NDD/Paper 2 - Stem cells Flashcards
Till and McCulloch 1961
Accidentally observed that IV injection of bone marrow cells in previously irradiated mice led to the formation of colonies of proliferating cells in the spleens of those animals.
The injected cells were blood-forming progenitors, able to fully regenerate into blood cells.
Thomson et al (1998)
Human ESCs were first isolated by Thomson et al (1998) in a landmark paper from the inner cell mass of a pre-implantation human blastocyst. They showed that these proliferate in an undifferentiated state in tissue culture indefinitely, and still maintained the potential to form derivatives of all 3 germ layers, by showing that they form teratomas in SCID immunocompromised mice
Chen et al 2014
Generated iPSC-derived motor neurons from pts carrying specific SOD1 mutations.
- The authors used genetic correction of both of these mutants as controls.
- The motor neurons demonstrated the spontaneous and progressive decrease in cell viability observed in humans and ALS-related morphological changes were observed in vitro (reduction in soma size and altered dendrite length).
- Moreover, they were able to reveal novel disease mechanisms - showed that in spinal motor neurons, mutant SOD1 sequesters neurofilament-L mRNA, thereby altering the proportion of NF subunits - leading to protein aggregation and axonal loss.
Bernnand 2011
Published in Nature. Worked on SCZ.
- Reprogrammed fibroblasts from SCZD patients into human iPSCs and subsequently differentiated these disorder-specific hiPSCs into neurons.
- They found these neurons showed:
- diminished neuronal connectivity → Using trans-neuronal spread of rabies virus (stronger synapse = better spread) as an assay of neuronal connectivity, they found this was decreased
o Does not necessarily mean synaptic function is decreased (could be limitations fo synaptic activity assays) - decreased neurite number
- PSD95-protein levels
- glutamate receptor expression
- Almost 600 genes had expression altered more than 1.3-fold, 25% had been previously implicated in SCZ -upport idea of SCZ model where many different combinations of gene dysfunction may disrupt the key pathways affected in SCZ
- diminished neuronal connectivity → Using trans-neuronal spread of rabies virus (stronger synapse = better spread) as an assay of neuronal connectivity, they found this was decreased
Thus their model recapitulated several hallmarks of SCZ
Limitations - Did not display many other SCZ features that are observed in post-mortem tissues and animal models. E.g. they did not observe defects in synaptic function, although this could have been due to technical limitations of the synaptic acivity assays. Furthermore - only small pt cohort so results might not generalise. SCZ is v heterogeneous
Future - Increase number of hiPSC cases and see consistent genes affected, and essential pathways will come to light
Moretti et al 2010
produced patient-specific iPSC models for long-QT syndrome, by taking dermal fibroblasts from a family with long-QT syndrome type 1 and healthy controls.
- They infected the cells with the OKSM factors to transform them into iPSCs, and directed their differentiation into functional cardiac myocytes that maintained the disease genotype of long-QT type 1.
- The team were able to study the disease, as the patient-derived cells exhibited the electrophysiological features of long-QT type 1.
- As expected, they observed long action potentials in the atrial and ventricular cells.
- Moreover, they could identify in long-QT type 1, that a dominant negative missense mutation occurring in the KCNQI gene (encoding the K+ channel mediating the delayed rectifier K+ current) was responsible.
- This was associated with altered cellular localisation of KCNQI due to a trafficking defect and a decreased K+ current. This is a profound example of how Moretti et al. uncovered fundamental aspect of disease pathophysiology by modelling using iPSCs.
Hu et al 2018
Studied the secretomes of human iPSC-derived neuornal models of AD.
- Secretomes = set of proteins expressed by an organism and secreted into the EC space.
- They assessed the ability of secretomes from iPSC-derived models of AD to disrupt LTP when delivered to the hippocampi of live adult RAT brains.
- They found secretomes from neurons with PSEN-1 mutations, APP duplications or trisomy 21 strongly inhibited high-frequency stimulation of induced hippocampal LTP.
- Disruption from PSEN-1 or APP was AB peptide-dependent, being prevented by immunodepletion of AB from the secretome.
- However, trisomy 21 was not - instead was inhibited by immunodepletion of EC tau.
- In all cases, LTP is not inhibited by any of the samples when antibodies against PrP are given.
- Suggest mechanisms converge at the level of cellular prion protein to induce synaptic dysfunction in vivo
- Caveats → Human proteins may affect rats different. Looked at LTP, rather than cell death.
Israel et al 2012
Generated iPSCs from fibroblasts of 2 familial (APP gene duplication) and 2 sporadic AD pts and wells as two controls - turned these into neurons. The two FAD and one of the SAD pts exhibited higher levels of AB, phospho-Tau and active GSK3beta. Beta-secretase inhibitors, but not gamma-secretase inhibitors decreased the levels of phospho-tau and GSK-3beta but not AB, suggesting that there may be multiple disease processes One of the most interesting parts of this study is the implication that on a molecular level, some sAD pts may exhibit similar pathology to fAD patients, although some are very different. They unfortunately did not test the inhibiots on the non-fAD-like sAD pt as this may have demonstrated whether or not these different phenotypes may have different drug responses, which would give a strong justification for phenotyping pts to stratify them prior to trialling treatments.
Qian et al 2016
generated forebrain-specific organoids from human iPSCs that recapitulated key features of cortical development. They then exposed the forebrain organoids to Zika virus, and performed quantitative analyses which revealed the preferential infection of neural progenitors. They showed infected neural progenitors become viral factories to produce more infectious viral particles, leading to a growing number of infected cells over time. They also uncovered that Zika virus infection resulted in increased cell death and proliferation, which decreased the neuronal cell-layer volume, resembling microcephaly. This validates the link between foetal Zika virus infection and birth defects. Moreover, the team showed that this Zika-induced neural progenitor cell death was much more prominent in forebrain oragnoids than monolayer cultures, showing that the 3D nature of organoids gives us insight into disease that monolayer cultures cannot provide. So they successfully modelled Zika virus infection using brain organoids and were able to gain new insights into the disease
Zhao et al 2020
APOEe4 allele strongly associated with development of AD
- Used iPSC-derived cerebral organoid - mimics brain with dif cells
- These came from cog unimpaired with APOEe3/e3 or e4/e4 or AD with either APOEe3/e3 or e4/e4
- Did immunostaining for a marker of apoptosis = more death in APOEe4 AD (only a marker though, did not actually visualise cell death)
- AB higher in AD regardless of APOE phenotype
- APOEe4 consistent with higher pTau regardless of disease
- Do healthy people have mechanism to counter ApoEe4 tau pathology?
- Gene editing to convert APOE4 to APOEe3 in organoids from AD reduced apoptosis markers, AB and tau - therapeutic strategy?
- Limitations →
- Did not actually visualise cell death, only a marker of it - could have used ethidium homodimer labelling or maybe flow cytometry
- They used RIPA buffer to lyse organoids before measuring AB - AB accumulates in stressed cells - could this be affecting it?
- Did not address heterogeneity between different organoids
- The core of their organoids were necrotic - should use a system where vascular cells or microglia are incorporated (immunoreactivity for apoptosis marker was only measured at the surface)
- Did not match sex in study groups (but did adjust for it)
Kondo et al 2013
Generated iPSCs from familial and sporadic AD patients and differentiated these into neural progenitor cells. They identified Aβ deposition in both spontaneous and familially derived neurons, and noted upregulation of oxidative stress associated genes and a downregulation of glycosylation associated genes, and upregulation of ROS suggesting ER and oxidative stress. These perturbations were reversed by β secretase inhibitors, suggesting that Aβ is responsible. DHA (docosahexanenoic acid) was effective in reversing many of the signals of intracellular stress, and partially rescued cell survival in one sporadic line but not the other; differential responses to treatment. Suggested DHA may be effective in a subset of pts.
Lee et al 2016
showed that 3D brain organoids allow for evaluation and selection of drugs in a personalised, patient-specific manner. They generated cortical organoids with β-amyloid plaques from sporadic Alzheimer’s disease (AD) patient-derived iPSCs. They showed that the pathogenic phenotype could be rescued by inhibition of β-secretase (BACE1) or γ-secretase, showing the drug-testing potential of this system. Interestingly, inhibition of these proteases was less efficient at reducing plaque load in organoids when compared to complementary 2D cultures, which strengthens the hypothesis that differences in diffusion between the two culture systems may account for this potentially more representative modelling of plaque formation. Moreover, they demonstrated significant variations in protease efficacy between five patient-derived brain organoids, even with similar initial plaque loads. By performing a proteome analysis of the five patient-derived organoids, they identified variations in specific protein levels (such as clathrin) that correlated with protease-induced plaque load reduction, hence this could provide a mechanism for the differences.
Hans Clevers, leader in the organoid field, and his team
Have demonstrated that patient-derived gut organoids are a powerful model for predicting treatment response. They developed a functional CFTR assay based on cAMP-induced organoid swelling combined (organoid would swell if the treatment was effective) with high-throughput screening imaging as a measure of CFTR function. Currently, they are attempting a nationwide effort to screen every CF pt in the Netherlands. Consequently, many pts with rare mutation, who otherwise would not have access to these drugs, have had positive results from organoid tests, and these pts now respond well clinically, so they have a treatment option that was not previously possible
Curt et al 2001
Transplanted embryonic mesencephalic tissue containing DA neurons bilaterally into the putamen of HUMANs, and found there was a signifcantly improvement in disease severity (Unified PD Rating Scale) in the transplant group as opposed to the sham operation group in patients under 60, but not over 60.
- This could be interpretted as meaning that in older pts there is a greater degree of degeneration and that it is harder for the implanted neurons to become functional, or that the degeneration has begun to affect other areas or cause maladaptive plasticity making restoring DA function insufficient to restore changes.
- Difficult to interpret as the study does not report pt outcome as a function of initial symptom severity, and so the reader is forced to assume that older pts are more likely to have advanced disease.
- The control group received a sham operation which did not penetrate the dura - this means the transplant group was likely to have neuroinflammation from the operation that was not adequately controlled for.
- Not only will this have neurobiological effects but may induce sickness behaviours which could alter the impact of placebo effect
Li et al 2008
Report two cases of pts who received embryonic mesencephalic tissue containing DA neurons > 10 years ago. Many implanted neurons appeared healthy and had survived over this extended period. They also noted that some of these neurons had a-synuclein and ubiquitin-positive Lewy bodies and the alpha-syn was shown using antibody staining to be phosphorylated at Ser129 - indicating pathological post-translational modification. This has been proposed to support the idea that host alpha-syn induced misfolding in the transplanted neurons (prion-like properties). Alone this is not sufficient to support this conclusion - alpha-syn misfolding is common and occurs sporadically especially in stressed neurons. Neuronal stress may be increased for a number of reasons, e.g. by the microglial activation associated with PD, lack of neurotrophic support, epigenetic history of the cells, the differentiation process or an unfavourable transplant environment
Cruz et al 2018
Published results of a phase 1 clinical trial in Nature, in which they engineered a RPE patch comprising a monolayer of RPE derived from hESCs, on a coated synthetic membrane. They delivered this patch into the subretinal space of one eye in two pts with severe AMD. The patch survival and surpassed primary endpoints - after 12 months there was a visual acuity gain of 29 and 21 letter in the two pts Cav - Need to long-term local immunosuppression.
Hanna et al 2007
Demonstrated the utility of this method in the treatment of a humanised SCA mouse model with a mutation in the B-globin gene. They generated autologous iPSCs which they differentiated into haematopoietic progenitors and corrected the beta-globin gene to the WT using homologous recombination. The mice were irradiated and the progenitors were transplanted into the mice. This resulted in the substantial improvement in the disease phenotype, indicating i§PSCs have a therapeutic application in regenerative medicine
