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