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ANAT 381 > Stem Cells 2 > Flashcards

Stem Cells 2 Flashcards

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1
Q

Describe what stem cell markers have to be

A

Linked to stem cell functions and useful for prospective stem cell studies

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2
Q

What are pluripotent stem cells derived from

A

Embryos
Story of testicular teratomas, a type of germ cell tumour and embryonic stem cells

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3
Q

Name the 3 types of embryo derived pluripotent stem cells

A

Embryonic stem cells
Embryonic germ cells
Embryonic carcinoma cells, stem cells of teratomas

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4
Q

What are embryonal carcinomas

A

Aka teratomas (benign) —> teratocarcinomas

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5
Q

Describe exp about teratomas

A

Strain 129 mice = some mice start having spontaneous teratomas —> chaotic tumour mass, includes various cell types=cells come from 3 emrbyonic germ layer (endo, Meso,ecto) = started mating, inbred line = mouse strain 129sv = specific, more tumours 1-2% in mostly gonads

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6
Q

What was historical assumption made about teratomas

A

Assumption made that came from twin they absorbed, not true tho
Pair partner twins

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7
Q

DESCRIBE germ cell development

A

Proximal posterior epiblast, comes out through primitive streak
Comes to bottom of allantois (umbilical cord) then proliferates and as embryonic dev, hind gut invaginates —> cells includes in here, in hindgut
Then move —> genital ridge, towards font sides
9.5 dpc embryo = when embryo starts turning around, left turn, actively migrating from gut —> genitalia ridge, through mesentery

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8
Q

Describe 10.5 day embryo

A

Hind limb bud, forelimb bud, cuts here and insert forceps and then scoop
Genital ridges with mesentery and aorta = aorta-gonad mesonephros region
Hematopoietic stem cells originate from same place, proliferate here, genital ridge

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9
Q

Describe 12.5 day embryo (genital ridges)

A

See emergence of kidney and mesonephros
Genital/testicular cord starts emergining so can sex them

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10
Q

Describe experimental set up for testicular teratomas and germ cell dev exp - stevens

A

Procedure = implant pre implantation embryos or embryonic gonads into testes of adult mice= took out genital ridge and transplanted into adult testes
Thought it was embryonic origin
Testes of mice accessible and easy, also where teratomas form often
Stevens made a series of expirements and determined that teratomas arise from early embryos or pcgs in 1950s-60s
Saw that teratomas emerge wheen grafted whole embryo as easily as 4 embryonic days, frequency increased until 6 emrbyonic days, also when genital ridges of 10-12 days old embryos grafted = teratomas
Predicted derivations of all types of pluripotent stem cells of embryonic origin

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11
Q

Describe experimental set up for testicular teratomas and germ cell dev exp - stevens RESULts

A

Measured occurrence of teratomas, didn’t find anything
During early embryogenesis = embryos can transform to teratomas
Germ cell specification and migration = small numbers and then migrate so location and number of them is small = couldn’t find info here
Pcg specification = quantitative restrictions and lack of anatomical knowledge caused him to be unable to reproduce teratomas by trasnplantation

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12
Q

What do teratomas arise from

A

Germ cells

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13
Q

What do teratomas arise from - describe experiemnt

A

Use spontaneous mutant mouse called slj = steel, sterile only VERY FEW germ cells, mutation on steel locus of genome, c-kit ligand growth factor, mutant causes lack of spermatognesis/gametognesis bc environmental issue, if 2 locus mutant = infertile
(C kit ligand= growth factor secreted by testicular somatic cells, germ cells genetically normal)

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14
Q

What do teratomas arise from - describe experiemnt - was it fair

A

Exp compared wt wt, slj wt, slj slj
Slj wt and wt wt normal 159 genital ridges implanted 74% teratoma formation
Slj slj defective 56 genital ridges implanted and 2% teratoma formation
Concluded = origin of teratomas = germ cells
Yes FAIR = since Mendelian, so mutant 1/3 of other 2

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15
Q

T or F = teratoma cells die very fast and cannot be maintained for a long time

A

FALSE
Teratoma cells can be maintained for a long time

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16
Q

Describe exp involving teratoma cell maintenance

A

Teratoma cells dissociated - enzyme, makes single cell suspension —>
Inject teratoma cells into abd cavity, peritoneal of immune compatible adult —> teratoma cells grow and from aggregation = embryoid body (makes sphere, can see tissues and cell types from all 3 germ layers, looks like blastocyst/early morula)
Also so can regenerate embryoid body - if harvest, make into single cells, and re injectt into abd cavity of new mice

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17
Q

What exists in teratoma cells

A

Mouse exp = long term self renewal and regeneration ability, indicating that a stem cell pop exists in teratoma cells

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18
Q

What can from a teratoma

A

A single cell

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19
Q

What can from a teratoma - describe exp

A

Enzymatically digest teratoma cells (from embryoid bodies), and encapsulate a single teratoma cell on a glass capillary (just one), confirmed that capillary has one cell, and implant capillary subcutaneously
Waited and see if teratoma = did exp over 1700 times and 2-3% showed dev of teratoma

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20
Q

What does a single cell have the ability to o

A

Has ability to generate diffent cell types derived from 3 embryonic germ layers
= pluripotency
Clonal descendants of one single cell = concept of clonogenecity
Pluripotency= has ability to produce 3 germ cell layer, Alec ell types of body

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21
Q

Can teratoma cells be integrated into normal embryogenesis

A

Eyeyeyeyeys
Yes

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22
Q

Can teratoma cells be integrated into normal embryogenesis - DEscrIBE EXP

A

Inject teratoma cell injected into blastocyst
One cell injected into blastocyst and inject into pseudo pregnant females (not acc pregnant, induced) —> pup born with chimeric stripe on skin (severest immune reaction)
White mouse, inject cells into it, tip fo needle reaches blastocyst, embryo collapses then come s back (bc physical damage), teratoma cels from black coat strain = tumour cells visible without any tools
Tumor cells can become part of embryo and life and not cause cancer
Studies relie on cell function = presence of stem cells was recognized retrospectively

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23
Q

WHAT CAN teratomas arise from

A

Early embryos and pgcs
Most clinical cases of teratomas reprsent germ cell cancers

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24
Q

What do teratomas represent

A

Chaotic structures, everything chaotic
Embryoid body when make tumour mass

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25
What can teratoma cells do - 3
proliferate indefinitely (long-termself-renewal) integrate in normal embryonic development (differentiation) exhibit this dual function as a single cell (clonogenicity)
26
Describe stem cells in teratomas
Embryonal carcinoma cells Can be maintained in vitro for long time without losing teratoma formation ability
27
What happens when teratoma cells injected into blastocysts
Can from chimeras CANNOT enter germline, bc of tumor origin, always cells aneuploid so cannot enter germ cells, to ensure genetic inetgrity of offspring Results obtained in 50s through 70s forms basis for establishment of emrbyonic stem cells in 80s
28
What are embryonic stem cels
Of mouse, 1980s
29
What are embryonic stem cells derived from
Icm of blastocysts - preimplantation (E~3.5)
30
Can embryonic stem cells be maintained indefinitely in culture
Yessss Fragile karyotype tho, so if culture for long = often become aneploud and not suitable for practice application
31
Can embryonic stem cells integrate into normal blastocyst cells
Can integrate into normal developmental processes, when injected into blastocyst and produce all cell types in body Including germ cells
32
What can embryonic stem not produce
Placenta = no trophectodermal lineage = emrbyonic stem cells are not totipotent -just pluripotent
33
Can embryonic stem cells form teratomas
Yes When transplanted into immunocompatible adult mice - cells from 3 embryonic germ layers Not between species tho, but yes mouse to mouse
34
Describe properties of embryonic stem cells
Long term self renewal and differentiation ability
35
Describe entire process of production of embryonic stem cells
Culture medium contains 10-20% of serum, with feeder cells = non proliferative (embryonic fibroblasts, treated chemically irridiated so do not proliferate anymore), give essential factors needed for cells Take 3.5-4.5 day old embryo icm, sticks to feeder cells, forms and separates a bit = te and icm Physically dissociated and separate and digest and seed onto new feeder cells ~4days = reseed onto new feeder cells and separate icm vs te, then pick icm cells individually, pick up withnpipettte and disget and seed onto new feeder cells —> 5-6 days and can establish emrbyonic stem cells Have to redo every few days = digest and re seeds No G0 CELL cycle phase so divides every 16 hrs
36
How similar are embryonic stem cells to inner cell mass - exp
Tetraploid embryogenesis approach Do it biologically and functionally = morphology and cell Behaviour, is daughter cell identical to each other or mother Lacz positive 2 cell embryos, shock embryos at 2 cell stage and disrupts bilayer pm and nuclei fuse = 4n cell, starts dividing Can maintain these lacz + embryonic stem cells but wont become embryos, but can become placenta So do aggregation and mix with normal diploid dells, and transfer to surrogate (pseudo pregnant mice) = embryos develop, umbilical cord and placenta blue = makes aggregation chimera
37
How similar are embryonic stem cells to inner cell mass - conclusions
Embryos generated solely from embryonic stem cells through tetraploid chime risk Whole embryo came from tumour cells, functionally embryonic stem cells identical to icm cells Demonstrate regeneration activity = production of 3 germ layers and production of embryo
38
Describe origin, derivation and differentiation of embryonal carcinoma cells
Origin = teratoma Derivation = serum,feeder Differentiation = 3germ layer, no germline, no placenta
39
Describe origin, derivation and differentiation of embryonic stem cells
Origin = icm 3.5 dpc blastocyst Derivation = serum, feeder (lif) Differentiation = 3 germ layers, germ line, no placenta
40
Describe origin, derivation and differentiation of embryonic germ cells
Origin = pgcs 8.5-12.5 dpc Derivation = serum, feeder (lif), KitL, fgf2 (growth factors only needed for derivation, once established = maintained in same way for esc cells) Differentiation = 3germ layer, germline, no placenta
41
Compare differentiation of all types of mouse pluripotent cells
All can make teratomas and Cannot produce placenta on own and have 3 germ layers Escs and egcs once established as pluripotent germ cells = almost identical
42
Describe uniqueness of pluripotent stem cells
Embryo derived stem cells - escs, egcs, eccs, are pelicular stem cells (cancer cells, artificial) They are artificial stem cells that do not exit in body after birth and They are tumour cells with an ability to integrate into normal development If icm after born = cancer
43
Describe embryonic stem cells for gene targeting
Use cells more practically
44
What happens when ko bmp7
Deletion of bmp7 gene, null mutation, gene ko = no eyes and less kidney
45
Describe embryonic stem cells for trangenesis - whole process of ko - basic steps
Homologous recombination in emrbyonic stem cells Selection of embryonic stem cells modified properly Injection into blastocyst Transplantation in pseudo pregnant females Chimeric offspring
46
Why are emrbyonic stem cells needed for gene targeting
Way to put plasmid dna sequence - get it into genome and nucleus, only time when they can = during cell divsion since nuclear envelop dissolves Es cells do not have G0 phase, 16hrs, = more chance for trans gene to encounter host genome, prolfierate rapidly = neomycin selection can be done easily Es cells can integrate into embryonic dev, genome of offspring easily Can also enter germline = transmit transgene to babies Es cells very effective in this type of genetic manipulation,= reverse genetics
47
What do teratomas contain
Solid tumours containing a number of diff cells types, derived from 3 embryonic germ layers Occurs most in gonads
48
CAn teratomas enter the germ line
Nawh
49
What do primordial germ cells represent
One source of teratomas
50
How can teratomas be induced
Experimentally by transplanting early embryos or embryonic gonads into testis - biphasic origin
51
HOW IS THE STEMNESS OF EMBRYONICAL STEM CELLS REGULATED
Mechanism of maintenance of stem cell state = extra cellular (extrinsic) and intrinsic (intracellaulr)
52
DESCRibe extrinsic cues that maintain mouse emrbyonic stem cell self renewal in vitro
Mouse esc, not human (since cells respond diff) Feeder cells secrete LIF Mouse esc culture also requires serum
53
Describe regulation of mouse embryonic stem cell self renewal - name and describe extrinsic factors
Soluble glycoproteins, growt factors Serum derived factor = bmp4 Feeder derived factor = lif (leukaemia inhibitory factor)
54
Describe LIF signalling pathway
Lif binds receptor = lifr (single spanning membrane receptor) Binding recruits gp130 Jak = specific sequence in gp130, intracelllualr domain = captures jak protein kinase in cytoplasm, ligand receptor binding takes place and jak phosphorylates stats 3 (signal transduction and activator of transcription) on tyrosine and induces dimerization of stat3 = brings protein to nucleus, then controls transcription of target gene —> induces self renewal
55
What if actions of life is removed from emrbyonic stem cell culture
Widthdrawal of lif (feeder cells) or shut down stat3 action = differentiate to endoderm/ mesoderm (- or deactivate jax or over activate stat and see what happens when lif over activated)
56
What does activated stat3 promote
Pluripotency downstream of lif
57
Describe exp of activated stat3
With serum, no feeders, then start differentiating = chaotic, use antibody staining to se germ layers Stat3ER= put together transgene in emrbyonic stem cells, stat3 combined with estrogen receptor No factor - normal cells and stat3Er = differentiate Lif = normal culture cells Add 4ht and no lif, ht = tamoxifen, weak estrogen= differenate as same as factor one Maintain normal es morphology, even tho no lif feeder cells,reason = tamoxifen, is an estrogen and binds to estrogen receptor of transgene and dimerizes and enters nucleus and actives stat3 = initiation of signaling cascade downstream of lif
58
Describe exp of activated stat3 - conclusions
Lif maintains es cel pluripoentcy via jak/stat3 pathway Lack of lif signalling induces endodermal/mesodermal differentiation = lif blocks endo/meso differeniation
59
Describe bmp4 signaling - extrinsic signalling mechanism
Bmp4 comes from serum Smads = intracellualr signllaing molecule and helps transcription of target genes
60
What if actions of bmp4 removed from emrbyonic stem cell culture
Remove and see what hapepjns Withdraw or block bmp4, w/o lif = differentiate to endodermis/mesoderm Withdraw or block bmp4, w/ lif = differenatiate to neural fate (neuroectoderm)
61
Describe exp = with serum and lif
Bmp and life synergistically promote es cell pluripotency Introduction of antagonist smad into escs = induces neural differentiation Useed drugs to shut down smad signalling cascade = tuj1 neuron maker and sox1 neural precursor marker Lack of smad signalling induces neuroectodermal differentiation- found emergence of neurons = Smad signalling blocks neuroectodermal differentiation
62
Describe exp = with serum and lif = conclusions
Lif and bmp4 can replace feeder + serum to maintain escs at undifferentiated state, normal state
63
Describe summary of extrinsic factors
Lif/stat3 blocks endoderm, mesodermal Bmp4/smad blocks neuroectoderm Blocks differentiation rather than stimulate self renewal Differentiation path blocked= no choice but to remain as stem cells
64
Describe embryonic stem cells for trangenesis - whole process of ko - gen
Bmp7 gene, specific target gene, use restriction endonuclease and clone bmp7, inset neomycin resistance = make bacteria plasmid construct, mutation in protein become dysfunctional, even after transplantation, also expressed neomycin resistance genes Insert plasmid construct into es cells and can bind to genome = Then culture and add neomycin to culture medium, many cells do not have target gene incorporated = integration efficiency not good Inject blastocyst into uterus Formation of chimeric mice = Born Mate and make diff genotypes of bmp7 mutant mice,= hetero, homo, cross hetero, 3 genotypes, can have homo mutant that lacks eyes and kidneys

ANAT 381 (24 decks)

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