Stem Cells 5

Question 1

Describe targets of stem cell based therapeutic strategy

Answer 1

Stem cell or (micro) environment = niche

Question 2

Describe modes of manipulation of stem cell based therapeutic strategy

Answer 2

Exogenous = in vitro, ex vivo
Endogenous = in vivo

Question 3

Describe exogenous stem cell based therapeutic strategy

Answer 3

Pluripotent or adult stem cell —>
stem cell isolation = to increase stem cells by positive selection or by eliminating unessecary cells
stem cell propagation = small fraction of cell lineage, need to expand pop - recall not many stem cells —>
Transplantation (to site of body where tissue needs to be regenerated) —> regeneration or
Differentiation modification (induce differentiation at a Certain level)—> transplantation —> regeneration (hope that regeneration occurs)

Artificial niche —> sc manipulation —> transplantation (into body or pure artificial niche in diseased site)

Question 4

Describe endogenous stem cell based therapeutic strategy

Answer 4

Often used pharmacological manipulation - drugs, to control fate of stem cells (or viruses = many things) =
Scs = renewal, differentiation, mobilization, death (in cases of cancer stem cells)
Or
Niches = activation, inhibition, control, death

Question 5

What is stem cell therapy based on - what does it target

Answer 5

Targets cause of pathology not symptoms —> not manipulating symptoms or handle them
Issues tho = stem cells small and do not proliferate much, pluripotent stem cells tumorigenic, but adult stem cells are not

Question 6

Describe muscle stem cells

Answer 6

Satellite cells = have regeneration ability, sit on muscle fiver
Basal membrane= on top and surrounding muscle fiber

Question 7

Describe mesenchymal stem cells

Answer 7

Looks like fibroblast cells, very ambitious mesenchymal stem cells are derived from bone marrow stromatolites
And can produce many types of cells = bone, cartilage, muscle, stroke, tendon, adipocyte
Useful for clinical applications

Question 8

Describe bone fractures generally

Answer 8

Can be complex or cause large gap = difficulty to treat
One leg shorter than other, by >10 cam diff = handicap,
Can be due to car accidents, developmental conditions

Question 9

Describe an approach to fix bone gaps

Answer 9

Fill gaps with ceramic cylinder = rigidity corresponds to bones
Has pores so cells, bv and neurons can induce the pores
Enter scaffold to fix complex fracture of bone

Question 10

Describe canine model exp = Gen

Answer 10

Implantation of porous ceramics loaded with mscs = canine model
Ceramics was used as scaffold for regeneration by stem cells

Question 11

Describe canine model exp = no ceramic cylinder

Answer 11

Regeneration but still huge gap even after 4 months,
Little or no new bone formed, no union occurred

Question 12

Describe canine model exp = With a ceramic cylinder no mesechymal stem cells

Answer 12

Bone fusing to ceramics and bone marrow invades cylinder, gap artificially closed
Union occured at host bone implant interfaces
Bone formation did not occur throughout entire implant
Not super stable

Question 13

Describe canine model exp = With ceramic cylinder and mesenchymal stem cells

Answer 13

Dipped cylinder in ecm (cells, collagen, laminin), so whole ceramic column covered then dunk into mesenchymal stem cells = sticks to ecm inside and out
Heavier appearance on x ray = indicate more bone regeneration = calcium precipitation
Scaffold embedded in and integrated = has more stability
Substantial callus formed around implants
Union at host bone - implant interfaces is evident
Better, stronger

Question 14

Did the canine exp require immunodeficient dogs

Answer 14

A cellular scaffold made immunosupression unecessary while allowing stem cells to seed and start regeneration

Question 15

Describe conclusions of canine exp

Answer 15

Engineering of stem cell niches is an approach to regenerative therapy using stem cells
Used in humans too, almost in lungs = to replace removed tissue, like trachea

Question 16

What controls stem cell fate

Answer 16

Matrix elasticity
Ability of stem cells to communicate with environment and direct themselves to a certain fate

Question 17

Describe exp - stem cell changes based on matrix elasticity - gen

Answer 17

Substrate is polyacrylamide gel (acellualr) with diff elasticity and is coated with ecm - collagen 1 - so cells can touch gel comfortably
Put msc in culture dish = plastic goatee with gel

Question 18

Describe exp - stem cell changes based on matrix elasticity - specific hardness levels

Answer 18

Can change rigidity based on concentration of gel
Soft = cells branch out cytoplasm
Medium = cylindrical shape, not spreading, between the soft and hard types
Hard = spread wide cell, long
Cells have diff morphology based on gel - evolves over time

Question 19

Describe exp - stem cell changes based on matrix elasticity - branches graph

Answer 19

As gel gets harder = loses branches, more widespread tissue shape
Neurons = showed pattern of branches similar to what mesenchymal cells show
= soft gel supports cells to take neuronal morphology

Question 20

Describe exp - stem cell changes based on matrix elasticity - spindle factor graph

Answer 20

At a certain elasticity = medium level, cells become muscle cells = myoblasts
If too hard = declines
Substrate elasticity induces stem cells to specific direction

Question 21

What are differentially expressed depending on elasticity

Answer 21

Cell type specific genes are differnitally expressed depending on elasticity
Anti body staining against specific proteins
Beta3 tubulin = neuroncell, soft gel
MyoD = TF, Muscle origin, expressed at medium softness only
CBFalpha1 = osteoblast, localized proteins in nucleus, hard gel

Question 22

Describe what happens to factors as substrate elasticity increases

Answer 22

P-nfh - neuro = suppressed as gel gets harder
Medium on = myod
Bone specific starts being expressed when gel harder
Mesenchymal stem cells can feel what they are on, based on hard/softness
Bone exp = hard ceramic so makes bones

Question 23

What can give cells different feels of stiffness

Answer 23

Thickness of substrate
Ex = under gel = plastic, so thicker gel = will feel softer
500nm gel = thinner so bone
70um gel = thicker to less characteristic of osteoblasts

Question 24

Describe sustaining potential of skeletal muscle stem cells - how and Gen exp setup

Answer 24

Muscle stem cell isolation —> purified —> 7 day culture musc (on substrates with diff elasticities)—> transplantation into damaged tissue (killl muscle cells first with toxin) —> histology to see

Question 25

Describe sustaining potential of skeletal muscle stem cells - hard gel

Answer 25

If culture on v hard gel = do not generate muscle = lose muscle characteristics Needs specific elasticity fo substrate to maintain stem cells

Question 26

Describe sustaining potential of skeletal muscle stem cells - softer gel

Answer 26

More stem cells transplanted = the more regeneration occured Need specific in vitro culture to maintain cell abilities

Question 27

Wha can stem cells do

Answer 27

Converse with substrate and coordinate their fates according to environment Fate already determined so want to encourage to keep their functions Modification of environment contributes to improvement of stem cell manipulation efficiency Space of engineering attempts —> concept can be used for bioengineering

Question 28

Describe sphere culture system

Answer 28

Applicable to diff stem cell types Neural stem cells, Mammary gland stem cells, retinal stem cells, skin derived progenitor cells, neuro/glioblatoma stem cells, breast cancer stem cells Can all becomes sphere cells = cell structures can be applied for drug screening

Question 29

Describe neuropshere for drug discovery - 1

Answer 29

Made neuorsphres containing nscs - culture and exposed to many test compounds

Question 30

Describe neuropshere for drug discovery -2

Answer 30

New or approved compounds - applied certain concentration of drugs (If find good candidate compound = to treat certain conditions or disease or manipulate stem cells = do not need approval) See viability’s of cells Some encourage proliferation - top At bottom = to kills them, like if came from cancer cell

Question 31

Describe neuropshere for drug discovery - generating lead compounds

Answer 31

Compound libraries = can have 10 000 compounds Can be fda approved compounds or naturally derived compounds or can make small molecules by collaborating with chemical engineers

Question 32

Describe neuropshere for drug discovery - high content analysis - high throughput screening

Answer 32

384 well plate screening, can do 384 compounds if do 10 = 3840 compounds = everything automatic Automatic system to measure parameters Automated plate delivery, auto focus, expose and acquire - fluorescence based, automated image analysis, automatic data archival, automatic data spooling to the server, instantaneous data display, analysis of results

Question 33

Describe neuropshere for drug discovery - 3

Answer 33

See what it looks like Good survival or no

Question 34

Describe neuropshere for drug discovery- 4

Answer 34

Specific assay for a compound

Question 35

Describe stem cell based cell and gene therapy

Answer 35

Therapy based on stem cell targets = cause of pathology not symptoms Modify genes before transplantation

Question 36

Describe use of retroviral vectors for stem cell based gene/cell therapies - deliver

Answer 36

Deliver a missing gene in stem cells = Retrovirus (inclu lentivirus)= delivers a gene of inserts into host cell genome, integrates, permanent expression of genes Ex= adenosine deaminase to hscs (required for function of lymphocytes)

Question 37

Describe use of retroviral vectors for stem cell based gene/cell therapies - viral vectors

Answer 37

Use of viral vectors as delivery tools = efficient, can deliver gene into genome Words in vitro an in vivo Targets many cells at once Can be permanent = since genomic integration of transgene

Question 38

Describe use of retroviral vectors for stem cell based gene/cell therapies- risks

Answer 38

Risk of genetic modification Random integration = do not really know where goes - randomly Immune reaction = some cases (Covid - adenovirus was used to deliver spike protein, causes more side effects since we all have antibodies against adenovirus)

Question 39

Uni of Pennsylvania 1999, exp - describe

Answer 39

Ornithine transcarbamylase deficiency (affects urinate system = urea, usually treat with diet and medicines v restrictive) - Massive immune response to an adenoviral vector (injected into blood of patient and had short v aggressive immune response that attacked organs) - Multiple organ failure (boy dead ar 18, was shown in animal models but researchers ignored it)

Question 40

Necker sick children hospital, Paris, 2002 - describe

Answer 40

Severe immunodeficiency (delivered genes to overcome this) - Radom integration of a therapeutic gene in the genome of patients’ cells, using a retroviral vector for gene delivery (immune systems recovered) - Emergence of rare T-cell leukemia 3 years after the gene therapy. (Random integration of retroviral genes of kidneys - into genome) Buds outcomes bolted gene therapy trials

Question 41

Why escs needed for gene targeting

Answer 41

Aggressive proliferation Non viral approach - more frequent/sucessfully done But adult stem cells do not divide like es cells so difficult

Question 42

Describe Gen editing - gen

Answer 42

Immunological defence mechanism of bacteria When virus comes in = use this sequence to bind genome of virus Not gene targeting

Question 43

Describe Gen editing - specifics

Answer 43

Crispr = site recognition - loop shape of sequence derived from bacterial virus, so use sequence to bind genome of virus and destroy it Cas9 = enzyme, cleavage - done using guide rna, can modify rna Can induce diff functions Site specific - non specific recognition of target protein does not happen bc super specific Technically more straightforward than other methods

Question 44

Describe Gen editing - what can you do with crispr/cas9 system

Answer 44

- Introducing insertion/deletion mutations - Replacing one sequence with another - Introducing base substitutions - Adding a new sequence - Editing multiple genes at the same time - Modifying/controlling gene transcription - Modifying/controlling epigenetics - Manipulating RNAs.

Question 45

Describe anemias caused by genetic disorders affects erythrocytes

Answer 45

Sickle cell anemia = point mutations in globin gene B thalassemia = insufficient production of globin - due to mutation in enhancer

Question 46

What is therapeutic strategy to treat anemia - 5 steps

Answer 46

1. Harvest hematopoietic stem cells (HSCs) from a patient. 2. Interrogate the HSC genome and correct the mutation in the globin gene ex vivo by gene editing or gene targeting. (Identify mutation by examining genome) 3. Transfer the modified HSCs back into the patient. 4. HSCs engraft (no immune problems) and continuously produce erythrocytes with normal globins. 5. A complete and permanent cure, that requires no pharmacological intervention. (or, the therapy may need to be repeated if HPCs were modified)

Question 47

Why is b thalassemia harder to treate

Answer 47

Sickle cell = 1 point mutation But b thalassemia = >350 diff mutations, to target in each patient = very hard and labour intensive Both caused by mutations in beta globin gene and have same clinical symptoms

Question 48

Describe concept of strategy to help anemia

Answer 48

Adult globin gene is mutation (bc adults can be exposed to malaria) - FETAL GLOBIN GENE IS NOT Forced expression of fetal globin (gamma globin), leading to production of fetal hemoglobin in addition to adult hemoglobin (wake up gamma globin, capacity of carrying oxygen a bit Lower than adult one) Gene editing in hscs/hpcs that allows for gamma globin in erythrocytes in adult patients

Question 49

What induced gamma globin gene

Answer 49

Knocking down bcl11a - tf regulator, binds and suppressed gamma globin gene in erythroid lineage Sirna = breaks down bcl11a = inhibit translation of protein, less expression so gamma globin expressed Transcriptional suppression of bcl11a should lead to fetal globin and production of fetal hemoglobin in adults

Question 50

Describe editing bcl11a enhancer - Gen

Answer 50

Diff types = show position of guide rnas, needed to confirm one works Gata = site where specific tf binds to stimulate gene - target here, important so know to target here - target enhancer introduction of mutations in a specific region of bcl11a enhancer region, using crispr-cas9 technology If hit 1617 = most effective to stimulate gamma globin Rise in gamma globin should correspond to supresssion of bcl11a expression More fetal type globin expressed as less bcl11mrna = inverse correlation

Question 51

Describe editing bcl11a enhancer - what does disruption of gata binding sequence in enhancer do

Answer 51

1) To reduce BCL11A expression; 2) To reactivate g-globin expression; 3) For both to occur in the erythroid lineage (because the GATA motif is critical in the erythroid lineage)

Question 52

Describe more preclinical testing done to check if anemia exp worked

Answer 52

1) Human hematopoietic stem/progenitor cells (HSPCs) were extracted from two SCD (sickle cell disease) patients. 2) Gene editing was done in vitro on the BCL11A enhancer in HSPCs using the CRISPR-Cas9 technique (using Sequence 1617). 3) Patients’ modified HSPCs were transplanted into “immunodeficient” mice. 4) Different types of human blood cells were harvested from mouse bone marrow 16 weeks later. 5) Various analyses done at the levels of transcripts (mRNA), proteins, and cells. (Took cells out of patients, edit and transplant to mouse and measure human cels in blood of mouse)

Question 53

Describe engraftment of edited cells - exp results

Answer 53

After editing = no effect really - still same % of human cells and induced mutation % Wel maintained after transplantation, similar prevalence of induced mutation, nearly 100%

Question 54

Describe erythroid specific effects protein - exp results

Answer 54

Bcl11a in B CELLS = not affect by genetic editing Bcl11a in eyrthoid cells = gene editing resulted in reduction of protein expression of bcl11a specifically in blood cell lineage - inverse correlation

Question 55

Describe gamma globin expression - exp results

Answer 55

Expressed for after gene editing

Question 56

Describe erythroid specific effects protein - biological assay

Answer 56

Mbs = sodium metabisulfite test- diff osmotic pressure such that rubs will show sickle shape if not edited = unedited So can see normal morphology maintained of edited cells

Question 57

Has anemia exp even done irl

Answer 57

Applied clinically but must treat with bone marrow meds (one to damage bone marrow)- if have to do to kids in Africa, = more sensitive maybe Similar approaches used- device and developing but sucessful cases have been reported

Question 58

Describe what stem cell based therapies do - summary

Answer 58

Stem cell-based therapies (cell therapy, regenerative medicine) can overcome a disease state and achieve a complete cure, rather than alleviating symptoms. Gene editing adds a precision to stem cell-based gene therapy. (if it is indeed safe) Next generation stem cell therapy

Question 59

What does gene therapy need to satisfy

Answer 59

Does it work Is it safe = most important Does it pay off (if too costly, or hard to apply)