Stem Cells 5 Flashcards
Describe targets of stem cell based therapeutic strategy
Stem cell or (micro) environment = niche
Describe modes of manipulation of stem cell based therapeutic strategy
Exogenous = in vitro, ex vivo
Endogenous = in vivo
Describe exogenous stem cell based therapeutic strategy
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)
Describe endogenous stem cell based therapeutic strategy
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
What is stem cell therapy based on - what does it target
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
Describe muscle stem cells
Satellite cells = have regeneration ability, sit on muscle fiver
Basal membrane= on top and surrounding muscle fiber
Describe mesenchymal stem cells
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
Describe bone fractures generally
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
Describe an approach to fix bone gaps
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
Describe canine model exp = Gen
Implantation of porous ceramics loaded with mscs = canine model
Ceramics was used as scaffold for regeneration by stem cells
Describe canine model exp = no ceramic cylinder
Regeneration but still huge gap even after 4 months,
Little or no new bone formed, no union occurred
Describe canine model exp = With a ceramic cylinder no mesechymal stem cells
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
Describe canine model exp = With ceramic cylinder and mesenchymal stem cells
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
Did the canine exp require immunodeficient dogs
A cellular scaffold made immunosupression unecessary while allowing stem cells to seed and start regeneration
Describe conclusions of canine exp
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
What controls stem cell fate
Matrix elasticity
Ability of stem cells to communicate with environment and direct themselves to a certain fate
Describe exp - stem cell changes based on matrix elasticity - gen
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
Describe exp - stem cell changes based on matrix elasticity - specific hardness levels
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
Describe exp - stem cell changes based on matrix elasticity - branches graph
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
Describe exp - stem cell changes based on matrix elasticity - spindle factor graph
At a certain elasticity = medium level, cells become muscle cells = myoblasts
If too hard = declines
Substrate elasticity induces stem cells to specific direction
What are differentially expressed depending on elasticity
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
Describe what happens to factors as substrate elasticity increases
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
What can give cells different feels of stiffness
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
Describe sustaining potential of skeletal muscle stem cells - how and Gen exp setup
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
Describe sustaining potential of skeletal muscle stem cells - hard gel
If culture on v hard gel = do not generate muscle = lose muscle characteristics
Needs specific elasticity fo substrate to maintain stem cells
Describe sustaining potential of skeletal muscle stem cells - softer gel
More stem cells transplanted = the more regeneration occured
Need specific in vitro culture to maintain cell abilities
Wha can stem cells do
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
Describe sphere culture system
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
Describe neuropshere for drug discovery - 1
Made neuorsphres containing nscs - culture and exposed to many test compounds
Describe neuropshere for drug discovery -2
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
Describe neuropshere for drug discovery - generating lead compounds
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
Describe neuropshere for drug discovery - high content analysis - high throughput screening
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
Describe neuropshere for drug discovery - 3
See what it looks like
Good survival or no
Describe neuropshere for drug discovery- 4
Specific assay for a compound
Describe stem cell based cell and gene therapy
Therapy based on stem cell targets = cause of pathology not symptoms
Modify genes before transplantation
Describe use of retroviral vectors for stem cell based gene/cell therapies - deliver
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)
Describe use of retroviral vectors for stem cell based gene/cell therapies - viral vectors
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
Describe use of retroviral vectors for stem cell based gene/cell therapies- risks
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)
Uni of Pennsylvania 1999, exp - describe
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)
Necker sick children hospital, Paris, 2002 - describe
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
Why escs needed for gene targeting
Aggressive proliferation
Non viral approach - more frequent/sucessfully done
But adult stem cells do not divide like es cells so difficult
Describe Gen editing - gen
Immunological defence mechanism of bacteria
When virus comes in = use this sequence to bind genome of virus
Not gene targeting
Describe Gen editing - specifics
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
Describe Gen editing - what can you do with crispr/cas9 system
- 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.
Describe anemias caused by genetic disorders affects erythrocytes
Sickle cell anemia = point mutations in globin gene
B thalassemia = insufficient production of globin - due to mutation in enhancer
What is therapeutic strategy to treat anemia - 5 steps
- Harvest hematopoietic stem cells (HSCs) from a patient.
- 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)
- Transfer the modified HSCs back into the patient.
- HSCs engraft (no immune problems) and continuously produce erythrocytes with normal globins.
- A complete and permanent cure, that requires no pharmacological intervention.
(or, the therapy may need to be repeated if HPCs were modified)
Why is b thalassemia harder to treate
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
Describe concept of strategy to help anemia
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
What induced gamma globin gene
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
Describe editing bcl11a enhancer - Gen
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
Describe editing bcl11a enhancer - what does disruption of gata binding sequence in enhancer do
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)
Describe more preclinical testing done to check if anemia exp worked
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)
Describe engraftment of edited cells - exp results
After editing = no effect really - still same % of human cells and induced mutation %
Wel maintained after transplantation, similar prevalence of induced mutation, nearly 100%
Describe erythroid specific effects protein - exp results
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
Describe gamma globin expression - exp results
Expressed for after gene editing
Describe erythroid specific effects protein - biological assay
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
Has anemia exp even done irl
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
Describe what stem cell based therapies do - summary
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
What does gene therapy need to satisfy
Does it work
Is it safe = most important
Does it pay off (if too costly, or hard to apply)
