Fibrosis readouts

[Amritha Varshini]

Hi TooT team!

We just received reticulin and CD34 IHC stained slides for our organoids, and we are not seeing any positive signal in either case. We have TGFb treated and MF sample engrafted conditions, which should both show some level of positivity here. We used BU-8 cells, and other than the organoids being a bit dense, everything else seemed fine with all other readouts (H&E, flow). The staining was done by our histology core using their standard protocols. We were wondering if you had any insight into why this might be an issue for us? Did you use longer staining times/any modifications to the staining protocols?

Thanks,

Amritha

[Bethan Psaila]

Dear Amritha

 

Did you also do RT-PCR for fibrosis markers eg collagen 1 and ACTA2 (aSMA)? That is usually very robustly increased with TGFB stimulation, so it would be surprising if these markers were not upregulated at RNA level. We generally do both RNA and IHC together, as the RNA is less subjective / variable between sectioning planes. 

 

Patient cell engraftment is usually pretty robust too, but obviously a little more variable between samples.

 

What concentration of TGFb did you use and for what duration?

 

I can’t recall that we ever tested the BU lines in fibrosis assays, but we have good data now for a fibrosis response with the stemcell tech line and the MCDN10 line (from an NIH collaborator), as well as the original Gibco line.

 

For the CD34 IHC – did you see no staining at all, or just no difference with TGFb treatment?

 

You can find suggested TGFB treatment protocol and IHC methods in the Nat Protocols paper if helpful.

 

Beth 

 

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[Bethan Psaila]

Yes, that’s the SCT line we are using now and have extensively characterised

 

It sounds like a staining issue then if you don’t have any positive signal at all. An example here of nicely stained CD34+ vasculature and reticulin fibres from a recent experiment.

 

I would suggest running RT-PCR alongside your IHC protocols (it’s faster turnaround too) and including no treatment, TGFb (eg 25ng/ml for 3 days) and if you are screening potential inhibitors then we also find SB431542 useful as a control which should give robust TGFB inhibitor.

 

 

 

 

 

From: Amritha Varshini <amrithava...@gmail.com>
Date: Tuesday, 16 April 2024 at 17:06
To: Bethan Psaila <bethan...@ndcls.ox.ac.uk>
Cc: Bone Marrow Organoids <morerg...@googlegroups.com>
Subject: Re: [BMO Group] Fibrosis readouts

Hi Beth,

 

We haven't done any RT-PCR on this batch of samples, but will definitely do that with our next batch. We don't have any leftover material at this point, unfortunately. 

 

We did 25 ng/ml of TGFb for 72 hours because we wanted a good positive control, but maybe there's something about BU-8 that makes them less prone to fibrosis. For both reticulin and CD34, we did not see any staining at all in any of the conditions, which makes me wonder if the staining protocol needs to be modified. The positive controls that our core uses are livers, which show lots of fibrosis and that probably won't be the case with organoids. 

 

Are these the stem cell tech lines you're using? The Gibco line is discontinued, as you know. And the NIH line is not on their public repository. We're thinking maybe we should switch to one of these since you have validated the fibrosis in them.

 

https://www.stemcell.com/ipscdirect-healthy-control-human-ipsc-line-scti003-a.html

 

https://www.stemcell.com/healthy-control-human-ipsc-line-female-scti003-a.html

 

Best,

Amritha

 

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Amritha Varshini

[Bethan Psaila]

We outsource our reticulin staining to a core facility too so don’t have a specific protocol. Retic stains are hard to get right. If it is working well on bone marrow (murine/human) then it should be good. We’ve used two core facilities and neither needed any specific optimisation as far as I am aware

 

 

 

From: Amritha Varshini <amrithava...@gmail.com>
Date: Tuesday, 16 April 2024 at 20:03
To: Bethan Psaila <bethan...@ndcls.ox.ac.uk>
Cc: Bone Marrow Organoids <morerg...@googlegroups.com>
Subject: Re: [BMO Group] Fibrosis readouts

Sounds good, we're starting up a new batch this week, so we'll definitely do the RT-PCR this time around.

 

I was wondering if you could take a look at the reticulin staining protocol from our core to see if we need to modify it in any way to match your protocol. Attaching it here. We still have the paraffin blocks and unstained slides, so we would love to troubleshoot staining with these if possible. 

 

Thank you so much for your help!

 

Best,

Amritha

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Amritha Varshini

[Salim Atakhanov]

Hi all,

for qPCR, do you just digest cells with collagenase and then isolate RNA or did you try to also sort purify a certain population? I wonder if I will see significant changes in the fibrosis markers if I just do RNA from the whole organoid without sorting. Sorting will also decrease the yield, too...

Best,

Salim

[Abdullah Khan]

We've generally just done a digest and then isolated the RNA (Aude has been using this Fibrosis extraction kit with great success). We are discussing purifying a population now but as you say, the problem is likely complexity + yield. 

The data in the paper is all without sorting, and where there is a clear effect/phenotype like with SB inhibition it's clear, but where effects are more subtle it might be worth a sort or, as Aude suggested, something like a CD45- bead approach. 

RNA from sorted fibro/endothelial cells is likely to be very low yield so you would need to upscale significantly! 

Abs

[Salim Atakhanov]

Yes exactly, those are the points I am also considering. Could you tell please what the fibrosis extraction kit is or share the link to the kit? so that I could read about it.

If you will test CD45 depletion using MACS on BMOs faster than me, could you tell me what the outcome was :D ? Same, if I will test before you, I could also update you guys. 

Thanks a lot!

Salim

[Amritha Varshini]

Hi Beth,

We haven't done any RT-PCR on this batch of samples, but will definitely do that with our next batch. We don't have any leftover material at this point, unfortunately. 

We did 25 ng/ml of TGFb for 72 hours because we wanted a good positive control, but maybe there's something about BU-8 that makes them less prone to fibrosis. For both reticulin and CD34, we did not see any staining at all in any of the conditions, which makes me wonder if the staining protocol needs to be modified. The positive controls that our core uses are livers, which show lots of fibrosis and that probably won't be the case with organoids. 

Are these the stem cell tech lines you're using? The Gibco line is discontinued, as you know. And the NIH line is not on their public repository. We're thinking maybe we should switch to one of these since you have validated the fibrosis in them.

https://www.stemcell.com/ipscdirect-healthy-control-human-ipsc-line-scti003-a.html

https://www.stemcell.com/healthy-control-human-ipsc-line-female-scti003-a.html

Best,

Amritha

On Mon, Apr 15, 2024 at 6:18 PM Bethan Psaila <bethan...@ndcls.ox.ac.uk> wrote:

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Amritha Varshini

[Amritha Varshini]

Sounds good, we're starting up a new batch this week, so we'll definitely do the RT-PCR this time around.

I was wondering if you could take a look at the reticulin staining protocol from our core to see if we need to modify it in any way to match your protocol. Attaching it here. We still have the paraffin blocks and unstained slides, so we would love to troubleshoot staining with these if possible. 

Thank you so much for your help!

Best,

Amritha

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Amritha Varshini