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ANAT 381 > Stem Cells 2 > Flashcards

Stem Cells 2 Flashcards

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
Q

What can teratoma cells do - 3

A

proliferate indefinitely (long-termself-renewal)
integrate in normal embryonic development (differentiation)
exhibit this dual function as a single cell (clonogenicity)

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26
Q

Describe stem cells in teratomas

A

Embryonal carcinoma cells
Can be maintained in vitro for long time without losing teratoma formation ability

27
Q

What happens when teratoma cells injected into blastocysts

A

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
Q

What are embryonic stem cels

A

Of mouse, 1980s

29
Q

What are embryonic stem cells derived from

A

Icm of blastocysts - preimplantation (E~3.5)

30
Q

Can embryonic stem cells be maintained indefinitely in culture

A

Yessss
Fragile karyotype tho, so if culture for long = often become aneploud and not suitable for practice application

31
Q

Can embryonic stem cells integrate into normal blastocyst cells

A

Can integrate into normal developmental processes, when injected into blastocyst and produce all cell types in body
Including germ cells

32
Q

What can embryonic stem not produce

A

Placenta = no trophectodermal lineage = emrbyonic stem cells are not totipotent -just pluripotent

33
Q

Can embryonic stem cells form teratomas

A

Yes
When transplanted into immunocompatible adult mice - cells from 3 embryonic germ layers
Not between species tho, but yes mouse to mouse

34
Q

Describe properties of embryonic stem cells

A

Long term self renewal and differentiation ability

35
Q

Describe entire process of production of embryonic stem cells

A

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
Q

How similar are embryonic stem cells to inner cell mass - exp

A

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
Q

How similar are embryonic stem cells to inner cell mass - conclusions

A

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
Q

Describe origin, derivation and differentiation of embryonal carcinoma cells

A

Origin = teratoma
Derivation = serum,feeder
Differentiation = 3germ layer, no germline, no placenta

39
Q

Describe origin, derivation and differentiation of embryonic stem cells

A

Origin = icm 3.5 dpc blastocyst
Derivation = serum, feeder (lif)
Differentiation = 3 germ layers, germ line, no placenta

40
Q

Describe origin, derivation and differentiation of embryonic germ cells

A

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
Q

Compare differentiation of all types of mouse pluripotent cells

A

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
Q

Describe uniqueness of pluripotent stem cells

A

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
Q

Describe embryonic stem cells for gene targeting

A

Use cells more practically

44
Q

What happens when ko bmp7

A

Deletion of bmp7 gene, null mutation, gene ko = no eyes and less kidney

45
Q

Describe embryonic stem cells for trangenesis - whole process of ko - basic steps

A

Homologous recombination in emrbyonic stem cells
Selection of embryonic stem cells modified properly
Injection into blastocyst
Transplantation in pseudo pregnant females
Chimeric offspring

46
Q

Why are emrbyonic stem cells needed for gene targeting

A

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
Q

What do teratomas contain

A

Solid tumours containing a number of diff cells types, derived from 3 embryonic germ layers
Occurs most in gonads

48
Q

CAn teratomas enter the germ line

A

Nawh

49
Q

What do primordial germ cells represent

A

One source of teratomas

50
Q

How can teratomas be induced

A

Experimentally by transplanting early embryos or embryonic gonads into testis - biphasic origin

51
Q

HOW IS THE STEMNESS OF EMBRYONICAL STEM CELLS REGULATED

A

Mechanism of maintenance of stem cell state = extra cellular (extrinsic) and intrinsic (intracellaulr)

52
Q

DESCRibe extrinsic cues that maintain mouse emrbyonic stem cell self renewal in vitro

A

Mouse esc, not human (since cells respond diff)
Feeder cells secrete LIF
Mouse esc culture also requires serum

53
Q

Describe regulation of mouse embryonic stem cell self renewal - name and describe extrinsic factors

A

Soluble glycoproteins, growt factors
Serum derived factor = bmp4
Feeder derived factor = lif (leukaemia inhibitory factor)

54
Q

Describe LIF signalling pathway

A

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
Q

What if actions of life is removed from emrbyonic stem cell culture

A

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
Q

What does activated stat3 promote

A

Pluripotency downstream of lif

57
Q

Describe exp of activated stat3

A

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
Q

Describe exp of activated stat3 - conclusions

A

Lif maintains es cel pluripoentcy via jak/stat3 pathway
Lack of lif signalling induces endodermal/mesodermal differentiation = lif blocks endo/meso differeniation

59
Q

Describe bmp4 signaling - extrinsic signalling mechanism

A

Bmp4 comes from serum
Smads = intracellualr signllaing molecule and helps transcription of target genes

60
Q

What if actions of bmp4 removed from emrbyonic stem cell culture

A

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
Q

Describe exp = with serum and lif

A

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
Q

Describe exp = with serum and lif = conclusions

A

Lif and bmp4 can replace feeder + serum to maintain escs at undifferentiated state, normal state

63
Q

Describe summary of extrinsic factors

A

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
Q

Describe embryonic stem cells for trangenesis - whole process of ko - gen

A

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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