Loss of Pluripotency and Regulation of Differentiation Flashcards
How does EB formation facilitate differentiation?
- ES/iPS cells form EBs in the absence of cell-substrate adhesion → gives aggregates containing differentiated cells
- In mouse, EBs differentiate in 2-4 days, forming an outer layer of PrE like cells with an inner core of epiblast like cells
- EBs mimic the early embro – the cavity is similar to the proamniotic cavity and expression of stage-specific differentiation makrers roughly follows normal development
- Human EBs take around 10 days – makes sense as human development in utero takes longer than mouse
- EBs form derivatives of all 3 germ lineages after re-plating in the presence of serum
- Differentiation occurs in patches in no particular arrangement
- Differentiation into all 3 germ layers is more efficient if cells go through the EB phase rather than just removing pluripotency associated factors.
Why does EB formation promote differentiation?
• Adjacent cell populations reciprocally interact during lineage commitment, as in the embryo
• Reciprocal signaling between PrE and core ICM promotes further differentiation in core cells
Step 1: FGF-4 induces differentiation into extraembryonic endoderm
• FGF4 produced by the ICM under control of Oct-4 and Sox-2
• in FGF4 KO mice, XE does not form
• Two pathways are crucial:
− PI3K/AKT
− ERK/MAPK
• Forced knockdown of Nanog also induces XE
− Nanog inhibits Gata6 (necessary for XE formation) – giving Epi cells
− FGF4 inhibits nanog, strengthening Gata6 expression in XE cells.
Step 2: Reciprocal interaction back to the epiblast induces formation of ectoderm epithelium
• Requires formation of a laminin containing BM between ectoderm and endoderm (similar in EBs)
• Ectoderm will give rise to endoderm, mesoderm and neurectoderm at gastrulation
• Ectoderm requires low Oct-4 and absence of BMP
What are the interactions between TF and GF signalling?
- Level of Oct-4 regulates lineage specification for hESCs
- In serum containing medium:
- In mouse → increase Oct-4 by 50% gets XE, decreasing Oct-5 by 50% gives TE
- In human → Nanog knockdown gives TE and XE differentiation
What happens in the absence of serum?
• Normal levels of Oct-4 (for hESCs) and no BMP → self renewal of pluripotent stem cells
• Normal level of Oct-4 and added BMP → get mesendoderm
• Knock down of Oct-4 and no BMP → embryonic neurectoderm
• Knock down of Oct-4 and added BMP → XE and TE lineages
− This seems different to what we see in serum containing medium (O4 KO gives TE, not extraembryonic neurectoderm)
− Serum may contain some BMP, which is why we see this.
• After BMP treatment in absence of serum, microarray analysis reveals many TE markers increase 3 fold:
− TFs → Gata2, Gata3, Msx-2, SSI-3
− TE differentiation associated proteins → hCG, LH
- Oct-4 binds to the BMP-4 promoter, probably negatively regulating it and switching BMP off in early epiblast and hESCs. Later, it comes on to regulate mesendoderm and primitive streak formation.
- Nanog overexpression leads to complete blockage of neural differentiation through inhibition of Gata6
- In serum containing medium (and in the embryo) knockdown of Nanog leads to appearance of TE and XE lineages, but in defined medium, KO of Nanog in hESCs leads to appearance of neural markers
- Sox-2 overexpression reduced definitive endoderm markers
How can ectoderm be directly induced (differentiation of cells to neurons)?
• ES cells tend to differentiate spontaneously to neural phenotypes → idea is that neural is the default speciied pathway, and this differentiation needs to be blocked by BMPs/IDs (mouse) or Nodal (human) to achieve other cell types
• Retinoic acid has been shown to increase the proportion of neurons from mESCs through activation of Wnt signaling
• Human embryo bodies can be induced to form neurospheres by addition of insulin, heparin, GDNF and FGF2
− From neurospheres to neural stem cells by adding FGF2 and laminin
− Get neurons by adding FGF2, BDNF and GDNF
− Get astrocytes by adding BMP and CNTF
− Get oligodendrocytes by adding NT3 and SHH
How can endoderm be directly induced?
Experiment D’Amour et al, 2005:
• In the presence of low serum, high Nodal/Activin (100nm/ml) induced first a mesoderm intermediate (Brachyury) then endoderm precursors (expressing FoxA2, Sox-17B transcripts)
• No EB intermediate is formed
• Mesoderm isn’t formed
• You see time dependent expression of endoderm genes
• Serum is titrated to give the effect – you only get endoderm at low serum
D’Amour 2006 improved on these protocols:
• High activin pushes cells to form mesendoderm, then you need to add an array of other factors to sequentially push them through all the different lineages
➢ High activin → Mesendoderm
➢ Activin → definitive endoderm
➢ FGF-10 and cyclopamine → primitive gut tube
➢ RA → foregut endoderm
➢ DAPT and extendin-4 → pancreatic endoderm
➢ IGF-1 and HGF → endocrine beta cells
• Throughout this, you have to block PI3k signaling, as this drives self-renewal
• However, these resulting beta cells have problems
− Only 12% were insulin secreting cells
− C-peptide is released on stimulation, however they are not responsive to glucose! they are immature → no good as a therapeutic or disease model
− many synthesise more than one hormone → are polyhormonal endocrine cells, not just a beta cell.
− Large amounts of pro-insulin secreted (the uncleaved, unactivated form) → suggest similar to immature fetal pancreatic cells
• Therefore had to further develop the protocol
− PSC differentiated beta cells do secrete insulin in response to high glucose
− These ‘Hues 8 hESCs’ have a much more similar pattern of insulin secretion to the natural adult beta cells compared to the polyhormonal cells
− These cells also rescue an insulin deficient mouse when implanted under the kidney capsule → so they work in vivo too!
How can mesoderm be directly induced?
• Start off much the same as the D’Amour procotol – put activins and Wnt in to push ESCs to primitive mesoderm
➢ Then add BMP-4, FGF2 and Follistatin to push to mesoderm (follistatin blocks endoderm)
➢ Then add GDF-5
• In 14 days get aggregages of cells that are immature chondrocyte progenitors
− They produce collagen 2 – key collagen of cartilage
− Express condroitin sulphate – key glycosamino glycan of cartilage
− Express aggregan – key proteoglycan of cartilage
• Can produce them in about 95-97% purity demonstrated by flow cytometry towards the key TF Sox-9
• Injecting these cells into the joint of a rat repairs defective cartilage
Differentiation to cardiomyocytes:
Dambrot et al, 2011
• First approach is based on EB formation but includes multiple induces, usually GF and repressions known to influence heart development in the embryo
− Can occur in regulator growth medium containing FBS and is first evidenced by contractile areas of rhythmically beating cardiomyocytes
− Addition of GFs can further enhance EBs to a more directed differentiation
− A method where EBS were placed in hanging drops on a petri dish had limited success with hESCs compared with mESCs, but differentiation in hESCs was increased using defined GF conditions and spin EBs (EBs created from precise cell numbers)
− Crucial additions include:
− TGF-b
− BMP
− Wnt
− FGF
− p38 MAPK
− SCF
• Second approach based on stromal cell co-culture, exploiting the influence of endoderm for cardiac differentiation.
− Co-culture with endoderm like cell lines or their conditioned medium
− Use of mouse END-2 cells have been used with hESCs
− However In the END-2 culture system, 85% of cardiomyocytes produced are ventricular, whereas other methods produce atrial and ventricular types
• Third method uses a high densitiy monolyer supplemented with BMP4 and activin → reported to be very efficient.
