Its interesting that the myogenic program involved in skeletal muscle regeneration and in functional hypertrophy, despite a lot of similarities, appears to differ in its dependance on satellite cells.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3152922/

http://dev.biologists.org/content/138/17/3647.long

Very good point Lucas - thanks for that.

In studies of Esser's lab, they ablated Pax7(+) satellite cells. Rudnicki's lab demonstrated that these cells are essential for the proper muscle regeneration after injury. In Esser's studies they identified the satellite cells using immunohistochemistry and real-time qRT-PCR for Pax7. The more rare Pax7(-)/Pax3(+) satellite cells were not quantified. I was wondering if this population of satellite cells is sufficient to sustain the functional hypertrophy.

Is only a proposition, but what do you think? Maybe a conditional ablation of both Pax7+ and Pax3+ cells could answer this question?

http://www.ncbi.nlm.nih.gov/pubmed/16391000

Hi David. I definately think that it is still worth investigating satellite cell activity in especially humans! To date most of our knowledge is based on animal or in vitro experiments. Most human studies still focus on the identification of SC's. I believe there is still a large gap in our knowledge especially regarding the interaction between the acute phase immune response and regeneration.

Hi Mari. Important observation, which sateltites adaptations of cell in humans? So I think we should continue investigating the cells satellites.

Mari van de Vyver - great response. I think you are right we just need to develop a human model to test the contibution that satellite cells make to growth. However, I doubt ethics would ever get cleared to gamma-irradiate human muscle in vivo. So perhaps and ex vivo/in vitro model is required.

Lucas - great response and great point. My feeling is though that if the satellite cell population was truely required in these animals for hypertrophy there there would at least be a dosing type effect i.e. you would see some growth that would be in proportion to the amount of contribution from the Pax3+ cells. As far as I know this doesn't occur.

While we are on the topic of satellite cells. Pax7 is most commonly used as marker and we as researcher assume that an increase in Pax7+ SC per fibre is indicative of proliferation. Yet our muscle research group have failed to detect any significant changes in other MRF's, especially myoD and myogenin following high intensity eccentric exercise in humans. So I'm starting to think that this increase in Pax7+ SC following eccentric exercise is not an indication of proliferation but merely an increase in the expression of Pax7.

So I agree with David we definately need an ex vivo model. I believe that there is a group at the University of Kwazulu Natal that is currently working on the development of an 3D in vitro model (Carola Niesler's group).

Dear David

You could have a look at the following recent review paper

Physiol Rev 93: 23–67, 2013 doi:10.1152/physrev.00043.2011

SATELLITE CELLS AND THE MUSCLE STEM CELL NICHE

Hang Yin, Feodor Price, and Michael A. Rudnicki

Mari,

Indeed in a recent paper of Tajbakhsh's group they clearly demonstrated that the level of Pax7 expression reflects muscle Stem Cell heterogeneity.

A Subpopulation of Adult Skeletal Muscle Stem Cells Retains All Template DNA Strands after Cell Division. Rocheteau et al.

http://dx.doi.org/10.1016/j.cell.2011.11.049

Not to tout our own work, but we published a recent review on this and related issues: see Trends Mol Med. 2012 Oct;18(10):599-606. doi: 10.1016/j.molmed.2012.07.004. Epub 2012 Aug 8.

Stem cells in the hood: the skeletal muscle niche.

Pannérec A, Marazzi G, Sassoon D.

We also have a manuscript under revision to be re-submitted very soon on this issue. It would appear that satellite cells are not really necessary for myostatin-block induced hypertrophy as a few others have reported, HOWEVER, it is important to note that the genetic models in mice in which satellite cells are depleted initially result in a marked decrease in fiber size. There is also evidence that an initial short term hypertrophy of fibers is satellite cell independent whereas maintenance of this hypertrophy may require satellite cells. We also see a crucial interaction for the myogenic population of PICs that are not satellite cells and do not express wither Pax3 nor Pax7 unless recruited into the myogenic lineage.

My oversimplified view is that satellite cells are not the whole story and that other cells in the muscle tissue are centrally involved in 'growing' the muscle and giving it plasticity. This would make sense if you consider that exercise induced hypertrophy also involves increased vascularization, increased connective tissue and other adaptive tissues changes that support the fibers and their satellite cells.

Hi David, I cant say that satellite cells are required for hypertrophy, but i believe they contribute. This may also depend on if the hypertrophy stimulus cause some degree of muscle injury. A few years back we published a paper showing evidence of muscle regeneration in the synergist ablation model of overload-induced hypertrophy which was associated with increased satellite cell proliferation. Here is the link if you are interested. http://www.ncbi.nlm.nih.gov/pubmed?term=beta%202%20integrins%20contribute%20to%20skeletal%20muscle%20hypertrophy

It still an issue under investigation!!!!!!!!!!!!!!!!11

To add to the remarks by David Sassoon; Inflammatory cells as well.... Looking forward to reading your upcoming publications

In one hand, the main problem I see in the studies with the Pax7-DTA mice is that they claim that hypertrophy is independent of myonuclear accretion (of any cell type) but they did not demonstrate whether functional hypertrophy can be maintained for long periods without myonuclear accretion...

In the other hand, it is clear that satellite cells proliferate and fuse to the pre-existing fibers during overloading-induced hypertrophy as it has been shown by many independent labs (including our lab).

Humm, controversial no? I agree whith Pallafacchina et al talking about this issue in a recent review ( http://dx.doi.org/10.1016/j.numecd.2012.02.002 ):

"Indeed, conditional ablation of

>90% of satellite cells was recently obtained in mature

skeletal muscle using tamoxifen inducible expression of

diphtheria toxin A-chain in Pax7 expressing cells [32].

Overload hypertrophy and increase in force of the plantaris

muscle after removal of the synergist gastrocnemius was

unchanged in these mice supporting the notion that satellite

cells are not necessary for functional muscle hypertrophy

[32]. However, in a physiological context, satellite

cells do undergo proliferation and fusion during overload

hypertrophy and it remains possible that they do contribute

to functional hypertrophy: adaptive changes in the protein

synthesis/protein degradation balance within the myofibers

might occur in the absence of satellite cells thus leading to

an apparently normal hypertrophy process [11]. Indeed,

a complex cross-talk between myofibers and satellite cells

take place during muscle hypertrophy, a shown by the

finding that deletion of serum response factor (SRF)

specifically in myofibers and not in satellite cells blunts

overload-induced hypertrophy and impairs satellite cell

proliferation and recruitment to pre-existing fibers".

this is a great discussion and a great place for it. I want to clarify that the work in the Development paper was driven by John McCarthy and Charlotte Peterson and not me but I was a collaborator. I do think satellite cells are very important cells but I would like to see the investigations open up beyond hypertrophy. There is a lot literature from the 80-90s that showed quite a plasticity in satellite cell number/myonuclear number in different models (running, chronic stimulation as well as hypertrophy). So my bias has been that they are doing something but tying them so tightly to fiber size has limited our understanding ... as well as our research designs.

HI all, a great discussion point, and one that i think will be ongoing for many years to come. I think the key phrase from Davids original question is 'Physiological exercise', as a lot of recent work which suggests that satellite cells are not necessary for skeletal muscle hypertrophy/adaptation has used models which fail to represent physiological conditions e.g. synergistic ablation/genetic/tamoxifen induced ablation of satellite cells/chemical muscle injury etc in rodents. In addition, many of these studies also examine a relatively short time course of response. Whilst this data is insightful and allows strong mechanistic understanding of the satellite cells involvement in muscle adaptation they cannot truly be classified as 'physiological exercise'. The majority of data in humans shows that following exercise there is an increase in satellite cell numbers (see link) and there are data to show that chronically this is accompanied by myonuclear addition. Indeed, it is likely that, particularly following bouts of chronic resistance type training there will need to be increases in myonuclei to support the needs of the fiber growth.

As a final caveat however, it is worth noting that these responses may differ between individuals (responders/non-responders), and there are certainly many human studies needed to answer all of our questions.

Certainly an area worth continued investigation!!

http://www.cellularandmolecularexercisephysiology.com/index.php/CMEP/article/view/12

Thanks to everyone who has weighed in on this discussion so far - a few highlights:

@Karyn Esser - thanks so much for your input on this topic. I really appreciate it.

@Eusebio Perdiguero and @David Sassoon - thanks to you too for such in depth answers and for the references.

@Neil Martin - you make a great point here about the human exercise and about the addition of myonuclei. But one thing has always bothered me on this is that slow fibres have the smallest myonuclear domains and to me this suggests that slow fiber function is probably more dependant upon a highly regulated myonuclear domain than are the intrinsicly larger fast fibres - any ideas? But you are right most of the satellite cell ablation studies were then tested with either compensatory overload or wing weighting. However the most recent DTA mouse study was carried out using hind limb suspension induced atrophy followed by recovery induced regrowth - a much more physiological model of muscle growth. This study had similar results to the first - i.e. satellite cell recruitment is not required for that degree of hypertrohpy to occur and I think that this argues quite strongly that in rodents at least the recruitment or expansion of satellite cells are not required for physiological growth - but in humans this is still very much an open question (in my mind anyway).

@Lucas Guimarães-Ferreira makes a great point on the difference on the dependance of growth vs regeneration on satellite cells. And it is this concept that I find the hardest to reconcile - why is it that regeneration following injury has such a high dependance upon satellite cell recruitment but in loading induced growth this is dispensible????

I almost forgot. Here is a great link to a point-conterpoint in JAppPysiol from a

few years ago and is a very interesteting read on the topic for anyone who hasn't seen it. But I imagine everyone here has likely read it already :-)http://jap.physiology.org/content/103/3/1099.full.pdf+html

Well, this was fun and nice to see old colleagues and meet new ones.

Thanks David. I think it could well be that slow fibres are more tightly regulated by the myonuclei as you suggest. perhaps slow fibres have smaller ceiling sizes (if this exists), and they therefore must acquire new nuclei at smaller fibre sizes than fast fibres. Indeed, slow fibres of rodents have been show to posses higher satellite cell numbers than fast fibres, and this gives a larger pool for myonuclear addition which would be essential if frequent fusion was neccesary. There is a paucity of literature on fusion kinetics/satellite cell differences between fibre types however.

I totally agree with David. We also have some data on Pax 3/Pax7 negative cells resident in skeletal muscle that we hope to publish soon too. Pax7 positive satellite cells are not the whole story in muscle in terms of progenitors.

I also agree with Prof David Sassoon and Anita. We also published the myogenic cell source distinct from satellite cells in the interstitial spaces of skeletal muscle as the multipotent stem cell for over 10 years ago. Then, we have proofed it persistently through several publications. During these processes, we frequently experienced complete deny of our data by the reviewers who believe all myogenic events in skeletal muscle depend on satellite cells. Some of them also believe that the satellite cells are multipotent. For these researchers, it is certain that the satellite cells wholly contribute to muscle hypertrophy. However, in our standing position, it is certain that some of the stem and/or progenitor cells share their roles, including satellite cells, and then, establish muscle hypertrophy, and with hyperplasia and regeneration, associate with reconstitution of vascular and nervous system. This difference in the vector-strength for the satellite cells between us and satellite cell researchers complicate the story. Recently, publications about stem and/or progenitor cells in the skeletal muscle have increased. Therefore, I think that consisting of the concept about them is fairly necessary as the next step, such as equal, different and similar in function and/or location. These studies may also lead to re-evaluation of satellite cell function.

@Tetsuro Tamaki - thanks for the great contribution here. If you post the links here to the relevant papers on your research gate page I will share those links on twitter to help spread the word.

@Anita Quigley - thanks for some great input. When you publish that data let me know and again I'll share the link to the publication on twitter.

@Tetsuro--I feel your pain in trying to establish the potential contribution of other non-satellite cells. It took us 18 months to get our paper accepted to Nature Cell Biology and I think that was thanks to at least one open minded reviewer. Otherwise, there is quite a bit of dogma in the field. Perhaps we should make a new post here to try to gather colleagues with similar data and put together a comprehensive review on this single topic. As DH requested, post the references you were referring to?

Some good points Tetsuro. We have hesitated to publish some of our results due to resistance from reviewers too, but we feel the time is right now as publications (as you mentioned) about stem cells in skeletal muscle have been on the increase and I also think there is a dogma about satellite cells that is starting to change, hopefully leading to better understanding of muscle physiology and disease states such as Muscular Dystrophy (current therapeutic approaches for Duchenne Muscular Dystrophy include myoblast/progenitor transplantation..see Jaques Tremblay's (from Quebec) work, I am sure many of you are aware of this work).

Getting back to David's initial question, In my opinion we still don't know enough about "satellite cells" or stem cells in the muscle to divert our attention away from this area. This is a fantastic discussion everyone!!

I agree Prof Sassoon. I would love to be involved in a new post on this subject, I think a comprehensive review is needed.

Thank you for your kind suggestion DH. However, I am not sure. How can I post the papers in this link, but once I will try to use “Add attachment file button” and upload one pdf files (review article) which cannot download from my research gate page. If this is not a right way, please let me know.

I don't disagree with much that has been posted, but I will continue to push on the concept that linking satellite cells and/or other myogenic precusors cells to hypertrophy maybe limiting the discussion and missing some important roles for satellite cells in adult muscle maintenance. No one argues that during synergist ablation induced hypertrophy there is satellite cell fusion into muscle but I will say that their requirement to simply drive the growth response does not seem to be required. Whether they are needed for long term maintenance is a good question but growth and maintenance maybe two different mechanisms. I also am familiar with several papers demonstrating satellite fusion into adult muscle with run training and this is not a model in which the muscle fibers are getting bigger. So from my perspective as someone that has lived in the muscle adaptation field for a number of years, these findings suggest that tying satellite cell fusion solely to muscle hypertrophy maybe missing other very important roles for satellite cells in adult muscle (beyond regeneration).

Hi TT - just tweeted your paper. The download worked perfect, thanks for this.

Thanks for another great response KE. The satellite cell fusion in response to running/ablation has always made me wonder if it plays a more important role in endurance adaptions or fiber shifting than growth per se. Especially since TypeI fibres have the smallest myonuclear domain sizes. But there are people here much more expert on that than me :-)

Hi Everyone,

Sorry to enter the discussion so late, but this topic is so fascinating I hope to revive it. I would welcome any comments on the criteria we are using to define satellite cells. Finding them in histological sections is one thing, but the 2009 Stem Cells paper by Siegel, Cornelison and colleagues

(Stem Cells. 2009 Oct;27(10):2527-38. doi: 10.1002/stem.178.

3D timelapse analysis of muscle satellite cell motility.

Siegel AL, Atchison K, Fisher KE, Davis GE, Cornelison DD.) makes me question what a satellite cell is, because a satellite cell is so dynamic and mobile, and does not appear to be confined to an anatomic space...and this was a defining criteria of satellite cells by Mauro!

In essence, I guess I am asking if there are agreed-upon ways to find satellite cells wherever they may be found. To extend the ramifications of the paper above, it seems to me that using anatomic criteria to define satellite cells may be inadequate, particularly following extensive muscle injury. I'm also interested in cell surface markers of satellite cells--so they can be isolated with precision, without destroying them.

Hi Chad,

To thinking the satellite cells and the other myogenic cells, I think that the ability of basal lamina (BL) formation is important, especially following extensive muscle injury. The satellite cells remaining in the parent fiber BL does not need to form BL. However, if some of them departed from parent fiber (this may be an accidental case, because of the strength of BL), how can they do? Of course, the satellite cells can move, as presented in the paper (Stem Cells 2009), but it was basically limited along the BL, and this was in the in vitro condition. Therefore, it seems natural that the satellite cells mainly working in the basal lamina of parent fibers, and this should be reflected the anatomical feature.

TT--Thanks for your quick and helpful answer!

With regards to surface markers of satellite cells, Amy Wagers' lab has used the following surface marker profile, and I welcome anyone to comment about your experience with isolation of satellite cells:

CD45-,Sca-1-,CD11b-,CXCR4+,Beta1 integrin+

to Chad, I would say that whatever markers you use, you should perform a cytospin on the freshly isolated cells to see what % are Pax7+. It should be close to 100% or else you got something else. There is a very rare Pax7- but M-Cad+ population that we see histologically (which are also PW1+) but always hard to know if this is simply due to low Pax7 levels.

And so, let's start this new line of discussion re: non-satellite myogenic cells.

Thanks David, and I appreciate your suggestion. As far as non-satellite myogenic cells perhaps we could narrow that down to cells occurring in vivo (as opposed to myogenic iPS cells or embryonics that have been manipulated towards a myogenic fate).

That makes me think of a 2007 paper by Christov et al, titled Muscle satellite cells and endothelial cells: close neighbors and privileged partners. More specifically, it brings up the issue of whether pericytes and mesoangioblasts could either directly become muscle cells, or if they are indirectly instrumental, in creating a microenvironment that is favorable for myogenesis. Several labs have studied pericytes and mesoangioblasts in this respect, as possible cell therapy for muscular dystrophy.

Another thought is that bone has osteoclasts, which are generally accepted as having originated from macrophages. If memory serves, both satellite cells and "alternatively activated" macrophages express the mannose receptor, CD206. Thus, does skeletal muscle have tissue-resident macrophages that might be a step away from being myogenic?

Looking forward to everyone's thoughts.

A quick answer to the last part of your question is my 'personal' take...namely, the expression of any particular cell surface marker does not ascribe that cell to any lineage. Recall that most of these markers are 'borrowed' from another lineage (blood) in any case.

It is always important to remember that the repair or the hypertrophy is occurring and that while satellite cells may or may not drive these processes under certain physiological or pathological conditions, we should not forget that cellular adaptation is ocurring and that we need to understand the processes, regardless of the terminology or the mearkers used to asign a specific cell type. A really interesting paper was published in the mid 90s from the group of L Garcia, investigating the role of bone marrow derived stem cells in the repair of muscle damage in dystrophic animals - this fascinated me at the time as it provided critical evidence, almost 20 years ago, to show that various cells are key to the processes of hypertrophy and repair. Therefore deciding in advance what cells have an important role to play under control or stressful conditions will hamper our research progress. I think what Karyn says is absolutely right "linking satellite cells and/or other myogenic precusors cells to hypertrophy maybe limiting the discussion and missing some important roles for satellite cells in adult muscle maintenance". Similarly, I think we may be missing important roles for cells, which are not satellite cells but also have a critical role to play in these processes.

Thanks Dave as always for a brilliant question and to get people debating in an open arena - always important!

Hi David, I have one question. About the freshly isolated cells, did you mince the muscle before enzymatic treatment? I never experienced close to 100% of Pax7+ cells in the freshly isolated cells. In our experiment, we never minced the muscle, and used intact whole muscle to enzymatic treatment. Thus, our freshly isolated cells included less than 1 % of Pax7+ cells. This low inclusion of Pax7+ cells were also confirmed by single-cell RT-PCR. If this large discrepancy depended to the isolation method (mince or non-mince), I think this may be a big key for the non-satellite myogenic cells.