Stem Cells 3 Flashcards
Describe cross talk between signalling pathways
Lif —> stat3
Erk - inhibited by dmp4–>smad
Erk = map kinase pathway, Imogene activated protein kinase = inhibits self renewal so induces differentiation
Lif and bmp4 encourage self renewal so inhibit differentiation
Describe mapk signalling
Mapk signalling (pretty strong) mediated by Erk, predisposes mouse Escs to differentiation Commitment fate
Describe exp - questions about mapk
What if suppress mapk signalling in ICm cells, can it make esc derivation more efficient?
Would it keep escs at undifferentiated state?
Can it have same effect as bmp4 and Lif
Can embryonic stem cells be established without lif and serum
Yeee
When Erk suppressed by combo of Erk inhibitors =so must repress mapk signalling
Describe exp - Erk inhibitors
Used 3 types of Erk inhibitors - were added to culture
Each inhibits diff step of map kinase - signalling cascade
As long as mapkinase signalling repressed - even if no feeder or serum, can still induce and maintain embryonic stem cells
Describe extrinsic signalling mechanism -4
1- Lif and bmp4 important extrinsic factors that promote Mesc self renewal
2 - Lif blocks mesc differenitaion to primitive endoderm and mesoderm
3- bmp4 blocks mesc differentiation to neuroectodermal fate
4- Lif and bmp4 counteract action of mapk signalling (stimulated by fgf4) that induces differentiation commitment (inhibit mapk = directs esc to maintain stem cell fate)
Describe regulation of esc self renewal by intrinsic factors
Mechanisms inside cells
Oct4, nanog and sox2
Tfs - oct4 and nanog - homeobox proteins
Sox2-related to sry
Act together to maintain stem cells from inside cells
Describe oct4-neg embryos
Differentiate to te in vitro
Needed for maintenance es cells in mouse and humans
If Ko oct4 = cannot maintain icm, loses icm and only primitive endoderm remains - mixture of cells
Functional importance of oct4
Oct4 as an intracellular marker of escs
Describe oct 4 in morula and compacted morula
Morula = detect oct4
1st differentiation occurs =
Compacted morula = oct4 downregulation = te lineage (placental, reduces expression significantly), only icm cells express oct4,
Describe oct 4 expresion in blastocyst
Oct4 transient upregilation = trigger for formation of primitive endoderm
Still oct4 expressed in icm
Oct4 overexpression = when icm generates primitive endoderm
None in te
(Oct 4 important to maintain stem cell stage of es cells but over expression in Vivo = leads to differentiation icm)
Describe how emrbyonis stem cells behave depending on expression level of oct4
Increased oct4 expression by 50% = primitive endoderm, mesoderm
Normal = pluripotent stem cell
Hyperinsufficiency = remove one allele, expression decrease by 50% = te
When is oct4 required
At particular levels to maintain pluripotency
Overexpress = commit to differentiation
Reduce by half = cells are te, express cdx2
Describe altering the gene dosage of nanog and oct4 - gen
Modifies pluripotent phenotype of mescs
Were trying to see if can make es cells from adult seen cells - did not work
Importance of oct4 and nanog activities - what happens if one missing
Coordinated action of tfs - is nessecary to maintain stem cell fate
Describe altering the gene dosage of nanog and oct4 - specifics mods
Normal, conditions seen in vivo
Nanog over expression = Lif independent es cell
Oct4 over expression = endoderm and mesodermal
Oct4 ko = te, oct 4 inhibits te differentiation
Nanog ko = just like overexpresion oct4 4 = primitive endoderm
Describe overview of intrinsic factors mediating mouse es cell self renewal
Oct4/sox2 inhibit te differentiation
Nanog inhibits primitive endoderm
Acting together = maintain stem cell state, block differentiation = similar to cell extrinsic mechanisms
Describe genes bounds and regulated by oct4, nanog and sox2 in human es cells
A number of genes linked to esc differentiation are regulated by oct4, nanog and Sox2
Chip set= identify promoter that tfs bind to and downstream genes regulated
Active = stimulate self renewal genes, maybe the result of the expression of these genes
Silent = neurogenesis genes, ectoderm differenation, mesoderm production genes
Also upregulate themselves = positive feedback of gene expression
What is self renewal of emrbyonic stem cells regulated by
Intrinsic and extrinsic factors
Describe what the 3 tfs do generally = oct4, nanog, sox2
Constitute signalling circuit for self renewal of both mouse and human escs
Surpress genes important for lineage commitment - endoderm, mesoderm, ectoderm and extra embryonic tissues
Also ipregulate themselves and each other = positive feedback
Describe how tfs link extrinsic to intrinsic factors
Not well understood
Describe pros of esc application in clinical settings
Indefinite source of tissue engineering (long term renewal and proliferation)
Any cell types can be produced
Genetic manipulation/gene therapy
Tissue regeneration = compete fix, not handling symptoms only - can fix damage tissues and cell lineage
Can enter germ line and affect offspring
Describe cons of esc application in clinical settings
Tumorigenicity = induction of complete differentiation, selection of differentiated cells = can induce teratomas
Low efficiency/low purity of differentiated target cells,improvement of induction protocols
Imunnocompatbility = esc back, manipulation of escs to make them naive - if genotype diff= will reject cells that are transplanted even after complete differentiation
Ethics —. Patient specific escs (best to do), but would have to kill a bunch of embryos
Describe animal cloning - dolly
White faced= mammary cells from udder and took cells form nucleus
Black faced = took oocyte and removed nucleus = oocyte without dna
Inserted white nucleus (adult derived) nucleus into space between zona and oocyte
Zapped with electric shock = disrupt lipid belayer and nucleus went into egg
Cultured in vitro until blastocyst then put into surrogate ewe (black sheep, pseudo preg)
ONE SUCESSFUL BIRTH
CRITICAL Step = synchronize adult cells and oocyte cell cycle - pickup g0 (before s phase, quioessent) cell cycle adults and pt it in
Describe biological significance of animal cloning
1 = differentiated cells retain a complete set of genetic info - which can become offspring (most cells = have appropriate stiff for embryonic dev, but mostly shut down (so liver cell can act as liver cell), not all cells have complete set = T cells, plasma cells - can only make one type of antibody, make T cell repceptor so do not have complete set dna)
2 = oocyte cytoplasm can reprogram somatic nuclei, (somtiac nuclei can be reprogrammed)- into embryonic state so that dna can contribute and enter embryonic dev
What can oocyte cytoplasm do
Reprogram somatic nuclei- to totipotent cell
Describe exposure of fibroblast derived from skin to oocyte cytoplasm
Leads to generation of escs - in culture with squashed oocyte to exposed to cytoplasm
Can we produce escs from committed or terminally differentiated cells
Factors needed to reprogram adult cells —> embryonic state
Can we produce escs from committed or terminally differentiated cells - experimental design
Attempted to convert fibroblast cells to es cells
Mouse embryonic or adult fibroblast — made so genome carries Trans gene = b-geo (b gala conjugated w/ neomycin = if activated = blue and neo resistant),
Promoter = fbx15 must be activator to induce fusion protein but if ko = es cells normal - happens in this exp
Made 24 retroviruses and looked at genes, each Carries one type of gene
Retrovirus = capable of integrating viral gene into genome of host and infect cells and liver gene into host cells
1- 24 diff genes selected from genes - expressed in both oocytes and escs - (only 24 wells)
2- all were introduced into fibroblasts
3- one or combo of genes omitted
Can we produce escs from committed or terminally differentiated cells - results
Exposed fibroblast to retrovirus juice and were able to develop cells
From here = removed one at a time, if many colonies = absence has nothing to do with es cell production
All factors worked the best then created negative selection strategy
10 genes selected, one omitted at a time then selected 4 factors (produce same results as 10 factors), if omitted 1-2 of these = not good results
Efficiency better = improve success rate but. Also narrowing down which tfs important
Resulting cells produced - methods work for mouse, humans and other live stock species = IPS INDUCED PLURIPOTENT STEM CELLS
What are Yamanaka factors
OCT4, SOX2, C-MYC, KLF4 (4 factors needed to induce ipsc)
What do yamanaka factors do
Oct4 and sox2 = restore and maintain pluripotency
Myc = disrupts chromatin - dna-protein complexes - and allows oct4 and sox2 to access genome (also v well known oncogene)
KLF4 - serves as a cofactor for oct4 and sox2, also inhibits cell death (also oncogene)
Is nanog a yamanaka factor
NOOO
Nanog is dispensable = not needed
Nanog not needed as exogenous trans gene but can drive expression of nanog from host cells = endogenous nanog expressed
Driven by positive feedback
*once ipsc completely equivalent to es cells (reprogrammed) = viral genes shut down and express nanog, sox2 and oct4 from endogenous host cell genome
How similar are escs to ipscs
Look at biological functions
Fibroblast derived es like cells robustly proliferate for a long time - similar = long term self renewal activity demonstrated
What do pluripotent fibroblasts contribute to
Tissues of developing fetus
When inject into blastocyst = integrated into normal embryonic dev and contribute to 3 embryonic tissues of germ layer origins - endoderm, mesoderm, ectoderm, neuroextdemr
If integrate gfp = embryo has high rate of chimerism - can see with donor ipsc that carried gfp
Can stain with antibody against donor ips cells
Are ips cells identical to es cells
NAWWHWHWHWHWh
1=no live birth of chimeric offspring = all were aborted, or mice moms ate baby
2= global gene expression patterns diff, microarray Analysis diff
3= dna methylation patterns diff
= probably why no live birthday of chimeric offspring
Describe exp generation of germline competent induced pluripotent stem cells
Exp where used nanog as promoter
= 4 transgenes were strikingly silenced in nanog ips cells = obtained adult chimeras
Found gene expression patterns and dna meth similar to es cells
Also germline competent but procured tumours in 20% of offspring - by reactivation of c myc by chance
Describe generation of induced pluripotent stem cells without myc from mouse and human fibroblasts
Removed c myc - way less efficient but no tumours and live births achieved
Describe intrinsic signllaing mechanism that sustains esc self renewal
By manipulating cell intrinsic and signalling network = can induce reprogramming
Oct4, nanog and sox2 constitute major transcriptional regulation network for esc self renewal
Genes positively control expression of each other
What happens when force activation of network
Using oct4, sox2, c myc an fklf4 leads to reporgramming fo somatic cells to Iscs = using yamanaka factors
Can push back to embryonic stage
Describe reprogramming process entirely
GRADUal - not sudden at all
One trans gene with all 4 yamanaka factors, with teto promoter (test on = seq bound by tetracycline homolog, so that when add doxycycline activated all 4 genes), integrated tars gene to genome of fibroblast = can turn on and off yamanaka factors = can change length of expresure
Increase duration of gene = increase rate of success of es cell formation
Before 14 days = ipsc not maintained- differentiate
Have to wait 2 weeks to see expression of self sustaining es cells - if shorter = cannot maintain
BC = THIS IS WHEN endogenous tfs expression = nanog, oct4, 2 weeks post exposure tetracycline
Describe how yamanaka factors can be used to reprogram - gen
Need to be expressed for short period of time = at least 2 weeks, to induce ips from adult cells
= transgenes do not need to be integrated into host cell genome - can be shorter (good since not usually safe to overly manipulate genome)
Describe how yamanaka factors can be used to reprogram - transfer of factors
Using =
Adenoviruses = no integration into host cells
Modified protein = can go through bilayer pm
Small meolcuels = spontaneously penetrate cell membrane - use in clinical trials
Describe what are affected by cells of origin in reprogramming
Levels of dna meth and his tone mods - epigenome, origin of cell
What remains after reprogramming
Original cell signatures remain —> tendency to differentiate into cell type - preferentially
If only partially reprogrammed = still carry dna meat, cells have strong tendency to go back to same cell type
Although these ipscs stilll show robust differentiation ability
Are all ips liens equal in differentiation protocol
Some Ipsc lines = maybe not completely reprogrammed
A series of glitches in reprogramming process/protocol
Not all Ips lines will be equal in differentiation potential
If make ips from own cells and transplant/inject into body = body will recognize and will become teratoma, if want to use cells = needs good level of differentiation induced
Describe stem cells of diff animas
First es cell = mice, next cattle, mink, humans,
Rats= spontaneously differentiate so cannot maintain rat es cells, but established using inhibiting factor
Could only maintain after discovered inhibitng factor - stop differentiation
