Progress Update: FSHD myofibers cultured from human xenografts

by Robert J Bloch PhD
See grant: FSHD myofibers cultured from human xenografts

Our research is devoted to creating mice that have human skeletal muscles, and then using them to study FSHD and therapeutic approaches to treating FSHD. We create the mice by eliminating their Tibialis anterior muscles (hindlimb muscles that in humans raise the foot when the ankle is kept on the ground) and preventing them from regenerating. We do this by X-irradiating the hindlimbs, to prevent regrowth of mouse cells, and then killing the muscle with proteins from the venom of a cobra. Then we inject the hindlimb with myogenic cells (cells that can produce muscle tissue when fully differentiated) and allow them to engraft and grow. We stimulate these processes by delivering electrical pulses to the hindlimbs several times a week. When we follow this procedure with healthy myogenic cells or cells from an FSHD patient, we get very nicely formed skeletal muscle tissue that is essentially free of any mouse tissue and that contracts when the motor nerves are stimulated. We have followed three lines of investigation in studying these grafts, called “xenografts” because a foreign (“xeno”, or ξένο, in Greek) source of tissue is introduced into the mice.

In the first, we are trying to make the xenografts bigger, so that we can perform physiological experiments on them, to study the changes in their ability to contract in response to injuries, drugs or gene therapies.  We have found several modifications to our methods that increase the sizes of the xenografts significantly, including injecting more myogenic cells, allowing the mice to exercise in their cages, and providing the grafts with growth factors. We plan to combine several of these improvements to see if we can form xenografts that are as big as the mouse’s Tibialis anterior muscles that they are replacing. As of now, we are almost half-way there.

In the second approach, we have been exploring methods to place the individual myofibers that form in the xenografts into tissue culture, so that they can be studied in isolation. These kinds of experiments have the potential to reveal, under the microscope, many of the physiological and morphological deficits associated with FSHD as they are initiated, and so we have invested a considerable effort to develop the necessary methods. Although we have met with only limited success so far, we continue to investigate several alternative approaches.

In our third line of investigation, we have been examining the genetic and epigenetic status of the grafts to learn if they accurately reflect the status of mature FSHD and healthy muscles. We were very fortunate to be able to collaborate with Drs. Takako and Peter Jones on these experiments.  Our results have shown that FSHD grafts express significant amounts of DUX4 and several of its downstream gene targets, including ZSCAN4, TRIM43 and MBD3L2, but that control grafts, prepared from healthy cells, do not. Furthermore, the FSHD grafts show much lower levels of CpG methylation, the epigenetic marker associated with activation of gene expression in FSHD. These grafts therefore provide an outstanding experimental model in which to study the effects and specificity of different therapies for FSHD in mature human skeletal muscle.


Xenografting human myogenic cells into mice: A new model for FSHD
Mueller, A.L., O’Neill, A., Llach, A., Stadler, G., Wright, W. and Bloch, R.J.
Muscular Dystrophy Association Clinical Conference, Arlington, VA, March 2016;
First prize, $500.

Xenografting Human Myogenic Cells into Mice: A New Model for FSHD
Llach A., Mueller A.L., Jones T.I., Sakellariou P., O’Neill A., Jones P.L. and Bloch R.J.
New Directions, Orlando, FL, June 2016:
First prize, $1000.

Modelin FSHD by Xenografting Human Myogenic Cells into Mice
Mueller A.L., O’Neill A., Llach A., Stadler, G., Wright, W. and Bloch R.J.
FSH Society International Consortium, Cambridge,MA, Nov., 2016

Methods for Improving the Quality of Xenografts in Mice by Control and FSHD Myogenic Cells
Llach A., Mueller A.L, O’Neill A.and Bloch R.J.
FSH Society International Consortium, Cambridge,MA, Nov., 2016