Stem Cells Flashcards
What are the stages of the cell cycle and what happens in each stage
The cell cycle is the series of events that lead to cell division and replication. It consists of several stages, each with specific processes. The cell cycle can be divided into two main phases: Interphase and the Mitotic (M) Phase.
- Interphase (Preparation for Division)
Interphase is the longest phase of the cell cycle, where the cell grows, performs its normal functions, and prepares for division. It is divided into three stages:
G1 phase (Gap 1):
The cell grows in size and carries out its normal metabolic activities.
The cell synthesizes proteins and organelles needed for cell function and division.
It checks for favorable conditions to proceed to the next phase.
S phase (Synthesis):
DNA replication occurs. Each chromosome is duplicated, forming two sister chromatids connected by a centromere.
This ensures that when the cell divides, each daughter cell receives an identical copy of the genetic material.
G2 phase (Gap 2):
The cell continues to grow and prepares for mitosis.
It synthesizes proteins and organelles required for cell division.
The cell checks the newly replicated DNA for errors and repairs them if needed.
After the G2 phase, the cell enters the M phase (Mitosis).
- Mitotic (M) Phase (Cell Division)
The M phase consists of two key processes: Mitosis (nuclear division) and Cytokinesis (cytoplasmic division).
What is meant by potency
What do stem cells do
What are the different types of stem cells
What are embryonic stem cells
What are the three germ layers*
The three germ layers are the primary layers of cells that form during early embryonic development.
The ectoderm is the outermost layer.
It gives rise to:
The nervous system (brain, spinal cord, and nerves).
The epidermis (outer layer of skin).
Hair, nails, and sweat glands.
Parts of the eyes (such as the lens).
The inner ear.
Some parts of the pituitary gland.
The mesoderm is the middle layer.
It gives rise to:
Muscles (skeletal, smooth, and cardiac).
Bones and cartilage.
The circulatory system (heart, blood vessels, blood cells).
The kidneys and gonads (ovaries and testes).
Connective tissues such as ligaments and tendons.
The lining of body cavities (peritoneum, pleura, and pericardium).
Endoderm
The endoderm is the innermost layer.
It gives rise to:
The epithelial lining of the digestive system (stomach, intestines, liver, pancreas).
The respiratory system (lungs, trachea, bronchi).
The urinary bladder and parts of the urethra.
Parts of the thyroid gland, parathyroid glands, and thymus.
The inner lining of the ear.
Together, these three germ layers form all the tissues and organs of the body through a process called organogenesis.
What cell types would you generate from ES cells
and why?*
Embryonic stem (ES) cells are pluripotent, meaning they have the potential to differentiate into nearly any type of cell in the body.
E.g
Neurons can be derived from ES cells to study neurodevelopment, neurodegenerative diseases (
How can embryonic stem cells be expanded in vitro and driven to differentiate into particular cell types*
ES cells are commonly cultured on a layer of feeder cells (such as mouse embryonic fibroblasts) or molecularly defined extracellular matrices (e.g., Matrigel or Laminin) that help maintain the cells’ undifferentiated state. Feeder cells provide growth factors and molecules that promote ES cell proliferation and pluripotency.
To direct the differentiation of ES cells into a specific cell type, specific growth factors or small molecules are added to the culture media. The combination of signals controls the activation or repression of key transcription factors, pathways, and genes that are required for differentiation into a particular lineage.
What is the ethical issue of using human embryonic stem cells
How can a differentiated cell be made undifferentiated again
the process is called reprogramming
iPSCs are generated by reprogramming somatic (differentiated) cells into pluripotent cells.
To generate iPSCs, four key transcription factors—OCT4, SOX2, KLF4, and c-MYC—are introduced into the differentiated cell using e.g viral vectors. These factors are known as Yamanaka factors and are capable of reprogramming adult somatic cells back to a pluripotent state.
OCT4: Maintains pluripotency and regulates self-renewal.
SOX2: Works with OCT4 to maintain pluripotency.
KLF4: Promotes cell proliferation and contributes to pluripotency.
c-MYC: A proto-oncogene that helps in cell reprogramming but is usually not necessary for all reprogramming methods due to its potential to cause cancer.
What are the advantages of iPSCs over ES cells?
eliminates the need for embryos in their creation. This bypasses many of the ethical concerns associated with using embryonic stem cells
iPSCs can be derived from a patient’s own cells, which reduces the need for donor embryos and makes it possible to generate personalized cell lines - reducing the risk of immune rejection
What are iPSCs*
iPSCs (induced pluripotent stem cells) are a type of pluripotent stem cell that are created by reprogramming differentiated somatic cells (such as skin cells, blood cells, or fibroblasts) to a state similar to embryonic stem cells (ESCs). These cells have the ability to divide indefinitely and can differentiate into almost any cell type in the body
What is the teratoma test*
If stem cells can form a teratoma, it suggests that they have the potential to differentiate into a variety of cell types representing all three germ layers. This indicates the cells retain their pluripotent characteristics
Undifferentiated stem cells (ESCs or iPSCs) are injected into the subcutaneous tissue or testicular tissue of immunodeficient mice (e.g., NOD/SCID mice, RAG mice, or NSG mice) that lack a functioning immune system. The absence of an immune system is crucial, as it allows the foreign stem cells to survive and grow without being rejected.
Teratoma Formation: Over several weeks, if the stem cells are pluripotent, they can differentiate into a mass that forms a teratoma. The tumor is typically composed of tissues from all three germ layers
After a teratoma forms, it is removed and analyzed histologically (using microscopy and staining techniques) to confirm the presence of tissues from all three germ layers
How do cells decide whether to undergo mitosis*
This checkpoint is controlled by cyclins and cyclin-dependent kinases (CDKs), which are proteins that drive the cell cycle forward when activated.
If favorable conditions are met (e.g., adequate size, sufficient nutrients, and no DNA damage), cyclin-CDK complexes activate transcription factors that allow the cell to move into the S phase and begin DNA replication.
How can the CreLox technique be used for lineage tracing of cells*
Cre recombinase is an enzyme that can recognize and bind to LoxP sites, which are specific DNA sequences.
The LoxP sites are typically inserted into the genome of the organism or cell in question, flanking a particular region of DNA.
Cre recombinase, when expressed, causes recombination between the LoxP sites, leading to the deletion, inversion, or translocation of the DNA sequence between them.
A reporter gene (e.g., GFP, RFP, or lacZ) is inserted into the genome of the organism such that it is silenced by a floxed sequence (a DNA sequence flanked by LoxP sites). This silencing ensures that the reporter gene is not expressed in cells unless recombination occurs.
Cre Recombinase Expression Line: Another genetic construct is used to express Cre recombinase in specific cells or tissues of interest. The expression of Cre can be controlled by a tissue-specific promoter or in response to an inducible system (e.g., Tamoxifen-induced Cre), so it can be activated at a specific time or in a particular cell type.
Cre Activation: When Cre recombinase is activated, it recognizes the LoxP sites in the reporter line and induces recombination, which leads to the activation of the reporter gene. This can involve:
Removal of a STOP sequence (in the case of floxed STOP construct) that is preventing expression of the reporter gene.
Inversion or excision of a sequence that was blocking reporter gene expression.
Once the reporter gene is activated in a cell, the cell and all of its descendants will express the reporter gene, allowing researchers to track their lineage.
Give some Applications of stem cell research
What was found with Rett iPSCs
What is trans differentiation *
also known as direct reprogramming, is the process by which a differentiated cell transforms into a different type of differentiated cell without first returning to a pluripotent or stem cell state
Transdifferentiation typically involves the introduction of a specific set of transcription factors that reprogram the gene expression profile of the cell. These transcription factors act as master regulators of cell identity, switching on genes that promote the fate of the new cell type.
These processes may involve modulation of epigenetic modifications, cell signaling pathways, and the activation or silencing of key genes responsible for cell identity
What are tissue specific stem cells*
Tissue-specific stem cells, also known as somatic stem cells or adult stem cells, are a type of stem cell found in various tissues of the body that have the ability to self-renew and differentiate into specialized cell types specific to the tissue in which they reside. These stem cells play a crucial role in tissue maintenance, repair, and regeneration throughout an organism’s life
What is a stem cell niche
What are the body axes and planes of sections
Outline the development of CNS
10) myelination by oligodendrocytes
11) remodel as required throughout life ( synaptic plasticity)
How does neurulation work*
Neurulation is the process during early embryonic development in vertebrates where the neural tube forms, which later develops into the brain and spinal cord
a region of the ectoderm (the outer layer of the embryo) thickens and forms the neural plate
The neural plate begins to fold inwards along its central axis, creating folds on either side. The edges of the plate, called the neural folds, begin to move towards each other, while the area in the middle, called the neural groove, deepens
The neural folds gradually converge and fuse together at the midline, forming a tube-like structure called the neural tube. This closure starts in the middle of the embryo and progresses both anteriorly (towards the head) and posteriorly (towards the tail).
The neural tube will later give rise to the brain and spinal cord.
How can Different neural cell types develop from neural stem cells
stem cells of the nervous system that can produce neural cells including
neurons and oligodendrocytes and astrocytes in the embryo and the adult CNS
(potency varies with species and developmental stage)
= a type of ‘tissue-specific’ stem cells aka ‘adult stem cells’ later
What does neuro- to gliogenic switch
involve
What are organoids and what can they be used for
How is the midbrain formed during brain development
What changes during development
What can spatial omics be used for
Can postnatal and adult
mammalian brains make new neurons
What is a satellite cell
What is the basic pathway of skeletal muscle formation
What is the clinical relevance of satellite cells
What is meant by asymmetrical division of satellite cells
Why are satellite cells important
What keeps skeletal muscle stem cells quiescent until activated
How to test whether quiescent adult stem cells controlled by miRNAs
YFP+ve SCs (that had had Dicer removed)
spontaneously exited quiescence and entered the cell cycle and were depleted
So miRNA-489 can supress Dek and keep satellite cells G0
(Dek protein promotes proliferation and expansion of myogenic progenitors)
How would a sustained expression of miRNA-489 affect the response to muscle injuries?
Experiment :
1) Injure lower hindlimb muscles.
2) An miRNA-489 expression plasmid and a control electroporated.
3) qRT-PCR carried out.
☞ a high level of miRNA-489 expression in the muscle with plasmid.
4) Allow some time for regeneration in this muscle.
miRNA-489 is conserved between many species
(Friedman, R. C. et al. 2008)
It is up-regulated in quiescent cells
Suppresses the Dek oncogene to regulate satellite cell quiescence
miRNA-489
How are satellite cells activated
How is daughter cell differentiation controlled
What happens in a normal muscle compared to a dystrophic muscle*
What changes occur in ageing muscles*
Aged muscle fibres express more Fgf 2 and this
drives satellite cells out of quiescence and
depletes them
Perhaps the muscle fibre is expressing more FGF2 to try to mitigate age-related changes
What is the structure of skin*
What is the different functions of skin*
outer epidermis = keratinocytes
with stem cells in basal layer
dermis = connective tissue with
lots of ECM-secreting fibroblasts
What is the niche of Epidermal stem cells in basal layer
Adhesion to the basement
membrane is important, regulates if
a daughter cell can ‘delaminate’
and another can stay as a SC
What are Hair follicle stem cells (HFSCs)
Explain the Hair follicle stem cells cycle
Stress can affect this e.g., reduce anagen length
and hair gets shorter and weaker
Bulge-HFSCs are in G0
Primed-HFSCs are more easily activated
are under the bulge in the hair germ
What Cell activity occurs in the hair follicle cycle
What different cell types are present in and outside the hair follicle bulge
Outside the bulge and hair germ:
dermal fibroblasts
adipose cells
muscle cells
immune cells
nerves
blood and lymphatic systems
How is the hair follicle cycle regulated
How do Hair follicle SCs help shape their own niche
How can the environment influence hair follicle stem cells behaviour
What is the effect of ageing on the hair follicle stem cell niche*
What are some Applications of skin stem cell research*
Gene therapy in epidermal skin cells to replace diseased skin
