Growing Skeletal Muscle in the Laboratory


Skeletal muscle – that type of voluntary muscle that allows movement – has proven difficult to grow in the laboratory. While particular cells can be differentiated into skeletal muscle cells, forming a coherent, structurally sound skeletal muscle is a tough nut to crack from a research perspective.

Another problem dogging muscle research is the difficulty growing new muscle in patients with muscle diseases such as muscular dystrophy or other types of disorders that weaken and degrade skeletal muscle.

Now research groups at the Boston Children’s Hospital Stem Cell Program have reported that they can boost the muscle mass and even reverse the disease of mice that suffer from a type of murine muscular dystrophy. To do this, this group use a combination of three different compounds that were identified in a rapid culture system.

This ingenious rapid culture system uses the cells of zebrafish (Danio rerio) embryos to screen for these muscle-inducing compounds. These single cells are placed into the well of a 96-well plate, and then treated with various compounds to determine if those chemical induce the muscle formation. To facilitate this process, the zebrafish embryo cells express a very special marker that consists of the myosin light polypeptide 2 gene fused to a red-colored protein called “cherry.” When cells become muscle, they express the myosin light polypeptide 2 gene at high levels. Therefore, any embryo cell that differentiates into muscle should glow a red color.

(A) myf5-GFP;mylz2-mCherry double-transgenic expression recapitulates expression of the endogenous genes. myf5-GFP is first detected at the 11-somite stage. mylz2-mCherry expression is not observed until 32 hpf. Scale bars represent 200 mm. (B) myf5-GFP;mylz2-mCherry embryos were dissociated at the oblong stage and cultured in zESC medium. Images were taken 48 hr after plating. Scale bars represent 250 mm.
(A) myf5-GFP;mylz2-mCherry double-transgenic expression recapitulates expression of the endogenous genes. myf5-GFP is first detected at the 11-somite
stage. mylz2-mCherry expression is not observed until 32 hpf. Scale bars represent 200 mm.
(B) myf5-GFP;mylz2-mCherry embryos were dissociated at the oblong stage and cultured in zESC medium. Images were taken 48 hr after plating. Scale bars
represent 250 mm.

Once a cocktail of muscle-inducing chemicals were identified in this assay, those same chemicals were used to treat induced pluripotent stem cells made from cells taken from patients with muscular dystrophy.  Those iPSCs were treated with the combination of chemicals identified in the zebrafish embryo screen as muscle inducing agents.

Zebrafish embryo culture system

The results were outstanding.  Leonard Zon from the Division of Hematology/Oncology, Children’s Hospital Boston and Dana-Farber Cancer Institute and his colleagues showed that a combination of basic Fibroblast Growth Factor, an  adenylyl cyclase activator called forskolin, and the GSK3β inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs).  Furthermore, these muscle cells produced engraftable myogenic progenitors that contributed to muscle repair when implanted into mice with a rodent form of muscular dystrophy.

Representative hematoxylin and eosin staining (H&E) images and immunostaining on TA sections of preinjured NSG mice injected with 1 3 105 iPSCs at day 14 of differentiation. Muscles injected with BJ, 00409, or 05400 iPSC-derived cells stain positively for human d-Sarcoglycan protein (red). Fibers were counterstained with Laminin (green). No staining is observed in PBS-injected mice or when 00409 fibroblast cells were transplanted. Because the area of human cell engraftment could not be specifically distinguished on H&E stained sections, which must be processed differently from sections for immunostaining, the H&E images shown do not represent the same muscle region as that shown in immunofluorescence images. Scale bars represent 100 mm, n = 3 per sample.
Representative hematoxylin and eosin staining
(H&E) images and immunostaining on TA sections
of preinjured NSG mice injected with 1 3 105
iPSCs at day 14 of differentiation. Muscles injected
with BJ, 00409, or 05400 iPSC-derived cells
stain positively for human d-Sarcoglycan protein
(red). Fibers were counterstained with Laminin
(green). No staining is observed in PBS-injected
mice or when 00409 fibroblast cells were transplanted.
Because the area of human cell engraftment
could not be specifically distinguished on
H&E stained sections, which must be processed
differently from sections for immunostaining, the
H&E images shown do not represent the same
muscle region as that shown in immunofluorescence
images. Scale bars represent 100 mm, n = 3
per sample.

Zon hopes that clinical trials can being soon in order to translate these remarkable results into patients with muscle loss within the next several years.  Zon and his co-workers are also screening compounds to address other types of disorders beyond muscular dystrophy.

This paper represents the application of shear and utter genius.  However, there is one caveat.  The mice into which the muscles were injected were immunodeficient mice whose immune systems are unable to reject transplanted tissues.  In human patients with muscular dystrophy, an immune response against dystrophin, the defective protein, has been an enduring problem (for a review of this, see T. Okada and S. Takeda, Pharmaceuticals (Basel). 2013 Jun 27;6(7):813-836).  While there have been some technological developments that might circumvent this problem, transplanting large quantities of muscle cells might be beyond the pale.  Muscular dystrophy results from disruption of an important junction between the muscle and substratum to which the muscle is secured.  This connection is mediated by the “dystrophin-glycoprotein complex.”  Structural disruptions of this complex (shown below) lead to unanchored muscle that cannot contract properly, and eventually atrophies and degrades.

Dystrophin-glycoprotein complex. Molecular structure of the dystrophin-glycoprotein complex and related proteins superimposed on the sarcolemma and subsarcolemmal actin network (redrawn from Yoshida et al. [5], with modifications). cc, coiled-coil motif on dystrophin (Dys) and dystrobrevin (DB); SGC, sarcoglycan complex;SSPN, sarcospan; Syn, syntrophin; Cav3, caveolin-3; N and C, the N and C termini, respectively; G, G-domain of laminin; asterisk indicates the actin-binding site on the dystrophin rod domain; WW, WW domain.
Dystrophin-glycoprotein complex. Molecular structure of the dystrophin-glycoprotein complex and related proteins superimposed on the sarcolemma and subsarcolemmal actin network (redrawn from Yoshida et al. [5], with modifications). cc, coiled-coil motif on dystrophin (Dys) and dystrobrevin (DB); SGC, sarcoglycan complex;SSPN, sarcospan; Syn, syntrophin; Cav3, caveolin-3; N and C, the N and C termini, respectively; G, G-domain of laminin; asterisk indicates the actin-binding site on the dystrophin rod domain; WW, WW domain.
This is a remarkable advance, but until the host immune response issue is satisfactorily addressed, it will remain a problem.

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Published by

mburatov

Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).