ASTM F3369-19
(Guide)Standard Guide for Assessing the Skeletal Myoblast Phenotype
Standard Guide for Assessing the Skeletal Myoblast Phenotype
SIGNIFICANCE AND USE
5.1 This guide describes markers involved in myoblast differentiation that can be used to screen stem cells to help define myogenic capacity. Stem cells include pluripotent and multipotent stem cells capable of differentiating into several different mesenchymal cells, including skeletal muscle myoblasts.
5.2 To assess myogenesis in cells derived and not derived from muscle, markers are measured to accurately define the changes in transcription and structural proteins that regulate differentiation, fusion, and myotube formation. Discussion of these markers is important to understand why they are recommended.
5.3 Myogenic Differentiation:
5.3.1 Myogenic differentiation is a highly regulated process controlled by paired box (Pax) transcription factors and the myogenic regulatory factor (MRF) family. During early differentiation in adults, myogenic progenitors such as activated satellite cells or myoblasts express Pax3 and Pax7. Pax3 and Pax7 transcription factors switch the cells toward a myogenic fate, and repress myocyte differentiation (2), priming the cell for later MRFs. To form muscle, the family of MRFs is required to terminally differentiate myoblasts and form myofibers. These regulatory proteins belong to a superfamily of basic helix-loop-helix transcription factors that consists of myogenic differentiation factor 1 (Myod1), myogenic factor 5 (Myf5), myogenin (Myog), and myogenic factor 6 (Myf6). In the initial stages of myogenic differentiation, Myod1 and Myf5 are the first MRFs to be expressed, and trigger increased production of Myog and Myf6 (3). Increased intracellular Myog and Myf6 induces terminal differentiation of myoblasts into myocytes, leading to fused myotubes.
5.4 Forming Myotubes:
5.4.1 While myogenic markers describe differentiation, fusion into multinucleated myotubes is an important factor in muscle biology. Myoblasts differentiate into a fusogenic phenotype characterized by multiple fusion markers. One marker of note is m-c...
SCOPE
1.1 Myogenic differentiation is a process regulated by specific transcription factors and signaling molecules that have been shown to induce a myogenic phenotype. Transcription factors mark the stages of myogenesis and act as benchmarks for use in myogenic assays.
1.2 This guide applies to mammalian cells but does not apply to non-mammalian cells as the myogenic markers for non-mammalian cells can be different than those described here.
1.3 This guide proposes appropriate markers to measure when conducting myogenic differentiation assays. This guide describes the stages for multipotent stem cell differentiation toward myoblasts and myotubes. This guide provides information about the appropriate methods to determine myogenic differentiation. This guide does not provide information about media, supplements, or substrates that drive differentiation toward a myogenic phenotype.
1.4 The purpose of this guide is to act as an aid for work performed in the area of skeletal myogenesis. Using this guide, researchers should be able to understand which skeletal muscle markers are best suited for experiments. This guide will improve consistency for studies of myogenic differentiation of multipotent stem cells by identifying appropriate markers for each stage leading to myocyte differentiation. It should be noted that myoblast differentiation in vitro may not be predictive of results that may be obtained in vivo.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Gu...
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Designation: F3369 − 19
Standard Guide for
1
Assessing the Skeletal Myoblast Phenotype
This standard is issued under the fixed designation F3369; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
1.1 Myogenic differentiation is a process regulated by
specifictranscriptionfactorsandsignalingmoleculesthathave
2. Referenced Documents
been shown to induce a myogenic phenotype. Transcription
2
2.1 ASTM Standards:
factors mark the stages of myogenesis and act as benchmarks
F2312Terminology Relating to Tissue Engineered Medical
for use in myogenic assays.
Products
1.2 This guide applies to mammalian cells but does not
apply to non-mammalian cells as the myogenic markers for 3. Terminology
non-mammalian cells can be different than those described
3.1 Unless provided otherwise in 3.2, terminology shall be
here.
in conformance with Terminology F2312.
1.3 This guide proposes appropriate markers to measure
3.2 Definitions:
when conducting myogenic differentiation assays. This guide
3.2.1 myoblasts, n—myoblasts repair or replace damaged
describes the stages for multipotent stem cell differentiation
muscle fibers by differentiating and fusing either with an
toward myoblasts and myotubes.This guide provides informa-
existing muscle fiber or with each other to form multi-
tion about the appropriate methods to determine myogenic
nucleated myotubes. Proliferating myoblasts are able to self-
differentiation. This guide does not provide information about
renew, but lose that ability once they exit the mitotic cycle.
media, supplements, or substrates that drive differentiation
3.2.2 myogenic cells, n—myogenic cells express a class of
toward a myogenic phenotype.
markers that suggest a muscle cell lineage fate or a cell’s
1.4 The purpose of this guide is to act as an aid for work
involvement in muscle fiber formation, or both.
performedintheareaofskeletalmyogenesis.Usingthisguide,
3.2.3 myogenic differentiation, n—myogenic differentiation
researchersshouldbeabletounderstandwhichskeletalmuscle
refers to the normal process by which early muscle precursor
markers are best suited for experiments. This guide will
cells and myoblasts become more specialized cells capable of
improve consistency for studies of myogenic differentiation of
fusing with mature myoblasts or existing myotubes.
multipotent stem cells by identifying appropriate markers for
Furthermore, the process of differentiation can be measured
each stage leading to myocyte differentiation. It should be
using markers to help describe cell behavior during culture.
noted that myoblast differentiation in vitro may not be predic-
This guide will describe those different markers.
tive of results that may be obtained in vivo.
3.2.4 myogenicterminaldifferentiation,n—myogenictermi-
1.5 This standard does not purport to address all of the
nal differentiation refers to the normal end stage of myoblast
safety concerns, if any, associated with its use. It is the
maturation, where myoblasts can no longer proliferate and are
responsibility of the user of this standard to establish appro-
able to fuse into myotubes. Experimental data to quantify this
priate safety, health, and environmental practices and deter-
stage of differentiation is necessary in gaining additional
mine the applicability of regulatory limitations prior to use.
information regarding cell behavior during culture.
1.6 This international standard was developed in accor-
3.2.5 myotubes, n—myotubes are long tubular structures
dance with internationally recognized principles on standard-
with multiple nuclei, originating from mature myoblasts that
ization established in the Decision on Principles for the
fusedtogether.Nucleiarecentrallylocatedfollowingtheinitial
Development of International Standards, Guides and Recom-
fiber formation and as the myotube matures into a functional
fiber the nuclei translocate to the periphery.
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This guide is under the jurisdiction ofASTM Committee F04 on Medical and
2
Surgical Materials and Devices and is the direct responsibility of Subcommittee For referenced ASTM standards, visit the ASTM website, www.astm.org, or
F04.43 on Cells and Tissue Engineered Constructs for TEMPs. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Current edition approved Feb. 1, 2019. Published March 2019. DOI: 10.1520/ Standards volume information, refer to the standard’s Document Summary page on
F3369-19. the ASTM website.
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