Questions regarding BMO protocol and cell population analysis

[Yoonjoo Kang]

Hi everyone,

I am currently working on bone marrow organoid (BMO) generation and had a few questions while following the protocol.

1. EB number and initial setup 

The paper mentions using around 80-100 EBs per well.

I would like to confirm whether this is the recommended range, or if it typically requires optimization depending on batch or conditions.

Also, from your experience, how critical is EB size control during mesoderm induction for downstream differentiation efficiency?

2. Hydrogel removal at D13 separation

After hydrogel embedding at D5, sprouting structures for, and at D13 the protocol suggests removing the hydrogel as much as possible.

However, in practice, removing the hydrogel often leads to loss of sprouting structures as well. In this case, which would you prioritize:

1) maximizing hydrogel removal, or 

2) preserving the sprout structures?

Any practical tips or criteria you use during this step would be greatly appreciated.

3. Difficulty detecting expected hematopoietic / endothelial populations

Although I am following the protocol, I am having difficulty detecting the expected populations:

1) CD235abmidCD71- CD45+ CD34+

2) CD45-CD31+

I am trying to determine whether this issue is due to 

1) suboptimal diffenetiation, or

2) staining /  FACS gating strategy

I will also share my gating strategy any feedback or troubleshooting suggestions would be greatly appreciated.

Thank you in advance for your help!

Best,

Yoonjoo Kang

[Abdullah Khan]

Hi Yoonjoo - responses below. 

1. EB number and initial setup 

The paper mentions using around 80-100 EBs per well.

I would like to confirm whether this is the recommended range, or if it typically requires optimization depending on batch or conditions.

Recommended range, we tend to keep it stable between batches and conditions to minimise variation. 

Also, from your experience, how critical is EB size control during mesoderm induction for downstream differentiation efficiency?

If your EBs are too big then you seem to get poorer sprouting + are more likely to get necrotic codes.

2. Hydrogel removal at D13 separation

After hydrogel embedding at D5, sprouting structures for, and at D13 the protocol suggests removing the hydrogel as much as possible.

However, in practice, removing the hydrogel often leads to loss of sprouting structures as well. In this case, which would you prioritize:

1) maximizing hydrogel removal, or 

2) preserving the sprout structures?

Any practical tips or criteria you use during this step would be greatly appreciated.

What makes you say you are losing the sprouting structures? Once you extract/remove from the gel the external sprouts tend to collapse, but you retain an internal vasculature. It really depends on what your downstream use is, but you should have a vessel network for a few weeks after removing from the hydrogel. 

3. Difficulty detecting expected hematopoietic / endothelial populations

Although I am following the protocol, I am having difficulty detecting the expected populations:

1) CD235abmidCD71- CD45+ CD34+

2) CD45-CD31+

I am trying to determine whether this issue is due to 

1) suboptimal diffenetiation, or

2) staining /  FACS gating strategy

I will also share my gating strategy any feedback or troubleshooting suggestions would be greatly appreciated.

Your data looks really good! A couple of questions: 

- What time point are you looking at in your flow data? 

- Your Ery fraction looks good, are you looking specifically for an early Ery progenitor population which is CD34+? 

- Your endo fraction is convincing just a bit small, have you done any imaging to look at the vessel network? You still have a sizeable CD45- population which looks very MSC/fibro, so the imaging would help get a sense of what the cell distribution is. 

- The only population that looks very small is your HSPC population - can you gate on your CD34s in the CD45- population? You should get some bright CD34+ endothelial cells there, which will help you trouble shoot 

Thanks for Tooting! Have a good easter weekend. 

Abs 

Thank you in advance for your help!

hydrogel 

[Yoonjoo Kang]

Thank you so much for your helpful feedback.

The flow data are from D18.

I had a few follow-up questions.

First, do you have a rough expected or acceptable range for the total percentages of CD45+, CD31+, and CD34+ populations that you would consider indicative of good BMO differentiation? I understand that this may vary between batches, but any general guideline would be very helpful for troubleshooting.

Second, regarding imaging, could you explain in more detail how imaging can help assess cell distribution within the organoids? For example, would you recommend focusing on CD31 or VE-cadherin staining to evaluate the vessel network, and co-staining with CD45 or MSC/fibroblast markers to examine the spatial distribution of different cell populations?

Third, regarding the D13 hydrogel separation step, we noticed that when stronger pipetting is used to remove the hydrogel, the external sprout-like structures tend to disappear or collapse. In this case, would you recommend optimizing the separation condition to preserve these external sprouts, or is it acceptable as long as the internal vascular network and overall organoid integrity are maintained?

Thank you again for your advice.

2026년 4월 6일 월요일 오후 10시 46분 37초 UTC+9에 Abdullah Khan님이 작성:

[Abdullah Khan]

First, do you have a rough expected or acceptable range for the total percentages of CD45+, CD31+, and CD34+ populations that you would consider indicative of good BMO differentiation? I understand that this may vary between batches, but any general guideline would be very helpful for troubleshooting.

Depends on time point but I would like to consistently see 60% CD45+ and 40% stromal distributions in house! At later time points you get more haem expansion. 

Second, regarding imaging, could you explain in more detail how imaging can help assess cell distribution within the organoids? For example, would you recommend focusing on CD31 or VE-cadherin staining to evaluate the vessel network, and co-staining with CD45 or MSC/fibroblast markers to examine the spatial distribution of different cell populations?

Depends a lot on your research question - we've always used that to validate that the structure has some architecture but haven't gone into more detail than that. We have some projects where the Q is where are cells localising. 

Third, regarding the D13 hydrogel separation step, we noticed that when stronger pipetting is used to remove the hydrogel, the external sprout-like structures tend to disappear or collapse. In this case, would you recommend optimizing the separation condition to preserve these external sprouts, or is it acceptable as long as the internal vascular network and overall organoid integrity are maintained?

We've mostly moved on to the granular method in the comBO paper - but as a rule pipetting that damages morphology should be avoided as it will likely affect your viability down the line. Use a pasteur or wide bore tip to avoid this. 

Thanks for tooting! 

Abs