New vs established techniques Flashcards
Thomson et al (1998)
Isolated ESCs from ICM of 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.
Gurdon in 1962
- Another type of PSC that has been around for a long time is those generated by somatic cell nuclear transfer (SCNT), a technique developed by Gurdon in 1962 in Xenopus, for which he won the Nobel Prize in Physiology and Medicine many years later in 2012. He showed that implanting the somatic nucleus into an enucleated donor oocyte produced a blastocyst with ESCs genetically identical to the original somatic cell, therefore providing an allogenic source of ESCs.
Takahashi and Yamanaka (2006)
who screened a library of 24 genes, and through trial-and-error, identified 4 factors: Oct4, Sox2, Klf4 and c-Myc (the OKSM factors) that together induce reprogramming of adult mouse + subsequently human fibroblast cells into PSCs
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
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
2019 CRISPR Cas trial
Excitingly, in 2019, the first successful clinical trials using CRISPR-Cas9 for gene editing in haematopoietic stem cells were completed for 2 patients suffering from the red blood cell disorders β-thalassaemia and sickle cell anaemia, both of which are caused by mutations in the β-globin gene (or HBB), resulting in red blood cells that are inefficient at carrying oxygen.
- Individuals with severe β-thalassaemia and sickle cell anaemia require lifelong blood transfusions and chelation therapy to remove the extra iron that results from the blood transfusions.
- In this clinical trial, blood stem cells were removed from the patients, treated with CRISPR to boost the production of beta-haemoglobin, and the engineered cells were then re-infused back to the patients. The CRISPR edits create a deletion in the BCL11A gene, which encodes a transcription factor that otherwise represses foetal haemoglobin synthesis.
17th century Italy, Marcello Malpighi
was the first to use the light microscope to study tiny biological entities widely considered the founder of histology
- Analysed wide variety of organisms, including bats and frogs
- When studying lung structure under the microscope, he identified membranous alveoli and hair-like connections between arteries and veins, which he called capillaries
o Important discovery it established how oxygen breathed in circulates through the bloodstream and provides for the body
Braak et al. (1991)
light microscopy -demonstrated very little neuroanatomical and temporal correlation between amyloid plaques and neurodegeneration; plaques were mainly found in the precuneus and frontal lobes, while neuronal death began and occurred most readily in the entorhinal cortex and hippocampus.
Trzaskoma et al. (May 2020)
- They combined scanning electron microscopy with in situ hybridisation to visualise 3D chromatin folding at targeted genomic regions with ultra-resolution
- They used this technique on human lymphoblastoid cells and were able to visualise many distinctive 3D chromatin folding structures
- They uncovered significant heterogeneity of chromatin folding ultrastructures in individual nuclei, indicating these 3D chromatin states are very fluid need to try and further understand these dynamic changes and their potential role in gene expression
Zhang 2020
demonstrates an exciting direction that the EEG/MEG field is heading**
• In this paper, they showed that a machine-learning algorithm the authors designed can predict antidepressant response in major depression by analysis electroencephalographic signatures!
o A big challenge in treating depression is that only ~30-40% of patients respond to antidepressant monotherapy, leaving 60-70% of patients who do not respond optimally, partly because clinical diagnosis of depression encompasses biologically heterogeneous conditions
o So ability to predict treatment response could help match patients to appropriate treatment more rapidly
o Here, the authors designed a machine-learning algorithm tailored for resting-state EEG and applied it to data from the largest image-coupled, placebo-controlled antidepressant study, which involved 309 participants
o From the EEG alone, this algorithm was able to robustly predict symptom improvement for sertraline (an SSRI) versus placebo
o This sertraline-predictive EEG signature both reflected general antidepressant responsivity and related differentially to a rTMS (repetitive transcranial magnetic stimulation) treatment outcome
• Future algorithms are likely to be more complex, predict response to multiple drugs
• Provides an avenue for personalised treatment of depression by combining computational methods with EEG recordings!
Claussnitzer et al. (2015)
they used multi-omics to gain new insight into the pathophysiology of obesity.
- They analysed the FTO locus, which has the strongest association with obesity in GWAS
- To identify the cell type in which the causal variant acts, they studied chromatin state maps of this region in 127 cell types that had been profiled previously by the Roadmap Epigenomics Project, which directed them to further explore a long enhancer that was active in mesenchymal adipocyte progenitors
- They then studied the long-rang 3D chromatin (Hi-C) interactions between this enhancer, which revealed two genes, IRX3 and IRX5, the expression of which was associated with the risk haplotype
o They carried out experimental manipulations in adipocytes and mice.
For example, knocking down IRX3 or IRX5 in primary adipocytes from people with the risk allele increased thermogenesis 7-fold, whilst overexpressing these genes had the opposite effect
- So from an association in a GWAS, they were able to identify that a pathway in adipocyte thermogenesis involving IRX3 and IRX5, which when manipulated, had powerful pro- and anti-obesity effects.
Bojkova et al. (14th May 2020
- A highly relevant example that was published just 2 weeks ago in Nature by Bojkova et al. (14th May 2020) is using proteomics of SARS-CoV-2-infected host cell to discover new therapy targets.
- Given the rate at which this virus is spreading globally and the lack of treatment options and a cellular understanding of SARS-CoV-2, identifying drug targets is currently a global health priority
o Here, the authors established a human cell culture model for infection with SARS-CoV-2 clinical isolates
o Infected cells with SARS-CoV-2 and cultured them for 2-24h and compared proteome changes in mock-infected samples
o Using pathway analysis they identified increases in pathways involving including splicing, carbon metabolism, translation and nucleic acid metabolism
o They tested small molecule inhibitors against these pathways, which prevented viral replication of host cells – e.g. showed that cycloheximide (inhibitor of translation elongation) inhibited SARS-CoV-2 replication at concentrations that are not toxic to human Caco-2 cells - Thus, using omics to understand the cellular infection profile of this virus enables the authors to identify drugs that could specifically inhibit viral replication
- This work provides extremely important insight for the development of therapy options for COVID-19.
