Graziano Martello

16/
Thank to the collaborators @pgrumati.bsky.social, Davide Cacchiarelli, Paolo Martini. This project has been supported by ERC, Armenise Harvard foundation, University of Padova, Telethon foundation, @novonordisk.bsky.social , @wwtf.at, @viennabiocenter.bsky.social, @lundbeckfonden.bsky.social.

15/
We believe this approach will expand the use of naive hPSCs for large-scale studies, making it easier to explore early human development and disease modelling.

14/
In summary, serum coating is a scalable, cost-effective, and reproducible alternative to MEFs for naive hPSC culture. It maintains developmental potential, reduces DNA mutations, and eliminates MEF-related confounders.

13/
Mass spectrometry of the serum coating revealed key ECM proteins like vitronectin, fibronectin, and collagens. These proteins likely play a crucial role in supporting naive hPSC culture and differentiation.

12/
Plus, exome sequencing revealed fewer DNA mutations compared to MEF cultures. 🧬

11/
Blastoids generated from multiple naive hPSC lines - after expansion on serum - formed both embryonic and extraembryonic cell lineages.

10/
and also high blastoid formation efficiency

9/
Naive hPSCs on serum retained high trophectoderm potential

8/
Naive hPSCs on serum coatings also showed efficient developmental progression via capacitation and germ layer specification

7/
Serum coating eliminated fibroblast contamination from RNA sequencing analyses. 🧹✅

6/
Here’s what we found:
Growth rate, clonogenicity, and global gene expression profile on serum coating were comparable to MEF-based cultures.

5/
Yes! We adapted naive hPSCs (both induced and embryonic origins) to a feeder-free, serum-based coating in PXGL medium. 🧫
This method worked across 8 hPSC lines in 5 labs, showing robust growth for more than 20 passages! 💪

4/
Adding a small amount of fetal bovine serum improves the culture of mouse ESCs under feeder-free conditions. Serum-coated substrates have also successfully supported conventional hPSCs without feeders (e.g. CDM medium in @vallierlab.bsky.social ). Could this work for naive hPSCs too? 🤔

3/
Did you know serum has been used to enhance cell adhesion since the mid-20th century? Serum is rich in extracellular matrix (ECM) proteins like vitronectin and fibronectin, which promote cell adhesion and growth.

2/
However, culturing naive hPSCs typically relies on mouse embryonic fibroblasts (MEFs) as a feeder layer. MEFs are variable, resource-intensive, and can confound experimental results. 🐭
How can we improve this system? Let’s explore! 👇

1/
In the past decade, naive human pluripotent stem cells (hPSCs), representing a pre-implantation stage, have revolutionized developmental biology. They differentiate into both embryonic and extraembryonic lineages and self-organize into blastocyst-like structures (blastoids).

With pleasure we share our preprint reporting robust and cost-effective expansion of naive human Pluripotent Stem Cells without feeder cells. A fantastic collaborative effort with @leeboratory.bsky.social @jzylicz.bsky.social @pasquelab.bsky.social and Nicolas Rivron.

tinyurl.com/5n6z9rt9