at hearts of people born at different times relative to the spike 14C to see if they were making new CMs
Results
- It was found that we do make new CMs but it is very low (<1%) and the rate decrease with age
If someone was born in 1965 and died in 2022 what would the 14C levels in their CMs and skin cells be?
- in the CM cells there would be a lot of 14C in their DNA when they were born because there was a lot of 14C in the atmosphere
- Once they die there will be less 14C in the atmosphere but they will still have a lot of 14C in their DNA because there isn’t a lot of cell division
- In the skin cells of a person born in 1965 would have a lot of 14C in them but as the cells proliferate the 14C is dilute out over time
- When they die the skin cells have low levels of 14C
If someone was born in 1965 and died in 2022 what would the 14C levels in their CMs and skin cells be?
- in the CM cells there would be a lot of 14C in their DNA when they were born because there was a lot of 14C in the atmosphere
- Once they die there will be less 14C in the atmosphere but they will still have a lot of 14C in their DNA because there isn’t a lot of cell division
- In the skin cells of a person born in 1965 would have a lot of 14C in them but as the cells proliferate the 14C is dilute out over time
- When they die the skin cells have low levels of 14C
What happens when a heart attack happens?
- a coronary artery normally brings oxygenated blood to the heart bringing nutrients and oxygen
- when a heart attack occurs the coronary arteries don’t receive oxygen and die → this causes inflammation and scar formation
How can heart attacks be treated with stem cells? What are the methods?
- 1) cell replacement
- Pluripotent stem cells → add signals → new cardiomyocytes implanted into the injury
- replace dead cells
- signals might be released that lead to cells being made to support repair as trophic support
- Concerns? Will the new CM connect electrically with the old ones (early studies show arrhythmias)
- Pluripotent stem cells → add signals → new cardiomyocytes implanted into the injury
- 2) trophic support
- Use MSC or other support cells → offer trophic support to the injury
- they will release growth factors, reduce inflammation, and increase the formation of new blood vessels (angiogenesis)
- Use MSC or other support cells → offer trophic support to the injury
- 3) Directly apply/ implant trophic support
- no need to worry about MSCs not differentiating correctly
What is an example of trophic support in pigs and rats?
- Injury was induced in rat/pig hearts → A patch (synthetic polymer scaffold) w/ support cells on it that are found in the heart (NOT stem cells) → They release trophic signals
- results:
- Less cells death
- increase angiogenesis (heart vessel formation)
- increase in ability to pump blood
What would a paper be retracted?
Scientific misconduct
- incorrect data (falsified)
- unethical use of human subjects or animals
- plagiarism
Self correction
- If the author can’t reproduce results
- contaminated/bad reagents
Why does scientific misconduct happen?
- hype around stem cells
- Hard to publish negative results (no incentive)
What are the skeletal muscle cells called?
They are called muscle fiber: They are long multinucleate cells
Where are skeletal muscles found? What is their function?
skeletal muscles control the movement of the body by moving the bones they are attached to
What are satellite cells (potency?) and their functions?
- they are quiescent in non-injured adult muscle cells (They DO NO divide) → triggered to divide by injury
- are unipotent
How are satellite cells mobilized?
During injury signals are released into the microenvironment that activate satellite cells
- growth factors from the ECM, signals from immune cells and chemicals released by damaged fiber cells activate satellite cells
- Cells are then sent to the site of the injury →
- Some satellite cells divide symmetrically and proliferate (make more copies of themselves)
- Some do asymmetric cell division to create myoblast (progenitor cells) which then differentiate into myocysts (Muscle cells)
- Multiple myocytes can line up together and fuse to create new muscle fiber w/ multiple nuclei
- Myocytes can also add nuclei to injured muscle fibers (fuse together) → increasing nuclei = increase in protein synthesis = repair injury
How does lifting weights make muscles stronger?
Weightlifting → injures muscles (micro tears) ⇒ increase in active satellite cells/ increase in protein expression in muscle fibers → increase in active satellite cells → increase in myocyte fusion → bigger and stronger muscles
How do chromatin change when satellite cells differentiate into myocytes?
- satellite cells → Myoblast (progenitor cells) → myocyte (differentiated cells)
- in muscle genes the chromatin are closed → once MyoD (transcription factor) is expressed → the Pioneer factors, MyoD and Co-activators will open the chromatin (there will be an increase in muscle genes including MyoD gene)
- Postive feedback loop → expression of MyoD make more MyoD
→ Fibroblast can have forced expression of MyoD and that will make them muscle cells (transdifferentiation)
How do chromatin change when satellite cells differentiate into myocytes?
- satellite cells → Myoblast (progenitor cells) → myocyte (differentiated cells)
- in muscle genes the chromatin are closed → once MyoD (transcription factor) is expressed → the Pioneer factors, MyoD and Co-activators will open the chromatin (there will be an increase in muscle genes including MyoD gene)
- Postive feedback loop → expression of MyoD make more MyoD
→ Fibroblast can have forced expression of MyoD and that will make them muscle cells (transdifferentiation)
How is cultured meat made?
ESC or satellite cells from animals → grow culture (2)
- 3D cell culture; self organize into muscle tissue (not structurally sound) → Lab grown meat
- Grow on a scaffold, add signals → Muscle tissue → lab grown mear
- Is the scaffold edible? or reusable?
- what cells should be used to make the muscle? should they have fat cells? blood vessels? what ratios of multicellularity?
What are some advantages and limitations of making lab grown meat?
Advantages
- Reduce animal harm
- can eliminate pathogens
- better for the environment
- engineer better, healthier meat
Disadvantages
- Concerns about hazardous byproducts (food safety)
- Regulations: FDA (food) and USDA (agriculture)
- “not natural” → how to market?
- Is it vegetarian?
- Economic impacts?
- How will moving away from farms affects jobs?
- expensive to make
What is the external urinary sphincter and what happens when the external urinary sphincter is injured?
- Its a skeletal muscle in the urethra that is controlled by motor nuerons (open and close it)
- If it is injured you can’t control when you urinate
What is an autologous treatment for the urinary sphincter injury? What are the results?
Biopsy (take out) from quadriceps of the injured patient →
- Isolate the myoblast (progenitor cells) →
- Grow in a lab →
- Inject into the sphinceter of the injured patient
- No adverse effects
- Don’t have to worry about teratoma or tumors because they are using myoblast (only differentiate into the cells needed) - All patients improved
- more cells injected= better improvement - Presumably once in the patient, the myoblast are differentating into myocytes
What is muscular dystrophy? And what is Duchenne Muscular dystrophy?
- Genetic diseases that affect skeletal muscle function that results in progressive degeneration of skeletal muscle → (can also affect the heart)
- Mutation in dystrophin (on x chromosome, X linked disease, effects males)
- Protein that dystrophin encodes for is important for the structure of skeletal muscle
How do “normal” satellite cells do cell division versus cells with Duchenne Muscular dystrophy?
- Normal Satellite cells division
- Symmetric cell division (satellite cells divides into two satellite cells) and Asymmetric cells division (satellite cells divide into one satellite cella and one myoblast (progenitor cell))
- When there is an injury both types of cell division need to occur - Cells with dystrophin mutation (in all cells)
- Symmetric cell division occurs → Increase in satellite cells (normal)
- Decrease in asymmetric cell division → Fewer myocytes (progenitor cells) (abnormal)
How can stem cells be used to treat traumatic skeletal muscle injuries?
Which cells will you use? From where will you get them? How will you grow them?
How will the cells be given to the patient?
What are some things that could go wrong with the procedure?
Sample treatments
Fund donor ESCs → Differentiate the cells into satellite cells /myoblast → Implant into patient that is undergoing immunosuppresent treatment (autologous)
Problems: Patient need to be given immunosuppresents, where/which muscles should be treated? How often?
Use donated umbilical cord (since they express low levels of HLA markers there is no need for immunosuppresents) → MSC → Inject into different affected muscles→ Improves temporarily because of trophic support (inject every 4 months)
What are the two glands of the pancreas?
Exocrine pancreas and Endocrine pancreas
What is the Exocrine pancreas’ structure and function?
Endocrine pancreas
- small “islands of cells” dispersed over the pancreas → Islets of Langerhans
- Produce hormones that are secreted into blood vessels
- Control blood glucose levels (insulin and glucagon)
- Islets are comprised of 5 cell type:
- Alpha cells → Secrete Glucagon (20%)
- Beta cells → Secrete Insulin (70%)
Islet cells → Blood vessels→ Liver
What is the Endocrine pancreas’ structure and function?
- Exocrine pancreas
- 98% if total cells
- Acinar cells and Ductal cells
- Produce digestive enzymes that are secreted into the pancreatic duct
What are the cell lineages in pancreatic cell development? What are the transcription factors they are positive for?
1) Endoderm stem/progenitor cells
2) Pancreatic progenitor (Pdx1 & Sox9)
3) Endocrine Progenitor cells (Pdx1 & ngn3)
4) Mature beta cells (Pdx1 & Insulin)
4) Mature Alpha cells (Brn4 & Glucagon)
4) other mature islet cells
3) Mature Ductal cells (Sox9)
3) Exocrine progenitor cell
4) Mature exocrine or Acinar cell (Sox9 & digestive enzymes)
What regulates blood glucose? (2)
By two classes of cell surface receptors that function through different signal transduction pathways→ Insulin receptor and glucagon receptors
How is blood glucose regulates? What are the pathways?
meal → increase in glucose → triggers beta cells that secrete insulin into the liver (takes in/stores glucose) → reduce blood glucose
No meal → Low levels of glucose → triggers Alpha cells that secrete glucagon into the liver → produces and secretes glucose → Increases blood glucose levels
What is type one and type two diabetes?
Type one:
- Autoimmune disease
- destructive of beta islet cells
- Treatment: Insulin injections
Type two:
- range from reduced insulin to receptor mutations to post receptor processes
- Treatment: Diet, exercise, pathway specific drugs, insulin
What are some treatments for diabetes? And what are some issues?
Organ transplants
- Can restore proper glucose regulation
- BUT surgeries and immunosuppression required and can be dangerous
- Risk of Immunorejection, stability and function of transplanted organ
Islet transplantation
- difficult to find donors that are compatible, getting new islets to survive, finding experienced islet isolation teams and side effects of medication to prevent rejection
What are some strategies to generate new beta cells and functional islets? (3)
- Differentiation of ESC or other types of stem cells
- ESC or iPSCs (in vitro) →(direct differentiation, multistep process) → pancreas progenitor cells → Differentiate into new beta cells→ (proliferation/ cell-cell interactions) → Beta cell islets
- Bioengineering of pancreatic tissue using multiple cell types
- Reprograming differentiated pancreatic cells or other cell types
- Adult beta cells (and other cell types) → proliferation, bioengineering/ cell-cell interactions → Beta cells islets
What are cells comprise and islet?
- Alpha cells
- Beta cells
- Delta/Epsilon/PP cells
What is the function of Alpha cells?
Secrete glucagon
What is the function of Beta cells?
Secrete insulin
What is the function of Delta/Epsilon/PP cells?
each secretes a different hormone
What are some experimental issues to consider when generating function beta-islet cells and islets?
- In vivo vs in vitro outcomes
- Multiple step process; gene expression, differentiation signals, and cell-cell interaction
- functionality of newly generated beta cells
- Goal: Glucose stimulated insulin production
- Formation, functionality, and stability of bioengineered beta cell islets
How is the production of insulin in beta-islet cells that are dispersed versus clustered?
- beta islet cells that are dispersed secrete low to moderate levels of insulin per cell
*Clustered beta islet cells secrete high levels of insulin per cell →cell-cell contact triggers signaling that allows insulin to be produced
How are multiple cell types Bioengineered into beta-islet cell tissue? What are some issue/limitations?
- Differentiation strategies: Reprogramming using gene expression and hormonal signals , In vivo vs In vitro
Need: Combination of differentiated cells and stem cells
-Pancreatic beta islet cells
- Endothelial cells
- Mesenchymal stem cells (drives self condensation/possible trophic support)
In vitro:
-Co culture 3 cell types w/ hormone signals to drive blood vessel formation in ECM made of collagen
-over time self condensation (migration and proliferation of blood vessel cells) will form pancreatic beta islet like tissue
In vivo:
- pancreatic beta islet like tissue that secretes insulin in vitro is transplanted into hyperglycemic mice
- this increase the levels of glucose uptake in the liver (secretion of insulin in vivo)
Issues: Low numbers of original beta-islet cells, low levels of insulin secretion, inefficient response to glucose
How do you reprogram pancreatic cells to form beta islet cells in vivo?
-When there is an injury in the pancreatic duct (in vivo in mice) the neighboring ductal cells become Pdx1 + /ngn3 + expressing cells (transdifferentiation into endocrine progenitor-like cells)
- There was an observed increase in the number of beta islet cells near the ducts
Capability of cells in vivo to transdifferentiate
How can ductal cells be transdifferentiated into endocrine cells in vitro?
- (mice) Adult ductal cells cultures in vitro
- they are forced to express Pdx1, ngn3 and other genes (growth factors/hormone signals)
- the cells secrete insulin (beta islet-like cells cultured in vitro)
(no attempt to from islet like structures was made in this experiment)
How are exocrine cells transdifferentiated into endocrine cells? What are some key issue?
- (mice) Acing pancreatic cells
- they are forced to express Pdx1, ngn3 and other genes (growth factors/hormone signals)
- the cells turn into insulin producing beta like cells
- transplant the cell into diabetic mice (did not form islet like structures)
- there was a several in diabetic state (drop in blood glucose levels)
Key issue
- tissue stability
- islet formation
-regulation by glucose
