Hi all, I'm trying to isolate VSMCs from aortic samples, and whilst there are protocols available for isolation and culture, the identification of VSMCs from myofibroblasts is difficult as they share various markers. Is/are there any specific surface marker(s) I can possibly use to live sort for VSMCs with FACs?
Hi Wei Wen,
h-caldesmon seems interesting to look at. Below are the link for the papers.
http://www.ncbi.nlm.nih.gov/pubmed/16625070
http://www.ncbi.nlm.nih.gov/pubmed/11068549
Hope this is useful.
Thanks for the input Shiv! :)
Not sure if the markers for VSMC and SMC may be different in terms of h-caldesmon. I may be wrong but h-caldesmon is a protein that regulates cellular contraction with actin and tropomyosin and hence intracellular?
I'm looking for cell surface markers of VSMCs that would be useful for live cell sorting with FACs for subsequent culture, but most surface markers such as PDGFRa/b (and many more intracellular markers) may also be expressed in (myo)fibroblasts.
Some have used CD90 (Thy1) and/or ALDH as a positive marker for fibroblasts and negative marker for VSMCs (Weber et al., Ped Res, 2011; Holdsworth-Carson et al., Mol Hum Reprod, 2014) but am not sure if that is really specific for VSMCs and works as well in human aortic tissue?
Hi Wei Wen
Take a look at this article. I believe it might help.
http://www.nature.com/pr/journal/v70/n3/full/pr2011165a.html
According to it you can make a negative selection and gate on vWFnegCD90neg
Hi Wei Wen Lim
How did this work out for you? Did you find a resolve?
Best wishes,
Cengiz
Hi Cengiz Akbulut !
I've used negative selection after tissue explant-culture for enriching VSMCs by excluding for CD90 (common fibroblast and mesenchymal marker) and CD144 (specific EC marker) following an adapted protocol by Weber et al. (2017). Protocol is outlined in our group's recent publication below. Cultured VSMCs are spindle-shaped and demonstrate high expression of contractile markers and "hill and valley" characteristics at confluence.
In my experience, to gather as much viable SMCs as possible for downstream cell culture, I'd recommend MACS over FACS for cell sorting. And I'd suggest to perform experiments at early passages, as cultured VSMCs tend to lose their contractile markers with prolonged subculture.
Hope that helps!
References:
1) https://www.nature.com/articles/pr2011165
2) https://www.nature.com/articles/s41598-020-74944-7
Hi Wei Wen Lim !
Thanks so much for the reply!
That really is helpful, we are presently using surgical biopsies and allow for SMCs to migrate out the tissue on to laminin which we later characterise at low passage. The cells often have a decent expression of markers, but sometimes cells dont migrate out of the tissue and the method can take several months. As a benefit though we do get a lot of vials compared to doing a digestion.
I guess the negative selection suggests an affirmation that the actual VSMC is too heterogenous and not really well defined hence its easier to say what isn't it, then what it is!
Many thanks,
Cengiz
Hi Cengiz Akbulut , glad to be of help!
In my experience, I've had aortic biopsies samples that migrate extremely slowly as well or stop migrating totally after some time (aortic samples in particular migrate out much slower compared to mammary arterial samples). In this setting, I usually set an arbitrary cutoff for explant-culture of ~1 month...if they have not migrated out by then they tend not to grow well later and they appear senescent early (cells look flat and do not proliferate). Also, as I mince the tissue and plate them spread across the collagen coated dish, if there are explants that do not grow well, I'd remove them from the dish to make space for those that do. After ~4 weeks, I'd remove all the explants and subculture the cells which should reach confluence rather quickly (usually within a week per passage depending on seeding density).
As the initial explant culture tend to reap mix cells (ECs, Fibs, VSMCs) despite our use of SMC specialized media, I opted for negative selection for ECs and Fibs, which unlike VSMCs, have known cell surface markers. ECs tend to stop growing after a couple of passaging in SMC media, but Fibs which are morphologically similar to VSMCs are a common contaminant and share many similar intracellular markers with VSMCs (especially myofibs). In regards to VSMC heterogeneity (a common limitation with explant-culture given that synthetic VSMCs tend to proliferate/migrate faster), you may wish to try the smooth muscle differentiation supplement (S0085, Gibco) to prime for contractile phenotype before experiments (ref below).
Reference:
https://www.nature.com/articles/srep33407
Cheers,
Weiwen
Hi Wei,
we inventoried all cell markers in order to better define the vascular mesenchymal landscape. Based on these data it appears that the simple discrimination between VSMC and myofibroblasts is not possible (see attached paper)
Hi Jan,
Thanks for providing the paper. Truly a large and important body of work for the field, congratulations!
It does seem that the vascular landscape in situ is much more complex than previously thought. Another amazing paper I've came across recently also suggest the same (see below) i.e. 20-40% of myofibroblast-like ACTA2+ fibrous cap cells are non-SMCs!
Cheers,
Weiwen
https://www.nature.com/articles/s42255-020-00338-8
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