Stem cells in drug development

PSCs → mesoderm →

cardiac muscle, skeletal muscle, kidney tubule cells, RBCs and gut smooth muscle

PSCs → endoderm →

alveolar, thyroid and pancreatic cells

PSCs → ectoderm →

epidermal skin cells, neurons and pigment cells

outline the creation of iPSCs for in vitro screening

• patient somatic cell sample

• → iPSCs

• → 2D target cell cultures/3D organoid systems

what somatic cells are taken from the diseased donor?

squamous epithelial cells, keratinocytes or fibroblasts

What are the 4 Yamanaka factors?

Kif4, Myc, Sox2 and Oct4

Yamanaka factors

upregulate embryonic stem cell genes. Activate proliferation, loosen chromatin structure and downregulate differentiation genes

Fibrodysplasia ossificans progressiva (FOP)

progressive condition of soft tissue to bone

what were FOP patient iPSCs differentiated into?

pre-somite as this is where the cell would then differentiate into either bone or dermal cells

What were FOP iPSCs used for?

drug screening - 3/4892 were selected and 2/3 taken on to FOP model preclinical testing

drugs found to be effective against FOP

AZD and TAK - reduced bone volume from approx 100mm^3 to 40mm^3

iPSC derived motor neurons as a model of sporadic ALS/MND

showed branching of neurons followed by degradation in neuron structure

How many compounds were ALS model iPSCs used to screen

1232 - wanted to find compounds which preserved neurite length and had minimal side effects

How was drug efficacy evaluated in iPSC ALS drug screening?

via FUS aggregates, G3BP aggregates, LDH leakage, cleaved caspase 3 and pTDP 43 inclusions

FUS

fused in sarcoma - RNA binding protein

G3BP

Ras GAP

ropinirole

drug found through ALS iPSC screening - already used in Parkinsons and restless leg syndrome

traits of ropinirole

high phenotypic rescue, able to cross blood-brain barrier, targets both familial and sporadic ALS cases

gene knockout in mice embryonic stem cells

• electroporation carried out on stem cells

• homologous recombination to make gene non functional

• KO cells selected using resistance marker

how are knockout stem cells used to create a new mouse

KO cells injected into embryo’s blastocoel cavity and implanted into pseudo-pregnant female → chimaeric mice. Chimaeric mice bred to produce litter with 25% homozygous KO mice, 25% WT controls

GM-CSF KO mice

chronic pulmonary Mycobacterium abscessus model used for preclinical testing of antimicrobial agents as mice typically able to clear infection quickly

GM CSF KO mice infection

approximately a week long and could be cleared using antibiotics