LEC57: Cell Differentiation, Stem Cells, and Reprogramming Flashcards
what is a differentiated cell?
cell that expresses a genetic program
proteins are characteristic and specific to that cell type, and confer on that cell cell type-specific structure and function
what is the idea of progressive acquisition of identity?
at each point when a cell undergoes cell division, the cell responds to an environmetal signal, and expresses a regulatory protein that’s distinct for the fate chosen
how does combinatorial gene control in development occur?
begin with an embryonic cell
a regulatory protein acts on it, and cell divides, 1 has regulatory protein incorporated, 1 doesn’t
then induce 2 new regulatory proteins into cell division
if do this as go down cell mitoses, will end up with many different nuique cell fates
what type of proteins are the regulatory proteins that control combinatorial gene control in development?
transcription factors
uncommitted precursor cells?
cells with >1 possible fate
determination?
process whereby a cell commits to a single lineage and acquires competence to undergo terminal differentiation
cell differentiation?
process whereby a cell acquires cell type-specific properties (structure, function)
what do ventral somites become
axial skeleton
what do dorsal somites become
1) myoblasts > **skeletal myofibers **
2) dermoblasts > dermis
what do skeletal muscle cells derive from?
precursors found in dorsal halves of somites
what are the events that occur for myoblast cell differentation?
1) exit from cell cycle, stop dividing
2) express muscle-specific genes
3) fuse into multinucleated myofibers
4) express ACh receptors and other proteins of the neuromuscular junction (so they can be innervated by motor neurons)
name the MRFs of the MyoD family
what basically are they?
muscle regulatory factors of the MyoD family of proteins, master regulators of the skeletal muscle lineage
MyoD, Myf5, Myogenin, MRF4
what does it mean that the MyoD family of MRFs are master regulators?
they’re **necessary and sufficient **muscle-specific txn factors **capable of reprogramming non-muscle cells to the muscle cell lineage **
what are the specific functions of the different MyoD MRFs?
Myf5, MyoD: determination of somitic cell > myoblast
Myogenin, MRF4: differentiation of myoblast > terminally differentiated myofiber
what is MyoD family of protein’s function re: genes?
MRFs work w/ other txn factors to drive muscle-specific gene expression
what is the structure and function of muscle creatine kinase (MCK) gene enhancer?
located at -1250 to -1050 relative to txn start site
has binding sites for MRFs, MEF2 factors, other txn factors
MRFs and MEF2 bind directly to DNA at to each othere at this enhancer to drive expression of MCK and other muscle-specific genes
when is MCK present?
only in terminally differentiated myofiber, not in the myoblast - myobaslt doesn’t have muscle-specific genes on
what is the function of positive feedback loops in muscle fiber development?
what’s the determination step in positive feedback in txn regulation of myoblast differentiation?
beacuse MCK isn’t expressed until terminally differentiated muscle cell, this happens in embryo to induce expression of Myf, MyoD, which’re involved in differentiation
once turned on by Wnt and Shh, MyoD or Myf can maintain its own expression; it binds to its own enhancer, keeps itself turned on; cellular memory of that signal causes stable phenotype so myoblast remains in absence of Wnt and SHH
what achieves the determined state for myoblast differentation?
MyoD autoregulation
what happens upon growth factor removal in myoblast differentiation?
Myf5 and MyoD induce expression of differentiation MRFs and MEF2 which then auto- and cross-regulate each others’ expression, amplifying and amintaining the stimulus to differentiate
what does amplification and maintenance of myoblasts during differentiation?
myogenin
MRF4
what induces expression of Mef2 factors
myogenin and MRF4
describe the process of transcriptional regulation of myoblast differentiation
what brings a developing muscle cell into G1 or G0? why?
MyoD is only expressed in an adult muscle cell, at which point, do not need to divide any more
so when you remove growth factors, this increases MyoD, which increases p21, which prevents Rb phosphorylation and so does not enter into S phase
causes cell to exit cell cycle
what does this image show?
many examples of ways to take generic cells and make them into terminally differentiated cell type we want by using a transcription factor that’s a master regulator
what is transdifferentiation?
changing a cell from a differentiated cell state to a cell of another lineage
what is dedifferentiation?
reprogramming a differentiated cell to pluripotent cell state - stem cell state
what is a stem cell
cell that can self-renew, that is make a daughter w/ the properties of the parent, and can* *generate several differentiated cell types
what is unique about the zygote stem cell?
it’s the ONLY totipotent stem cell - can form all of the cells of the embryo, the critical extraembryonic tissues to support the embryo, the placenta, etc., from that cell type
what are embryonic stem (ES) cells
pluripotent stem cells that derive from the inner cell mass of the blastocyst that have been placed into culture
they can form every cell of the body, differentiate into all lineages of the 3 germ layers in vitro and in vivo
they are immortal in culture - divide forever
what a unpotent stem cells/eg?
ability to form a single cell type or lineage
e.g. testes stem cells
multipotent stem cell def/eg?
ability to give rise to multiple cell types
blood stem cells
pluripotent? eg?
ability to give rise to all cells of the embryo
ES - embryonic stem - cells
totipotent? eg?
ability to give rise to all cells of an organism, including embryonic and extraembryonic tissues
eg zygotes
what kind of stem cells are muscle, nerve, bone, and other tissue cells?
some are multipotent; some are unipotent
what is somatic cell nuclear transfer?
SCNT: transfer of a somatic nucleus into an enucleated oocyte
first step to generating a pluripotent stem cell
reproductive cloning?
generation of a new organism following SCNT, somatic cell nuclear transfer, or other means
i.e. dolly the sheep
what is therapeutic cloning
generation of a blastocyst following SCNT from which ES cells are isolated and used as a source of differentiated cells for therapy
what are induced pluripotent (iPS) cells
reprogramming of somatic cells to an embryonic stem cell-like phenotype
by adding the Yamanaka 4-factor cocktail, gain expression of pluripotency genes Oct4, Sox2, Klf4, c-myc
what are the benefits/drawbacks of somatic cell nuclear transfer vs. induced pluripotency methods of nuclear reprogramming to pluripotent stem cells?
SCNT: laborious, extremely inefficient
Induced pluripotency: Easier; inefficient, but getting better
still unclear which is better for generating cells for therapy
how can a patient’s iPS cells be generated?
iPS: induced pluripotent stem cells
can take a skin biopsy from a patient; incubate w/ 4 Yamanaka txn factors; isolate the patient-specific iPS cells
what are the potential therapeutics from generating a patient’s iPS cells?
1) directed differentiation: use patient’s iPS cells, differentiate in vitro to affected cell type, screen for therapeutic compounds, treat with disease-specific drugs
2) use patient’s iPS cells, use gene targeting to repair disease-causing mutation, do invitro differentiation into healthy cells, transplant genetically matched healthy cells into patient
challenges to use of iPS as a therapeutic tool?
1) yamanaka factors can be oncogenic
2) vectors can be muatgenic, cause insertional mutagenesis
3) low efficiency of reprogramming
4) cells may acquire genetic changes during reprogramming to iPS cells
how are pluripotency factors regulated?
core factors autoregulate their own expression, directly control pluripotency transcriptional program
yamanaka factors?
pluripotency genes Oct4, Sox2, Klf4, c-myc
transform adult somatic cells into reprogrammed pluripotent stem cells
