Midterm 2B

Question 1

Stem cell potency

Answer 1

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

Question 2

Bone marrow stem cells

Answer 2

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

Question 3

hematopoeisis

Answer 3

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

Question 4

Collection of HSC

Answer 4

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

Question 5

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

Answer 5

• 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

Question 6

long term bone marrow cultures

Answer 6

• 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

Question 7

Bone marrow transplants

Answer 7

• 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

Question 8

leukemia patients and autologous bone marrow transplants

Answer 8

• 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

Question 9

other stem cells for regenerative medicine

Answer 9

• Umbilical stem cells
• adult stem cells

Question 10

Umbilical stem cells vs bone marrow stem cells

Answer 10

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

Question 11

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

Answer 11

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

Question 12

factors for induced pluripotency in adult stem cells

Answer 12

SOCK acronym

SOX2
Oct4
cMyc
KLF4

Question 13

Origins for transplant tissue (3 kinds)

Answer 13

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

Question 14

requirements for tissue/organ growth in vitro

Answer 14

stem cells
scaffolding
Growth/adhesion factors
vasculature

Question 15

organ scaffolding

Answer 15

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

Question 16

types of scaffolding creation

Answer 16

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

Question 17

growth/adhesion factors for organ growths

Answer 17

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

Question 18

inducing vasculature in vitro organ growth

Answer 18

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

Question 19

in vitro organ growths - bioreactors

Answer 19

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

Question 20

examples of in vitro organ/tissue transplants

Answer 20

skin grafts
urethral tubing
prototype hearts

Question 21

skin grafts

Answer 21

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)

Question 22

urethra tubing transplants

Answer 22

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

Question 23

heart transplant prototypes

Answer 23

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

Question 24

baculovirus - As a pesticide + life cycle of infection

Answer 24

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

Question 25

occlusion bodies

Answer 25

formed by baculovirus secondary infections protects virions from host cell in vivo and protects from ECM/env conditions

Question 26

baculovirus - for GOI expression

Answer 26

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

Question 27

first example of baculovirus GOI expression

Answer 27

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

Question 28

baculovirus GOi expression - the method

Answer 28

- 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

Question 29

Antibodies - basics

Answer 29

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

Question 30

Mouse-Human Ab chimerization

Answer 30

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

Question 31

production of human ab

Answer 31

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

Question 32

phage display production of Ab

Answer 32

- 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

Question 33

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

Answer 33

- 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)

Question 34

Ab applications

Answer 34

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

Question 35

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

Answer 35

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

Question 36

eggs for attenuated/inactivated vaccines

Answer 36

diff virusses infect diff spaces in the cell --> diff innoculation sites aniotic cavity yolk allantoic sac chloroallotoic sac

Question 37

Criteria for cells used in vaccine expression

Answer 37

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)

Question 38

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

Answer 38

low cost vs eggs + easier to purify vaccine

Question 39

human cell lines for vaccine expression

Answer 39

WI38 + MRC5 (human lung fibroblasts)

Question 40

virus quantifications

Answer 40

plaque forming assay - basically CFUs cell lawn + serial dilution of virusses --> count # of plaques/clearing and factor in dilution

Question 41

covid vaccines

Answer 41

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