Regeneration (Stem Cells) Flashcards
Features of NSCs
How can they be derived from MSCs
Differentiate into neurons, astrocytes, oligodendrocytes
Can be derived from MSCs in EGF/bFGF conditional medium through SOX1 activation
Development of NSCs from ESCs
ESC
Preliminary NSC
NSC (FGF/EGF responsive)
Definitive ESC
How does proliferation change with progression through development
Proliferation/cell turnover increases with increased specialisation
A slower turnover is associated with cell health and telomere length
Main neurogenic niches
Subventricular Zone
Subgranular Zone of Dentate gyrus
Central Canal zone of SC
Differentiation of NSCs
ChIP-quantitative PCR: HDAC2/3 bind genes associated with development/differentiation e.. Cebpb, Hoxd4, Ovol2, Zfp7
Contribution of factors neuron development
Secretory factors control dorso-ventral, rostro-caudal differentiation
Forebrain: alpha-BMP, Wnt
Midbrain: FGF8
Spinal Cord: RA
Inputs involved in purkinje cell differentiation
Balance of pro and inhibitory signals:
Fgf2
ROCK, TGFbeta (inhibitory)
Granule cells impact maturation/ development of a full phenotype
NSCs in hypoxic-ischaemic stroke
Hypoxic-Ischaemic stroke assc. with microglial activation and tissue loss
Hypoxia inducbile factors (HIF1, WntBeta, Catenin, IFN-y) are key mediators of O2 dependent mechanism of NSC proliferation/differentiation
NSCs in chronic hypoxia
Chronic hypoxia has less of an inflammatory response
Recovery impaired due to glial scar formation, vs capacity of progenitors
Increases in cell proliferation and glutamatergic progenitors expressing the transcription factor Tbr2 in the SVZ following a hypoxia insult
Parkinsons Disease- features
Loss of dopaminergic neurons in the Substantia Nigra
NSCs in Parkinson’s disease
NSCs can stimulate de-differentiation of astrocytes, and release of exogenous GFs
Inhibition of microglia, slow PD progression by micro-environment modulation
Akerud 2001- GDNF effects in PD
Akerud 2001: GDNF-releasing NSCs grafted into a mouse 6-Hydroxydopamine model of PD, preventing DAergic neuron degeneration and reducing behavioural impairments
Qiu 2017- transplanting mDA progenitors
Transplantation and induction of mDA progenitors at 3 time points of differentiation caused increased PSA-NCAM labelling, and labelling of neuronal markers Increased maturation (TH+ cells) and satisfactory behavioural functional recovery
Manipulating endogenous SCs in SCI
Neurospheres
Corns 2015
Cells cultured from the CCZ proliferate in vitro in response to EGF and bFGF, forming neurospheres
PNU (and Donepezil) increasing EdU labelling in CCZ and GM/WM, with increased CL for PANQKI, HUCD, Sox2
Manipulating endogenous SCs in the hippocampus
Donepezil increases survival of newborn neurons labelled with BrdU/EdU in vivo, increasing CL with MBP
Cholinergic signalling
Alzheimers- due to reduced cholinergic signalling due to Amyloid beta plaques binding alpha 7
Beta 2 KOs have significantly smaller dentate gyri than age-matched WTs
What can CSCs differentiate into?
Cardiomyocytes
Endothelial Cells
Smooth Muscle Cells
Fibroblasts
Potential applications of CSCs
Recovery from cardiac injury
Increasing cell senescence with age
BUT Discrepancies as to source, potential turnover of CSC populations
Evidence for CSCs in recovery
Ellison 2013
Isoproterenol induced injury model (causes diffuse CM death without affecting tissue architecture)
-Cre Recombinase mice (YFP in ckit+ cells)
Showed:
- Increased BrdU labelling which co-expressed ckit+ YFP
- Increased expression of GATA4 and Nkx2.5 (early TFs of cardiac lineage)
- High tropism for cardiac tissue when eCSCs injected into tail vein
- Ablation of ckit+ eCSCs w/5FU caused cardiomyopathy, hypertrophy, and HF
- Transplanted eCSCs isolated after regeneration retained SC properties in vivo
Evidence against CSCs in recovery
Van Berio 2014
MI Model
-Numbers of ckit+ CMs after injury very low
Loss of kit gene completely prevented CM formation from ckit+ cells
Age-dependent effects of CSCs
Jesty 2012
Neonatal infarction in ckit-GFP hearts:
- Expansion of ckit cells
- Increased Nkx2.5 expression
- Partial infarct regeneration
- Adoption of myogenic and vascular fates
Adult mice: Modest ckit induction, no change in Nkx2.5, adopted a vascular fate
Optimising CSCs with Pim1 Kinase
Pim1 Kinase: enhances proliferation, metabolic activity, differentiation, preserves mitochondrial integrity
Pim1 manipulation in hCPCs isolated from LVAD implantation patients
- Decreased population doubling time, increased proliferation rate, reduced staining for senescence assc. beta-galactosidase, elongation of telomere length
- p53/p16 has opposite effects
SCIPIO trial of CSCs
CSC treatment increased LVEF, reduced infarct size
Over 4 months, and increasingly over 12 months
Tang 2010- effects of exogenous CSCs
Injection of CSC-eGFP showed low numbers, but increased BrdU labelling increased
- % of ckit+/BrdU+ cells increased even in those with no EGFP+ cells, suggesting a paracrine action
CADUCEUS Trial
Cardiosphre derived cells reduced scar size and mass
- Did not affect myogenesis- evidence of paracrine action
Exosomes in paracrine activity of CSCs
Exosomes: nanovesicles shed from cells which can pass on markers of disease, and cell survival/generation
Exosome-treated hearts:
- Improved LVEF, decreased hypertrophy, dilated cardiomyopathy
- Lower cytokine levels
-Exosome release suppression abrogated CDC benefits
Targetting the endogenous CSC secretome
Adult CMs co-cultured with ckit+ CSCs attentuated apoptosis and increased caspase 3, IGF-1 expression
Using MSC secretome
RCT of hMSCs demonstrated their safety
Increased LVEF, reduced arrhythmias
MSCs and CSCs additive benefits
Bone marrow-derived stem cells
Improvements in LVEF, end-systolic/diastolic volumes, reduced risk of death
-Benefits may last more than 12 months in acute MI
How does aging affect stem cell niches
Changes in efficiency of regeneration (prolonged inflammatory/fibroblastic activity, reduced MSC activation)
-Changes in pro-senescence/quiescence signals e.g. STAT3
e.g. systemically conjoining mice impairs muscle regeneration in the young, but improves in aged mice
What is Quiescence?
Cells temporarily leave the cell cycle to exist in a sustained G0 phase
Influenced by factors such as p21, p27
What is senescence?
What is SASP
Evidence of effects of p16 (Baker 2016)
Permanent, but not irreversible removal of cells from the cell cycle
Influenced by DNA damage, telomere erosion (p53, p21), oncogene activation (p16)
SASP: Senescence Associated Secretory Phenotype- senescent cells which go on to secrete pro-inflammatory molecules e.g. Interleukins
Baker 2016: Selective apoptosis in p16 inkA cells (accumulate in the heart with age)
-Reduced cardiac hypertrophy, increased resistance to beta-adrenergic stress, extended lifespan
How does senescence contribute to inhibitory signalling in SC niches?
Inflammasome activation e.g. cytokine secretion SASP Exosome release Mitochondrial dysfunction DNA damage Reactive Oxygen Species formation
Contribute to cell cycle arrest
Injury may exacerbate this in CSC niches, with CM death, scarring/ stromal infiltration, ECM stiffness
How is TNF alpha involved in inhibitory signalling in SC niches?
May have an inhibitory effect on CSCs
- TNFR1 KO: massive upregulation of Mef2c and increased proliferation following MI
- TNFR2 KO: similar response in smooth muscle/ endothelial cells
- Endogenous TNF: blunted Nestin downregulation and suppressed beta3 tubulin expression
How does aging affect inhibitory signalling in SC niches?
MuSCs isolated from aged mice implanted into young mice cannot rejuvenate an injury Treated MuSCs (p16/Jak2-Stat3 inhibition) transplant into young and old mice leads to improved regeneration
Price 2014: JAK-STAT targets in 18m mice are expressed higher than younger mice
- JAK2-STAT3 KD/ pharmacological inhibition stimulated satellite stem cell division and enhanced repopulation ability
Methods of manipulating the SC niche
Creating an in vitro niche
- Issues with increased generation of ROS
Manipulating niches
GF/ cytokine administration, engineered ECM, O2 gradient, electrical pacing, cyclic straining etc.
Hydrogels, decellularised ECMs, synthetic matrices
Effects of Exercise on CSCs
High intensity exercise increased BrdU labelling of ckit+ CSCs and CMs in the LV of exercising animals
- Increased IGF-1, TGF-beta 1 (implicated in myocardial hypetrophy)
- Increased expression of Neuregulin 1, Periostin, BMP-10 (regulating hypertrophy/ myocyte replacement following injury)
Effects of Exogenous Growth Factors on CSCs
IGF/HGF in pig infarction model
- Dose-dependently activated ckit+ CSCs, increased myogenic differentiation and improved CM survival
- Reduced fibrosis and CM reactive hypertrophy
- Reduced infarct size, increased LVEF 2 months after MI
Effects of MSC secretome on NSCs in PD model
Increased expression of beta-actin and TH
- Drove increase in proliferation
- Improved behavioural effects of PD (spatial learning ability, apomorphine induced rotational asymmetry)
