comBo Pre-Print and protoocol

[Abdullah Khan]

Hi all, 

Sharing this here: https://www.biorxiv.org/content/10.1101/2025.02.16.638505v1

This is the pre-print for our 'next gen' approach. A lot of you have reached out since the ISEH webinar so pleased to finally share it. 

The methods section is pretty comprehensive but I'll try and roll out a step by step protocol in the next couple of months. I have a couple of grant deadlines that are taking most of my time until April, but will try and do this by then! By a similar token may not be as responsive as usual on here ! 

Hope you're all keeping well! 

As always, thanks for TooTing, 

Abs

[Salim Atakhanov]

Hi TooT,

congratulations on such a mind-blowing manuscript! 

I have few questions about this new protocol:

1) Is there a reason for you to change the composition of the hydrogel in "bulk hydrogel" ? In this you mention about fibrinogen and thrombin in addition. Did you face any problems in the hydrogel which doesn't contain fibrinogen and thrombin?

2) Did I understand correctly that on day 35 you have some calcified tissue, but on day 80 you have mature bone? Or how good is the bone ossified on day 35?

3) As you annotated osteoclasts in your single cell data from comBO protocol, does the BMOs undergo bone turnover (bone resorption-bone formation)? 

4) From day 14 onwards, you don't add any VEGF, this means that  the heterogenous ECs don't need those cytokines anymore? From day 20 onwards you just add EPO and TPO. I get the idea behind it, but is there a negative effect (less stem cells, for example, or engrafting primary CD34+ cells will yield in MEP population and less into myeloid if culturing only with EPO, TPO)? Also, did you culture BMOs until day 80 just with these exogenous cytokines or did you add anything else to stimulate bone formation? 

You guys are trailblazers in the field! Fingers crossed for a smooth revision and fast publication of this important manuscript!

BW,

Salim

[Abdullah Khan]

Hi Salim, 

Thank you so much for the kind words - from all of us here it means a lot that you guys like and can reproduce the work for your own research. 

Answers below: 

1) Is there a reason for you to change the composition of the hydrogel in "bulk hydrogel" ? In this you mention about fibrinogen and thrombin in addition. Did you face any problems in the hydrogel which doesn't contain fibrinogen and thrombin?

The fibrinogen and thrombin should form fibrin which we introduced originally to support the vasculogenesis and ossification (fibrin gels are used quite a lot in on-chip vascular models and Liam Grover's group showed that fibin + col1 helps with ossification). 

We had been shifting towards fibrin in gels as well as a bulking agent and to make our base layers cheaper, but we haven't tested the fragmented method in the absence of Fibrin. My suggestion would be to keep it in if you can, it's quite cheap (thrombin less so, but you don't need a lot to polymerise), though if you choose not to I suspect the main difference might just be in ossification.

2) Did I understand correctly that on day 35 you have some calcified tissue, but on day 80 you have mature bone? Or how good is the bone ossified on day 35?

In the paper we have deposits of osteopontin, osteocalcin, and col1 that look like early bone on day 35, but no actual mineralization. This takes a bit longer (we will do a time course in the next few weeks to map it out properly). 

I've been testing a refined media formulation to accelerate this - we used low levels of L-ascorbic acid and glycerophosphate deliberately so we didn't mess too much with the haematopoiesis, but in new formulations with higher L-Ascorbic and B-glycerophosphate pentahydrate things look good. It will take me a bit of time to pull this data together. 

My suggestion if ossification is of interest is to increase the L-Ascorbic Acid + B-glycerophosphate content after day 21 and look around day 42 (if you email me in a month or so I will have a better idea of these conditions). 

3) As you annotated osteoclasts in your single cell data from comBO protocol, does the BMOs undergo bone turnover (bone resorption-bone formation)? 

We haven't assayed for this yet, but hopefully! I am reasonably convinced by the osteoclasts because of the changes in those clusters after myeloma engraftment. 

4) From day 14 onwards, you don't add any VEGF, this means that  the heterogenous ECs don't need those cytokines anymore? From day 20 onwards you just add EPO and TPO. I get the idea behind it, but is there a negative effect (less stem cells, for example, or engrafting primary CD34+ cells will yield in MEP population and less into myeloid if culturing only with EPO, TPO)? 

These are good questions - my philosophy has been that if it is a true ex vivo niche we should ween off exogenous (non BM derived) growth factors and so I have stuck to this. There are 2x questions here one on the endothelial fraction, the other on the stem cells. 

For the endothelium : the pericyte like MSC express VEGF etc quite nicely, but we do see a steady decline in endothelial fraction after day 21. I think you can probably maintain them a bit longer if you supplement with VEGFA + C at low level (5 or 10ng/mL) from day 21 onwards BUT i suspect the major obstacle to maintaining the vasculature ex vivo is going to be dynamic flow (working on this!) 

In our data so far you get a pretty full commitment of your iPSC derived HSC fraction so everything is fully diffed out by day 42. It's possible to optimise these conditions to maintain HSPCs/HSCs for longer, and I have some data on this, but it's not the main focus of the work at the moment. We haven't done a primary cell engraftment where we add CD34s without any exogenous factors, but typically only the first few days will have the additional factors (day 0 and day 3/4 feeds will have SCF, FLT3, IL7, TPO, EPO, IL3, IL6 before going down to EPO/TPO). Usually this gives you a nice broad range of differentiated cells and some CD34s. Does this answer your question? 

Also, did you culture BMOs until day 80 just with these exogenous cytokines or did you add anything else to stimulate bone formation? 

Until d80 it was done as described (EPO + TPO in media with L Ascorbic acid etc) - if you want to accelerate the bone formation I woudl suggest using Lonza's osteogenic medium ! It's very expensive, but does work very well. 

Hope this helps + thanks for Tooting, 

Abs

[Salim Atakhanov]

Hi Abs,

thanks for your reply! 

Is it possible if you write the detailed preparation of the new hydrogel (bulk)? You reconstitute fibrinogen in 0,9%NaCL ? (how much do you add and for how long can this be stored?) 

Thrombin - which company do you get it from? How do you prepare this?

So you fully got rid of matrigel in the bulk hydrogel? 

You mentioned about the Lonza's osteogenic medium, did you try this already? Would you just use this instead of StemPro and add cytokines there or how would you do this? 

BW,

Salim

[Abdullah Khan]

Hi Salim, 

I've got a couple of grant deadlines looming - can you pop me an email in a couple of weeks and I can send these details through? I will forget for sure so a reminder is very welcome. I will have much more bandwidth after 10th April. The fibrinogen prep should be in the comBO methods section, if there are specific details missing can you email me those and I can try and get it together sooner rather than later. My plan in April/May is to put the formal protocol up here but don't have time for it for a few weeks.

Abs

[CAMILLA TOMBARI]

Hi Abs,

 
thank you for all the information you share in this group. I know you are now very busy with some deadlines, and I really wish you good luck.
In the meantime, can I ask you a couple of quick questions?
Could you tell us the catalogue number and company of the thrombin you used?
Also, since the granular microgel protocol saves a lot of material, including the collagen mixture, which is quite expensive, how do you handle the leftover? In the Olijnik et al., you state that storage at 4°C is fine for up to 6 weeks after reconstitution. Have you by any chance tried to use the collagen mix after a longer period of time?

Thanks again!

Camilla

[Abdullah Khan]

Hi Camilla, 

Thank you - really appreciate it. 

Q1. Of course - thrombin detail here, we've used two in the past few months because of some stock issues and both worked: 

Sigma - T4393-100UN

Merck- 605157-KU (I think this is cheaper and is what we are currently using). 

Q2. We haven't, usually because we now do fairly big batches so it is rare for a vial of Collagen I:IV to last more than a month in our lab! According to the manufacturer: https://advancedbiomatrix.com/typeiv-2.html it is good for 3 months after reconstitution. I wouldn't suggest pushing past that date, just not really worth the risk.... 

Hope this helps, and as ever, thanks for TooTing! 

Abs

[Cecilia Reyneri]

Hi TooT,

I have a quick question about the ComBo protocol, regarding the seeding on Day 5:

when seeding the aggregates into 96-well plates, do you use a multichannel pipette, or do you seed them one by one? I am worried that, using a multichannel pipette, I will end up with some empty wells and some wells with 2-3 aggregates. And if that's the case, have you seen lower efficiency in the wells with multiple aggregates? Should we separate them and make sure that we have individual aggregates in each well?

Thank you!

Cecilia

[Abdullah Khan]

HI Cecilia, 

It's a good question - this is a bit knacky but you will get the hang of it. 

We use multi-channel pipettes, the trick here is to only work with relatively small volumes as the aggregates settle, to keep mixing, and assume a degree of loss (so always have an excess of aggregates). Having more than one in a well does not seem to cause a problem, they just merge as the branching occurs. 

So for example I had 600 aggregates to embed yesterday. I planned for 4x96 well plates. Once my granular gel/media was ready, I resuspended my aggregates. Using a 10mL Stripette, I mixed everything in a 50 mL falcon and transferred 5 ml at a time to the reagent reservoir, and distributed 50uL per well whilst mixing in the reservoir as quickly as possible. 

This approach works well for me - if you use larger volumes they settle too quickly and that's usually when things become uneven. 

Hope this helps. 

Thanks for TooTing. 

Abs

[CAMILLA TOMBARI]

Hi TooT,

I would like just a confirmation regarding the ComBO protocol and the cytokines concentrations which are slightly different compared to the Nat Protocol.

It seems to me that the concentrations of EPO and TPO, from d5 to d10, are significantly reduced compared the Nat. Protocol and that, from d 10 to d14, EPO and TPO are no longer added. However, from d14, TPO is present in the medium again and, from d21, EPO has also been reintroduced.

I wonder if the concentrations reported in Suppl. Fig 1 are correct.

Thank you very much.

Camilla

[Abdullah Khan]

Hey Camilla, 

Yes those are correct - the idea is that we are trying to avoid over-committing to the myeloid lineages in the early phases. We add TPO back in as soon as we can as it's needed for stem cell development, and then add EPO later once we have nice lymphoid development alongside the myeloid. 

Does that make sense? 

Abs

[CAMILLA TOMBARI]

Hi Abs,
we set up a parallel BMO culture to compare the protocol described in Shen et al. (following the cytokines concentrations indicated) and the Nat protocol applying the hydrogel embedding and the granular microgel technique.
We ended up with nice-looking BMOs in the microgel, maybe a bit smaller.
However, several BMOs plated in granular microgel protocols had cells surrounding the BMOs, during the culture at the end of the differentiation. Those surrounding cells (or sometimes even clumps) form a "crown", although the BMOs appear intact.
Have you ever experienced something similar?
I attach a picture to show what I mean.

Thank you so much!
Camilla

[Abdullah Khan]

Hi Camilla, 

Yes this is perfectly normal - I suspect that your haem cells follow an oxygen gradient and eject from the BMO after a certain poiint. Just something to bear in mind when you do analysis (make sure you aren't losing the suspension cells). 

Well done! looks great. 

Thanks for TooTing, 

Abs