What I'm asking is rather why are other myogenic populations are necessary whether satellite cells are excellent in regenerating muscle injury?

What is the real contribution of these cells in-vivo?

There is a real advantage in recruiting other myogenic cells or is it just a side effect?

This is a great area. A hot topic that is dealing with several issues.

The two main recent points in my opinion are the "discovery" of the so-called, FAP, fibroadipogenic progenitors: (see Nat Cell Biol. 2010 Feb;12(2):153-63. doi: 10.1038/ncb2015. Epub 2010 Jan 17. Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis. Joe AW1, Yi L, Natarajan A, Le Grand F, So L, Wang J, Rudnicki MA, Rossi FM)

Also, note the recent demonstration of hypertrophy, even with knockdown of about 80% satellite cells (Effective fiber hypertrophy in satellite cell-depleted skeletal muscle.

McCarthy JJ, Mula J, Miyazaki M, Erfani R, Garrison K, Farooqui AB, Srikuea R, Lawson BA, Grimes B, Keller C, Van Zant G, Campbell KS, Esser KA, Dupont-Versteegden EE, Peterson CA. Development. 2011 Sep;138(17):3657-66. doi: 10.1242/dev.068858.

PMID: 21828094 [PubMed - indexed for MEDLINE]

Talk about good stuff for discussion! These are complex experiments with complex interpretations…

Rick

Perhaps the role of some of these non-satellite cells is as support e.g. secretion of paracrine factors to assist in the normal process of regeneration, and that while these cells are more limited in giving rise to new muscle fibres they can do so when the extent of injury/disease is extreme or when the satellite cells themselves have been adversely affected by disease progression.

Almost certainly, but the key is when and which ones. Those are tough experiments to do!

In the muscles of after a week with massive and severe damage, two patterns of muscle fibers, which had small diameters and central nuclei could be found together. One is regenerating fibers, and another is newly formed myofibers. On first viewing, there are same, but actually different. It is reported that the key point is basal lamina shape, and this difference may represent the role of satellite cells and non-satellite myogenic cells in vivo. Importantly, massive muscle damage could not regenerate only myogenic cells. Therefore, multipotent stem cells are necessary in the interstitium of skeletal muscle (of course including their endocrine/paracrine capacities), and there are likely to not satellite cells, because of their location.

Thanks, Dr. Tamaki. Is this work published?

Dear Dr. Lieber

Yes, this is including in the following papers (Tamaki et al., Histochem Cell Biol 2009; Curr Pharm Des 2010). I think there can be downloaded via Research Gate page. If not, please let me know or request through RG.

In my opinion in muscle there are several other myogenic precursor playing central role in muscle regeneration, among these perycites have shown a double activity differentiating into satellite cells ( Dellavalle et al., 2011) and of course generating new perycites for blood vessel stabilization. Recently we published a work on Frontiers in Physiology (Fuoco et al., 2014) showing muscle derived perycite activity in promoting huvec capillary like structure formation in vitro and generating ectopic muscle tissue when implanted subcutaneously on the mice back in vivo.

Thus my question: is it possible that in pax7 depleted mice experiments impairing muscle regeneration it could be depleted the intermediate state needed for other myogenic progenitor to generate new myoblast for myofibers reformation?

Hi Cesare: I think that Pax7 knockout mice with no and/or quite low satellite cells have skeletal muscle, is one of the answers of your question.

My question regard regeneration in pax7 ko mice, for what I know muscle regeneration si completely abolished, thus the reason is that satellite cells are essential fro muscle regeneration or there is also the impairement of pax7+ intermediate state of activated myogenic precursor?

Can someone please guide me to the strongest evidence out there that demonstrates non satellite cells contributing directly to adult muscle repair?

I don't think it exists. The Pax7 conditional knockdowns only get rid of 80-90% of the SCs and still seem to regenerate fine. Great question. Someone needs to do some serious lineage tracing in these models.

@Richard: You're right - 95% of SCs are gone (based on marker expression and other characteristics) but the cells are still there and the remaining could do the job under "normal" regenerating conditions... On the other hand we found an impairment of regenerating capacity when we applied TAM during regeneration. An interpretation could be that some cells did not express Pax7 until start of regeneration process that could be other progenitors...

On the other hand we could also show that the majority of SCs originate from Myf5 lineage and abolishing of Pax7 in those lineage revealed a strong regenerating phenotype - so at least, if there is a role of the "others" in regeneration they should have a myogenic origin. But for sure this is not an exclusion argument. Moreover we still found also a few number Pax7-ve cells in the Myf5 dependent Pax7 KO mice in MyoD KO background (no Pax7 in Myf5 lineage and no MyoD lineage al all). But if this is due to the imperfection of the genetic system or real "non-myogenic" precursors is hard to say...

@Stefan: I guess killing adult Myf5+ cells (which would include Pax7 expressing cells) would help to further uncover the answer.

This is a great experiment. Is anyone doing that?

Not that I'm aware, but there is a TAM inducible Myf5cre allele that would make it possible. With any of these experiments imperfections in deletion efficiency severely limit the strength of the interpretation.

Richard: regarding hypertrophy after depletion of satellite cells, I think it is important to point out that hypertrophy was not normal - although there was an increase in fiber volume, there was no corresponding increase in nuclei, i.e. this study does not constitute evidence for non-satellite cells contributing to muscle fibers. In addition, the studies in this paper evaluated muscle 2 and 6 weeks post synergist ablation. Data at later time points (presented by Charlotte Peterson at the 2013 Gordon myogenesis conference) showed a reduced hypertrophy in the absence of satellite cells.

I really believed and has been published (Dellavalle et al., 2011) that perycites can undergo satellite lineage and differentiate into muscle. Now is the two phenomena are connected, it means that myogenic differentiation is consequence of pax7 intermediate state is to investigate yet, nevertheless at least the perivascular compartment derived cells are able to perform the transition into satellite cells.

Although I was one of the biggest skeptics, Denis Guttridge's 2013 paper on the massive expansion of Pax7+ myogenic progenitors in cancer cachexia convinced me that in some circumstances Pax7- cells in adult muscle can give rise to Pax7+ cells: http://www.jci.org/articles/view/68523/pdf.

These progenitors are truly myogenic (but not in a cancer-bearing mouse) and were not labelled by an earlier pulse of Pax7-creERTm, although this pulse, applied prior to tumor implantation, labelled almost all satellite cells.

Article NF-κB–mediated Pax7 dysregulation in the muscle microenvi...

I'm trying to get an appreciation of the potency of non-satellite cells to make satellite cells (as defined by Pax7). Michael has highlighted the Guttridge paper (beautiful work) as it relates to Cancer cachexia. This is very compelling in my opinion. I'm still left wondering how often this mechanism is activated. How rare are these events in the broader context on satellite cell maintenance and injury response?

Andrew, as I think you know, we have some evidence that cells with myogenic potential indeed exist that are Pax7- and PW1/Peg3+ (see Mitchell et al., Nature Cell Biol). This stated, our most recent evidence is that in order to participate in vivo, they need the satellite cell population. Regardless, the satellite cell is the major (if not unique) contributor to muscle following injury. As others have stated, lineage marking is required, and in our case, we are hunting for the marker first. 

I think the whole field will benefit greatly from a lineage marker that positively tracks non satellite cells (SCs) and their contribution to muscle in the presence and absence of SCs. David, do you know if the contribution of the non SC constellation stops, once mice reach an adult post growth state i..e . 4 months or over? Most work to date, if I'm not mistaken tracks non SCs from maturing mice. I wonder if these cells are transient populations

If you see our paper, Pannerec et al (Development), we see these cells in the adult. The difference between this report and our initial report is that the Sca1 med population is transient. In the PW1+/Sca1+ cells, retained in the adult (but low in old age), we see 2 populations. One is PDGFRa + corresponding to FAPs. All FAPs are PW1+. The second, present at roughly equal levels are what we now call myoPICs. We have NO marker for these cells, only the absence of PDGFRa and the expression of PW1. We are performing RNAseq to find one. Ongoing now. 

Thanks for clarifying. 

There are three populations of non-satellite stem cells that exist within skeletal muscle connective tissues (endomysium, perimysium and epimysium) that contribute to its repair. All three populations are located outside the basal lamina, while satellite cells reside inside the basal lamina, but outside the plasma membrane. These three populations are mesodermal stem cells (CD90+/CD13+/Thy-1+), pluripotent stem cells (CD10+/SSEA+), and totipotent stem cells (CD66e+/CEA+).

Dear Dr, Young

I have read most of all your papers about the markers of the stem cells in the skeletal muscle interstitium, and I would like to ask you as follows directly. One is whether the expressions are detected in freshly isolated, cultured or in vivo state? If there are freshly isolated state, how isolated; such as hardly minced with relatively higher enzyme concentration or mild treatment without mincing. I think there are potential affectors for the cell surface marker expression. Expressions are changeable under these conditions.

Dear Dr. Tamaki,

I understand your concern. We had similar concerns as well. We initially discovered the markers using FACS analysis from the freshly isolated state. We then looked at cultured cells, both short term and long term, and finally in cryostat sectioned frozen material. In all instances they matched. 

We also noted that if we induced the stem cells out of their uncommitted state into lineage commitment we would lose those particular cell surface markers but gain others in the process. For example, if we start with a totipotent stem cell with the CD66e+(human)/CEA+(non-human mammal) "fingerprint", as it differentiates it progresses through a series of cell surface markers denoting a transitional state between totipotent stem cells and pluripotent stem cells, i.e., CD66e+/CEA+ to CD66e+/CEA+/SSEA+/CD10+; to pluripotent stem cells, i.e., SSEA+/CD10+; to a transitional state between pluripotent stem cells and germ layer lineage stem cells, i.e., SSEA+/CD10+ to SSEA+/CD10+/CD90+/Thy-1+; to mesodermal stem cells having the Thy-1+/CD90+/CD13+ fingerprint.

We also tested the differentiation potential of the cells with these particular cell surface marker profiles. The CD66e+/CEA+ (totipotent stem) cells could be induced to form 66/66 potential cell types across all three germ layer lineages as assessed by our inductive phenotypic expression/ELICA assay system.  The CD10+/SSEA+ (pluripotent stem) cells could be induced to form 63/66 possible cell types across all three germ layer lineages. Whereas the Thy-1+/CD90+/CD13+ (mesodermal stem) cells formed 37/66 possible cell types across only the mesodermal germ layer lineage.

We also used different isolation paradigms to extract the cells from skeletal muscle as well as from other tissues, i.e., collagenase/dispase digest, collagenase digest, mincing followed by antibodies bound to magnetic beads in columns or panning, cryopreservation, Ficoll gradients, and differential centrifugation. 

The above analyses were tested in both human cells and in cells from non-human mammals. 

Dear Dr. Young

Thank you for your kind response with a detailed and referential explanation. Do your means 66 potential cell types are in the Table 1 (subtract some of non-cell surface marker) of your recent review article (MOJ 2014)?

Dear Dr. Tamaki,

We used multiple markers (109) to identify 66 distinct cell types, based on phenotypic expression. The particular markers used are listed in Table 1 of the MOJ 2014 article.