, pluripotency
sstteemm cows
cells that differentiate into other In mammals there two broad types of stem cells
Biological can ,
a re :
embryonic stem cells which isolated from the
types of cells and can divide to produce more of ,
a re inner
the same
type of stem cells (self renewal) cell mass of blastocyst s ,
and adult stem cells
In vivo loss of potency
2nd lineage decision :
ecto ,
endo and mesoderm
to -4 potent differentiation placenta and organism pluripotent
1
to -4potent : trophoblast and inner cell mass not most interesting ; can also form extraembryonic tissue
pluripotent : capable of differentiation into all three germlayers
multipotent :
progenitor cells (hennapoetic cell fate decisions are epi genomic based
run , potent : differentiate into one cell only because all cells contain the same DNA
Types of pluripotent cells
-
Embryonic stem cells (ESCs) : cells derived from embryo pluripotency is transient in the embryo ,
-
Epiblast stem cells (EP1 5CS) : cells derived from epiblast in culture phenomenon you fix them
-
Embryonot carcinoma cells €6s) : cells from terato carcinoma to stop differentiating
-
Embryonic Germ cells (EG Cs ) : cells derived from primordial germ cells
-
Induced pluripotent stem cell (PSG) : cell made from somatic cell
Test for pluripotency
-
Multilineage differentiation
differentiate into and mesoderm In human multi
→ ecto ,
endo
lineage differentiation in vivo not possible .
Extensive proliferation For absolute proof teratoma with differentiated cells of
germ layers
-
→ cells keep could be performed
growing in mice
Known marker /
genes proteins
-
→
when present it is a pluripotent cell
-
Produce germ line fhimensm)
inject stem cell with EGFP epiblast and from cell
→ in
grow organism single
-
Teratoma
if tumor with different cell
→ see
types is
being made
,Application of ES cells
-
model for
embryonic development Replacing cells is useful in
→
study gene regulation and characteristics -
stroke : loss of muscle cells
-
regenerative medicine -
duchenne muscle
:
degeneration
→
grow large quantities and differentiate into any tissue -
Parkinson loss of dopamine
:
generating cells
of KO
-
generation mouse
all cells to effect tissues
→
grow mouse with mutation in see on
Dangers
disease model
graft rejection
-
-
→ differentiate into desired specialisation -
graft versus host
tetracarcinoma
-
cytotoxicity tests -
Maintain pluripotency
cultured cells in dish are prone to differentiate
to inhibit differentiation vitro
ways
:
in
-
Feeders + serum
→ classic ,
mouse
embryonic fibroblast + cow serum
Life and batch dependent
serum cow serum
very undefined
-
+ is
→ Leahemea inhibitory factor
-
zi + Lf
→ 2 kinase inhibitors + 41 , very defined
Regulatory network
3 have autoregulated loop and activate themselves and eachother
→
upregulate pluripotency and self renewal
-
→ down
regulate developmental regulators L1F and Bmpu activate
pathways that directly talk
stem cells often have master regulators to
pluripotency network
→ bind promoters and enhancehers
→
regulate on
many
levels to sustain pluripotency
chromatin structure
chromatin modifiers a re important in development EUchromat.in Heterochromatin
→ cells need to change for differentiation H>Kume's H3K gmez
chromatin modifiers can assist
reprogramming Hzkzbmez Hskgmez
→ make it faster efficienter . or make it fat H3K 7g mes Hukzomes
HsKghaAc methylation
ES cells
-
globally decondensed (accessible)
enriched active histone marks (
green)
-
in
-
loosely bound chromatin proteins
Differentiated cells
-
condensed heterochromatin
decrease
adkennttcchhrroommaak-unn-stab.ly
-
silent histone marks in
acetylation B3W
bound chromatin proteins of bound to histone proteins
segments DNA . ,
that and
have both
repressing activating
epigenetic regulators in the same
region
, R
eprogwwaaddddxnnggttoonn.be#ggeenneeEtxccramming
marbles sample the
landscape is a metaphor for
and
how
at rest
gene regulation
at the lowest point
modulates development
the cell fate
the slope
groove on
,
come
,
.
Chromatin state corresponds with potency
→
pluripotent cells have flexible chromatin structure
→ differentiated cells have compact and specific chromatin state
somatic cell nuclear transfer Cell
reprogramming
nucleus from somatic cell The of mature specialised cells
I.
get process reverting ,
2. transplant in
oocyte without nucleus into induced pluripotent stem cells .
Erasure of
cell development
→
zn nucleus will develop like
embryo epigenetic marks during germ .
-
Technically challenging
-
Oocytes are
necessary
keepsakes ggeennee
A attached to regulatory sequence
gene
a
of of interest Fluorescence expressed
gene .
is
when
gene is
being expressed .
criteria for pluripotency
Differentiation capacity •
unlimited
growth
In UW0
morphology
- •
teratoma's under skin nude pluripotency markers
→ when ipscs
injected
•
mice
-
in vitro •
differentiation capacity ( in vivo and vitro)
→
embryo d bodies look for all layers
•
chimera /
germline transmission (not human ipscs)
, :
germ
IPS Cs
generation
four transcription factors can convert somatic cell into 1ps
Sequential event of
reprogramming
1. Initiation : add transcription factors
→ epithelial transition
mesenchymal - :
morphology change
fibroblast repressed and IPSC
genes induced
→
genes are
2. maturation Pioneer ffaaettorr
3. stabilization Transcription factor that can
directly bind to
→ I Pscs are stable : cells do not
stay in non - differentiate state condensed chromatin .
Recreate other factors
trans and histone modification enzymes and
→
genes are not necessary
anymore methylation
05k as pioneer
i. octy can bind to closed chromatin
and recruits factors to open heterochromatin
2. establishes pluripotency network
gene
sstteemm cows
cells that differentiate into other In mammals there two broad types of stem cells
Biological can ,
a re :
embryonic stem cells which isolated from the
types of cells and can divide to produce more of ,
a re inner
the same
type of stem cells (self renewal) cell mass of blastocyst s ,
and adult stem cells
In vivo loss of potency
2nd lineage decision :
ecto ,
endo and mesoderm
to -4 potent differentiation placenta and organism pluripotent
1
to -4potent : trophoblast and inner cell mass not most interesting ; can also form extraembryonic tissue
pluripotent : capable of differentiation into all three germlayers
multipotent :
progenitor cells (hennapoetic cell fate decisions are epi genomic based
run , potent : differentiate into one cell only because all cells contain the same DNA
Types of pluripotent cells
-
Embryonic stem cells (ESCs) : cells derived from embryo pluripotency is transient in the embryo ,
-
Epiblast stem cells (EP1 5CS) : cells derived from epiblast in culture phenomenon you fix them
-
Embryonot carcinoma cells €6s) : cells from terato carcinoma to stop differentiating
-
Embryonic Germ cells (EG Cs ) : cells derived from primordial germ cells
-
Induced pluripotent stem cell (PSG) : cell made from somatic cell
Test for pluripotency
-
Multilineage differentiation
differentiate into and mesoderm In human multi
→ ecto ,
endo
lineage differentiation in vivo not possible .
Extensive proliferation For absolute proof teratoma with differentiated cells of
germ layers
-
→ cells keep could be performed
growing in mice
Known marker /
genes proteins
-
→
when present it is a pluripotent cell
-
Produce germ line fhimensm)
inject stem cell with EGFP epiblast and from cell
→ in
grow organism single
-
Teratoma
if tumor with different cell
→ see
types is
being made
,Application of ES cells
-
model for
embryonic development Replacing cells is useful in
→
study gene regulation and characteristics -
stroke : loss of muscle cells
-
regenerative medicine -
duchenne muscle
:
degeneration
→
grow large quantities and differentiate into any tissue -
Parkinson loss of dopamine
:
generating cells
of KO
-
generation mouse
all cells to effect tissues
→
grow mouse with mutation in see on
Dangers
disease model
graft rejection
-
-
→ differentiate into desired specialisation -
graft versus host
tetracarcinoma
-
cytotoxicity tests -
Maintain pluripotency
cultured cells in dish are prone to differentiate
to inhibit differentiation vitro
ways
:
in
-
Feeders + serum
→ classic ,
mouse
embryonic fibroblast + cow serum
Life and batch dependent
serum cow serum
very undefined
-
+ is
→ Leahemea inhibitory factor
-
zi + Lf
→ 2 kinase inhibitors + 41 , very defined
Regulatory network
3 have autoregulated loop and activate themselves and eachother
→
upregulate pluripotency and self renewal
-
→ down
regulate developmental regulators L1F and Bmpu activate
pathways that directly talk
stem cells often have master regulators to
pluripotency network
→ bind promoters and enhancehers
→
regulate on
many
levels to sustain pluripotency
chromatin structure
chromatin modifiers a re important in development EUchromat.in Heterochromatin
→ cells need to change for differentiation H>Kume's H3K gmez
chromatin modifiers can assist
reprogramming Hzkzbmez Hskgmez
→ make it faster efficienter . or make it fat H3K 7g mes Hukzomes
HsKghaAc methylation
ES cells
-
globally decondensed (accessible)
enriched active histone marks (
green)
-
in
-
loosely bound chromatin proteins
Differentiated cells
-
condensed heterochromatin
decrease
adkennttcchhrroommaak-unn-stab.ly
-
silent histone marks in
acetylation B3W
bound chromatin proteins of bound to histone proteins
segments DNA . ,
that and
have both
repressing activating
epigenetic regulators in the same
region
, R
eprogwwaaddddxnnggttoonn.be#ggeenneeEtxccramming
marbles sample the
landscape is a metaphor for
and
how
at rest
gene regulation
at the lowest point
modulates development
the cell fate
the slope
groove on
,
come
,
.
Chromatin state corresponds with potency
→
pluripotent cells have flexible chromatin structure
→ differentiated cells have compact and specific chromatin state
somatic cell nuclear transfer Cell
reprogramming
nucleus from somatic cell The of mature specialised cells
I.
get process reverting ,
2. transplant in
oocyte without nucleus into induced pluripotent stem cells .
Erasure of
cell development
→
zn nucleus will develop like
embryo epigenetic marks during germ .
-
Technically challenging
-
Oocytes are
necessary
keepsakes ggeennee
A attached to regulatory sequence
gene
a
of of interest Fluorescence expressed
gene .
is
when
gene is
being expressed .
criteria for pluripotency
Differentiation capacity •
unlimited
growth
In UW0
morphology
- •
teratoma's under skin nude pluripotency markers
→ when ipscs
injected
•
mice
-
in vitro •
differentiation capacity ( in vivo and vitro)
→
embryo d bodies look for all layers
•
chimera /
germline transmission (not human ipscs)
, :
germ
IPS Cs
generation
four transcription factors can convert somatic cell into 1ps
Sequential event of
reprogramming
1. Initiation : add transcription factors
→ epithelial transition
mesenchymal - :
morphology change
fibroblast repressed and IPSC
genes induced
→
genes are
2. maturation Pioneer ffaaettorr
3. stabilization Transcription factor that can
directly bind to
→ I Pscs are stable : cells do not
stay in non - differentiate state condensed chromatin .
Recreate other factors
trans and histone modification enzymes and
→
genes are not necessary
anymore methylation
05k as pioneer
i. octy can bind to closed chromatin
and recruits factors to open heterochromatin
2. establishes pluripotency network
gene
