#2 of our laminin highlights series. This time I want to draw attention to two papers, both from March this year. On their own, each of these are pretty cool, asking important questions but, taken together, they contribute important observations to the laminin field that may not be fully appreciated individually. Put simply, when it comes to alpha4 containing laminins, which beta chain is present makes a functional difference.
1 paragraph intro before we get into the meat (want a more extensive laminin intro? check our friendly intro here).
All laminins are heterotrimers consisting of one alpha, one beta and one gamma chain (LM411 is laminin α4, β1, γ1). The alpha chains contain most of the binding sites for other proteins, most notably the C-terminus of alpha chains contain the strongest binding sites for most cell-surface receptors including integrins and dystroglycans, whereas the N-terminus of all three chains is generally considered more important for network assembly and binding of extracellular matrix proteins. The beta and gamma chains are more than just essential parts of the trimer. Some seminal work (Taniguchi et al) has demonstrated that although the alpha chains contain the binding sites, the C-terminus of the beta chains influences the affinity of the alpha chains for a subset of their integrin receptors. The two papers described below have investigated the functional implications of this, though that’s not really what they set out to do!!
Making a hepatocyte: LM411 might be your best bet
Imaging-Based Screen Identifies Laminin 411 as a Physiologically Relevant Niche Factor with Importance for i-Hep Applications – Ong and Serra et al – March 2018 – Stem Cell Reports
In this study the authors attempted to improve the technique used to drive induced pluripotent stem (IPS) cells down a hepatocyte lineage through identifying the optimal extracellular matrix protein coating for their culture. To be able to objectively and systematically screen a range of different matrix conditions, they developed a screening platform based on the phenotypic characteristics: using nuclear morphology, cell morphology and albumin expression and a bit of machine learning they scored populations on how close their phenotype was to adult primary hepatocytes.
Using this platform a panel of 58 proteins were screened, these were selected based on a combination of Matrisome project data and those which they had access to (i.e. commercially available). 8 proteins came through the first screen as being pretty decent, of these LM411 performed the best of all. Some of the other laminins also performed reasonably well, including LM211, LM521 and LM111. Col 2 also looked decent, however LM411 was about 20% better than the rest.
They then performed a range of other experiments using LM411 compared with a pretty rubbish substrate of col 1 (rubbish with respect to their own data and hepatocyte maintenance). LM411 continued to perform well not only for IPC to hepatocyte cultures but also for IPSC derived anti-trypsin deficiency cultures.
The take home.
If you want to convert IPS to Hepatocyte, use LM411 (along with the rest of your ideal culture conditions). Previously it was shown that LM521/LM111 was pretty good at driving embryonic stem cells down a hepatocyte lineage therefore the strength of this study was really their semi-hypothesis independent screening approach.
There are lots of papers out there and more coming all the time that directly compare different laminin coatings for their ability to support/differentiate different cell lines. In this case the IPS cells will have contributed to the matrix as they begin to differentiate, therefore the initial coating is likely needed just for a short term push in the right direction.
Why is this about beta chain specificity?
Intriguingly the screening data showed that LM421 was much worse than LM411 at driving cells toward a hepatocyte-like phenotype. Indeed, LM421 came out of their screen as being no different from their control conditions. These data show that there is a beta2 vs beta1 chain specificity in terms of IPS differentiation response.
Although this paper is a useful resource for those wanting to work with hepatocytes, the LM411 vs LM421 difference seemed to me to be an obvious question to follow-up and one where the answer would not only be interesting but could also be useful. Knowing what drives this difference could provide a route toward synthesis of a smaller, easier to manufacture in GMP conditions, and cheaper alternative.
And that leads me on to…
Want to keep your endothelial cells young? Better choose LM421
Switch in Laminin β2 to Laminin β1 Isoforms During Aging Controls Endothelial Cell Functions – Wagner et al – March 2018 – Arterioscler Thromb Vasc Biol
As I was pondering the hepatocyte question, this paper came out that directly explores the functional difference of LM411 and LM421 albeit not in the context of hepatocytes.
Here, the authors compared heart endothelial cells isolated from younger and older adult mice using RNAseq. Amongst other age associated changes, there was a clear transcript level switch from laminin beta2 expression in the young mice to laminin beta1 in the older animals without changes to alpha or gamma chains present. A similar switch in expression was also observed following injury.
So what does this expression switch mean in terms of function?
Well the authors next used either LM411 or LM421 as dish coatings and plated umbilical vein endothelial cells or coronary artery endothelial cells upon them. Short-term adhesion assays revealed less adhesion to LM411 than LM421, while tube formation and migration were also relatively poor on LM411. However, the opposite was observed when it came to markers/phenotypic observations consistent with endothelial to mesenchymal transition in the presence of TGFb2; suggesting that LM411, in this context, aids this conversion. Ageing/injury are also associated with increased expression of endo to mesenchymal transition.
Some interesting data is also included in the supplemental figures where they compare the secretome profile on cells cultured on the two matrices. This analysis reveals dramatic differences in thrombospondin-2 and PDGF (low on 411) and PIGF, VEGF-C (high on LM411). This paper is genuinely really nicely done and it’s a bit of a shame that it is in short form and these parts of the findings are only really brought in as tantalising aside in the discussion!
OK cool, cells behave differently, but how/why?
Next they looked at integrin beta1 activation and saw less activation in cells cultured on LM411 rather than LM421 as you would expect based on the older studies described above. Knocking down integrin b1 and plating on LM421 recapitulates the phenotype of plating cells on LM411. To be fair, this doesn’t completely prove that it is a differential activation of beta1 integrin containing integrin heterodimers that is making the difference, but it certainly does support a model where this could be the reason.
Together these two papers demonstrate well that the beta chain matters. Of course they do; evolution wouldn’t have selected for two structurally similar proteins to be differentially expressed if they didn’t add something to this great adventure that we call life. At this point, I’d point out that there is some b1 integrin activity in the cells cultured on LM411. the difference is a titration of the effect rather than a binary activation/non-activation. But when is comes to signalling hubs, small differences can have large downstream consequences…. they might make the difference between becoming a hepatocyte or not!
See the previous Laminin Research Highlights – focusing on laminin therapies here