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Biol 483 > Midterm 2B > Flashcards

Midterm 2B Flashcards

Lemieux slides - Lect 5 - 8

1
Q

Stem cell potency

A

Pluripotent stem cells (PSC) obtained from blastula inncer cell mass –> $$$$ + prone to p53 mutations when grown in vivo

multipotent stem cells (MSC) –> responsible for tissue regeneration –> an become iPSC

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2
Q

Bone marrow stem cells

A

induce MSC to form hematopoeitc stem cells (HSC)
form all blood cells –> complete bone marrow regen can be done with only 10K HSC

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3
Q

hematopoeisis

A

process of HSC forming new blood cells
lymphoid progenitors –> B and T cells
myeloid progenitors –> eryth + macrophage + granulocytes + platelets

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4
Q

Collection of HSC

A

ingect pt with cytokines to illicit HSC into bloodstream –> collect sample
Use pAb to clump unwanted cell types
ficoll centrifugation –> mass gradient –> extract HSC layer
plate on soft agar –> allow colonies to form

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5
Q

Hematopoetic colony forming assay - tracking differentiation+ early vs late progenitors

A
  • HSC colonies on soft agar
  • track colony morpholgy
  • early progenitors = BFU-E
  • late progenitors = CFU-E
  • microscopy of cells to track degree of differentiation –> recall diff characteristics are cumulative the further differentiated the cell is
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6
Q

long term bone marrow cultures

A
  • extract bone marrow tissue OR culture from scratch
  • scratch culture from HSC using correct cultivation to induce loosely packed bone marrow structure formation
  • allow marrow culture to produce blood cells –> provided for patients with destroyed bone marrow (eg leukemia, rad/chemo the
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7
Q

Bone marrow transplants

A
  • formation of bone marrow transplant = slow –> need LOTS of tissue for transplants
  • transplant must be immunogenically matched to host –> otherwise GVHD (transplant tissue immunogenically attacks host)
  • autologous transplant = grown from pt’s own tissues –> no GVHD
  • allogenic transplant = grown from someone else’s tissue –> potential GVHD –> require immunosuppresants
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8
Q

leukemia patients and autologous bone marrow transplants

A
  • extract HSC from leukemia patient –> culturing
  • leukemic mutated cells will have inefficient growth –> outcompeted by healthier HSC
  • use healthy non-leukemia cells to grow autologous bone marrow
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9
Q

other stem cells for regenerative medicine

A
  • Umbilical stem cells
  • adult stem cells
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10
Q

Umbilical stem cells vs bone marrow stem cells

A

More HSC rich
More readily proliferate
autologous
expensive to extract/maintain

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11
Q

Limitation of adult stem cells for regenerative meds + work around soln

A

prone to in vitro mutations –> to maintain long term (accumulate mutations over time)
Solved by becoming iPSC (embryonic state) if given correct factors

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12
Q

factors for induced pluripotency in adult stem cells

A

SOCK acronym

SOX2
Oct4
cMyc
KLF4

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13
Q

Origins for transplant tissue (3 kinds)

A

autologous - own tissue
allogenic - other’s tissue
xenogenic - animal tissue

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14
Q

requirements for tissue/organ growth in vitro

A

stem cells
scaffolding
Growth/adhesion factors
vasculature

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15
Q

organ scaffolding

A

biocompatible
biodegradable (replacable w/cells)
natural materials - collagen
synthetic materials - polymers

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16
Q

types of scaffolding creation

A

PEG-based hydrogels
electron spun - use high V to form polymer nanoweave

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17
Q

growth/adhesion factors for organ growths

A

needed for encouraging stem cell seeding + tissue formation

binding
adsorption - (coating)
covalent linkage
blended - embedded into scaffolding material –> slow release as scaffold is gradually biodegraded and replaced
coaxial spin - forming scaffold as hollow tube filled with factors

18
Q

inducing vasculature in vitro organ growth

A

use angiogenesis factors
vasc required for 3D structures for nutrients delivery + waste removal

19
Q

in vitro organ growths - bioreactors

A

in vivo - scaffold transplantation –> allow host cells to seed –> used for guided tissue regeneration (wound repair)

in vitro - grow into thick mass + vascualture –> beware waste accumulation

xenotransplantation - disable pig’s organogenesis abilities –> provide human iPSC –> allow in vivo human organs to develope –> harvest from pig (allogenic)

bioprinting - prototyping of using bioink (cells) 3D printed –> passage through nozzel prone to cell stress/death

20
Q

examples of in vitro organ/tissue transplants

A

skin grafts
urethral tubing
prototype hearts

21
Q

skin grafts

A

skin structure = epidermis (dead cells + keratinocytes) + dermis (fibroblasts)
keratinocytes create the dead cells, dermis creates ECM/collagen binding agents

grow from iPSC or neonatal foreskin cells
fibroblast layer –> add keratinocuytes on top –> allow epidermis to form
harvest epidermis + small bit of fibroblasts –> transplant

limitations of skin grafts
in vitro skin –> no sweat glands + less able to wound constriction (wound doesn’t close up as readily)

22
Q

urethra tubing transplants

A

structure: uroepithelial interior lining surrounded by smooth muscles
historically used basic silicone tubing –> lacking muscles = poor mechanical control

use tubular scaffold –> seeding with uroepithelial + smooth muscle
scaff = biodegradable polymers or polyglycolic acid w/collagen binding

23
Q

heart transplant prototypes

A

deceased rat –> decellularized –> obtain ECM scaff + vasc –> seedin with human cells

24
Q

baculovirus - As a pesticide + life cycle of infection

A

used as natural pesticide
virus = HIGH target specificty therefore low ecological damage

infection
primary infection by receptor-endocytosis –> lysis with new virions
secondary infection causes virions to form occulsion body –> replicaiton inside occlusion –> release of occlusion into env

25
Q

occlusion bodies

A

formed by baculovirus secondary infections

protects virions from host cell in vivo and protects from ECM/env conditions

26
Q

baculovirus - for GOI expression

A

viral promoter = STRONG –> ideal as an expression vector when transduced into cell culture
Baculovirus = insect virus –> requires insect cells for culturing –> sf9 cell line

27
Q

first example of baculovirus GOI expression

A

INF-Beta (interferon) protein –> imperfect glycosylation (almost human compatible)

28
Q

baculovirus GOi expression - the method

A
  • Ecoli genome modified with baculoviral genome –> bacmid
  • transform with helper plasmid + donor plasmid (carrying GOI)
  • EC/virus bacmid recombines with donor –> aided by helper plasmid proteins –> create baculovirus transmission vector w/GOI
  • EC clones the bacmid –> bacmid harvested
  • transfection of bacmid into host animal cells –> expression of GOI
29
Q

Antibodies - basics

A

macrophage presents Ag frags on MHCII –> T(H) cells recognize –> activate B cells –> Ab expression

FC region = leukocyte binding domain
Fab = epitope binding determined by FV region (encodes specific CDR genes)

heavy chains –> 5 versions of IgX (MADGE) –> mutations/gene shuffling of Ab after 1st exposure creates Ab variations based on diff heavy chain –> therefore diff binding affinity to Ag

30
Q

Mouse-Human Ab chimerization

A

only mouse = momab
chimerized human heavy w/mouse light = ximab
human w/mouse CDR = zumab
only human = umab

31
Q

production of human ab

A

transgenic mouse (xenomouse)
normal Ag exposure to human pt
phage display

32
Q

phage display production of Ab

A
  • phages linked with random FV domain/scFAB
  • screen on Ag exposure –> identify which FV/scFAB binds to Ag strongest
  • purify and sequence
  • construction of mAb
33
Q

production of Ab in animal cell culture- What factor is most important for determing the specific host cell to use? example cell lines?

A
  • diff cells –> diff post trans mods –> diff Ab made
  • application of Ab determines how Ab is made (industrial uses can be imperfect/less strongent. medical requires very specific Ab forms and must be pure)
  • CHO cells = human Ab
  • HEK293 and Perc6 = PERFECT human ab (low expression rate)
  • Sf9 insect cells = imperfect glycosylation but low cost
  • Ecoli = when the glycosylation doesn’t matter (usually industrial application)
34
Q

Ab applications

A

ELISA - Anchored primary Ab to detect Ag directly or use anchored Ag to screen for primary Ab (indirect Ag detection)

anchoring to column chromatography –> purification of specific proteins –> elute at low pH (ab is reused)

passive immunotherapy

cancer cell targetted treatments

35
Q

vaccine synthesis - producer cell options (types, not cell lines)

A
  • embryonic sacs (eggs) - used for attenuated/inactivated vaccines
  • animal cells –> expression of subunit/peptide vaccines
  • human cells
36
Q

eggs for attenuated/inactivated vaccines

A

diff virusses infect diff spaces in the cell –> diff innoculation sites

aniotic cavity
yolk
allantoic sac
chloroallotoic sac

37
Q

Criteria for cells used in vaccine expression

A

subunit/peptide vaccines transmitted by viral vector into the cells (viral promoter = STRONG)

  • compatible with viral vector
  • must be genetically stable as to not mutate the vaccine (therefore HeLa is not usable)
  • high expression yield
  • cells can be cryopreserved (for long term manufacturing)
38
Q

animal cells for vaccine expression - Why use them instead of eggs?

A

low cost vs eggs + easier to purify vaccine

39
Q

human cell lines for vaccine expression

A

WI38 + MRC5 (human lung fibroblasts)

40
Q

virus quantifications

A

plaque forming assay - basically CFUs

cell lawn + serial dilution of virusses –> count # of plaques/clearing and factor in dilution

41
Q

covid vaccines

A

novavax - baculoviral transduction of moth cells
adenovirusses - donor cell carrying vaccine

Biol 483 (5 decks)

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