Exam 2 Flashcards
1
Q
Sketch a diagram and explain reprogramming, transdifferentiation and dedifferentiation
A
look at pic
2
Q
Mention 1 example (cell type) for reprogramming, dediff and transdiff.
A
Reprogramming/Retrodifferentiation: Differentiated cell (skin) -> iPSCs
Dedifferentiation: Unipotent (skin) ->Multipotent (adult SC)
Transdifferentiation: One terminal cell (skin) -> another terminal cell (neuron)
3
Q
FGF functions in a cell
A
COME
a. Embryonic Development
b. Organogenesis
c. Cell migration
d. Metabolism
4
Q
Mediators in FGF signaling
A
a. MAPK – mitogen activated protein kinases (adds phosphate)
b. STAT – Signal transducers and activators of transcription
c. TGF-B signaling – transforming growth factor
d. GSK-3 – Glycogen synthase kinase
5
Q
Video – FGF Signaling
A
FGF2 ligand binds to FGFR (transmembrane receptor)
Dimerization of the receptor (come together)
Autophosphorylation of FGFR (self-phosphorylates) & intracellular tyrosine kinase adds phosphate)
SOS (son of sevenless, GTPase) and Grb are activated
Ras and Raf are activated (serine threonine kinase)
P- MAPKK (mek)
P-MAPK (erk)
Enters nucleus and activated TFs (PSC maintenance) by phosphorylation
6
Q
Lif pathway
A
LIF (Leukemia Inhibitory Factor) binds to LIF-R (gp130) activates both JAK1/PI3K & STAT3/AKT
enters nucleus and activates Klf4/Tbx3
activates CTF
7
Q
BMP Pathway
A
BMP4 binds to BMP4R
Smad 1/5/8
enter nucleus and activated CTF
8
Q
TGF-b pathway
A
TGF-b/activin bind to TGF-R
Smad 2/3
enters nucleus and activates CTF
9
Q
Wnt Pathway
A
Wnt binds with Frizzled
b-Catenin
enter nucleus and activates TCF/LEF
activates CTF
10
Q
What does FGF stand for?
A
Fibroblast growth factor
11
Q
what does GSK-3 stand for?
A
Glycogen synthase kinase -3
12
Q
What does JAK/STAT stand for?
A
Janus Kinase & Signal Transducer and Activator of Transcription
13
Q
What does MAPK-ERK stand for?
A
mitogen-activated protein kinases, extracellular signal-regulated kinases
14
Q
What does NF-Kb stand for?
A
Nuclear Factor Kappa - Beta
15
Q
what does PI3K-AKT stand for?
A
phosphatidylinositol 3-kinase
16
Q
what does TGF-B stand for ?
A
Transforming Growth Factor - Beta
17
Q
What does SMAD stand for?
A
fusion of Caenorhabditis elegans Sma genes and the Drosophila Mad, Mothers against decapentaplegic
18
Q
What does BMP stand for?
A
Bone morphogenetic protein
19
Q
What does Wnt stand for?
A
Wingless and lnt-1
20
Q
Laureates in physiology and medicine 2022
A
Svante Paabo - DNA from fossils
21
Q
Where are NSCs found in the brain?
A
Hippocampus and ventricular/sub-ventricular zones
22
Q
Draw a structure of a neuron (4 parts)
A
Dendrite, nucleus, soma and axon
23
Q
3 types of cells derived from NSCs and their function
A
a. Neuron
b. Astrocyte – helps maintain inflammation
c. Oligodendrocyte – wraps around neuron (insulate), faster relaying of signals
24
Q
What is an Ependymal cell? Their function?
A
ciliated-epithelial glial cells
Helps maintain the cerebrospinal fluid, Ionic balance, antioxidant, regulates neurotransmitters, water transport
25
Uni, bi and multipolar neurons, pyramidal cells (functions and related disorders)
a. Uni – sensory neuron (cell body on side)
b. Bi – interneuron (cell body in center and 2 extensions) (connect one part of brain to another part) - (schizophrenia/bipolar disorders)
c. Multi – Motor Neuron, Most common, makes up brain, multiple dendrites
d. Pyrimidal – help in relaying signals faster
26
Origination of NSCs and how they change with age
a. Originate from neuro-epithelial progenitor cells through symmetric division
b. De-differentation occurs to produce NSC
c. Neurogenesis occurs -> glial, nuerons (pruning and apoptosis – tells cells to stop)
AGE: As they age the self renewal capacity stops around 45-55 years
Neural cells exhibit asymmetrical division and this is lost as they get older so they lose the neural stem cell pool
27
What are Neural Stem Cells (NSCs)?
multipotent, self renew, proliferate without limit, and produce progeny cells that terminally differentiate into neurons. Non-stem cell progeny is Neural Progenitor cells.
28
What is a Neural Progenitor Cell?
Can proliferate and differentiate into more than one cell type.
They are unipotent, bipotent or multipotent and have limited proliferative ability and no self renewal.
29
What is a Neural Precursor Cell (NPCs)?
a mixed population of cells consisting of all undifferentiated progeny of neural stem cells (neural progenitor cells and neural stem cells) usually derived from ESCs and IPSCs.
30
What is a Neuroepithelial Progenitor Cell?
earliest neural cell type. Neuroepithelial to NSC pools is dedifferentiation then to neurogenesis (glial, neurons, apoptosis (programmed cell death) and pruning (early stages prune the brain and spinal cord caused by apoptosis)
31
How does the young and old niche of NSCs differ?
Young niche – neurogenesis, neuroblasts, quiescent NSC, Neuroprecursors, and nueroprogenitor cells
Old niche – inflammatory cytokines, ependymal cells become quiescent and lose cilia, activated microglia start eating cells (ependymal), Tc cells kill all cells in path even activated neural cells
32
Factors needed to differentiate iPSCs to neurons
1. Add FGF-2 and EGF
2. Grow iPSCs in 2D
3. Remove EGF
4. dd NT-3 (neurotrophic factor – 3) , NT-4 and reduce FGF-2
5. Add Shh (sonic hedgehog) and FGF-8
33
Factors needed to differentiate iPSCs to glial cells.
1. Add FGF-2 and EGF
2. Grow in 2D/3D
3. Remove the EGF
4. Add BMP (bone morphogenetic protein), CNTF (ciliary neurotrophic factor)
5. Then add NGN (neurogenin), PDGF (platelet derived growth factor), cAMP (cyclic AMP), and FGF-2
34
What do you get when you add BDNF, Sonic hedgehog and RA to Neurons? Know the abbreviations of growth factors
1. BDNF -> GABA neurons
2. Sonic hedgehog + FGF-8 -> Dopamine neurons
3. RA -> Motor neurons
35
What are neurospheres? How do they differ from 2D neurons?
Grown in 3D environment of a single cell type. Similar to normal human nerve tissue.
36
Differences between qNCSs and aNSCs. Why are they important as we age?
qNSCs – low metabolic needs, lysosome/autophagy, low ATP levels
aNSCs – high metabolic needs, proteasome, ER barrier
37
Disease that could be treated using NSCs: ALS
(What cell is lost, how to treat and symptoms)
ALS – amyotrophic lateral sclerosis
Loss of cortical and spinal motor neurons
New neurons from NSC transplants which would grow new axons or NSC derived astrocytes to protect remaining motor neurons
Difficulty walking and weakness in legs/feet/ankles
38
Disease that could be treated using NSCs: Huntingtons
(What cell is lost, how to treat and symptoms)
Loss of inhibitory GABAergic neurons
Replacement or protection of striatal GABAergic neuron using NSC derived cells to slow spread of degeneration or grafting of NSCs into multiple sited in striatum and cortex
Involuntary jerking or slow and unusual eye movements
39
Disease that could be treated using NSCs: Parkinsons
(What cell is lost, how to treat and symptoms)
Loss of dopamine production in basal ganglia
Dopamine producing neurons derived from ES cells to replace those that are lost
Tremors and slowed movement
40
Disease that could be treated using NSCs: Spinal Cord Injury
(What cell is lost, how to treat and symptoms)
Severed long-distance connections between brain and limbs
Human ES cells derived NSCs to generate new oligodendrocytes for remyelination of remaining damaged axons
Extreme back pain and weakness or paralysis in any part of body
41
Disease that could be treated using NSCs: MS
(What cell is lost, how to treat and symptoms)
Multiple Sclerosis
Oligodendrocyte degeneration leading to demyelination of axons
Transplantation of pre-differentiated NSCs (targeting the CNS) for remyelination and renewed motor function
Fatigue and vision problems
42
Different routes of administering NSCs.
Best non-invasive methods?
Intracerebral, intraarterial, intraperitoneal, intravenous (non-invasive), and intranasal (non-invasive)
43
How can you prevent chronic neurodegeneration?
Dietary intervention - Calorie restriction and intermittent fasting
Increase neurogenesis and improve olfaction /learning/ memory
44
What is a scaffold? Why is it important?
Biomaterials or growth factors that have been engineered to make/mimic tissues in vivo for tissue replacement
45
Name any 4 factors needed to make a biocompatible tissue scaffold.
a. Immobilized/mobile factors
b. Soluble growth factors
c. Auto and Paracrine signaling
d. Matrix
46
What is autocrine signaling/paracrine signaling ?
Autocrine - same cell secretes factors that signal to that cell
Paracrine – Cell secretes factors that signal to another cell
47
What is decellularized matrix? (steps)
How can this be used for tissue transplantation?
a. A matrix with no living cells present but the scaffold remains
b. Freeze then undergo homogenization using polyethylene glycol, Triton-X 100, and Sodium Dodecyl Sulfate to decellularize.
Then lyophilization to powder the tissue.
Add pepsin is used to digest the cells but scaffold remains. Neutralization to stop pepsin.
Finally coat with hydrogel to grow cells on the frozen tissue.
when cells are growing on matrix they grow to same shape of the previous tissue.
48
Which one is superior for cell growth and tissue fabrication, 2D or 3D? Why
2D – High stiffness, restricted to x-y plane, continuous layer of matrix, forced basal polarity
3D – Low stiffness, Adhesion distributed in all 3 dimensions, discrete matrix fibrils, No polarity
3D is better than 2D
49
Match the following:
a. ESCs, MSCs, Lung cells/spinal discs, Bone, Skin, Matured Neurons/bones, Neural Stem Cells/MSCs, Cancer Stem Cells
b. aerogels, collagen, gelatin/fibronectin, hydroxyapatite, bioink, microcarriers, hydrogel/3D spheriods, matrigel/vironectin/laminin511
a. Bone – hydroxyapatite
b. Skin – collagen
c. Lung cells, spinal discs – aerogels
d. MSCs – gelatin, fibronectin
e. ESCs – matrigel, vitronectin, laminin 511
f. Cancer stem cells – hydrogel, 3D spheroids
g. Neural stem cells, MSCs – microcarriers
h. Matured neurons and bones – bioink
50
Briefly describe biomaterials currently used for stem cell transplants. Mention any 2 examples each.
a. Nanofibers – include silver, gold, carbon- based fibers, single and multi-walled, could be toxic
b. Microcapsules – adding cells in small spheres in large amounts. Nutrients and growth factors are able to enter. Get stuck around sphere or porous micro carriers, grow cells in 3D
c. Protein-based – collagen (synthetic or from cadavers) and Fibrin (blood clots)
51
Mention any 4 applications of microfluid/microchip devices.
1.Immune regulation for A.I diseases (mixing more than 1 type of cells – T cells on pancreas cells)
2. Metabolism/Physiology of drugs (sodium – potassium ion channels)
3. Toxicology
4. Kidney functions
52
What are aerogels
A 2D/3D air/liquid interface to help grow defined cell types which are good for 3D constructs (bone/cartilage/and spinal discs)
53
What is cGMP?
Certified good manufacturing practice standards
54
Epigenetic influencers on humans
a. Psychological state
b. Diet
c. Drugs
d. Exercise
e. Social interactions
55
Four levels of epigenetic regulation in PSCs
1. DNA methylation
2. Histone Modifications (covalent)
3. Micro RNA Modifications
4. 3D genome architecture
56
What is eu and heterochromatin?
a. Euchromatin – active gene region of DNA (open space)
b. Heterochromatin – inactive genome region (packed)
57
Nucleosome, histone proteins (core histones and linker histone)
a. Nucleosome formed when 8 core histone subunits come together (2x H2A, H2b, H3, and H4) linked with H1 linker protein
b. Multiple nucleosomes stack up to form chromatin fiber
c. This is then looped and packed into chromosomes
d. Chromosomes are found in the nucleus
58
Epigenetic modifications to histones by adding functional groups: Acetylation
Acetylation – addition of CH3CO- group to activate the gene – Acetylation at H3K9 activates genes (lysine residues)
Writer – histone Acetyltransferases (HATs)
Erasers – Histone Deacetylases (HDACs)
59
Epigenetic modifications to histones by adding functional groups: Methylation
Methylation – adding CH3 group (Lysine and Arginine residues) – arginine methylation of H3 and H4 activates / lysine methylation at H3K4me3 activates but inactivates at H3K9me3 and H3K27me3
Writers – Histone methyltransferases (HMTs) and protein arginine methyltransferases (PRMTs)
Erasers – Lysine Demethylases (KDMs)
60
Epigenetic modifications to histones by adding functional groups: Phosphorylation
Phosphorylation – addition of a phosphoryl group (Serine, tyrosine, and threonine residues)
Writers – kinases
Erasers – phosphatases
61
Epigenetic modifications to histones by adding functional groups: Ub
Ubiquitination – tagging with ubiquitin to destroy proteins and inactivate genes for a long period of time (silencing) (lysine residues)
62
Mechanism of DNA methylation, CpG islands, know active and inactive states of a gene
Methylation – add methyl groups to DNA sequences at cytosine residue to inactivate (silenced) or remove to activate (transcription)
CpG islands – where DNA methylation takes place
63
DNMTs involved in methylation and demethylation
DNA methyltransferases (methylation) – DNMT1, 3a, and 3b
DNA demethylases – tet family of proteins involved in activating genes
64
Activation / inactivation of histone 3 via Methylation
a. H3K4me3 – active
b. H3K9me3 - inactive
c. H3K27me3 - inactive
d. H3K79me2 - active
65
BONUS all 20 amino acids
alanine
arginine
asparagine
aspartic acid
cysteine
glutamine
glutamic acid
glycine
histidine
isoleucine
leucine
lysine
methionine
phenylalanine
proline
serine
threonine
tryptophan
tyrosine
valine
66
Nc-RNAs (6 types)
1. Micro-RNA (gene silencing)
2. Piwi-interacting RNA (silence transposons)
- can make a trihybrid complex
3. Small interference RNAs (gene silencing)
4. Long non-coding RNAs (H3K4me3, H3K36me3 – gene silencing)
- from intronic region of DNA
5. Enhancer RNAs (active gene expressions)
6. Promoter assisted RNAs - gene expression active/inactive
- binds to promoter region by recruiting proteins)
67
Slide 10 – DNA/lnc RNA triple helix, miRNA, lnc RNA with protein, lncRNA with mature mRNA
triple helix - trihybrid complex with LNC and binds right before promoter region
miRNA - binds to LNC to inhibit another repressor
LNC w/ protein - conformational change by directly inhibiting protein function by binding it
LNC w/ Mature mRNA - no ribosomes can attach to it
68
States of chromatin and chromatin binding proteins in ESCs and differentiated cells
ESCs – chromatin is more open and less chromatin proteins bind to DNA
Differentiation – More chromatin proteins bind to DNA tightly repressing transcription and heterochromatin dominance
69
What are bivalent domains and their mechanism (slide 14 and 15)? Which cells have more bivalent domains and which ones don’t?
Bivalent Domains: chromatin regions co-occupied by H3K4me3 (on) and H3K27me3 (Off)
Exclusive to PSCs and ESCs, differentiated cells do not
70
Heterochromatin organization in ESCs and NPCs
NPCs have more heterochromatin domains with more genes silenced
ESCs less in number but less genes silenced
71
PcG and Trx group of proteins regulation of ESC pluripotency and differentiation (slide 16 and 17), their specific modifications
too much Trx: activates gene expression (endo, ecto, meso) - H3K4 methylation
too much of PcG (polycomb repressive complex): deactivates gene (silenced) - binds to H3K27me3
equal amounts of both: stays stem cell
72
Mechanism of Jumonji and G9a during PSC maintenance and differentiation.
Maintenance: TURN ON
1. Oct4 binds to Jmjd1a (takes off methyl group to activate gene) near promoter region
Differentiation: TURN OFF
1. Oct 4 inhibits itself by recruiting G9a (methyl transferase) to transfer methyl group
2. HP1/H3K9 bind to promoter region to shut off - inhibiting transcription
73
Differences in chromatin regulation in PSCs and differentiated cells
PSCs - bivalent chromatin domains (4/27), transcriptionally permissive, hyper-dynamic chromatin structure with open chromatin
Differentiated - maintenance of bivalent domains, more permanent repression of pluri. genes, more closed chromatin
74
3D genome organization (slide 20) – TAD and CTCF proteins for 3D localization in nucleus
TAD - topologically associated genes, remains closer to the nucleus, keep active genes closer to center
CTCF - CCCTC (activator/repressor) - bind to that sequence only. once a signal tells LAD or TAD (mediator)
75
What is LAD?
Lamina associated genes - they take DNA (inactive) and bind to Lamina so they don't float away
