pluripotent stem cells

mESCs LIF signalling BMP2/4 signalling Wnt signalling 3i and 2i conditions hESCs iPSCs Yamanaka factors organoids

stem cell definition

self-renew.

be clonal.

differentiate.

stem cell locations

fertilised egg.

early embryos.

umbilical cord.

adult tissues and organs (haematopietic and intestine).

cancers.

culturing mouse embryonic stem cells

requires specific growth factors to maintain undifferentiated cells.

indefinite proliferation is maintained in self-renewing conditions.

inner cell mass cells are cultured as embryonic stem cells as they are pluripotent.

maintenance of pluripotency in embryonic stem cells

promotion/enhancement of proliferation.

suppression/inhibition of differentiation, using extrinsic signals (growth factors/cytokines), signalling pathways, intrinsic factors (transcription factors).

original method of culturing mouse embryonic stem cells to maintain pluripotency

requires culture on feeder cells, e.g. fibroblasts, that produce leukaemia inhibitory factor (LIF).

adapted original method of culturing mouse embryonic stem cells to maintain pluripotency

LIF plus serum can be used in place of feeder cells.

serum has batch-to-batch variation and has to be tested prior to use.

LIF pathway contributes to maintenance of self-renewal.

difference between naive and primed state of pluripotency

naive - early cells isolated from blastocyst, pre-implantation.

primed - post-implantation, epiblast stage, primed to differentiate.

LIF signalling in mouse embryonic stem cells

activate STAT3 forms can maintain pluripotency without LIF, as it is downstream in the pathway.

c-myc is an oncogene.

ERK is an inhibitor of self-renewal.

what forms when implanting pluripotent stem cells?

teratomas - defined by seeing all three germ layers (endoderm, mesoderm, ectoderm).

alternative to serum for culturing mouse embryonic stem cells to maintain pluripotency

bone morphogenic proteins 2/4 (BMP2/4) could replace serum.

BMP2/4 with LIF maintain pluripotency.

bone morphogenic proteins 2/4 pathway for pluripotency

TGFbR2.

Smad (similar to mothers against decapentaplegic) 4 - transcription factor.

Id (inihibitor of differentiation) - transcription factor that prevents differentiation.

Wnt signalling (presence of Wnt ligand)

Wnt ligands are secreted molecules that bind to receptors (such as Frizzled).

Frizzled inhibits glycogen synthase kinase 3 (GSK3).

no phosphorylation of beta-catenin (substrate).

allows stabilisation of beta-catenin which accumulates and translocates into the nucleus.

TCF-3 binds to target sites on bet-catenin.

beta-catenine relieves TCF-3 repression of transcription and reinforces self-renewal.

alternative to using Wnt for maintenance of pluripotency

small molecule inhibitors of GSK-3 can maintain pluripotency instead on using Wnt.

Wnt signalling (absence of Wnt ligand)

GSK-3 phosphorylates beta-catenin which causes degradation at the proteasome.

allows TCF-3 to inhibit self-renewal.

alternative to using BMP2/4 or serum to culture mouse embryonic stem cells to maintain pluripotency

LIF plus inhibition of FGF receptor (using SU5402) and MAPK/ERK kinase (using PD184352) replaces requirement for BMP/serum.

3i conditions for pluripotency

inhibition of GSK-3 (using CHIR099021), in addition of FGFR (using SU5402) and MAPK/ERK kinase (using PD184352) inhibitors, improves viability and growth, as well as liberating cells from exogenous factors.

can derive and maintain Stat-3 null embryonic stem cells in 3i conditions.

2i conditions for pluripotency

refers to dual inhibition of MAPK/ERK kinase (PD184352) and GSK-3 (CHIR099021).

mouse embryonic stem cells culture in 2i conditions with LIF enter ground state (stable naive pluripotent state - reflective of blastocyst stage).

mouse embryonic stem cells cultured in 2i conditions and LIF are characterised by uniform expression of key pluripotency transcription factors (Oct4 etc).

ethical concerns of using human embryonic stem cells

requires the use of fertilised eggs.

only spare IVF eggs can be used in the UK.

donors must agree for the embryos to be used for research.

concerns that undifferentiated human embryonic stem cells could generate tumours if accidentally transplanted.

features of human embryonic stem cells

self-renewal, indefinite proliferation, and pluripotency.

more difficult to culture than mouse embryonic stem cells.

don’t like to be separated into single cells (can add ROCK inhibitors to solve this).

need the use of feeder cells for best growth.

can’t be maintained with LIF.

FGF2 and activin A (member of TGFb family) are the best combination of factors.

express Oct-4, Sox-2, and Nanog.

what was the inspiration for induced pluripotent stem cells?

Dolly the sheep.

Yamanaka factors

transcription factors

Oct-4 - key driver of pluripotency. test for expression through PCR or immunostaining.

Sox-2.

c-myc - oncogene.

Klf-4.

reprogramming mouse fibroblasts to pluripotency

achieved using using Yamanaka factors.

transcription factors needed to make induced pluripotent stem cells depends on original cells.

efficiency of reprogramming fibroblasts to pluripotency

for every 5×10^4 fibroblasts, approximately 10 embryonic stem cell colonies and 100 non embryonic stem cell colonies (we don’t want these).

approximately 1 in 5000 fibroblasts reprogram to embryonic-like cells.

30 day time span.

could be screening for drug compounds within 2-3 months.

an efficient process.

seed at low densities so non embryonic stem cell colonies don’t predominate.

steps for induced pluripotent stem cell derivation

choice of factors.

methods of factor delivery.

choice of cell type.

parameters of factor expression.

derivation conditions.

identification of induced pluripotent stem cell colonies.

expansion and characterisation.

how to choose transcription factors for induced pluripotent stem cell derivation?

depends on starting cell type expression.

use skin biopsies.

KOSM/Yamanaka factors.

e.g. if Sox-2 is present in starting cell then won’t need to for induced pluripotent stem cell generation.

what delivery can be used in induced pluripotent stem cell derivation?

lentivirus - don’t want long term integration into host genome so not best option.

retrovirus.

adenovirus.

plasmids.

RNA protein.

how to identify induced pluripotent stem cell colonies?

look for morphological changes.

look for markers of pluripotency (Oct-4 etc).

transplant cells into immunodeficient mouse to see if teratomas form.

make organoids from the cells, as alternative to the mouse.

take away the pluripotency and see how they differentiate.

stain for induced pluripotent stem cell markers.

applications of induced pluripotent stem cells

cell replacement therapies.

patient and disease specific induced pluripotent stem cells ‘disease in a dish’ models.

use in drug discovery.

cellular agriculture.

benefits to using organoids

reduces the need for animal experiments.

they are expandable.

can freeze/thaw them.