Midterm 3 Lec 25-36 Flashcards
1
Q
What are the cardiac muscle cells called? How are they connected? Why is this important to the function?
A
- 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
2
Q
Where are cardiac muscle found and what is their functions?
A
- They are found in the heart
- provide the force needed to pump blood throughout the body
3
Q
Do humans make new heart cells throughout their life?
A
- 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
4
Q
If someone was born in 1965 and died in 2022 what would the 14C levels in their CMs and skin cells be?
A
- 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
5
Q
If someone was born in 1965 and died in 2022 what would the 14C levels in their CMs and skin cells be?
A
- 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
6
Q
What happens when a heart attack happens?
A
- 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
7
Q
How can heart attacks be treated with stem cells? What are the methods?
A
- 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
8
Q
What is an example of trophic support in pigs and rats?
A
- 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
9
Q
What would a paper be retracted?
A
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
10
Q
Why does scientific misconduct happen?
A
- hype around stem cells
- Hard to publish negative results (no incentive)
11
Q
What are the skeletal muscle cells called?
A
They are called muscle fiber: They are long multinucleate cells
12
Q
Where are skeletal muscles found? What is their function?
A
skeletal muscles control the movement of the body by moving the bones they are attached to
13
Q
What are satellite cells (potency?) and their functions?
A
- they are quiescent in non-injured adult muscle cells (They DO NO divide) → triggered to divide by injury
- are unipotent
14
Q
How are satellite cells mobilized?
A
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
15
Q
How does lifting weights make muscles stronger?
A
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
16
Q
How do chromatin change when satellite cells differentiate into myocytes?
A
- 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)
17
Q
How do chromatin change when satellite cells differentiate into myocytes?
A
- 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)
18
Q
How is cultured meat made?
A
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?
19
Q
What are some advantages and limitations of making lab grown meat?
A
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
20
Q
What is the external urinary sphincter and what happens when the external urinary sphincter is injured?
A
- 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
21
Q
What is an autologous treatment for the urinary sphincter injury? What are the results?
A
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
22
Q
What is muscular dystrophy? And what is Duchenne Muscular dystrophy?
A
- 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
23
Q
How do “normal” satellite cells do cell division versus cells with Duchenne Muscular dystrophy?
A
- 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)
24
Q
How can stem cells be used to treat traumatic skeletal muscle injuries?
A
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)
25
What are the two glands of the pancreas?
Exocrine pancreas and Endocrine pancreas
26
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
27
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
Acinar cells → Duct → Small intestine
28
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)
29
What regulates blood glucose? (2)
By two classes of cell surface receptors that function through different signal transduction pathways→ Insulin receptor and glucagon receptors
30
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
31
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
32
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
33
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
34
What are cells comprise and islet?
- Alpha cells
- Beta cells
- Delta/Epsilon/PP cells
35
What is the function of Alpha cells?
Secrete glucagon
36
What is the function of Beta cells?
Secrete insulin
37
What is the function of Delta/Epsilon/PP cells?
each secretes a different hormone
38
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
39
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
40
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
41
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
42
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)
43
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
44
How are alpha cells transdifferntiated into beta islet cells?
- Alpha cells (make glucagon, increases blood sugar levels)
- place Transcription factor Pax4
- As a result you get beta-like cells that secrete insulin & glucagon (partial differentiation)
Why does it make both insulin and glucagon?
- Pax4 targets the promoted of the Pdx1 gene and induces the RNA →turns on beta like phenotype
- the Pdx1 chromatin structure is slightly open (no pioneer factor is needed to open) meaning Pax4 can turn on the RNA
45
How are human iPSCs differentiated into beta islet cells?
- Human iPSC are differentiated in vitro into pancreatic progenitor cells
- the cells are implanted into diabetic mice
- the mice differentiated beta islet cells are observed for 2-3 months
- there is no evidence of islet formation and there is reduced glucose levels (due to insulin production)
46
How are human embryonic stem cells used to produce functioning beta like cells?
- hESCs are cultured for about 20 days with specific sets of hormonal signals
- they differentiate into mixed cell population corresponding to different stages of differentiation including immature beta-islet like cells
- the cells are sorted to isolate the insulin producing cells then culture for 2-5 days
- aggregate into cell clusters (about 1000 cells per cluster)
47
What are some characteristics of beta islet like tissue?
- 99% of cells are endocrine (express Pdx1)
- similar overall gene expression compared to beta islet cells
- low sox2 expression (a marker for ductal/acinar cells)
-reverse diabetic state of mice (transplantation of human beta-islet like tissue is functional)
48
What are the next steps in the process of creating insulin producing beta islet like cells?
- in vivo transplantation
- islet function
- function and stability
-treat diabetic state (clinical trials)
-Gene editing to prevent detection by immune system
49
How does aging affect the function of hematopoietic cells?
* HSC are found in the bone marrow and give rise to red and white blood cells
Aging:
- they do more self renewal
-make more myeloid progenitor cells (red and white blood cells) and fewer lymphoid progenitor cells (lymphocytes/ immune system)
50
How does aging affect the function of satellite cells?
* satellite cells differentiate into myoblast/myocytes after skeletal muscle injury via asymmetric cell division
Aging:
- they do more asymmetric cell division and less symmetric cell division
- over time satellite cell numbest decrease and the body is less able to do muscle regeneration
explains why to some extent older people are more likely to get sick
51
What are the three main factors that affect cell function during aging? Which are intrinsic and extrinsic?
* Genetic mutation
- Intrinsic
* Epigenetic changes/ epigenetic drift
- Intrinsic
* changes to the stem cell niche environment
-Extrinsic
52
How do Genetic mutations affect cell function during aging?
- Intrinsic; in the DNA
-mistakes in DNA sequence during cell division/ DNA replication
- expression of DNA repair enzymes decreases/ is altered (can change cell function/ lead to cancer)
53
How do Epigenetic changes/ epigenetic drift affect cell function during aging
- Intrinsic; in the DNA
- histone modifications change which affect gene expression
- cause stem cells to proliferate/ differentiate differently compared to when they were younger
- mutations can affect enzymes involved in histone modification
- microenvironment can also affect enzyme function
EX: increase in Histone Methylation
-causes the chromatin to become compact at the lymphoid specific gene →shifts differentiation more toward lymphoid fate
can help explain why regeneration of tissue is impaired in older individuals despite the fact that stem cells are present
54
How did an experiment with mice demonstrate that epigenetic change are not permanent?
* Rejuvenation via epigenetic reprogramming
- researchers created a transgenic mouse line that could over express the 4 iPSC reprogramming factors; Sox 2, Oct 4, etc. →turned on with certain chemicals
- They turned on expression of the reprogramming factors cyclically (on and off) for 3 weeks
- they did this because if expression was left ton the whole time teratoma could form or the cells could lose function
- caused the old mice to have more satellite cells which were more capable of repairing injured muscle
- this experiment demonstrates that epigenetic changes are not permanent and can be altered to improve the regeneration potential of tissue
55
What are intrinsic factors?
something originating in the affected cell
56
What are extrinsic factors?
Something originating outside of the affected cell
57
What is a heterochronic transplant experiment ? (o→o)
Hetero=different / chronic= of age
1) old mouse HSCs transplanted into an old mouse (control) →the fate of the myeloid and lymphoid cells are normal
- # of transplanted cells o→o about the same myeloid and lymphoid cells
2) old mouse HSCs transplanted into a young mouse →
there is a slight decrease in myeloid cells and an INCREASE in lymphoid cells
This shows that the young microenvironment effects old HSCs because there is an increase in lymphoid cells in the young mouse that receives old HSCs
* intrinsic factors (like mutations and epigenetic) that affect stem cell function
58
What if the stem cells function like they did in the donor?
This indicated that there are intrinsic factors (like the microenvironment) that affect stem cell function
59
What is a heterochronic transplant experiment ? (y→y)
1) young mouse HSCs are transplanted into another young mouse (control)
- # of transplanted cells y→y about the same myeloid and lymphoid cells
2) young mouse HSCs transplanted into and old mouse→ there is an increase in myeloid cells and a DECREASE in lymphoid cells
This shows that the old microenvironment effects/impairs the function of your HSCs
In the aging HSC niche there is a signal from connective tissue in the bone marrow that causes more myeloid and less lymphoid cells
- this is probably caused by histone modification
* Extrinsic factors (like the microenvironment) affect stem cell function
60
What if the stem cells function like those in the recipient?
This indicates that extrinsic factor (like microenvironment) affect stem cell function
61
What is the heterochronic parabiosis experiment? what does it reveal about extrinsic aging signals?
- Parabiosis: The circulatory system of two different mice are connected together so the mice share blood and any signals contained in the blood
Parabiosis between an old mouse and a young mouse:
- Stem cells in the old mouse are rejuvenated
-increase in neurogenesis in the old mouse and improved memory
- increase in satellite cells and muscle repair
- improved heart function and blood flow
* even injecting young blood plasma effects and improved cognition in mouse model for Alzheimer's disease
62
What is a hair follicle?
The structure of cells that give rise to the growth of one hair
63
What are the stem cell niches in the hair follicle?
1) bulge: resides next to the growing hair. Contains hair stem cells and melanocyte stem cells
* hair stem cells →differentiate into keratinocytes → make keratin in hair
ALSO migrate to the skin when there is an injury
* melanocytes → differentiate into melanocytes → produce pigment that colors hair
2) Dermal Papilla: Located below the hair follicle.
*Contains mesenchymal stem cells that release growth signals to the hair follicle
64
What are the stem cells found in the bulge?
* hair stem cells →differentiate into keratinocytes → make keratin in hair
ALSO migrate to the skin when there is an injury
* melanocytes → differentiate into melanocytes → produce pigment that colors hair
65
What are the stem cells found in the Dermal Papilla?
*Contains mesenchymal stem cells that release growth signals to the hair follicle
66
The stem cells that make melanocytes decrease with age. What happens to the hair if there are no more melanocytes?
grey and white hairs
67
What are aging hair stem cells more likely to differentiate into? Why is that important?
-They are more likely to differentiate into skin keratinocytes and less likely to do self renewal
- the hair follicle becomes smaller and eventually disappears leading to hair thinning and balding
68
How is a human hair follicle bioengineered?
1) Researches obtain human hair follicles and isolate the stem cells under the hair (in the dermal papilla) and Keratinocytes
2) They grow the cells on a collagen scaffold with skin fibroblasts
3) Inject them into the skin of a mutant mice that doesn't grow hair
69
What is the likely role of the dermal paella in bioengineering hair follicles?
Contains mesenchymal stem cells that release growth signals to the hair follicle
70
How are stem cells used to create a skin organoid?
1) start w/ iPSCs or ESCs
2) place in Matrigel (3-D scaffold) with signals
3) differentiate them into a skin organoid that has hair follicles, pigmented hair, oil glands, nerves, etc.
3) transplant into mouse
* The hair follicle reoriented onto the right direction and are human hair
71
How does aging produce skin wrinkles?
- under the epidermal layer of skin, there is a layer of connective tissue made up of fibroblasts that secrete collagen and elastin (stretchy protein)
-ECM provides underlying structure to skin
Skin ages:
- Fibroblast produces less collagen and elastin
- some fibroblast die
- there is less fat under the skin
* all makes the skin less plump and more saggy
Fibroblast produce less collagen and elastin due to epigenetic drift and accumulation of mutations
72
Why do fibroblast produce less collagen and elastin?
-Fibroblast produce less collagen and elastin due to epigenetic drift and accumulation of mutations
-UV rays cause epigenetic changes and DNA damage
73
What are some current treatments to reduce wrinkles?
- injection of dermal fillers; put more material under the skin to plump it up
* can be made from
1) Collagen from cows
2) fat extracted from another part of the body and injected under the skin
74
What is cell assisted lipotransfer? What is the process?
1) Remove fat from the patient (autologous)
2) extract Mesenchyme stem cells using FACS from the fat
3) add the MSC to a small portion of the fat (increases the concentration of MSCs)
4) Inject the fat w/ MSCs into wrinkles
75
What are some potential problems with products that claim to have stem cells in them?
* Would plant stem cells benefit humans?
* regulations?
* exploitation of lack of knowledge
* Stem cells likely dead (if there are any) because they need a specific environment to survive
* Can the growth factors even get into the skin
* Expensive
76
What is the tissue structure of an ovary?
(inner to outer layers)
1) inner medulla: connective tissue, blood vessels, nerves
2) Cortex: Connective tissue, stroma with embedded follicles
2a) Follicle: contains oocytes
3) Connective tissue capsule
4) Epithelium
77
What are the oocyte cell types?
- Oocytes: eggs
- accessory cells: Theca cells and grandulosa cells
* produce steroids needed for oocyte development
- stroma cells
- Epithelial cells
- nerve cells, connective tissue cells, and blood vessels
78
What are the ovarian stem/progenitor cells?
VSEL: Very small embryonic like stem cells
OSC: Ovary stem cells (progenitor cells)
79
How does an oocyte develop?
1) Primordial germ cells (does self renewal and differentiation)
* pluripotent stem cell that expressed Sox2/Oct4
2) Oogonia (progenitor cells)
* Diploid (2n)
3) DNA duplication
4) Primary Oocyte
* Located in follicles in outer layer of ovary codex
* 4n DNA
80
What happens to the number of oocytes from birth to puberty?
There is a decline from 7 million primary oocytes (between conception and birth) to about 400,000 primary oocytes (after birth to puberty)
81
What is cancer?
a general term for more than 100 different diseases
a disease of stress and aging (gene mutations)
82
What are two characteristics of cancer cells?
1) cells proliferate in the presence of normal "stop" signals
2) No cell death (apoptosis)→ enhances cell survival
83
What are the types of tumors?
1) Benign tumor: NOT cancerous, tumors grow but will not spread, resemble the tissue of organ and are generally uniform cells surrounded by capsule
2) Malignant Tumor: Cancerous, tumors can grow and spread to other parts of the body, vary in size and shape
84
Over time what happens to normal cells?
There in an increasing number of genetic changes that affect the cell's ability to proliferate leading to cancer cells
85
What mutations occur and what are their effects on cancer stem/progenitor cells?
* Normal stem cells→ normal progenitor cells → mature cells
* Normal stem cells → Mutations → cancer stem cells → mutated progenitors → cancer cells
* Normal progenitors → muted progenitors → cancer cells
- Last two responsible for maintaining tumor/ a lineage of cancer cells
86
What is some evidence that supports that VSEL are the organs of cancer stem cells?
Have properties in common:
- Pluripotent
- symmetric and asymmetric division
- similar marker genes
-drug eflux (remove drugs/ become resistant to treatment)
- heterogeneity (diversity)
- Quiescent (dormant) (resistant to therapies)
87
How does heterogeneity arise?(2 models)
1) Stochastic model: Every cell can produce a new tumor
2) Hierarchal model/ stem cell model: Only once cell type produces a tumor (correct model)
88
How do cancer stem cells cause heterogeneity?
- Stem/progenitor cells in tumors are though to be involved in controlling the heterogeneity of tumor cells
Stem cell→ mutation emerges within stem cell or progenitor cells → multipotent progenitor cells →tumor initiation → many terminally differentiated cells within a tumor →tumor heterogeneity
89
What are cancer stem cells (CSC) and their properties?
* sub pop of cells within a tumor that have stem cell like property of self renewal and differentiation
-Can undergo asymmetric and symmetric cell division
* plays a role in the ability of a tumor to adapt and survive
- both symmetric and asymmetric division can occur in the same lineage
90
How does dormancy play a role in cancer cells ability to survive?
* CSC dormancy is associated with chemo resistance
1) A dormant cancer cell is surrounded by proliferating cancer cells
2) The patient undergoes therapies
* chemo therapy, radiation therapy, immune therapy, etc.
3) It kills all other cells except CSC which are dormant
4) Over time, signals are sent and the CSC leads to therapy resistant tumors
91
How does tumor metastases occur?
* metastases: New tumor growth
* The blood stream or lymphatic system carries cancer cells from the primary tumor to other parts of the body
92
How do tumors vascularize?
* During the proliferation of cancer cells there is a secretion of growth factors that lead to blood vessel formation
* Tumors without a blood supply are unable to grow. Growth factors attract nearby blood vessel cells to migrate to the cancer cell and form new branch of vascular system
93
How does a primary oocyte develop?
-The primary oocyte has gandulosa cells surrounding it
-Proliferation/maturation of follicle
* Hormonally driven: (E, LH, FSH)
-Mature follicle: (inner to outer layer)
* Primary Oocyte (4n)
* Grandulosa cell layer (converts testosterone to estrogen)
* Theca cells (convert cholesterol to testosterone)
* Zona Pellucida
94
How does an ovarian stem cells differentiate?
1) VSEL (analogous to primordial Germ Cells)→Pluripotent,
* produce Sox2 & Oct4
* Asymmetric division
* Quiescent until FSH (survive therapy)
2) differentiation (or self renewal)
3) OSC (ovary stem cells) progenitor cells (analogous to oogonia)
4) Differentiation and DNA duplication
5) Oocyte like cells and ovary cells
95
How does an oocyte develop from a primordial germ cell?
1) Primordial Germ cell (pluripotent)
2) Differentiation (or self renewal)
3) Oogonia (progenitor cells)
4) DNA duplication
5) Primary Oocyte
96
What is the difference between oocyte development versus Ovarian stem cell differentiation?
* In oocyte development oogonia undergoes DNA duplication to produce the primary oocyte
* In Ovary stem cells differentiation OSCs undergo differentiation AND DNA duplication
97
What cells are analogous to primordial Germ Cells?
VSEL stem cells
98
What cells are analogous to oogonia?
OSC
99
What do Ovary stem cells (progenitor cells) differentiate into?
* Multi-linage path way
1) Theca progenitor cell → theca cells
2) Stroma progenitor cell → Stroma cells (cortex)
3) Surface epithelial progenitor cell →surface epithelial cells
4) Gandulosa progenitor cell →Gandulosa cells
** Can get primary oocytes through DNA duplication within a follicle
- All cell types in ovary can be made
100
How are the primary oocytes produced from OSC?
- OSCs undergo massive proliferation (clonal expansion)
1) Clonal expansion of OSCs form germ cells nests of OCSs surrounded by endothelial cells
2) Meiosis occurs (DNA duplication, 4n) and Nest breakdown
3) Primordial follicle assembly with oocytes and grandulosa cells occurs
101
What experimental evidence is there for functional ovarian stem cells? (in vitro and in vivo)
In vitro
1) Perti dish in vitro → add VSEL or OSC
2) Add signals
3) In vitro → get oocyte like structures
In vivo
1) inject green fluorescent protein (GFP) injected into OSC (in vitro)
2) transplant OSC into ovary tissue
3) the OSCs initially grow and proliferate
4) If you cut into the tissue w/ each piece having an OSC (GFP)
5) transplant each piece into a mouse ovary
6) Remove after growth
* found a follicle with granulosa cells (GFP negative) surrounding an oocyte (GFP positive)
Conclusion:
* Ovarian stem cell can generate oocyte in vivo because oocyte is GFP positive
* The GFP negative ganulosa cells suggest the recipient can provide accessory cells (has the microenvironment)
102
How can human blood cells produce human oogonia?
1) Human blood cells (diploid cells)
2) Stemness genes/signals
3) Culture iPSCs in miniature ovary (bioengineered tissue)
4) four months later immature human oogonia is detected (precursor to human oocytes or eggs)
103
What is menopause? When does it occur?
- Aging of the ovary
- Loss of estrogen
- occurs between the ages of 45-55
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What is a strategy to mitigate menopause symptoms? What are the drawbacks?
- Estrogen replacement therapy has been used
- Risks:
* depending on the patient's age and medical history heart disease, stroke, blood clots, breast cancer, ovarian cancer
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What role does the microenvironment play in menopause?
* menopause associated with aging involves a compromised microenvironment or nice
-Granulosa cells play an important rile in establishing the microenvironment ; convert testosterone into estrogen
Aging:
- there is a signaling difference between the oocyte and the grandulosa cells
- the gransulosa cells lose the ability to produce estrogen
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What is the functional test of the row of the young microenvironment in follicle development?
* Ovarian stem cells from old ovary to young ovary
- normal oocyte development
* Ovarian stem cells from young ovary to old ovary
- compromised development of follicle, little/no oocyte development
!!! The microenvironment is responsible for the development of OSCs to oocytes
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What is some evidence that mesenchyme stem cells can form functional ovarian follicles? Why does it work?
1) mouse undergoes chemo ablation
2) kills oocyte in the mouse (mimics infertility)
3) implant mesenchyme stem cells into ovaries
4) oocyte development
5) fettle mice births mice
* The VSEL differentiate into oocytes and the mesenchyme stem cells were trophic support
* VSEL are quiescent; survive chemotherapy
108
What is premature ovarian failure?
* loss of normal function of ovaries before the age of 40
* Ovaries don't produce normal amounts of estrogen or release oocyte regularly
* causes menopause like symptoms
* Caused by stress, diet, genetics
