Stem Cells Flashcards

Question

What are the challenges of iPSCs

Answer 1

Control of differentiation – hard to drive a large number of stem cells to differentiate into a single cell type – will almost always get a mixture of cell types which is problematic if implemented into a patient

Answer 2

1. Take skin cells from patient - dissociate or leave them intact 2. Add Yamanaka factors -after 2/3 weeks some cells produce spheres of cells – sign of enhanced cell division hence pluripotency (during the 2/3 weeks, cell culture = very dense – so must frequently replenish medium + no contamination) 3. Use nanog, a TF, linked to GFP to report on pluripotency 4. Normally, reprogramming efficiency is low 5. iPSC can remember its original cell type from its epigenome (if has not been reset)

Answer 3

1. do they express genes usually expressed in ES cells (some heterogeneity - ES cells vary - don’t always have to express same genes) -by looking for proteins using antibodies 2. can they form teratomas w/derivatives of all 3 germ layers (teratomas = disordered embryos/tumours w/different cell types from across germ layers) 3. can the proliferate and form sphere cells (suggest self-renewal - indicate formation of stem cells - perform on cell plate or hanging drop experiment) 4. are pluripotency promoters or histones unmethylated (study the epigenome - bisulphite sequencing) 5. can iPSCs produce a whole embryo

Answer 4

-type 1 diabetes -chronic obstructive pulmonary disease -ALS -macular degeneration

Answer 5

> undifferentiated stem cells in a differentiated tissue or organ > can self-renew and produce daughter cells that can differentiate into cell types of the tissue > not pluripotent - multi/bi/uni potent > genome has become more restricted/less plastic - less open to influences and more committed to particular lineages

Answer 6

> In the embryo: go through a lot of cell division to generate enough cells for each tissue (can stay at G0 until cells are needed) > In the postnatal/adult: does not need to increase size of tissue so cells = mostly at G0 to wait for injury/ disease to trigger them to enter cell cycle and divide to repair and replace the damaged cells

Answer 7

> gene therapy: -harvest tissue-specific stem cells, genetically correct them, re-introduce back to patient to regenerate tissues with healthy cells -genetically create healthy cells from another donor and introduce into patient w/immunosuppressants > experiment: -harvest diseased tissue-specific stem cells & grow in vitro for better understanding of certain diseases

Answer 8

-after obtained cell sample, can culture in 2D (cell plate) or 3D (hanging drop, in gel, sculpted 3D printed set ups) -3D tells you more about stem cell behaviour -might observe spheres of cells develop: mixture of dividing and differentiating cells - indicate stem cells -check for stem cells eg. presence of genes, nanog markers, etc

Answer 9

-use antibody to check for antigens in sample of cells. antibody linked with enzyme or fluorescent dye for detection and labelling -use to check for proteins/components that stem cells are expressing -eg. staining pancreatic islet cells -therapeutic potential in treating type 1 diabetes -stain specific antibodies to locate insulin, glucagon, nuclear DNA -will get heterogeneity of cells expressing diff levels of proteins

Answer 10

-tiny, self-organised, 3D tissue cultures derived from stem cells -arise from self-reorganizing property of stem cells -mimic aspects of development, tissue structure and organisation (liver, lung, brain, etc)

Answer 11

1. embed pluripotent cells in ECM eg. Matrigel, to support the cells 2. add specific growth factors and proteins to mimic the in vivo environment and maintain the stem cell phenotype 3. based on the initial stem cell population and growth factors chosen, the matrix-embedded cells will self-assemble into 3D-organoid structures that behave similar to a specific tissue

Answer 12

> can study stages of organ development, mutant organs -therapuetic potential for studying diseases related to erroneous organ development > study the effect of virus or other factors on organs (eg. covid, air pollution on lungs)

Answer 13

pros: -easy to visualize, study, manipulate -easy to control variables in the experiment cons: -not representative of physiological conditions

Answer 14

> look for marker genes, mRNA, and/or proteins >identify diving cells in vivo: 1) label cells during DNA synthesis stage by antibodies/BrdU 2) pulse-chase experiment: shortly after introducing labeled compound, introduce excess of same but unlabelled substance into the environment 3) label-retaining cells could indicate SCs in G0 4) BrdU has disadvantages, can use other labels: Histone-2-GFP >transplant blood stem cell from bone marrow into recipient irradiated mouse (its own blood cells in bone marrow= dead); observe the SC repopulate the recipient mouse entire blood system (indicates multipotency of blood stem cells)

Answer 15

1. use Cre Lox transgenic mice line (express cre recombinase 1 under a cell-type specific promoter) 2. cross with Flox DNA sequence mice line -floxed sites = short DNA sequences which are a locus for frequent recombination -engineered w/EGFP for selection of successful recombination 3. results in mice line which when induced under specific cell type promoter, results in recombination only in specific cell type (eg. cardiomyocytes) 4. track marker & cell lineage 5. label, kill, manipulate specific cells at specific time points 6. tells about how the embryo was constructed over time & the dynamics of adult tissues

Answer 16

1. suspended cells in mixture are subjected to different fluorescent tags depending on the experimental setup 2. fluidics system channels the mixture of cells in a streamline fashion 3. as the cells flow through the stream of liquid, they pass through a laser-detector, that monitors the fluorescence and light scatter characteristics -scattered light is detected by forward or side data detector -forward scatter is prop. to size -side data scatter is prop. to complexity &/or granularity of the cell -plot data from forward and side scatter to group cells with different properties -can isolate diff. populations of cells 4. based on their characteristics, cells are separated in an electric field into different collecting tubes or multiwell plates 5. can separate out stem cells, culture them, transplant, perform omics, observe transcriptome/proteome

Answer 17

can transgenically label stem cells for imaging in physiological conditions

Answer 18

observe transcriptome of every cell w/o dissociating tissues 1. sample preparation: place slice of tissue in microprinted plate which has constructs that is absorbed by every cell in the tissue giving it a barcode 2. in situ PCR/collection of biomolecules to collect the barcodes of every cell eg. obtain tissue with protein markers/antibodies to collect all of their cDNA/proteins -profile every cell -can look at heterogeneity in tumour cell (compare healthy vs diseased cells)

Answer 19

>crypt base (Wnt & Shh signals) induce BMP4 (bone morphogenetic protein 4) expression in nearby connective tissue >BMP4 stops the epithelium becoming crypt

Answer 20

>EphB+ cells and ephrin+ cells repulse each other, keeping the populations in the correct place > if no EphB, cells can move up the villus

Answer 21

-by in situ hybridization, microdissection, PCR, omics -2+ populations of stem cells: -Lgr5+ stem cells at crypt base -active and replace epithelium daily -quiescent "reserve" stem cells, 4+ (4th position from the crypt base), activate to repair gut after injury

Answer 22

Lgr5+ cells = good at forming organoids can be used for drug screening, studying disease, in vivo studies of gut stem cell biology eg. effects of diet: how dietary fat promotes intestinal dysregulation

Answer 23

-the microenvironment surrounding the stem cell that contains signals that keep it as it is i.e undifferentiated, capable of self-renewing -can contain signals from daughter cells, supporting cells, ECM -cells in the niche can change to promote the stem cells' differentiation -cancer cells may escape niche control

Answer 24

-satellite cells = stem cells located under the basal lamina of muscle fibres -easily identified by the expression of pax7 (homeodomain TF)

Answer 25

-often sit in G0, but primed for activation (can re-enter cell cycle if needed) -genes for muscle differentiation switched off ~500 genes involved incl. cell cycle blockers & myogenic inhibitors eg. FOXO3 expressed in satellite cells in G0 -if delete, incorrect self-renewing and spontaneous differentiation increases. FOXO3 acts via regulating NOTCH signalling between neighbouring cells. Release of NOTCH intracellular domain can go on to regulate expression of different TFs -able to divide & migrate upon recognition of activating signal -express pro-muscle TF: myogenic TFs MyoD and Myof5 -fused to damaged myofibers or each other to form new myofibers -when terminally differentiate (can’t re-enter the cell cycle), will produce mature markers eg. myogenin

Answer 26

inflammation: after injury, activate them via TNF cytokine which activates p38 MAPK signalling -allows satellite cells to proliferate microRNAs: miRNA489 is conserved between many species & up-regulated in quiescent cells. Dek protein promotes proliferation and expansion of myogenic progenitors. miRNA489 can suppress Dek & keep satellite cells in G0.

Answer 27

asymmetrically; -1 daughter commits to myogenic lineage because it inherits myogenic TF -1 daughter self-renews and re-enters the G0 (does not lose stem cells over time)

Answer 28

Duchenne muscular dystrophy -dystrophin is expressed in satellite cells asymmetrically, leading to asymmetric division

Answer 29

don’t have muscle progenitors and not producing muscle daughter cells, so you can’t rescue damaged muscle so muscle will deteriorate over time

Answer 30

isolate the stem cells from the patient, genetically engineer to correct defects, re-introduce into the patient to rescue muscle regeneration or function

Answer 31

> by disrupting muscle cell quiescence: aged muscle fibres express more Fgf2, driving satellite cells out of quiescence & depletes them >old muscle stem cells switch from quiescence to senescence

Answer 32

possible via local and systemic factors

Answer 33

1. Some cells from ectoderm develops into nervous system (decides between differentiation into skin vs. neurons) 2. Those cells undergo cell division (still uncommitted in multipotency) 3. Regionalization occurs – signalling centers develop and signal out particular ligands which will have effects on nearby cells in a concentration-dependent manner (morphogens) * Brain development is coordinated with surrounding tissues ex. muscles, skulls 4. Mixing between diff. cell types restricted by selective adhesion (ex. cadherins adhere similar cell types together in a developing embryo) 5. Neurons generated (committed to neural lineage – nerve cells made before glial cells – neurons connect first & then regulating glial cells made to support those neurons) 6. Migration to final position – know that in right position by sensing ligands and ECM in the surrounding 7. Waves of glial cell production (oligodendrocytes & astrocytes) 8. Neurons connect and generate synapses 9. Cull excess neurons 10. Myelination by oligodendrocytes 11. Remodel as required (but not regenerated)

Answer 34

* TF: Hesx1 and Pax2 drive cells down a particular lineage (a particular neural/ glial cell type) -once plastic cells open to influences = will now activate a particular TF in response to local signals that will funnel their genome down a particular fate * Selective adhesion that prevents cell migration will result in cells continuously receiving the appropriate local factors so they will differentiate into the particular cell type needed at that location

Answer 35

* Cells in ectoderm decide whether will become skin or neural cells * Once neural plate is formed = committed to neural cells * Closing of neural groove = form neural tube * Signaling molecules (ex. Wnt or Sonic hedgehog) = important for organization of cells

Answer 36

* Neuroepithelial cells * Radial glial cells (has one end in ventricle, the other on opposite edge of tissue) o First make neurons, then glial cells to support neurons

Answer 37

- involves DNA methylation changes (epigenome) * Initially, genes that produce astrocytes = methylated, so not expressed & radial glial cells will produce neurons * Signals received by radial glial cells will result in methylation of genes that produce neurons and demethylation of genes that produce astrocytes to switch from neuron to glial cells production

Answer 38

dopaminergic neurons – only produced in the ventral mid brain * 2 main populations of dopaminergic neurons: o VTA neurons – involved in addiction/ reward behaviour o Susbtantia nigra pars compactor neurons – if die = cause Parkinson’s

Answer 39

The isthmus (A midbrain/hindbrain organising centre) is important for dopaminergic neuron production * Signalling factors – otx2, Wnt1, Gbx2, Fgf8 = involved in setting up position of isthmus which goes on to become a signalling centre to release more factors – release pattern will determine the fate of anterior/ posterior cells

Answer 40

* Neuroepithelial cells will give rise to dopaminergic neurons at the ventricular surface. They will then migrate along the radial glial guides to differentiate into their final position and send axons to release dopamine at the front of the brain. -Human adults (postnatal stage) = unable to generate new neurons * Adult mammalian brains able to generate new neurons = rodents * Two main regions in rodents: striatal subventricular zone (SVZ) and subgranular zone (SGZ) of the hippocampus:

Answer 41

* cells born in lining of lateral ventricle * divide to give neuroblasts (immature neurons) PSA-NCAM+ * Migrate to olfactory bulb via rostral migratory stream (RMS) (rodents) * integrate into the olfactory bulb i.e. contribute to an already established circuitry * also generate some oligodendrocytes

Answer 42

The neurons produced by SVZ neural stem cells are GABAergic & dopaminergic interneurons

Answer 43

-the subgranular zone (SGZ) of the dentate gyrus (DG, hippocampus) * DG transforms memory input (context, space, time) from entorhinal cortex layer 2 into outputs to CA3 neurons * New neurons seem to help separate new memories * Exercise boosts number, chronic stress can reduce it

Answer 44

* From production of neuroblasts * The cancer stem cell hypothesis = some tumours are seeded, maintained, and regrown from cancer stem cells (that can defectively produce huge amounts of neural and glial cells progeny) * Therapeutic potential: identify stem cell markers in tumors & induce them to differentiate, so will just be benign lump in brain instead of metastasizing into secondary tumor sites Ex. Glioblastoma Multiforme (GBM) * most common CNS tumour * median survival 14-16m despite optimal surgery, radiation and chemotherapy

Answer 45

* transplants – as treatment for Parkinson’s disease * As research tools

Stem Cells Flashcards

(69 cards)