Stem Cells Flashcards
Symmetrical division?
Morula to blastomeres. Early development.
True stem cells
Define stem cell
Undifferentiated cell
Able to self renew
Differentiate into many cel types
Asymmetrical division.?
Replaces self and produces daughter cell that develops.
Progenitor cells
Define epigenetics
Regulates gene expression and cell morphology without altering DNA sequence.
Epigenetic mechanisms
Chromatin condensation
Post-translational modifications of histones
DNA cytosine marks
Non coding RNA
What do histones variants do?
Affect chromatic structure and function
Post-translational modification of histones mechanisms
Acetylation
Phosphorylation
Methylation
Ubiquitination
What are DNA cytosine marks?
Methylation of cytosine in DNA
What do non coding RNA molecules do?
They are RNA molecules affiliated with Epigenetic memory. They REGULATE GENE EXPRESSION by binding to mRNA with antisense sequence.
Leads to transcriptional degradation or translational inhibition.
Therefore, adversely impacts protein expression.
Role of microRNA in Epigenetics?
Participate in establishment of de novo DNA methylation.
Therefore, critical in EPIGENETIC REGULATION.
Totipotency
Can form all cell types plus embryonic membranes
Pluripotentcy
Inner cell mass. Forms all three germ layers.
Nullipotency
Terminally differentiated
Roles of stem cells..
Development Tissue regeneration Cancer development Potential to treat incurable diseases Advance understanding of biological processes
Sources of stem cells
Embryonic stem cells
Adult stem cells
iPS cells
Why do we need to characterize iPS cells?
Reprogramming efficiency is low
Partially programmed cells common
Tendency to acquire abnormal karyotypes
Allows for cross line and cross lab comparisons
Persistent transgene expression (gives incomplete reprogramming and instability)
Criteria for characterization.
Morphological
Molecular
Functional
Egs of DIAD models
Familial dysautonomia
Long QT syndrome
Dyskeratosis congenita
Methods of stem cell delivery
Injection
Vascular route
Applied as cell layers
Seeded onto scaffolds
Stem cells require … When regenerating organs
Vascular supply
Scaffold
Site specific signals
(We need a means to identify SC - reporter genes, differentiation markers)
Issues with ESC
Teratoma
Immune rejection
Issues with iPS
Low cellular reprogrammed efficiency Epigenetic memory Oncogenic risks Cell line to cell line variation Low efficiency of cardiomyogenesis
Embryonic stem cell factors
Oct 3/4. Sox2. Nanog. C-Myc.
CD24. E-Cadherin. TRA-1-81. TRA-1-60
Haemopoeitic niche formed by
Subendosteal osteoblasts
Sinusoidal endothelial cells
Stroma (fiblastoid, adiposcytic, monocytic)
Where is cardiovascular stem cell niche?
RV free wall, atria, outflow tracts.
Characteristics of adult SC
Plastic
Transdifferentiate
Components of stem cell niche
Stem cells
ECM
Local signaling cells
Effective range covered by signaling
Main obstacle for adult stem cels
Renewable source needed
Hard to isolate and expand
Functional expansion needed - clinical potential
How does OSN establish pluripotency?
- Activates expression of other pluripotency associated factors.
- Simultaneously downregulates differentiation genes
- Activates own/ and each others gene expression
- Extensive protein-protein interaction
i. ESC transcription regulators
ii. Chromatin remodeling
iii. Modifies many factors
hES need to maintain pluripotency and self-renewal. FGF2 and Activin/Nodal signaling is needed. How does it work?
Phosphorylates proteins of signaling cascades (MAPK, Wnt)
Phosphorylates Oct 3/4 and Sox2
Cooperates with GFs to up regulate Nanog
Strategies for creating iPSCs
- Integrating vectors (rotavirus, lentivirus)
- Integrating excisable vectors
- Non-integrating DNA vectors (adenoviral, episomal, mini circle DNA)
- DNA free methods (Sendai virus, modified mRNAs, recombinant proteins/cell extracts)
What is needed for generating iPSCs
- Calendar and POA
- Inactivated feeder layers
- Conditioned medium
- Serum free media
- B-fibroblast GF
- Feeder-free cultures and medium.
