Skeletal Muscle Differentiation Kit

Differentiate human pluripotent stem cells to skeletal muscle

The world’s first protocol to differentiate human pluripotent stem cells to skeletal muscle with high yields and without cell sorting or genetic manipulation. Myotubes are contractile, express typical muscle markers and show striated sarcomeres. The process yields almost pure populations of myogenic cells; it is highly efficient and works robustly with all pluripotent stem cell lines – critical prerequisites for effective cell-based disease modeling and drug development.

Generation of Muscle Cells in 3 Steps

Muscle is generated from human pluripotent stem cells in 3 steps, via satellite-like progenitor cells and myoblasts that then fuse to multinucleated myotubes in the third step. This third generation of the process includes an optional enhanced fusion formulation, depending on your desired phenotype.

NameDatasheetPacksize Order
Myoblast Cell Culture Medium250 ml View
Myotube Cell Culture Medium250 ml View
Skeletal Muscle Induction medium250 ml View

Skeletal Muscle Differentiation Kit FAQs

Can non-human mammalian cells be differentiated with this media?

To date we do not have any data to support the differentiation of non-human mammalian cells, however we have no information to suggest this application would not work.

Can muscle tissue-isolated myosatellite cells be used with the Skeletal Muscle Differentiation Kit?

To date we have not tested muscle tissue isolate myosatellite cells, however we do not have any evidence to suggest it is not possible to successfully differentiate myosatellite cells. However, myosatellite cells are highly proliferative and relatively rare, therefore a good dissociation protocol and sufficient tissue quantity would be required to yield enough cells to input into the differentiation protocol.

Is it at all possible to perform the differentiation method on a matrix other than collagen?

Our customers have reported successful differentiation on alternative commercial ECM, including iMatrix Laminin 511 E8 (Cat. #AMS.892 011) and Matrigel.

Is it possible to expand and freeze cultures at any intermediate steps, i.e. satellite cells or myoblasts, and then thaw and continue the differentiation protocol?

Yes, precursors and myoblasts can be banked and thawed as needed. These cells are proliferative but we recommend generating large banks starting with pluripotent cells through stage I and/or II. The cells can be dissociated with trypsin/EDTA and preserved in either stage medium with 10% DMSO or CELLBANKER® 1 (Cat. #11888).

Which markers should be used and at what time point should you measure satellite cells numbers?

The expression of satellite cell markers Pax3 and Pax7 peaks around day 4/5 of stage 1 differentiation. In addition to those markers we have verified expression of CD56, CD34 and CXCR4. See Caron et al. 2016.

Is it possible to passage and maintain the cells after the final differentiation step? How long can myotubes be cultured?

Mature myotubes produced by the skeletal muscle differentiation kit can be maintained in the Myotube Cell Culture Medium for 2 weeks and the Myotube Fusion Medium for 3 weeks. However, mature myotubes will not passage well and at a certain point of maturation the cells will start to spontaneously contract which will cause them to detach from the ECM. Mature myotubes are not highly proliferative and cannot be easily frozen and recovered from cryopreservation. We recommend users cryopreserve their cells at either the progenitor or myoblast stage.

We are in the early stages of development on a culture system that will support long term culture of mature myotubes. It is based on a co-differentiation system in which the myotubes are innervated.

Do you have any recommendations for dyes for live-cell imaging of specific markers to confirm myotubes have been produced?

The AntiMHC by DSHB is a good marker for mature myotubes. Dystrophin is also a good marker.

What is the function of the Myotube Fusion Medium?

The myotube fusion media produces more robust myotubes with a higher degree of cell fusion. Myoblasts continue to proliferate in Myotube Cell Culture Medium and the fusion rate is slower.

Is it possible to use the Skeletal Muscle Differentiation Kit to differentiate Mesenchymal stem cells (MSCs) into the myogenic lineage?

To date, we do not have any data to support the differentiation of MSCs to myotubes.

Does the Skeletal Muscle Differentiation Kit work with fibroblasts?

To date, we do not have any data to support the trans-differentiation of fibroblasts to myotubes.

How long is the media stable for at +4°C, and can it be aliquoted and re-frozen?

The media is stable for 2 weeks at +4°C. It is also validated for one additional freeze/thaw cycle so it can be aliquoted & frozen and used by the printed expiration.