Lesson 17 Flashcards
if the sperm and egg fuse properly, they will form a single cel which is surrounded by what?
membrane pellucida
what is the function of the membrane pellucida?
protects the embryo from the maternal tissue
how long does the journey from the oviduct to the uterus take in humans?
5 days
when traveling from the oviduct to the uterus, the single cell starts dividing into what?
blastomeres
what do the blastomeres form?
the morula phase →each of the cells in this phase are able to give rise to an entire individual
as cells in the morula phase grow, what do they form?
a cavity forms and the embryo becomes a blastocyst
describe the blastocyst:
first developmental stage in which, within the embryo, there is a separation of the developmental potential of the cell
what are the flat cells that generate the walls the blastocyst?
trophoblast cells
what do trophoblast cells do?
protect a mass of about 50 internal cells called the inner cell mass (ICM)
what are the function of the cells in the ICM?
cells that give rise to the entire individual
what do trophoblast cells form?
give rise to the embryonic part of the placenta
what is another term for ICM cells?
pluripotent stem cells → they can give rise to everything, except the extra-embryonic (placental) tissue
what are cells from the ICM of the blastocyst called in vitro?
embryonic stem cells
the culture medium used to grow cells is composed of ions and and glucose necessary for growth, animal serum, and a molecule called Leukemia Inhibitor Factor (LIF), what is the function of the serum?
important in maintaining pluripotency
once the embryo develop to form a fetus, what can be harvested?
only adult stem cells - organ specific
how long are totipotent cells in the zygote?
between days 1-5
how long are pluripotent cells available?
between 5-10 days
what are adult stem cells considered as?
multopotent: they can differentiate into some cell types but very few
what is present in the embryo to generate the brain?
the neural tube
what does the neural tube form?
develops in the fetus to generate the anterior part of the brain and the spinal cord in the posterior
what is the neural tube generated from?
the neural plate
what is the neural plate generated from?
specified from the ectoderm which gives rise to neural progenitors
what two different tissues is the ectoderm split into during development?
neural plate and epidermal tissue
what will the neural plate produced from the ectoderm form?
all brain, spinal cord, and neural projections
what will the epidermal tissue formed from the ectoderm form?
the epidermis and the epidermal sheets
what are BMP factors?
bone morphogenic proteins: get expressed from the epidermis and specify the epidermal tissue
how are neural progenitors protected from the signal of BMPs?
fibroblasts growth factors (FGF-2)
what is the function of FGF-2?
block the action of BMPs and specify the ectodermal cells to become neural progenitors
what is a way to define the pluripotency of ESCs?
re-innoculoate them into animal cells (in-vivo)
how are ESCs re-innoculated?
they are taken from the dish and re-injected into a host blastocyst → inject about 20 ESCs into a single blastocyst and implant this into a mouse
if this implanted blastocyst implants, what does it create?
continue in embryonic development and generate an embryo and then a fetus → chimeric animal
chimeric animals are generated by two different cell types - what are they?
one cell type is the inner cell mass of the host blastocyst and the other are the ESCs cultured in vitro that we injected into the blastocyst
how can they tell which cell type was generated in the mice?
host blastocysts from come from mice with a white coat, while ESCs are generated from colored-coated animals
what occurs if you inject these cells as they are in vitro, to an adult animal?
they will generate a tumor of embryonic origin
what is the name of the tumor produced if these stem cells are injected into an adult mouse?
teratoma → they form large tumoral masses without metastasis and inside the tumors there are islands of differentiated cells of any kind
name the two chemical inhibitors found to be necessary to give the cells the ability to maintain their pluripotency?
PD0325901 and CHIR99021
what is the PD0325901?
small inhibitor of the FGF pathway
what is CHIR99021?
inhibits the GSK3 pathways which is part of the Wnt pathways
what other chemical inhibitor works in the same pathway along with PD inhibiting FGF4?
SU5402
what do FGFs cause?
cause the iPS cells to start to differentiate
pluripotency is not something that can be acquired - how must it be present?
something that occurs in the basal condition
Wnt molecules bind to the receptors on the membrane of a cell that are what?
frizzled
what do frizzled cells cause?
block the pathway → release functional β-catenin
what is the function of β-catenin?
a transcription
factor which is in the cytoplasm in its inhibited state, but when activated it’s internalized in the nucleus and it
works by activating a number of genes, which are the genes that are for factors of pluripotency
what is able to activate the same pluripotency in the same genes as β-catenin?
STAT1/3 → both activate the network of transcription facts that stimulate the pluripotency
what are the three pluriptency factors stimulated by β-catenin and STAT1/3?
Oct4, Sox2, Nanog
what is Oct4?
is the POU containing transcription factor → POU is the domain of the transcription factors which enables this protein to binding DNA to a specific target and activate or repress it
what is Sox2?
a transcription factor of a different class and binds the DNA to a different domain which is called HMG-box
what is Nanog?
a nuclear factor which has another domain, that binds the DNA to regulate its starter genes, which is a homeodomain
where is Oct4 specifically expressed?
in the cells of the inner mass because it is expressed to maintain pluripotency → if Oct4 is inactivated these cells cannot proliferate
what do these three transcription factors codify for?
three transcription factors which go into the nucleus and activate the promotor of their own genes
three transcription factors which go into the nucleus and activate the promotor of their own genes → what does this activate?
feed-forward loop because
their proteins bind to the promoter
of their genes and stimulate the
expression of their transcript and
therefore the production of more
proteins
why is the feed-forward loop essential?
when activated it maintains in a stable way the expression of this protein
what does the culture of human embryonic stem cells require that is different than mice?
ESCs do not contain LIF serum, but require FGF2 and active without serum to complete a different signal
describe the appearance of human ESCs in culture:
they are very flat
how must we culture human ESCs?
we have to expand them not to make
single cell culture but to make small aggregates because they don’t have the capacity as single cells to grow
what is the epiblasts stage?
a state where the inner mass already starts to generate an ectoderm
why are they called epiblast stem cells?
they are not generated from the inner cell mass but they are later derivatives from the inner cell mass, in particular the epiblast
why are epiblast stem cells pluripotent?
in vitro they can still give rise to everything but in vivo, they don’t because injected into the inner cell mass they don’t give rise to chimeras
epiblast stem cells are very similar to the human ESC, and what does this indicate?
the human embryonic stem cells are not generated from the inner cell mass but from an embryo that is a little bit older than the blastocyst and are derived from later derivatives of inner cells mass
in order to distinguish between embryonic stem cells and epiblast stem cells, what are the two different phases called?
the first are called primary or naive pluripotent stem cells and the second are called primed pluripotent embryonic stem cells
what is the concept of developmental timing?
the process by which the progenitor cells start to its differentiate process becoming mature, differentiated cells → in this process each cell initially is pluripotent and has a very large developmental potential
how did scientists clone fibroblasts into retroviruses?
they isolated these fibroblasts from a transgenic mouse which carried a
reporter which is called βgeo
which is inserted into the
FBX-15 gene locus
what did scientists discover when they cloned fibroblasts into retroviruses?
they found that very few cells infected with these combinations of factors changed the morphology
dramatically: they became very small, and they started to grow as island, very similar to the islands of the mouse embryonic stem cells
what did they find were necessary to start the conversion form adult fibroblasts into embryonic stem cells?
only four factors → iPS cells (induced pluripotent stem cells)
what are iPCs?
they reacquired the pluripotency: the ability to differentiate their cell type, to grow like mouse embryonic stem cells, but are induced from adult cells trough the forced expression of 4 factors
what four factors induce iPCs?
Oct4, Sox2, Klf4, and c-Myc → all genes that code for transcription factors
what are is the function of the four transcription factor that induce iPCs?
goes in the nucleus and bind to hundreds of targets and activate all the transcriptional pathways which are
necessary for pluripotent stem cells, and at the same time repress the genes which are active in the
differentiated cells
what did they use to prove IPS-cells were generated by the fully mature hepatocyte and not from progenitors in the cultures?
albumin → a factor expressed only in mature hepatocytes = Cre recombinases will only be expressed in mature hepatocytes
what happens when they cross a transgenic animal with an animal that has a constitutive promotor CAG (stop reporter)?
a constitutive promotor is expressed in any cell type - both embryo and adult
what was an important discovery made when studying if fully mature hepatocytes can be reprogrammed into iPCS cells?
fibroblasts are mature cells, but they don’t fulfill a very important metabolic process. While mature hepatocytes are fully well differentiated specialized
cells and yet these 4 transcription factors are sufficient to completely revert the identity from a metabolic cell
to an embryonic IPS-cell
what is the idea of cell reprogramming?
the idea that we can reprogram the identity of cells altogether
what is another name for Oct4, Sox2, Klf4, and c-Myc?
Yamanaka factors
what did they discover about derived IPS cells?
in the early stages of differentiation there still is a memory of the cells which they derived from
what must occur in order to completely cancel epigenetic memory of the cell of origin?
the IPS-cells need to be
stabilized in their reprogramming state in order to lose completely the epigenetic memory of the cell of origin
what does epigenetic memory mean?
the lasting genes of the cell of origin are still active in the IPS cell
