KJ studies Flashcards
Yamanaka and Takahashi
- Inserted a Bgal-NeoR reporter into Fbx15 locus
- RV transduced mouse fibroblasts with pluripotency factors = removed one by one to find the essential genes
- selected for NeoR to find pluripotent cells
- Found OKSM = Oct 4, Klf4, Sox2 and c-myc
- problem that it did not generate a chimeric mice
- tried again with Nanog
- reporter of GFP-puromycinR
- these generated chimeric mice
Modelling bipolar with iPSC
- reprogrammed skin cells from bipolar patients via OKSM reprogramming
- had isogenic controls
- differentiated into neuron cells
- RNA seq saw abnormal mitochondrial gene expression in response to treatment
- saw abnormal mitochondria and hyper-excitable AP in response to treatments
iPSC cardiomyocytes to study SARS-COV2
- human peripheral blood mononuclear cells were reprogrammed into iPSCs via OKSM episomal factors
- differentiated into cardiomyocytes via extrinsic factors
- infects with SARS-COV2 and saw that it could enter and replicate using ACE2
- saw cells stop beating in 3 days
- allowed the development of an ACE2 antibody
- check for markers such as ISL1
Reversing heart damage in mice with iPSC
- transient OKSM reprogramming in mice
- mice expressed OKSM in genome but were under an inducible cardiac specific promoter - promoter switched on with doxycycline
- reprogrammed cells into immature cardiomyocytes which could proliferate and repair the heart
- detected via cell surface markers such as ASMA
- if given doxycycline for 12 days then tumours formed and led to death
Modelling Alzheimers disease PSEN-1 with iPSC
- RV-OKSM hiPSC with PSEN-1 mutation compared to a sporadic AD
- differentiated cells into cortical neurones
- found a Tau peptide (eTau) was secreted in the media by PSEN-1 mutants
- eTau was collected and placed on healthy cells
- saw healthy cells secrete AB42, decreased soluble APPa
- also caused neuronal hyperactivity
Understanding SARS-COV2 with an organoid
- choroid plexus cells broken down via infection
- CSF containing virus and other inflammatory factors could pass the blood-brain barrier
- found it doesnt infect neurones or glia
Liver bud organoids for transplant
- use HLA matched iPSC which can differentiate into hepatic cells, blood vessel cells and mesenchymal cells
- self assemble into liver bud organoids
- GMP model
- transplant into liver failure mice showed recovery of hepatic function
Human cell atlas for studying retina development
- Zhuo et al (2024)
- Used single cell nucleus RNA seq and ATAC-seq
- used nuclei from 14 human embryos and fetuses aged 8 and 23 weeks post conception
- identifies all major retinal cell classes
- showed how retinal progenitor cells transition into neurogenic retinal progenitor cells
- identified spatial and temporal patterns
Human cell atlas for studying brain evolution across species
- Wang et al (2024)
- Compared mice, primates and humans
- looked at different cell times and pathways
Human cell atlas for studying Crohns disease
- Kong et al (2023)
- Took cells from the large intestine and the ileum from healthy and Crohns patients
- Crohns disease vs healthy patients
- saw composition changes in stromal and epithelium cells during disease
Human cell atlas for modelling disease
- Gabitto et al (2024) modelling Alzheimers disease
- Postmortem brain tissue was used
- STG and MTG tissue was used
- Used single nucleus RNA sequencing
- Showed different cell types are affected at different stages of AD – early on there is a selective loss of sst inhibitory neurones while at late stage there is widespread neuronal loss
- Identified key molecular pathways such as the down-regulation of genes involved in the ETC early in disease – possibly a protective response
- Spatial modelling of brain pathology
Human cell atlas use to study aging
- Kedlian et al (2024)
- Single cell RNA sequencing
- mapped the aging process in human intercostal muscles identifying cellular changes
- saw a decrease in ribosome biogenesis genes in a subset of muscle stem cells
Using CRT to treat EB
o Was used as a compassionate consideration for a 7-year-old boy with the condition
o Caused by a mutation in the LAMB3 gene preventing normal epidermal anchoring
o Took a biopsy of unblistered skin cells to establish a primary keratinocyte culture
o Transduced cells with a retroviral vector expressing the normal LAMB3 cDNA
o Cultivated on a fibrin substrate or plastic to produce an epidermal graft
o Transplanted grafts on boys body and saw that there was complete epidermal regeneration after one month
o Drawbacks – using retroviral vector so there is the chance of random integration into the genome causing cancer formation, growing grafts can be complex and time consuming
o Benefits – Uses own patient cells thus no chance of rejection and wont need to be on immunosuppressants, less invasive then other surgical interventions
Cell reprogramming to understand SARS-COV2
o Brain organoid with Choroid plexus cells – Lancaster et al
Generated using human pluripotent stem cells to create a brain organoid
Followed the Lancaster Protocol
* LV-OKSM iPSC
* Differentiate themselves in culture – self patterned
* Contains the forebrain, hindbrain and midbrain
Showed that infection caused the degradation of choroid plexus cells allowing CSF containing the virus and other inflammatory products to cross the blood-brain barrier
Showed little to no infection of neurones or glia
o Intestinal organoid:
Lamers et al (2020)
hiPSC have been used to make small intestinal organoids
Validated via TEM and cell surface markers (APOA1)
Found that it can infect enterocytes and be transmitted through fecal matter
o iPSC cardiomyocytes:
iPSC generated from human peripheral blood mononuclear cells reprogrammed via OKSM episomal vector
iPSC differentiated into cardiomyocytes via extrinsic factors
Infected with Sars-cov2
Saw it replicated and infected via the ACE2
Example of direct reprogramming
- Fallah et al 2023
- direct reprogramming of fibroblasts to osteoblasts
- RXOL cocktail used
- can be used for bone repair
