Stem Cells 3

Question 1

Describe cross talk between signalling pathways

Answer 1

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

Question 2

Describe mapk signalling

Answer 2

Mapk signalling (pretty strong) mediated by Erk, predisposes mouse Escs to differentiation Commitment fate

Question 3

Describe exp - questions about mapk

Answer 3

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

Question 4

Can embryonic stem cells be established without lif and serum

Answer 4

Yeee
When Erk suppressed by combo of Erk inhibitors =so must repress mapk signalling

Question 5

Describe exp - Erk inhibitors

Answer 5

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

Question 6

Describe extrinsic signalling mechanism -4

Answer 6

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)

Question 7

Describe regulation of esc self renewal by intrinsic factors

Answer 7

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

Question 8

Describe oct4-neg embryos

Answer 8

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

Question 9

Describe oct 4 in morula and compacted morula

Answer 9

Morula = detect oct4
1st differentiation occurs =
Compacted morula = oct4 downregulation = te lineage (placental, reduces expression significantly), only icm cells express oct4,

Question 10

Describe oct 4 expresion in blastocyst

Answer 10

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)

Question 11

Describe how emrbyonis stem cells behave depending on expression level of oct4

Answer 11

Increased oct4 expression by 50% = primitive endoderm, mesoderm
Normal = pluripotent stem cell
Hyperinsufficiency = remove one allele, expression decrease by 50% = te

Question 12

When is oct4 required

Answer 12

At particular levels to maintain pluripotency
Overexpress = commit to differentiation
Reduce by half = cells are te, express cdx2

Question 13

Describe altering the gene dosage of nanog and oct4 - gen

Answer 13

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

Question 14

Describe altering the gene dosage of nanog and oct4 - specifics mods

Answer 14

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

Question 15

Describe overview of intrinsic factors mediating mouse es cell self renewal

Answer 15

Oct4/sox2 inhibit te differentiation
Nanog inhibits primitive endoderm
Acting together = maintain stem cell state, block differentiation = similar to cell extrinsic mechanisms

Question 16

Describe genes bounds and regulated by oct4, nanog and sox2 in human es cells

Answer 16

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

Question 17

What is self renewal of emrbyonic stem cells regulated by

Answer 17

Intrinsic and extrinsic factors

Question 18

Describe what the 3 tfs do generally = oct4, nanog, sox2

Answer 18

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

Question 19

Describe how tfs link extrinsic to intrinsic factors

Answer 19

Not well understood

Question 20

Describe pros of esc application in clinical settings

Answer 20

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

Question 21

Describe cons of esc application in clinical settings

Answer 21

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

Question 22

Describe animal cloning - dolly

Answer 22

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

Question 23

Describe biological significance of animal cloning

Answer 23

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

Question 24

What can oocyte cytoplasm do

Answer 24

Reprogram somatic nuclei- to totipotent cell

Question 25

Describe exposure of fibroblast derived from skin to oocyte cytoplasm

Answer 25

Leads to generation of escs - in culture with squashed oocyte to exposed to cytoplasm

Question 26

Can we produce escs from committed or terminally differentiated cells

Answer 26

Factors needed to reprogram adult cells —> embryonic state

Question 27

Can we produce escs from committed or terminally differentiated cells - experimental design

Answer 27

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

Question 28

Can we produce escs from committed or terminally differentiated cells - results

Answer 28

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

Question 29

What are Yamanaka factors

Answer 29

OCT4, SOX2, C-MYC, KLF4 (4 factors needed to induce ipsc)

Question 30

What do yamanaka factors do

Answer 30

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)

Question 31

Is nanog a yamanaka factor

Answer 31

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

Question 32

How similar are escs to ipscs

Answer 32

Look at biological functions Fibroblast derived es like cells robustly proliferate for a long time - similar = long term self renewal activity demonstrated

Question 33

What do pluripotent fibroblasts contribute to

Answer 33

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

Question 34

Are ips cells identical to es cells

Answer 34

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

Question 35

Describe exp generation of germline competent induced pluripotent stem cells

Answer 35

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

Question 36

Describe generation of induced pluripotent stem cells without myc from mouse and human fibroblasts

Answer 36

Removed c myc - way less efficient but no tumours and live births achieved

Question 37

Describe intrinsic signllaing mechanism that sustains esc self renewal

Answer 37

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

Question 38

What happens when force activation of network

Answer 38

Using oct4, sox2, c myc an fklf4 leads to reporgramming fo somatic cells to Iscs = using yamanaka factors Can push back to embryonic stage

Question 39

Describe reprogramming process entirely

Answer 39

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

Question 40

Describe how yamanaka factors can be used to reprogram - gen

Answer 40

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)

Question 41

Describe how yamanaka factors can be used to reprogram - transfer of factors

Answer 41

Using = Adenoviruses = no integration into host cells Modified protein = can go through bilayer pm Small meolcuels = spontaneously penetrate cell membrane - use in clinical trials

Question 42

Describe what are affected by cells of origin in reprogramming

Answer 42

Levels of dna meth and his tone mods - epigenome, origin of cell

Question 43

What remains after reprogramming

Answer 43

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

Question 44

Are all ips liens equal in differentiation protocol

Answer 44

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

Question 45

Describe stem cells of diff animas

Answer 45

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