Stem Cells Flashcards
Regulation of Stem Cell Self-Renewal
Cell Intrinsic Regulation
- Sox-Oct-Nanog transcription factors that negatively regulate or repress transcription of genes that would normally allow cell to differentiate
- Ronin DNA binding protein suppresses transcription of differentiation inducing genes such as GATA4 and GATA6
Regulation of Stem Cell Renewal
Cell Extrinsic Regulation
- LIF ligand binds LIF receptor/GP130 dimer and works through STAT3 to inhibit MAPK pathway that causes cell differentiataion.
- BMP ligand binds to BMP receptor and works through SMAD to inhibit MAPK pathway which causes cell differentiation
Intestinal Crypt
-Crypt base columnar cells are the stem cells.
*Paneth cells are the niche for interstinal crypt cells
*removal of paneth cells results in loss of crypt cell
regeneration
- When they trans-differentiate, they give rise to transient amplifying cells.
- Transient amplifying cells make up different types of cells in the vilus
Adult stem cells
- Undifferentiated cells that can be found in differentiated tissue
- Can self-renew and differentiate into all specialized cell types of that tissue
- iPSCs are made from adult stem cells through process of de-differentiation
Examples:
- Hematopoietic Stem Cells in Bone Marrow that can form all blood cells in the body
- Stromal Stem Cells in Bone Marrow that give rise to bone, cartilage, fat, and connective tissue
Embryonic Stem Cells
Undifferentiated cells derived from a 5-day pre-implantation embryo that have the potential to differentiate into a wide variety of specialized cell types that arose from embryoblast
Cancer Stem Cells
Stem cells that renew, drive tumorigenesis, and give rise to differentiated progeny
Differentiated cancer
Cells are mature and look like the cells in the tissue from which it arose
Undifferentiated cancer
Cells are very immature and “primitive” and do not look like cells in the tissue from which it arose
*Gleason Grade 5 prostate cancer is very aggressive because these cells no longer look like epithelia but they have become more embryonic-like
Totipotent
Pluripotent
Mutlipotent
Oligopotent
Unitpotent
Totipotent
- Stem cells that are produced from the fusion of an egg or sperm as well as from the first few divisions.
- Can give rise to embryonic and extra embryonic tissues.
Pluripotent
- Descendants of totipotent cells
- They lose capacity to make certain structures (placenta) but still retain capacity to make any cell from the 3 germ layers
Mutlipotent
- Stem cells can differentiate into a number of cells but only those of a closely related family of cells.
i. e. hematopoietic stem cell can turn into five or six different types of blood cells.
Oligopotent
- Stem cells can differentiate into only a few cells
- i.e. lymphoid cell can only differentiate into B and T cells but cannot give rise to any blood cell
Unipotent
- Cells can produce only one cell type, their own
- Can renew indefinitely
- i.e skin
How are stem cells cultured in vitro to keep them as stem cells?
- Mouse Feeder Layer with confluent layer of non-replicating fibroblasts
- Secrete factors that prevent stem cells from growing - Via extrinsic and intrisic pathways that maintain stem cell niche
Intrinsic
a. Sox-Oct-Nanog transcription factors that prevent activation of differentiation genes.
b. Ronin DNA binding protein binds and inhibits GATA4 and GATA6 genes that induce differentiation
Extrinsic
a. LIF binds to LIF/gp130 dimer receptor and through Jak/STAT3 inhibits activation of MAPK which promotes differentiation
b. BMP binds to BMP receptor and through SMAD/ID, prevents activation of MAPK which promotes differentiation
Common Method for isolating Stem Cells
FACS (Fluorescence-activated cell sorting) or flow cytometer
- Can analyze 10,000 cells/sec
- Any cell that has fluorescence due to antibody with fluorophor binding specific antigen on stem cell, gets flicked into a tube.
- You end up with a population of cells that are pure SCs.
How are adult and embryonic stem cells stimulated to differentiate?
- Changing chemical composition of culture medium
- nestin-positive neuronal precursor cells –> removed beta-FGF causing cell to differentiate into a neuron instead
- nestin-positive pancreatic progenitor cellsà removed beta-FGF and added nicotinamide resulting in insulin-secreting pancreatic islet-like clusters - Altering surface of culture dish
- Making surface similar to bone will cause cells to differentiate into cartilage
- Making surface similar to brain causing cells to turn neuronal - Modifying cells by introducing certain genes
How can cells be maintained in differentaited state?
- Transcription factor whose gene is activated by a signal transduction cascade can bind to the enhancer of its own gene (positive feedback loop)
- Synthesize proteins that act on chromatin to keep gene accessible to transcription factors and machinery (epigenetic level)
- If differentiation is dependent on a particular signaling molecule, the cell can make both the molecule and its receptor.
- Cells can interact with neighboring cells such that each one stimulates the other to remain differentiated.
How can cell be reprogrammed into stem-like state aka how are iPSCs made?
- Nuclear transfer
- Cell fusion
- Genetic reprogramming- this is what is mostly done
- Data showed that Oct4, Sox2, Klf4, and cMyc were sufficient in making adult cell into iPSC.
Challenges of Reprogramming
- Low efficiency from use of retroviral vectors (0.01-0.1%).
- This challenge is being overcome by using plasmids rather than retroviruses.
- Genomic insertion can result in inserted genes gaining mutations
- Some genes you insert can cause mutations in other genes based on where they were inserted.
- Tumors can result since some of the reprogramming factors are oncogenes (c-myc).
- Use chemicals that mimic c-myc instead of genes
- Can get incomplete reprogramming such that they might not be truly pluoripotent
