BIOL 4507- Exam Flashcards
(291 cards)
1
Q
translational medicine
A
- “bench to bedside”
- a network to connect people working in labs to people working in hospitals
2
Q
describe the chain of people involved in translational medicine
A
PhD trained basic scientists -> MD, PhD trained clinician scientists -> regulatory, legal, and clinical trained specialists -> physicians
3
Q
describe the process of translational medicine
A
human disease -> hypothesis -> funding -> innovation and discovery -> publishing and patenting -> development pipeline (scaling and developing, pre-clinical assessments- animal models, etc.) -> clinical trials -> regulatory approval
4
Q
regenerative medicine
A
- developing and applying treatments to heal tissues and organs and restore function lost due to aging, disease, damage, or defects
- encompasses multiple areas of scientific inquiries, each of which is complex, but produce a powerful combination of technologies
5
Q
stem cell
A
- undifferentiated or partially differentiated cells
- retain the capacity to differentiate into various types of cells (“potency/potential”)
- can proliferate indefinitely to produce more the same stem cell (clonal expansion)
6
Q
what are the different levels of cells
A
totipotent, pluripotent, multipotent, unipotent, somatic
7
Q
totipotent
A
- zygote or morula cells
- can contribute to all of the cell types of embryonic development, including extra embryonic tissues
8
Q
name the 5 extra embryonic tissues
A
- placenta
- yolk sac
- amnion
- trophoblast
- extra embryonic endoderm lineages
9
Q
pluripotent
A
- have the ability to generate multiple classes of stem cells (e.g. embryonic stem cells can produce mesenchymal, hematopoietic, and neural stem cells) and give rise to all of the cell types that make up the body
- more restricted than totipotent (can’t produce extra embryonic tissues)
10
Q
multipotent
A
have the ability to differentiate into all the cell types within a particular lineage (more restricted)
11
Q
unipotent
A
can produce only one cell type but have the property of self renewal that distinguishes them from non stem cells
12
Q
somatic
A
body cells, can be reprogrammed into pluripotent SC (induced pluripotent SC)
13
Q
what are the sources of stem cells
A
- differentiated somatic cells
- adult tissues
- embryonic tissues
- fetal stem cells
- originally derived from miscarriages and abortions, restrictions on the use of fetal SC resulted in the development of human induced pluripotent SC
14
Q
how is stem cell therapy administered
A
ICV transplantation, intravascular infusion, intranasal delivery
15
Q
Parkinson’s case study
A
- 70 yr old patient with progressed Parkinson’s (lack of dopamine to coordinate fluid movements)
- fetal ventral mesencephalon precursor from fetal SC were transplanted into the region of the brain that receives dopamine and gave rise to dopamine producing neurons at maturity
- patient was able to coordinate fluid movement without medication
- following research looked into deep brain stimulation due to the difficulty of use and controversy around fetal stem cells
16
Q
research ethics
A
- new technological treatments require an ethical backup plan for if the research doesn’t continue progressing
- support for patients if the technology research does not continue
17
Q
molecular organization of cells
A
- multicellular tissues exist in one of 2 types of cellular arrangements:
- epithelial
- mesenchymal
18
Q
epithelial cells (4)
A
- regular columnar morphology
- cells are relatively static
- high degree of cell adhesion and cell-cell junctions
- produce a sheet of cells resting on a basal lamina with an apical surface
19
Q
mesenchymal cells (4)
A
- irregular, rounded, elongate morphology
- cells are highly motile
- bipolar, front-back polarity
- dynamic adhesions (lamellipoda and filopoda) and held together as a tissue within a 3D extracellular matrix (ECM)
20
Q
epithelial sheets (3)
A
- polarized
- rest on a basal lamina (ECM that serves as a foundation)
- can bend to form an epithelial tube or vesicle
21
Q
cell junction
A
- bind epithelial cells robustly to one another and to the basal lamina
- linker protein attaches to cadherin protein which will attach to another cell’s cadherin protein and link the cells together (dimerization)
22
Q
cadherin protein
A
transmembrane protein that spans the entire cell membrane
23
Q
adheren junction
A
initiation and stabilization of cell-cell adhesion
24
Q
tight junction
A
continuous intercellular barrier between epithelial cells
25
retinal neuroepithelium
- multipotent progenitors are located in the nueroblastic layer (NBL)
- differentiating neurons and glial cells are located in the inner neuroblatic layer and ganglion cell layer
26
glial cell
provides physical and chemical support to neurons and maintains their environment
27
ganglion cells
project info received by the photoreceptors to the brain
28
vimentin
- intermediate filament expressed in the mesenchymal cells that are differentiating
- involved in the non-NBL
- change the shape and polarity of the cell
29
epithelial mesenchymal transition (EMT)
rearrangement of cells to create additional morphological features
30
mesenchymal epithelial transition (MET)
the reverse process of EMT whereby cells coalesce into an epithelium
31
conversion of epithelial and mesenchymal cells
- the early embryo is structured as one or more epithelia
- in the adult organism, EMTs and METs occur during wound healing and tissue remodelling
- requires the coordinated changes of many distinct families and molecules
32
what are the main mechanisms that stimulate cells to transition into single migrating cells
- changes in cell-cell adhesion
- changes in cell- ECM adhesion
- changes in cell polarity and stimulation of motility
- inversion of the basal lamina
33
changes in cell-cell adhesion
- cells must detach from the epithelium in order to migrate away
- there are 2 main cadherins that mediate cell adhesion in epithelia:
- E-cadherin (part of the epithelia layer)
- N-cadherin (associated with mesenchymal cells, doesn't dimerize with E cadherin allowing the cells to move)
- often epithelia will down regulate E cadherin expression at the time of the EMT and express different cadherins, such as N cadherin to promote motility
34
integrin
- transmembrane protein
- 2 non covalently linked subunits that bind to ECM components (e.g. fibronectin, laminin, collagen)
35
changes in cell-ECM adhesion
- clustering of integrins on the cell surface affects the overall strength (avidity) of integrin- ECM interactions
- more integrins -> increased avidity and stronger ECM interactions
- different ECM components allow you to manipulate cells differently
36
changes in cell polarity and stimulation of cell motility
- epithelial polarity is characterized by cell-cell junctions:
- apicolateral domain (non adhesive)
- basal lamina (adhesive)
- changes in cell polarity helps promote EMTs
37
inversion of basal laminas
- in most EMTs, the emerging mesenchymal cells must penetrate a basal lamina (consists of ECM substrates- collagen type IV, fibronectin, lamina)
- mesenchymal cells may produce enzymes to degrade and breach the basal lamina (e.g. plasminogen activator)
38
how are EMT's controlled
- transcriptional control (transcription factors)
- posttranscriptional regulation
- molecular control (ligand receptor signalling, inflammatory signalling molecules, etc.)
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transcription factors
- regulate gene expression
- act in concert with one another to create large circuits
40
transcriptome
- a collection of transcription factors that define the cell and what it produces
- e.g. a stem cell is expressing a certain transcriptome, when it transitions to another type of cell it will express a different transcriptome
- requires a continuation of signalling that turns on specific profiles of transcription factors
41
post transcriptional regulation of EMTs (5)
- the activity of EMT transcription factors is regulated at the protein level
- translational control
- protein stability (targeting to the proteasome)
- nucelar localization (in order for things to function in the cell, they have to be in the appropriate location)
- non coding RNA (silences the ability of a gene to be expressed by binding just before the promoter and preventing transcription of the gene)
- RNA binding protein
42
musashi- 1
- RNA binding protein (post transcription) expressed in progenitor cells
- controls genes that define the transition from multipotent to TD
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ligand receptor signalling
- ligands (receptor complexes) interact with target cell receptor and starts a signalling cascade
- may be diffusible (floating) or expressed on the surface of another cell
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hierarchical organization of stem cells (3)
- self renewal (can clonal divide to self renew) vs terminally differentiated progeny (exited the cell cycle, no longer dividing, not considered a stem cell)
- progressive differentiation states
- vestige status
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self renewal vs TD progeny
symmetrical division and asymmetrical division
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symmetrical division
- clonal expansion
- SC -> 2 SC
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asymmetrical division
- can be clonal/TD or TD
- regulation can be intrinsic (i.e. transcription factors) or extrinsic (e.g. growth factors)
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progressive differentiation states
early stem cells, intermediate progenitors, terminally differentiated cells
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early stem cells
- long term renewal
- lots of potential to divide and more likely to be a self renewal potential event
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intermediate progenitor
- limited renewal
- more restricted in what it can generate
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terminally differentiated (TD) cells
no renewal
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vestige status (4)
quiescent, proliferative, intermediate, terminally differentiated
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quiescent
- inactive but ready
- not in the cell cycle but can reenter
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proliferative
- productive
- engaged in the cell cycle
55
intermediate progenitor (vestige)
- transient (between SC and TD)
- usually migrating and sometimes dividing
- lack pluripotency since they are not SC
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terminal differentiated (vestige)
cannot divide unless cancerous or reprogrammed
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criteria of pluripotent potential
Capacity to differentiate into cells of all 3 germ layers: endoderm, mesoderm, and ectoderm
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how we test for pluripotency
stem cells are injected into an immunodeficient mouse and eventually a tumor is produced -> the tumour is removed and examined -> tumors from pluripotent SC will have all three embryonic germ layers
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testing progenitor and SC heterogeneity
- 2 general approaches:
- transplantation protocols
- in vitro expansion and differentiation protocols (bulk culturing and single cell colony formation)
60
bulk culturing
the activity of each cell of the population is not reflected by the population average (unable to capture the activity of rare and critical cells and transiently amplifying other cells
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single cell colony formation
evaluate single cells with a high degree of comprehensiveness
62
stem cell sources (6)
single blastomere, morula, blastocyst, growth arrested embryo, somatic cell nuclear transfer, parthenogenesis
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morula
totipotent SC at the 16 cell stage, the whole clump of cells is taken
64
blastocyst
larger mass of cells than a morula (100-200 cells), has a population of different types of pluripotent SC
65
growth arrested embryo
- embryos are manipulated to guarantee they are only going to reach a certain stage of development
- zona pellucida is removed (protection during early development)
66
somatic cell nuclear transfer
cloning using a donor egg and donor nucleus
67
parthenogenesis
egg can develop into an embryo without being fertilized with sperm, can result in a lack of genetic diversity
68
single blastomere
a single cell removed from a blastocyst
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SC from the amnion (fetal SC)
- from the placenta and fetal membrane (i.e. amnion and chorion)
- less controversial source of SCs
- usually discarded after deliver and accessible during pregnancy through amniocentesis and chorionic villus sampling
70
amniocentesis
- prenatal test that take amniotic fluid around the baby in the uterus
- performed on women at higher risk of delivering a child with birth defects
71
sources of FSC
- amniotic epithelial cells (hAEC)- associated with the amniotic fluids/fluid membrane
- amniotic mesenchymal cells (hAMSC)- associated with the amniotic fluids/fluid membrane
- chorionic mesenchymal stromal cells- in the chorion
- chorionic trophoblastic cells- in the chorion
*all are candidates for use in transplantation
72
Wharton's jelly
a gelatinous connective tissue, insulates and protects the umbilical cord
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cord blood stem cells
- unrelated donor source of hematopoietic SC and amniotic epithelial cells
- accessing these cells allows you to modify them for later treatment
- previous dark history
74
clinical uses of CB (6)
- transplantation for hematological and non-hematological malignancies
- inherited metabolic disorders
- postnatal hypoxic ischemic encephalopathy
- cerebral palsy
- stroke
- autism (inflammatory theory)
75
induced pluripotent stem cells
introduction of 4 transcription factors can allow any somatic cell to be reprogrammed into an iPSC
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4 transcription factors (yamanaka)
oct 4, sox 2, klf4, l-myc
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describe the process of producing iPSC
- retrieve patient's somatic cells
- turn on yamanka transcription factors
- different protocols are used to induce the different tissue types
*positive feedback loop- transcription factors regulate the expression of a network of pluripotent associated genes
78
what are the 4 iPSC reprogramming techniques
retroviral transduction, Sendai virus, episomal vectors, introduction of synthetic mRNAs
79
retroviral transduction
- retroviruses have the ability to transform their single-stranded RNA genome into a double stranded DNA molecule that stably integrates into the genome of dividing target cells
- Myc is a proto-oncogene (can mutate into an oncogene)
80
sendai virus
- enveloped, single stranded RNA virus
- replicates directly into the cytoplasm of the host cell without any DNA intermediary
81
sendai virus in iPSC reprogramming (3)
- non integrating (replicates independent of the cell cycle)
- produces high copy numbers of the target gene (efficient)
- does not translocate to the nucleus and is diluted with each cell
82
episomal (bacterial) vectors
- non viral and non integrating
- plasmids are replicated and partitioned into daughter cells
83
advantages of episomal vectors (2)
- generation of iPSC in a single transfection
- plasmids are lost over time resulting in transgene free reprogrammed cell lines
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disadvantages of episomal vectors
can sometimes be integrated into the host genome, so the iPSC clones must be subject to genomic analysis
85
introduction of synthetic messenger RNAs
- mRNA is modified to overexpress pluripotent factors in somatic cells to reprogram them into iPSC
- substantial number of pluripotent factor expressions needed to drive cell transformation
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disadvantages of the introduction of synthetic mRNA
requires repeated transfections to induce over expression of pluripotent protein and further promote reprogramming due to transient (short lasting) expression
87
epigenetic
- changes in cell function that happen without changes in DNA sequence
- methylation state of an adult somatic cell must be remodelled during the reprogramming process to unlock access to the pluripotent associated gene network
- many genes are hypermethylated in donor cell types but hypomethylated in iPSCs
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epigenetic memory
- ability of cells to stably remain and transmit thier unique gene expression patterns to daughter cells
- epigenetic memories of iPSCs are short term and easily lost; time in culture reduces epigenetic differences among iPSCs
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methylation
suppresses gene expression
90
acetylation
encourages gene expression
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induced transdifferentiation
- direct transformation from one cell type to another
- a major mechanism in fish for regrowth
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advantages of transdifferentiation
- relatively fast in comparison to iPSC
- less likely than iPSC derivatives to contain residual undifferentiated cells that could produce teratomas
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disadvantages of transdifferentiation
- mature cell types have a limited capacity to divide
- require a large input population- cells are not going into the cell cycle, you're pushing them from one population to another
- finish the process with less cells than you started with
- population is clonally derived so genetic manipulation and characterization is difficult/not feasible
94
application of SC
disease modelling, personalized medicine, cell therapy, conservation of endangered species
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disease modelling
- positive results in animal models do not always translate to human studies
- not surprising since rodents diverged evolutionarily from humans 60 mya
- iPSCs allow scientists to study human diseases using human cells and dieases for which there is evidence of inheritance but no specific mutations identified
- iPSCS can be derived from patients with complex diseases that lack a clear genetic basis and preserve their genome
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personalized medicine
- many research methods tend to be similar ethnic backgrounds
- genetic background of an individual can determine the success or adverse effects of a particular drug
- can be used to create a customized medical plan or personalized regenerative medicine
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cell therapy
- autologous transplantation of iPSCs and their derivative is expected to be tolerated by the immune system
- banks of iPSCs that match a large percentage of the population have been created
- delivery of therapeutic cell types must be targeted to a region of interest
- IV injection is simple and easy, but often results in the cells being captured by the lungs or liver
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conservation of endangered species
- endangered organisms can be rederived to increase their population
- if the population is extinct and you have a cell sample or their genome, you could induce pluripotent SCs and create an embryo to implant into another species
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the hematopoietic system is a self renewal system
- divide and differentiate to produce maturing progeny
- divide to self renew to maintain a pool of SCs
100
the site of hematopoiesis changes during development
- placental
- fetal liver
- bone marrow (adults)
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hematopoietic SC therapies (6)
- bone marrow transplantation
- autologous/allogenic peripheral blood SC transplantation
- CB transplantation
- HSCs for severe combined immunodeficiency
- HSCs for autoimmune diseases
- HSCs for tolerance induction post transplant
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HSCs for tolerance induction post transplant
- used to prevent the reaction of transplanted organs/cells
- allogenic cells are mixed with host cells and injected months before the transplant to integrate them into the immune system
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mesenchymal SC sources (6)
- bone marrow compartment (best known source)
- adipose (accessible)
- dermal
- placenta
- CB and peripheral blood
- intervertebral disc (remnants of the notochord)
*high degree of potential
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multipotent adult progenitor cells
typically quiescent but possess some primordial potential to recapitulate SC activity
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types of ASCs
- hematopoietic/immunomodulatory, neural, bone, cardiac
- certain parts of the organ will have quiescent compartments
- brain creates new SC for the olfactory area (neurons are exposed and need to be replaced often and for the hippocampus (requrie new neurons for new memories and refreshing memories and synaptic connections))
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biomaterials
- any material, natural or synthetic, that comprises a whole or part of a living structure or biomedical device which performs, amplifies, or replaces the function that has been lost by injury or degenerative condition
- synthetically and pharmacologically inert
- used in a variety of sub disciplines (medicine, surgery, dentistry, vet med)
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conventional examples of biomaterials
- substitute heart valves (synthetic, xenotranspants)
- artificial joints
- dental implants
- fracture fixes
- skin regeneration templates
- contact lens
- kidney dialyzers
- blood vessel angioplasty
- cochlear implants
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3 main classes of biomaterials
- metals (e.g. orthopaedic screws)
- ceramics (e.g. dental implants)
- polymers (e.g. drug delivery)
- can be combined into composites
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properties of biomaterials (6)
- surface properties (rough, coarse, porous, reaction with water)
- corrosion resistance (electrical signals within the body may be corrosive)
- innate degradation (material will naturally break down over time)
- mechanical properties
- biochemical reactivity (is it going to react with the chemistry around it)
- radiation
110
related applications of biomaterials (4)
- implantation (is it inside or outside the body)
- hemocompatibility (a lot of chemistry occurs in the circulatory system- oxygen carrying capacity, coagulation, etc.)
- biodegration (body will break down the material- enzymes)
- immune surveillance (immune system will be reactive towards any foreign material)
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applications for biomaterials in regenerative medicine (5)
- cardiopulmonary organ replacements
- orthopaedic and dental implants
- surgical sutures and adhesives
- biological scaffolds
- nerve regeneration
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cardiopulmonary organ replacements
- cardiovascular implants and devices
- extracorporeal artificial organs
- artificial erythrocyte substitutes
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cardiovascular devices and implants
- artificial heart
- synthetic heart valves- used for training, potential future use for treatment
- coronary artery bypass surgery
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what do you need to consider when producing synthetic heart valves/vessels
- kinetic energy (heat)
- stretch
- diameter
115
coronary artery bypass surgery (CABG)
artery in the heart is blocked, disrupting functioning and rhythm of the heart -> artery is harvested from another part of the body -> donor artery is used to bypass the blocked section
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extracoporeal artificial organs
- provide mass transfer operations to support failing/impaired organ systems
- e.g. pace maker, insulin pump
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artificial erythrocyte substitutes
- perflurocarbon emulsions (lipid based liquid to contain perflurocarbon)/ microcapsules (more elaborate with a membrane wall)- molecules that can bind O2 (similar to hemoglobin- not good at removing CO2)
- encapsulated hemoglobin- requires hemoglobin source
118
dental implants
retropharyngeal space is linked directly to the pericardium- may introduce bacteria to the rest of the body
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bone and cartilage implants
- must consider the space between the impact (can facilitate bacterial growth)
- must consider porosity of the implant (is it rough enough for the bone to adhere to the implant)
120
titanium implants
- titanium is relatively non-reactive, lasts a long time, lightweight
- technology to detect substances improved (liquid chromatography mass spectrometry)- found that more titanium was leaching from implants than first thought
- titanium reduces osteogenic differentiation (bone growth), increases peri-implantitis (inflammation around the implant), increases osteoclast differentiation (bone breakdown), and decreases epithelial homeostasis
121
surgical sutures and adhesives (cyanoacrylate)
- accidentally invented by Harry Coover in 1942 (WWII) while he was working on making plastic lenses for firearm optics
- observed an acrylic resin that rapidly polymerized when reacted with hydrogen and strengthened into a long chain, poly-cyanoacrylate when exposed to water
- quickly and easily used to close lacerations
122
issues of cyanoacrylate
- curing is exothermic (produces heat) and could cause damage to surrounding tissues
- curing creates formaldehyde, which irritates skin, eyes, and respiratory systems
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alternatives to cyanoacrylate
- 2-octyl-cyanoacrylate and n-butyl-2-cyanoacrylate
- both are improved and less irritating medical adhesives
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biological scaffolds
- 2d and 3d scaffolds for in vitro growth
- 3d scaffold for mesenchymal SC tissue growth
- skeletal and cardiac muscle repair
- techniques include electrospinning, natural tissues, 3d printing, self assembly, and 3d bioprinting
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nerve regeneration
- peripheral NS can regenerate nerves relatively proficiently, but not always
- grafts can be used to bridge nerves together and reestablish connection and function
- chemotaxis and pathfinding
- when a neuron is injured and trying to regrow, the pathfinding molecule that it developed surrounded by are no longer present and the cell will likely grow in the wrong location (preferential motor nerve regeneration)
126
bioengineered bridging
synthetic material used to bridge the ends of nerves together and facilitate chemotaxis, pathfinding, regrowth, and cell survival mechanisms
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issues to consider when trialing biomaterials for prosthesis, implants, tissues, and fluids/gels
- biocompatibility- harm, dissolve, get encapsulated, bond with tissue
- hydrogels-cross linked polymers
- drug delivery, cell delivery, tamponade, wound healing
128
good manufacturing practice standards
- is the material you're putting in the body endotoxin (toxin anchored to the cell) free?
- dalkon shield IUDs
- composite biomaterial was created, the process of synthesis created endotoxins
- nylon retrieval string wicked in bacteria
- resulted in 18 deaths
129
cell-substrate interactions
- ECM, substrates, physical properties
- crystallinity, morphology, stiffness, compliance
- surface wet ability, surface charge, cell response
- can it be engineered/modified
- is it electrically conductive
- can its composition be altered
- cell adhesion, motility, proliferation/differentiation
- effects of topography and diffusible factor signalling
130
tissue clearing
transforms 3d tissue into a 3d nonporous hydrogel-hybridize form that is optically transparent and macromolecule permeable
131
CRISPR
clustered regularly interspaced short palindromic repeats
132
describe the origin story of CRISPR
- CRISPRs were first identified in E coli in 1987, when a series of repeated sequences interspaced with spacer sequences was accidently cloned
- in 1993, JD van Emden discovered that different strains of mycobacteria tuberculosis also had CRISPRs and other genomes were eventually discovered- thought to be a DNA repair mechanism at this point
- in the early 2000s, Mojica found similar spacer sequences in bacteriophages, viruses, and plasmids
133
CRISPR and adaptive immunity
- viruses cannot infect bacteria harbouring homologous spacer sequences
- suggests that these sequences play a role in the adaptive immune system in prokaryotes
134
natural mechanism for CRISPR based adaptive immunity
- virus infects a bacteria and inserts its own genome, hijacking the genetic expression mechanism and making all the components needed to reassemble itself
- if the viral genome is exposed to Cas protein, the cas protein will fragment the spacer sequences of the genome and clone them back into the bacterial genome creating a genetic record of immunization
- during a subsequent attack, the spacer sequences are transcribed to generate CRISPR RNA which forms an association with a Cas protein. the crRNA will guide the cas to bind to and cleave complementary DNA or RNA viral sequences, munching them up
135
describe the CRISPR-cas system
- CRISPR cas systems can regulate DNA (Cas9) and RNA (cas13)
- important for viruses, which can be made of DNA or RNA
- an endonuclease complex- has the ability to bind and cleave DNA
- requires a guide strand- RNA species that is going to associate with the Cas complex and act as a template to direct it to the target sequence
136
protospacer adjacent motifs (PAM)
- required for the cas complex to cleave DNA
- expressed on all things other than bacteria - cas cannot cleaves DNA without PAMs- ensures that only foreign viral nucleic acids are cleaved
- short (2-6 base pairs)
137
can CRISPR be used without PAMs
- no, not at the moment
- alternative variations on CRISPR editing innovations help add flexibility to this limitation
- different cas proteins or guide RNA species
138
improving CRISPR
- engineered CRIPSR systems to contain 2 components:
- CRISPR associated endonuclease
- synthetic guide RNA (sgRNA/gRNA)- short (20 nucleotides, targets specific regions of the genome
139
site specific endonuclease activity
- endonuclease activity- breaking DNA into 2 fragments
- the genomic locations that can be targets for editing by CRISPR are limited by the presence of nuclease specific PAM sequences
140
vector
- plasmid DNA ring
- DNA is continuous with itself
141
blunt end cloning
- insert from a vector is removed and ligase is used to ligate that insert into another vector
- ends of stand are straight across and may result in inversion
- the wrong direction is typically non functional
142
sticky end cloning
- insert and vector have overlapping ends (4-6 base pairs)
- prevents inversion and is more precise
143
restriction enzymes
- recognize specific sequences on the genome
- might make stick or blunt end cuts
144
non homologous end joining
- DNA is cleaved apart, may result in an insertion, deletion, or frameshift mutation
- more error prone and less precise
145
homology directed repair
- repair template telling the mechanism what to assemble
- less error prone and more precise
- create variations in the template sequence and create whatever mutations you want
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how is DNA inserted into cells (5)
microinjection, balistique gene gun, in vitro/in vivo electroporation, cationic lipids, viruses
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microinjection
teeny, tiny micropipette is used to inject individual cells using electric pulses
148
ballistic gene gun
gold particles are coated with the DNA of interest, packed into a bullet, and shot into cells- particles are coat in the cells and the coating is released
149
in vitro/in vivo electroporation
opens pores in the cell membrane or envelope allowing the material into the cell
150
cationic lipids
- mini membrane is formed in the presence of DNA/RNA
- membrane will capture DNA/RNA resulting in a loaded micro particle
- membrane will integrate efficiently into the target cell's membrane
151
viruses
- lentivirus- robust and indiscriminate, not worried about the long term outcome of the organism
- adenovirus- transient gene expression in both dividing and non dividing cells
- adeno-associated virus (AAV)- transient transgene expression in both dividing and non dividing cells
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challenges of CRISPR
delivery, ethics, off targeting, gene polymorphism in cancer, autoimmune response
153
delivery of CRISPR
- intramuscular
- systemic (circulatory system)- hitting more organ systems, may be too many (off target effect)
154
phase 1 clinical trial
- evaluates safety, side effects, and best dosage levels
- 1-2 dozen participants, typically less
155
phase 2 clinical trial
- evaluates safety and efficacy of the treatment (does it actually relieve, reverse, or stop a condition)
- hundreds of participants
156
phase 3 clinical trial
- evaluates long term adverse effects, how effective the treatment is compared to other standard treatments
- hundreds to thousands of participants
157
phase 4 clinical trial
- evaluating the safety of the treatment in the broader population, long term risks and benefits
- thousands of participants
158
CRISPR clinical trials
- trial phases may be combined to expedite testing a treatment
- all current CRISPR clinical trials target specific cells or tissues in individuals without affecting germ cells
159
CRISPR treatment areas (6)
blood disorders, cancer, inherited eye disorders, diabetes, infectious diseases, protein folding disorder
160
blood disorders and CRISPR
- SCD patients have sickle shaped RBCs which clog vessels and cause pain
- beta thalassemia (BT) patients lack sufficient hemoglobin levels
- CRISPR can deactivate the gene that prevents the formation of fetal hemoglobin, allowing cells to produce fetal hemoglobin and normal shaped cells
161
cancers and CRISPR
- leukemia and lymphoma
- Car-T immunotherapy involves genetically modifying a patient's T cells to have a receptor that allows them to recognize and attack cancer cells
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car-t immunotherapy trial results
- treatment was safe to administer and had acceptable side effects
- edited T cells took up residence in the bone marrow and remained at stable levels for the 9 months of the study
- T cells were able to find and infiltrate tumours
163
off target effects of car-t immunotherapy
- unintended edits at the target site
- 70% of cells showed at least one mutation at or near the target site during the T cell manufacturing process
- over time, the percentage of cells with mutations decreased
164
inherited genetic eye diseases and CRISPR
- Leber congenital amaurosis is the most common cause of inherited childhood blindness
- LCA10 gene is truncated, creating a mutant photoreceptor gene
- CRISPR replaces short gene with full length gene
- clinical injection of AAV with photoreceptor tropism- restores gene function
165
diagnostic challenges of LEB CRIPSR treatment
- difficult to evaluate attenuated loss of vision as a positive treatment- how do you tell if someone is getting les blind
- not restoring vision, just stopping the progression of blindness
166
diabetes and CRISPR
- type 1 is an endocrine disorder that occurs when pancreatic beta cells are destroyed, usually by an autoimmune reaction
- CRISPR is used to edit the immune related genes of these cells so the patient's immune system does not attack them
- edited cells are implanted into the patient's body in a special pouch and blood vessels grow along the outside of the pouch, bringing oxygen and nutrients and taking up insulin
- results- only in phase 1, no efficacy data yet, cells need to be isolated to prevent tumour growth
167
infectious diseases and CRISPR
- UTIs
- cocktail of 3 bacteriophages combined with CRISPR-cas3 designed to attack the genome of the three strains of e coli responsible for 95% of UTIs (destruction of genome kills bacteria)
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results of CRIPSR UTI treatment
- pending
- trial supported the safety and tolerability of the new therapy with no drug related adverse effects
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inflammatory diseases and CRISPR
- hereditary angioedema- patient has severe attacks of inflammation and swelling
- bradykinin regulates vasodilation (introduces more cells to an area, exacerbating inflammation) and is balanced by c1 inhibitor
- HAE patients have too much bradykinin, so reducing bradykinin restores the balance of C1: bradykinin
- results- pending, trial supported the safety and tolerability of the new therapy with no adverse drug effects
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protein folding disorder and CRISPR
- Hereditary transthyretin amyloidosis (hATTR) is a fatal disease causes by mutations in a single DNA base in the TTR gene
- mutated TTR gene encodes a misfolded protein that is sticky (amyloid fibrils- clump), and aggregates in organs and tissues
- results- most adverse effects were midl, all patients showed a reduction in TTR protein levels
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prion
- pathogenic agents that are transmissible and induce abnormal protein folding
- prion diseases are fatal degenerative brain disorder
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mad cow disease
- a progressive neurological disorder of cattle that results from infection by a prion
- industrial neocannibalism- dead diseased cows are steam treated and refed to cows
- if you continue reintroducing prions into a cattle community, they will eventually accumulate and cause disease
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future of gene editing
- "pasting"- inserting DNA to repair or replace harmful DNA
- multiplex editing- therapy to edit multiple genes at the same time
- treatment that uses base or prime editing- using CRISPR to directly change single DNA base pairs without making double stranded breaks
- turning on or off genes without changing DNA sequences
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bioprinting
- use of printing technology to bioprint tissues and organs
- aims to achieve reproducible, complex structures that are well vascularized and suitable for future clinical use
- human tissues and organs have arbitrary 3D shapes composed of multiple cell types and an ECM with functional organization
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why is vascularization important for bioprinting
- without vascularization, the centre cells of the mass will die (starved of resources) leading to a necrotic core and failed graft
- must create an environment that is recruiting in vascularization or printing the structure with vascularization
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computer aided design/manufacturing (CAD/CAM)
provides an automated way to replicate the 3D shape of a targeted tissue structure
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skin perforator flab surgery
- use of healthy autologous donor skin to fix another injured area of the body
- retains vascularization to ensure the graft doesn't die
- historically, cadavers were injected with radio-opaque dye and imaged to determine where perforator flags were located- some error since no one is going to perfectly align with a "generic human"
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steps to 3D print organs
imaging -> modelling and producing bioinks -> bioprinting
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magnetic resonance imaging (MRI)
- more detail of soft tissues, less detail of bony structures
- longer duration, higher cost
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computed tomography (CT)
- less detail than MRI, bony structures are more clear and detailed
- scan is simple and quick, less expensive
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sensory arrays
- tell the position of an instrument within the space of the room
- correlates with imaging
- combination of modalities are rendered together to form comprehensive imaging
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bioink
- hydrogels that can encapsulate and deliver cells and bioactive molecules through printing mechanisms
- cells are collected and cultured to provide the specific cells that you need
- can be cross-linkable into hydrogels (chemical, physical, enzymatic) before (more shear stress on cells) or after (less clogging, better resolution) printing
- must consider viscosity
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3D scanning
- collects 3D data of an object's shape
- best for proportionality and dimensions
- lacks detail that is deep within the structure
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reverse engineering a 3D scan into a CAD file
- start with interpolating points within and between image slices to improve the resolution
- generation of voxels (3D pixels) from the measured data
- extracting localized volumetric data from a targeted tissue structure to generate a surface model
- point cloud -> voxel model -> wire mesh model (smooth, more accurate surface)
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mechanisms of bioprinting
the effectiveness of each printing mechanism relies heavily on the choice of biomaterials and the targeted applications
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what are the main components of bioprinters
- 3 axis stage- stage controllers used to move the printer head in the x, y, z directions
- printing cartridges- usually in the form of a syringe, stores polymeric components of the scaffold or cellular/hydrogel components, contains the nozzle which determines the amount and rate of material dispensed
- dispenser- deposits the material
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what are the types of printers used?
inkjet, extrusion based, laser assisted, and hybrid
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inkjet printing
- create a localized temperature/pressure difference that expands material and pushes it down the nozzle
- includes inkjet and piezoelectric
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thermal inkjet printing
uses an electric heater (200-300C- heat will damage cells; not the preferred method) that generates small bubbles in the printhead, bubbles collapse to create pressure pulses that force droplets of liquid out the nozzle (4-10C rise in hydrogel temp)
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piezoelectric inkjet printing
- certain materials can generate electric charge in response to applied mechanical stress
- uses a polycrystalline ceramic to create a pressure pulse that ejects the droplet
- volume of liquid dispensed depends on temp gradient, frequency of pressure pulse, and ink viscosity
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extrusion based printing
- additive manufacturing that requires direct contact with a surface to print
- improved printing resolution, speed, and spatial control
- material is dispensed in a continuous string and includes the deposition of stacked 2D layers, as opposed to individual droplets
- may use pneumatic, screw, or piston dispensing
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pneumatic dispensing extrusion based printing
- uses air pressure to dispense bioink
- can deposit high viscosity materials
- delay in deposition owing to the time it takes to compress the gas in the cartridge
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piston dispensing extrusion based printing
- dispenses bioink with vertical mechanical forces
- provides more spatial control than pneumatic dispensing but with reduced max force capabilities
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screw dispensing extrusion based printing
- dispenses bioink with rotational mechanical forces
- provides more spatial control than pneumatic dispensing but with reduced max force capabilities
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laser assisted bioprinting
- focused laser pulse fired toward an absorbing layer, typically gold or titanium, to generate high pressure bubbles -> bubbles propel cell-containing bioinks towards a collector substrate
- have successfully transferred peptides, DNA, and cells
- nozzle free (avoids clogging), lots of control (increase or decrease laser power to change extrusion drop sizes, frequency, etc.)
- technology is very robust
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describe the applications of bioprinting in regenerative medicine
3D bioprinted vascular structures
in vitro tissue models
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three dimensional bioprinted vascular structures
- fully vascularized tissue constructs are required to attain long term cell survival and tissue functions
- 3D tissue constructs packed with metabolically active cells can rapidly form necrotic cores in the absence of a vascular network
- promote ingrowth of microvascular system into the implanted bioengineered tissue constructs in a timely manner
- generate pre-vascularized tissue constructs
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in vitro models
tumor models, drug screening systems, tissue engineering
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tumor models
- 3D bioprinting of cells as tumour models (can utilize other cell types- liver)
- tumour models are helpful for studying the interaction of immune and tumour cells for screening new treatments
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drug screening systems
- a high throughput system that produces a lot of data
- almost impossible to accurately screen tutor cells and predict the types of drugs that will affect a tumour
- biopsy tumour cells -> 3D print mini tutors -> place each tumour into a separate well -> high throughput small molecule drug screening program (a family of drugs is introduced to each well, families that affect tumour growth are retested individually to narrow down specific drugs)
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tissue engineering applications
- bone, cartilage, skeletal muscles and tendons, cardiac tissue and heart valves, skin
- the 3D printed tissue/device doesn't necessarily need to resemble the original organ- an artificial lung doesn't need to look like a lung to be functional
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bone bioprinting
bone graft scaffold that holds cells in the correct size and shape
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cartilage bioprinting
- 3D printing titanium for personalized joint implants
- 3D printing cartilaginous features, like noses and ears
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skeletal muscles and tendons bioprinting
- requires innervation (NS tissue), vascularization, correct order of actin and myosin
- must be able to generate tendons as well
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cardiac tissue and heart valves bioprinting
- requires cardiomyocytes, endothelial cells, epicardial cells, fibroblasts
- each cell type has different characteristics and needs to be treated differently
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skin bioprinting
- skin grafts create injuries to treat injuries (not ideal)
- materials that cover a wound and prevent infection have been created, but don't necessarily function like skin
- must consider that skin has lots of different layers and components
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ossicular chain prosthetics
- maleulus, incus, and stapes can be scanned and rendered in 3D
- allows you to create analogs that have the same functional characteristics and dimensions
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retinal prosthetics
- retinal implant stimulates sensation of vision, bypassing non functioning photoreceptors
- optical nerve implant- electrode array is placed around the optic nerve and stimulates the nerve to send info to the brain
- cortical implant elicits visual perceptions through direct stimulation of the visual part of the brain
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cell replacement therapy
includes transplantation (transplanting into the area it's going to integrate into) and implantation (uses long distance signalling, may not look like the original thing or be in the original location)
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experimental workflow of neural cell transplantation
- isolation or engineering or appropriate cell types
- safe enrichment or expansion of cells to generate sufficient numbers for transplant (cells must be healthy and not releasing toxic chemicals when transplanted)
- development of surgical delivery protocols (delivery will incur damage, so benefit of treatment must outweigh damage)
- management of cell viability, motility (you may want cells to migrate or not), integration, and safe functioning throughout the lifetime of the recipient
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factors to consider for transplantation (8)
- cell preparation and viability
- storage and handling (storage is very expensive, cells need to be durable enough to transplant and handle)
- delivery methods
- surgical placement (deposition shape, deposition cell delivery (how many cells, window of efficacy for cell density), location)
- immune response
- donor host interface
- vascularization
- are the cells programmed with a virus
- is the virus active? is everything used in the preparation of GMP? can you kill the transplanted cells if the need arises?
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autologous
cells/tissues obtained from the same individual that it will be transplanted to
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allogenic
- cells/tissues obtained from a genetically non-identical donor
- requires donor matching (MHC, rH, blood type)
- limited in the amount of material that can be harvested
- immune rejection is huge
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synergeneic
a type of allogeneic transplant in which the donor is an identical twin to the recipient
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xenogeneic
- cells/tissue obtained from one donor species to be transplanted into a different recipient species
- immune rejection is huge
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orthotropic (homotopic)
taking cells from a particular location in the donor and putting them in the same location as the recipient
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ectopic (heterotopic)
taking cells from a particular location in the donor and putting them in a different location int he recipient
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stem cell transplantation therapy workflow
- isolate, enrich, and count cells
- establish cell viability
- centrifuge cells to remove storage medium and resuspend in transplant medium
- final viability count
- wet ice transplant
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how are cells counted
- lift adhering cells from the bottom of the dish to get them moving within the slurry
- 1 micro litre of cells is placed into a hematocrit, dye that only enters dying cells is added, you can then determine the quantity of live cells
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how are cells resuspended into the transplant medium
- cells are centrifuged -> culture medium is siphoned off and replaced with the transplant medium
- repeated multiple times- laborious and hazardous
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SC sources for transplant
- human embryonic SC (derived from the inner cell mass of embryos)
- adult SC (least controversial, more restricted)
- fetal SC (ethical controversy)
- hiPSC (use yamanaka factors)
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SC transplant slurry composition (6)
- cells need to be delivered in a liquid medium that can support cell viability
- lots of nutrients- tailored to the cell type you're transplanting
- pH and salt buffered
- endotoxin free
- albumin (carries hormones, vitamins, and enzymes)
- hormones (fate (maintain differentiation trajectory), viability (e.g. insulin), inflammation suppressors)
- temp
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administration routes of cell transplant therapy
intratissue transplantation, intravascular infusion, intranasal delivery
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intratissue transplantation
- taking a population of cells/chunks of tissue and surgically implanting it into a particular location
- common
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intravascular infusion
- systemic injection
- blood brain barrier is limiting in regards to the CNS
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intranasal delivery
- brain perforates the skull and has free floating nerve endings in the olfactory epithelium; mucosa traps molecules and delivers them to neurons signalling directly into the brain
- acts like a super highway into the brain to administer cells
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delivery methods
- cells are interacting with the glass syringe and needle material
- cells may stick to the walls of the syringe (syringe can be pre-flushed with bovine serum albumin to coat the glass and prevent adhesion)
- cells may stick to one another (cohesion)
- sedimentation of cells may lead to inconsistent dosing
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stereotactic frame
fixed to the patient's head, provides reference points for targeting
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factors to consider for surgical delivery of cell replacement therapy (5)
- cell number
- hub (interface between string and plunger)- bevelled vs right angle
- cannula thickness (thicker needles have a lower gauge and create more injury but can deliver more cells)
- needle tip- bevelled (creates and incision and directs cells in a certain direction) vs blunt
- angle of delivery- changes how the cells sit in the syringe
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assessment of efficacy post transplant
- assessment is different in animal models vs human models (imaging is more challenging in a living organism)
- non-invasive in vivo imaging of transplanted cells
- fire fly luciferase gene + imaging substrate
- high energy wavelengths are only good for surface imaging; low energy wavelengths are good for deep tissue imaging
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describe firefly luciferase imaging
- fireflies have a gene that produces luciferase which interacts with a substrate and emits light
- luciferase can be put into transplanted cells and the substrate can be injected intravenously to cause the cells to glow
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managing immune response post transplant
- 0-6 hrs after transplant- acute response to surgical al trauma, platelets signal to neutrophils at the transplant site (clotting)
- 0-3 days after transplant- inflammatory response, complement proteins and platelets signal to neutrophils, neutrophils release cytokines and other responses, if this stage is overactive it will kill everything
- 3-10 days after transplant- innate response, macrophages and dendritic cells are recruited
- days to months after transplant- allograft rejection, T-cells recruited
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transplant medications
anti-rejection, anti bacterial, anti-viral, H2 blockers, steroids
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anti-rejection drugs
- prograf, mycolat
- lowers the body's ability to reject the transplant
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anti-bacterial drugs
- septrin
- fights bacterial infections
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anti-viral drugs
- Valcyte
- treat viral infections by inhibiting the development of viruses
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h2 blockers
- ranitidine
- decrease stomach acid produced to protect the stomach from other meds
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steroids
- prednisone
- reduce inflammation
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cellular kill switch
- combination of genetically encoded targets and drug administration
- targets are sensitive to drug treatment, which activates apoptosis or metabolic gene expression
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why would you need a cellular kill switch
- transplanted cells may have remnants (certain things integrate into the genome) of reprogramming/processing (hormonal/genetic)
- safety standards need to be employed to protect the recipient from deregulated donor cell activity
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pre-clinical applications for cell transplantation
- parkinsons and huntingtons- dopaminergic/glutaminergic cell transplant
- MS (autoimmune disorder that kills myelination in CNS)- autoimmune cell replacement (cells become myelin or signal existing myeline to stop dying)
- diabetes- pancreatic islet cell replacement
- stroke
- blinding diseases- fetal retinal sheets, photoreceptors, retinal pigmented epithelium
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what happens when we damage, but not kill, the cells in our body?
- main causes include motorcycle, ATV, and snowmobile accidents and falls from heights
- we hope that the death cascade (apoptosis/necrosis) doesn't kill remaining cells
- we hope that the surrounding microenvironment doesn't decide to phagocytose stressed cells and take them away (immune response)
- hope that cells manage to re-establish any unique cell modifications that are required fro function
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common causes of CNS injuries
spinal cord transection, olfactory bulb axotomy, optic nerve transection/ crush injury
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spinal cord transection
- displacement of the vertebral column that pinches or severs the spinal cord
- laminectomy (removing part or all of the vertebral bone) is used to attempt to relieve pressure on the spinal cord
- in some cases there is some restoration to function
- in almost all cases damage is complete and irreversible
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biotinylated dextran Amin (BDA)
- used as neuron tracers
- injected into a population of neurons and travels down axons to allow you to visualize where they are or are not travelling to see damage
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olfactory bulb axotomy
- abrupt impact on the forehead is going to damage the frontal and occipital lobe (brain jolts and hits the front and back of the skull)
- olfactory nuerons traversing the cribriform plate are going to be sheared
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optic nerve transection/crush injury
- retinal ganglion cells send their axons to the optic disc and exit the eye forming an optic nerve and becoming a tract in the brain
- if retinal ganglion cells become acotomjzed, the cells will go through a series of stages of cell death and attempts at regenerating
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progression of pathological response following optic nerve injury
- mechanical damage -> inflammatory response- first few hours, immune response mediates cell death cascade in CNS and Schwann cells encourage regrowth of axons in CNS (chemokine, cytokines, monocytes, neutrophils, myelination is broken down)-> macrophages and monocytes produce aggressive scarring that prevents regrowth
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how do neurons develop?
- neurons are born, migrate into a position, and send processes out to places they know they should be making contact
- if neurons reach out and touch the correct target cell, the target cell will release neurotrophic factors that travel retrogradallly through the axon and signal that it has been hooked up appropriately
- if neurons reach out and don't receive neurotrophic factors, the cell dies, ensuring the maintenance of good quality neural circuits
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target derived trophic support
pruning effect used during development to establish the circuitry of the NS
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growth cone
- swelling at the end of a process of an immature or damaged neuron
- uses motor proteins and cytoskeletal components to sense molecules in its environment and change shape
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pathfinding of growth cones during development
- location of the neuron during development determines where they will be located in the brain
1. neurons sends out axon to find the optic disc
2. axon fasciculate together to form the optic nerve, which grows into the brain and becomes the optic tract
3. optic nerve reaches the optic chiasm (decussation point- intersecting nerve fibres that form an X)
- nasal hemi-retina neuron moves contralaterally, temporal hemiretina moves ipsilaterally
4. neuron decides whether to fasciculate or de-fasciuclate
5. neurons decides which region it is targeting
6. neuron decides which layer to target
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pathfinding mechanisms
- adhesive substrate bound cues
- repellent substrate bound cues
- diffusible chemotropic cues
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adhesive substrate bound cues
- anchored to a specific region of the pathway
- tell the axons to stay within certain boundaries
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repellent substrate bound cues
- tell the growth cone not to go in a particular direction
- includes slits and ephrins, chondroitin sulphate proteoglycans
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diffusible chemotropic cues
create a concentration gradient that attracts icons and promotes movement in a certain direction
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regeneration
- regrowth rather than replacement (related to but fundamentally differ from cell replacement therapy
- can be applied to many organ systems, but the CNS and PNS constitute the prototypical model for tissue regen
- adult system has changed the enviro to be non-favourable to developmental programs- no guidance molecules or pathways to encourage things
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general methods for the stimulation of regeneration (4)
- direct drug (biomolecule delivery), cell-based molecular delivery, biomaterials, exploitation of endogenous substrates for regeneration
- every injury site that is imposed on the NS is going to have a scar that is non-permissive to axons trying to grow through the injury site
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direct drug (biomolecule delivery)
- done a lot in the early stages of regen
- dispensing a drug directly to cells to stimulate regrowth and aid in pathfinding for connectivity
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cell based molecular delivery
cell biomaterials that release synthesized factors and encourage cells to regrow
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biomaterials
- act as scaffolds that display substrate molecules for guidance
- can be laid out as a conduit and encourage regrowth across the damaged area
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exploitation of endogenous substrates for regeneration
turning on the genes of the original developmental program to promote regrowth
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regrowing a limb
- the goal of regenerative medicine
- lower vertebrates are able to regrow completely functional limbs in a short period of time
- involves the mobilization of a germal zone at the injury site
- lower vertebrates tend to retain adolescent features (e.g. progenitor cells left over from development that remain dormant until n injury, in which they reawaken and recapitulate what they did in development)
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slime molds
- innate capacity to explore its enviro and be very selective in decision making
- mold will send out different processes which probe around in search of food and retract anything that is not proficient
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oligodendrocytes
- associated with the CNS, can myeline multiple cells (efficient)
- non-permissive to regeneration- when a cell dies, it releases factors that create a pro-inflammatory response
- CNS is less likely to be injured than the PNS, thus are less likely to need regen
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Schwann cells
- associated with the PNS, can only myeline one cell
- can curb down the inflammatory response and control the type of phagocytic cells coming into the enviro
- can release trophic factors that encourage the cell to survive and grow
- can lay themselves out in a line and create a conduit for regrowth
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preferential motor neural regen
nerve successfully regrows, but pathfinding is all messed up and innervates a different structure changing function
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wound response and barriers to NS regen
- blood brain barrier
- reactive astrocytes and the glial scar
- non-growth signalling enviro of the adult NS
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blood brain barrier
- restricts access of phagocytic elements that can clean up debris
- restricts the entrance of other cell types that can signal regen
- breaching the blood brain barrier relieves the restriction of the immune system and inflammatory response allowing for the recruitment of things that shouldn't be entering
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reactive astrocytes and the glial scar
- hypertrophy- gets much larger and causes congestion
- proliferation- start dividing and changing the correct ratio of cells
- recruitment of microglia- tell microglia to start phagocytizing tissue (inflammatory response)
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non growth signalling extracellular environment of the adult NS
system doesn't have the correct cues to grow into the correct pathways
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primary injury
- damage to the tissue
- includes uncontrolled necrotic death, inflammation, ischemia (lack of blood flow), and hypoxia (lack of O2)
- processes persist for weeks and initiates a second wave of death
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secondary injury
- continued loss of tissue due to cascade of molecular and genetic changes post-primary injury
- includes apoptosis (neurons and oligodendrocytes), increase in injured tissue volume, formation of spindle shaped cystic cavity, insolation of injured tissue from the tissue environment by reactive astrocytes through the formation of a glial scar
- often described as a sequelae (consequence of a previous disease or injury)
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injury timeline
- injury event- primary injury
- intermediate- second to minutes, hemorrhage, decrease in ATP, increase in lactate (acidosis)
- early acute- minutes to hours, vasogenic and cytotoxic edema, micro vessel vasospasm, thrombosis, ion imbalance, loss of Na gradient, release of neurotic opioids, inflammation, lipid perioxidation, glutamatergix excitoxicity, formation of free radicals
- sub-acute- days to weeks, micoglial stimulation, macrophage activation, apoptosis
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clinical response to injury
- administration of steroids and anti-oxidant drugs
- release of any compression via surgery
- management of inflammatory response
- rehabilitation
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mechanisms of neurite growth
- filopodia and lamellipodia are mediated by motor molecules, actin, and cytoskeletal components
- different zones of signalling and extension/retraction allow for the growth cone's dynamic nature
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describe growth cone motility (5)
- mediated by actin polymerization and myosin contraction
- up and down regulation of each pathway allows for dynamic movement
- cytoskeletal component allows the growth cone to grow and navigate the enviro
- microtubule binding protein can stretch/coil and signal cascade of molecular response (senses contraction/extension)
- chemoattractant molecules guide the growth cone
- cell adhesion molecules allow the growth cone to move through the enviro
- filopodia molecularly couple and uncouple with cell adhesion molecules to pull itself forward and probe the enviro
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regenerative sprouting
occurs when the axons innervating a structure are severed and a growth cone is formed and regenerates new axons- more sprouting leads to more success
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zebrafish and spinal cord regen
- lower vertebrates face the same problems in regen as humans, but efficiently resolve them
- in mammals, nerve injuries result in a necrotic core that won't go away; zebrafish have the same cells, but they interact differently
- ependymo-radial glial cells (ERG) can efficiently create a pathway that cells can regrow through
- ERG cells are used during development in humans but are lost by adulthood p
- cell substrates remain a part of the CNS and mediate regenerating axons, remove the inflammatory response, and remove activated glial cells, allowing for regrowth
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biomolecule delivery and guidance conduits using biomaterials
- biomaterials allow you to choose viscosity, release molecules at a predictable rate, encourage cells to survive and regrow, etc.
- conduits/tubes allow axons to grow through or around an injury
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olfactory ensheathing cells
- peripheral cells that can replace the functioning of the Schwann cells and are very accessible
- protect the axons as they go through the cribriform plate
- robust, in constant contact with the external enviro- good at regenerating themselves
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transplanting olfactory ensheathing cells
- can be transplanted into many areas of the CNS and effectively encourage regrowth
- work like Schwann cells
- suppress the immune and inflammatory response, reduce fibrotic scarring, and create a substrate that provides a conduit for regrowth
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philipe monnier
rewrote a lot of the molecular signalling mechanisms that mediate the regrowth process
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repulsive guidance molecule
- released within the enviro
- signal the growth cone to collapse and regulate gene expression and neurite outgrowth and attraction
- allows you to direct regrowth and regeneration within the CNS
- clinical trials looking at variations of RGMs to direct regrowth in the context of stroke and traumatic brain injuries
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pre-context to xenobots
- all somatic cells contain the same genome; what creates variability in cell gene function is which genes are expressed and when
- germ cells are embedded within a population of other cells that are constantly communicating and secreting molecules that instruct them what to do
- all of the cells are responding tot he genetic programming of the genome, but deploying the communication in a very specific way
- what happens if you take the cells out of the enviro and let them behave how they want to?
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synthesis of xenobots (6)
1. supercomputer modelling
2. SC collection, dissociation, and agitation
3. pushing the fate of the SC into something that doesn't look like the original body plan
4. biobanking (storage)
5. assembly
6. shaping and using microsurgical tools (based on the computer modelling)
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applications of xenobots
- delivery of therapeutics
- direct repair of damaged tissue
- attack diseases
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characteristics of xenobots (6)
- programmable living beings
- communicate with each other
- can create other xenobots
- can change their cell expression
- create cilia (movement- can navigate their enviro)
- self healing
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