T4: Stem cells Flashcards
francollini
Stem cell definition
Undifferentiated cell that has the potential to differentiate into different somatic cells.
CHARACTERISTICS:
1. self renewal: unspecialised cells capable of renewal through cell division, sometimes after long periods of inactivity
2. Differentiation potential: under certain conditions they can be induced to become specialised cells.
Asymmetric division meaning
Stem cells divide asymmetrically: one daughter cell is identical to the parent cell (representing self renewal), and the other daughter cell is different (representing differentiation potential).
The 3 germ layers
In the development of a zygote, there is cell division to reach the blastocyst stage, and then the gastrula –> this gives rise to the three germ layers
The cells in each germ layer are responsible for differentiating to become different types of somatic cells:
1. ECTODERM: epidermis, neurons, pigment cells
2. MESODERM: cardiac/ skeletal/smooth muscle, kidney tubule, RBCs and bone marrow
3. ENDODERM: pancreatic cell, thyroid cell, alveolar lung cell
Levels of potency in stem cells from highest two lowest, and the source of these cells for each level
- TOTIPOTENT: differentiate into all 3 embryonic layer cells AND placenta (extra embryonic). Found in zygote and blastomere between 2/4 days of development. !only transiently present in organisms and can’t be maintained in culture (because they continue division. switching certain genes off)
- PLURIPOTENT: differentiate into all 3 embryonic layer cells but not placenta. Found in the inner cell mass of the blastocyst –> source of ESCs. !! only transiently present in organism BUT can be maintained in culture with the addition of certain transcription factors
- MULTIPOTENT: Adult somatic cells that can differentiate into some specialised body tissues
- UNI/BIPOTENT OR PROGENITOR: tend to differentiate into one one type of specialised cell, and have a hayflick limit.
Properties of embryonic stem cells (8)
-pluripotent, hence originate from the inner cell mass of blastocyst –> generate all types of embryonic tissue
-long term self renewal ability
-possess a stable diploid karyotype
-chimera formation
-express Oct4
-inducible proliferation and can shift to a state of differentiation
-lack the G1 checkpoint in the cell cycle
-lack X chromosome inactivation
3 Assays to prove pluripotency of a cell
- Formation of a teratoma upon ectopic injection
- Formation of a chimera
- In vitro differentiation into embryoid bodies
Teratoma formation details
-teratoma is a germ cell tumour containing several types of tissue (bone/muscle/hair) called pseudo organs
-ectopic injection: cells are not given any clear direction on how to differentiate
This means that the cells differentiate into many kinds of cells from the 3 germ layers, hence proving pluripotency
Formation of a chimera details
-chimera: any organism that has cells of 2 different genomes
-ESCs are injected into a blastocyst, integration of cells with the inner cell mass, insertion into a surrogate (eg. mouse), offspring are checked for chimeric properties (usually by labelling the initial ESCs with a fluorescent marker and seeing if the F1 mice are fluorescent)
!! chimera formation depends on the ability of initial ESCs to integrate with cells of inner cell mass of blastocyst (bcos both are pluripotent) so this is the exact cause of the proof of pluripotency
Differentiation in vitro into embryoid bodies details
-Embryonic bodies: 3D aggregates (clusters) of pluripotent stem cells caused by differentiation, and comprising of the three embryonic germ layers
-ESCs are allowed to differentiate in vitro (without a clear direction of differentiation) and this forms embryoid bodies which are stained to show markers for cells of the 3 germ layers
Markers of embryonic stem cells (intrinsic and extrinsic)
INTRINSIC SIGNALS:
1. TFs such as: Oct4, Nanog, Sox2
2. Membrane markers specific to the type of cell (whether is it mES or hES): surface antigens such as SSEA3/4 and tumour rejection antigens such as TRA160/181
3. LIN28: RNA binding protein that is best known for its roles in promoting pluripotency via regulation of microRNAs
EXTRINSIC SIGNALS:
CYTOKINES:
1. mES are LIF dependent (LIF/Jak/STAT1,3 pathway)
2. hES are LIF independent and rely on the WNT messenger and Frizzeld receptor
Process with which scientists discovered which genes and TFs were relevant in stem cell differentiation
KNOCK OUT METHOD: use of knock out mice where certain genes were abolished in each population
-Both Oct4 and Nanognegative mice stopped maturing past blastocyst phase,
Conclusion: Nanog and Oct4 are crucial to stem cell differentiation occurring at the blastocyst stage
Experimentation on Nanog expression and ability to maintain stem cell cultures
- mES + LIF –> self renewal
- mES - LIF –> differentiation
- mES - LIF with an over expression of nanog –> self renewal into stem cell
CONCLUSION: certain cells showed that even in the absence of LIF, stem cells were able to renew (case 3). This was due to an over expression in Nanog, which shows that Nanog’s role appears after LIF in the signaling pathway. (this is correct bcos LIF is the extracellular signalling molecule whereas nanog is the TF). HENCE: if nanog was supplied, mES can continue to self renew into stem cells even when there is no LIF present.
Discovery of hESCs
James Thompson (1998):
-extracted cells from the inner cell mass of a blastocyst and put them in culture
-monitored the karyotype of the cells to make sure it was stable and diploid
-checked for assays of pluripotency
-establishment of the ‘bona fide stem cells’
-led the way to principles of regenerative medicine
Regulations on hESC use in italy
-It is forbidden to use hESCs derived within own country, but it is possible to use
hESCs from other countries
-Max 3 embryos implanted for IVF
-Excess blastocysts generated in the past cannot be used to obtain ESCs (hence it is impossible to generate new ES cell lines)
Cloning of the first mammal experiment
Dolly the sheep, 1996
-uses somatic cell nuclear transfer (SCNT)
-same approach used by gurdon was used: transfers a nucleus of a somatic cell into an enucleated oocyte
-fusion of the egg and of the somatic cell nucleus occurred using a small electroshock.
-zygote became an embryo and was put back into the mother sheep uterus
!First example of reproductive cloning (because it results in the cloning of an entire organism)
