Myogenesis - Science topic

Dear all, anyone knows how to obtain pax7+/myoD- skeletal muscles progenitor cells (satellite cells) from mesenchymal stem cells? i am looking for and induction protocol without using transcription factors over-expression.

thank you ;)

Hi All,

I am facing some problem with beta actin as loading control in myogenesis western blots (not getting normalized). So is it obvious problem with beta actin or i should use another loading control for myogenesis western blots??

Please suggest.

Thanks in advance!!

I'm looking to do a short term analysis of the differentiation of C2C12 myoblasts. I've seen in some literature that MyoD1 is one of the initial proteins expressed, with a peak at around 24 hours after reaching confluency. I will be doing immunofluorescence on the cells and would like to clarify where in the cell should be the priority to look for MyoD1? Some images appear to show a localisation to the nucleus, whereas some show a more homogeneous and diffuse fluorescence throughout the cell. Any advice or tips would be welcome!

I am trying to differentiate the C2C12 myoblast cells to myotubes. I tried to grow the cells in glass bottom dishes. I found that the cells are getting elongated initiallly, but shrinking and becoming round in shape later. I used the differentiation medium supplemented with 2% Horse Serum and 1% of 1um insulin.

Myoblast transfection followed by differentiation is fine. However, some proteins inhibit differentiation itself (like dnAMPK). To study the roles of such factors in mature myotube functions I would appreciate a protocol for transfection of myotubes.

I am trying to culture the MDSCs from human skeletal muscle and the protocol I'm following is the one published by Eberli et al. in 2009 (http://www.sciencedirect.com/science/article/pii/S1046202308002016).

I'm using the same collagenase and dispase digestion, and ending up with a mass of hypercontracted fibres and mostly fibroblasts. Does anyone have any advice with respect to the handling and digestion of the muscle during the isolation so as to ensure myofibre viability? I'd be very grateful for your input!

I am trying to quantify the C2C12 differentiation. I have read that some programs like ImageJ can be used to calculate the fusion index (percentage of nuclei inside the myotubes) in Pappenheim or Jenner-Giemsa stained cultures. However, I haven't been able to find a specific protocol of how to do so, specially because I can't really see clearly the nuclei in the images I've acquired (see attachment). Has anyone done this or can anyone help me with hot to perform the image analysis? Many Thanks!

Upon performing reprogramming/differentiation over 3 days experiment, can desmin, MyoD or alpha actinin be detected this early?

I have a child with chronic hypothermia for few years, diagnosed recently with sero-negative myopathy on EMG.

I suspect hypothermia and myopathy are are linked by thyroxine effects on muscle or ACH receptors 

Because myoblast is differentiated to myotube with 2% horse serum,  what is the origin signal pathway of myoblast differentiation?  Cell stress or cell cycle proteins?

Recently I tried a western blot on various human muscle samples. Unfortunately, some of the samples migrated like the fourth sample you see in the picture. It seems like the proteins in the sample are too aggregated, but I really don't know what to do to "unpack" them and no one in my lab can give me any advice about it. I used RIPA lysis buffer to lyse the muscle sections, and broke them down by freezing and thawing them a few times.

Any advice would be immensely appreciated.

1) Can we quantify dystrophin thorugh skin biopsy? I mean can we know % of dystrophin present in the muscles/ body if any

2) Can we distinguish between endogenous and donor derived dystrophin if some one receives stem cell treatment 3) In case if no dystrophin staining is found post treatment can we know if the donor cells are engrafted in the host dystrophic muscles , but they didn't participate in myogenesis and form myotubes or repair dying muscle cells. I ask this because in one of the animal trials it was seen that though the muscle biopsy was not showing any dystrophin but the disease stabilized which was attributed to engrafted donor cells 4) Can we know the quantum of fibrosis , immune cells and inflammation by using respective markers for inflammation , etc like TNF -alpha , TGF -b , prostaglandins , cytokines like interleukin - 6 , interferons. If markers not available , at least can inflammation and fibrosis be quantified pre and post treatment, with skin biopsies 

3) Can we distinguish between endogenous and donor derived dystrophin if some one receives stem cell treatment

4) In case if no dystrophin staining is found post treatment can we know if the donor cells are engrafted in the host dystrophic muscles , but they didn't participate in myogenesis and form myotubes or repair dying muscle cells. I ask this because in one of the animal trials it was seen that though the muscle biopsy was not showing any dystrophin but the disease stabilized which was attributed to engrafted donor cells

5) Can we know the quantum of fibrosis , immune cells and inflammation by using respective markers for inflammation , etc like TNF -alpha , TGF -b , prostaglandins , cytokines like interleukin - 6 , interferons. If markers not available , at least can inflammation and fibrosis be quantified pre and post treatment, with skin biopsies

I am trying to find mesenchymal stem cell towards myogenic differentiation.. any suggestions will be highly appreciated.