Stem Cells 2 Flashcards
Describe what stem cell markers have to be
Linked to stem cell functions and useful for prospective stem cell studies
What are pluripotent stem cells derived from
Embryos
Story of testicular teratomas, a type of germ cell tumour and embryonic stem cells
Name the 3 types of embryo derived pluripotent stem cells
Embryonic stem cells
Embryonic germ cells
Embryonic carcinoma cells, stem cells of teratomas
What are embryonal carcinomas
Aka teratomas (benign) —> teratocarcinomas
Describe exp about teratomas
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
What was historical assumption made about teratomas
Assumption made that came from twin they absorbed, not true tho
Pair partner twins
DESCRIBE germ cell development
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
Describe 10.5 day embryo
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
Describe 12.5 day embryo (genital ridges)
See emergence of kidney and mesonephros
Genital/testicular cord starts emergining so can sex them
Describe experimental set up for testicular teratomas and germ cell dev exp - stevens
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
Describe experimental set up for testicular teratomas and germ cell dev exp - stevens RESULts
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
What do teratomas arise from
Germ cells
What do teratomas arise from - describe experiemnt
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)
What do teratomas arise from - describe experiemnt - was it fair
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
T or F = teratoma cells die very fast and cannot be maintained for a long time
FALSE
Teratoma cells can be maintained for a long time
Describe exp involving teratoma cell maintenance
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
What exists in teratoma cells
Mouse exp = long term self renewal and regeneration ability, indicating that a stem cell pop exists in teratoma cells
What can from a teratoma
A single cell
What can from a teratoma - describe exp
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
What does a single cell have the ability to o
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
Can teratoma cells be integrated into normal embryogenesis
Eyeyeyeyeys
Yes
Can teratoma cells be integrated into normal embryogenesis - DEscrIBE EXP
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
WHAT CAN teratomas arise from
Early embryos and pgcs
Most clinical cases of teratomas reprsent germ cell cancers
What do teratomas represent
Chaotic structures, everything chaotic
Embryoid body when make tumour mass
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)
Describe stem cells in teratomas
Embryonal carcinoma cells
Can be maintained in vitro for long time without losing teratoma formation ability
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
What are embryonic stem cels
Of mouse, 1980s
What are embryonic stem cells derived from
Icm of blastocysts - preimplantation (E~3.5)
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
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
What can embryonic stem not produce
Placenta = no trophectodermal lineage = emrbyonic stem cells are not totipotent -just pluripotent
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
Describe properties of embryonic stem cells
Long term self renewal and differentiation ability
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
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
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
Describe origin, derivation and differentiation of embryonal carcinoma cells
Origin = teratoma
Derivation = serum,feeder
Differentiation = 3germ layer, no germline, no placenta
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
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
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
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
Describe embryonic stem cells for gene targeting
Use cells more practically
What happens when ko bmp7
Deletion of bmp7 gene, null mutation, gene ko = no eyes and less kidney
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
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
What do teratomas contain
Solid tumours containing a number of diff cells types, derived from 3 embryonic germ layers
Occurs most in gonads
CAn teratomas enter the germ line
Nawh
What do primordial germ cells represent
One source of teratomas
How can teratomas be induced
Experimentally by transplanting early embryos or embryonic gonads into testis - biphasic origin
HOW IS THE STEMNESS OF EMBRYONICAL STEM CELLS REGULATED
Mechanism of maintenance of stem cell state = extra cellular (extrinsic) and intrinsic (intracellaulr)
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
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)
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
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)
What does activated stat3 promote
Pluripotency downstream of lif
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
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
Describe bmp4 signaling - extrinsic signalling mechanism
Bmp4 comes from serum
Smads = intracellualr signllaing molecule and helps transcription of target genes
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)
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
Describe exp = with serum and lif = conclusions
Lif and bmp4 can replace feeder + serum to maintain escs at undifferentiated state, normal state
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
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
