Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro.

TLDR: An optimized serum-free differentiation protocol to efficiently produce striated, millimeter-long muscle fibers together with satellite-like cells from human pluripotent stem cells (hPSCs) in vitro by mimicking key signaling events leading to muscle formation in the embryo is reported.

Abstract: Progress toward finding a cure for muscle diseases has been slow because of the absence of relevant cellular models and the lack of a reliable source of muscle progenitors for biomedical investigation. Here we report an optimized serum-free differentiation protocol to efficiently produce striated, millimeter-long muscle fibers together with satellite-like cells from human pluripotent stem cells (hPSCs) in vitro. By mimicking key signaling events leading to muscle formation in the embryo, in particular the dual modulation of Wnt and bone morphogenetic protein (BMP) pathway signaling, this directed differentiation protocol avoids the requirement for genetic modifications or cell sorting. Robust myogenesis can be achieved in vitro within 1 month by personnel experienced in hPSC culture. The differentiating culture can be subcultured to produce large amounts of myogenic progenitors amenable to numerous downstream applications. Beyond the study of myogenesis, this differentiation method offers an attractive platform for the development of relevant in vitro models of muscle dystrophies and drug screening strategies, as well as providing a source of cells for tissue engineering and cell therapy approaches.

Figures

Table 2: List of markers used to characterize the differentiating hPSC-derived myogenic cultures and corresponding antibodies for immunohistochemistry (Figures 2, 4, 5).

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Generation of human muscle bers and satellite-like cells
from human pluripotent stem cells in vitro
Jérome P Chal, Ziad Al Tanoury, Marie P Hestin, Bénédicte Gobert, Suvi P
Aivio, Aurore P Hick, Thomas Cherrier, Alexander P Nesmith, Kevin K
Parker, Olivier P Pourquié
To cite this version:
Jérome P Chal, Ziad Al Tanoury, Marie P Hestin, Bénédicte Gobert, Suvi P Aivio, et al.. Generation
of human muscle bers and satellite-like cells from human pluripotent stem cells in vitro. Nature
Protocols, Nature Publishing Group, 2016, 11 (10), pp.1833-50. �10.1038/nprot.2016.110�. �inserm-
01485521�

***Final Author Version*** for Publication doi:10.1038/nprot.2016.110
at http://www.nature.com/nprot/journal/v11/n10/abs/nprot.2016.110.html
Generation of human muscle fibers and satellite-like cells from human
pluripotent stem cells in vitro
Authors: Jérome Chal
1,2,3,4,#
, Ziad Al Tanoury
1,#
, Marie Hestin
2,3,4
, Bénédicte Gobert
1
,
Suvi Aivio
2,3,4
, Aurore Hick
5
, Thomas Cherrier
1
,
Alexander P. Nesmith
6
, Kevin K.
Parker
6
& Olivier Pourquié
1,2, 3,4,
*
Affiliations:
1
Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS (UMR
7104), Inserm U964, Université de Strasbourg, Illkirch Graffenstaden, France.
2
Department of Pathology, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur,
Boston, Massachusetts, USA.
3
Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston,
Massachusetts, USA.
4
Harvard Stem Cell Institute. 77 Avenue Louis Pasteur, Boston, Massachusetts, USA.
5
Anagenesis Biotechnologies, Parc d’innovation, 650 Boulevard Gonthier d’Andernach
67400 Illkirch Graffenstaden, France.
6
Disease Biophysics Group, Wyss Institute for Biologically Inspired Engineering,
School of Engineering and Applied Sciences, Harvard University, Cambridge, USA.
# equal contribution
* Contact Information: Olivier Pourquié, Ph.D.
Department of Genetics, Harvard Medical School and Department of Pathology,
Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, Boston, MA, USA.
email: pourquie@genetics.med.harvard.edu
Keywords: pluripotent stem cells, myogenesis, skeletal muscle, satellite cell,
bioengineering, directed differentiation, embryonic stem cell, iPS

Abstract
Progress towards finding a cure for muscle diseases has been slow due to the absence
of relevant cellular models and the lack of a reliable source of muscle progenitors for
biomedical investigation. Here, we report an optimized serum-free differentiation
protocol to efficiently produce striated, millimeter-long muscle fibers together with
satellite-like cells from human pluripotent stem cells (PSCs) in vitro. By mimicking key
signaling events leading to muscle formation in the embryo, in particular the dual
modulation of Wnt and Bone morphogenetic pathway (BMP) signaling pathways, this
directed differentiation protocol does not require genetic modifications or cell sorting.
Robust myogenesis can be achieved in vitro within 1 month by personnel experienced
in human PSC culture. The differentiating culture can be subcultured to produce large
amounts of myogenic progenitors amenable for numerous downstream applications.
Beyond the study of myogenesis, this differentiation method offers an attractive platform
to develop relevant in vitro models of muscle dystrophies, drug screening strategies,
and to provide a source of cells for tissue engineering and cell therapy approaches.
INTRODUCTION
Until recently little progress has been made to differentiate human PSCs toward a
myogenic fate. This in turn has hampered both the development of relevant human in
vitro models of muscle diseases and the assessment of cell therapy approaches. Here
we report an optimized directed differentiation protocol for in vitro production of mature
muscle fibers and their associated progenitors from human PSCs. This is a detailed
version of the protocol reported in Chal et al., 2015
1
with a number of improvements
including the expansion and cryopreservation of the hPSC-derived myogenic
progenitors. By mimicking the early signaling events occurring during paraxial
mesoderm specification in the embryo - in particular the simultaneous activation of Wnt
and inhibition of BMP signaling pathways - this protocol recapitulates the essential steps
of skeletal myogenesis in vitro in less than 30 days. The resulting fibers show
spontaneous contraction and provide a niche for associated Pax7-positive satellite -like
cells. An additional subculturing step allows for the preparation of proliferative myogenic

populations that can be further amplified, cryopreserved or differentiated into muscle
fibers. This protocol is readily amenable for multi-format in vitro cell assays and various
downstream applications. All together, the recapitulation of myogenesis in a dish will be
an invaluable tool for the muscle, stem cell, muscle physiology and pathology, and
developmental biology communities for both basic and applied research.
Development of the Protocol
Directed differentiation methods aim at exposing PSCs to differentiation cues allowing
the sequential recapitulation of key stages of paraxial mesoderm development and its
differentiation into skeletal muscle. Early attempts to generate skeletal muscles used
spontaneous differentiation of embryoid bodies, resulting in heterogeneous
differentiation and low efficiency
2,3
. Progress in 2D- culture systems was slow until
recently when manipulations of key signaling pathways involved in paraxial mesoderm
specification were incorporated to differentiation protocols. In the embryo, Wnt signaling
is required for paraxial mesoderm induction
4-7
(for review
8
). In embryos mutant for
Wnt3a or its targets T or Tbx6, ectopic neural tubes form instead of the paraxial
mesoderm in the posterior part of the embryo
7,9-11
. In PSC culture, Wnt signaling
promotes mesodermal differentiation
12-15
and several recent myogenic induction
protocols rely on early Wnt activation
1,16-19
. Wnt signaling also acts upstream of the
Fibroblast growth factor (FGF) pathway in paraxial mesoderm precursors of the tail bud,
by triggering expression of the Fgf8 ligand
20
. Later on, at the somite level, secreted
Wnts from surrounding tissues are also critical for dermomyotome specification (for
review
21
).
BMP signaling controls the medio-lateral identity of posterior mesoderm cells
22-26
. In
amniote embryos, high BMP leads to the specification of more lateral tissues such as
extraembryonic mesoderm, or lateral plate mesoderm (LPM), a tissue that contributes to
the limbs and body wall mesenchyme and to the long bones of the limbs but which does
not form skeletal muscle. Ectopic BMP signaling can divert cells fated to form paraxial
mesoderm to a lateral plate fate in vivo
22
. Conversely BMP loss of function mutation

leads to expanded paraxial mesoderm domain
27
. In vivo, the paraxial mesoderm is
protected from BMP signaling produced by the LPM by the inhibitors Noggin and
Chordin which are expressed by the notochord, the intermediate mesoderm and the
dorsal somite
24,25,28
. In vitro, in absence of proper BMP signaling inhibition, the early
paraxial mesoderm induced by Wnt signaling produces BMP4 and "drifts" by an
autocrine effect to generate LPM derivatives (J.C., Z.A.T. and O.P., unpublished
observation). At the heart of the protocol presented here lies the use of dual modulation
of the Wnt and BMP pathways to efficiently induce paraxial mesoderm from hPSCs.
This is a prerequisite for the production of skeletal myogenic progenitors in large
number. Other studies have shown that Wnt activation and BMP inhibition can produce
mesodermal fates from human PSCs, such as Intermediate mesoderm
29
and
chondrogenic mesoderm
30
which are closely related to the paraxial mesoderm. On the
other hand, activation of BMP signaling has been used to produce anterior LPM
derivatives such as cardiovascular cell types
31-34
. While little is known about the culture
of early embryonic paraxial mesoderm from primary cultures
35-38
, myogenic progenitors
and skeletal myoblast cultures have been extensively studied in vitro
39-43
.
The protocol described here stems from the transposition of a serum-free protocol
developed for adherent mouse ESC culture to human pluripotent cell cultures
1
(Figure1). At its core lays a dual modulation of Wnt and BMP pathways. It is based on
our understanding of paraxial mesoderm development in vivo, where Wnt is activated to
specify early paraxial mesoderm while BMP is inhibited to prevent the newly specified
paraxial mesoderm cells to drift to a lateral plate fate
22
. In absence of BMP inhibition,
Wnt activated- PSCs differentiate preferentially to LPM derivatives such as endothelial
progenitors
44
. For the hPSC protocol, the first step of the mESC protocol aiming at
producing epiblast stage cells, was removed as hPSCs are more related to mouse
EpiSCs
45
. Fine-tuning of the factors' concentrations and exposure time was also
necessary to obtain optimal paraxial mesoderm induction and subsequent skeletal
myogenesis (Figure 2). Long term differentiated hPSC-culture (>1 month) are very
dense, produce abundant extracellular matrix (ECM), but retain Pax7+ myogenic
progenitors. We noticed that dissociation and replating of these differentiated cultures
led to a significant enrichment in myogenic cells (Figure 3). This step allows to produce

Frequently asked questions

Q1. What are the contributions in "Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro" ?

Here, the authors report an optimized serum-free differentiation protocol to efficiently produce striated, millimeter-long muscle fibers together with satellite-like cells from human pluripotent stem cells ( PSCs ) in vitro. Beyond the study of myogenesis, this differentiation method offers an attractive platform to develop relevant in vitro models of muscle dystrophies, drug screening strategies, and to provide a source of cells for tissue engineering and cell therapy approaches. 

Q2. What is the role of Wnt signaling in the differentiation of skeletal muscle?

Wnt signaling also acts upstream of theFibroblast growth factor (FGF) pathway in paraxial mesoderm precursors of the tail bud,by triggering expression of the Fgf8 ligand20. 

Q3. What is the way to differentiate NCRM1 from other skeletal muscle?

Passaging allows for a homogeneousamplification of progenitors that in turn give rise to myocytes (Myogenin- positive) thatcan be differentiated to mature striated skeletal muscle fibers (Titin-positive). 

Q4. How many rounds of TrypLE Express can be replaced?

If 2 rounds of TrypLE Express incubation and mechanical fragmentation appears insufficient in dissociating the culture, TrypLE Express can be replaced by 0.25% Trypsin on the next round. 

Q5. What is the role of BMP in the differentiation of a skeletal muscle?

Inamniote embryos, high BMP leads to the specification of more lateral tissues such asextraembryonic mesoderm, or lateral plate mesoderm (LPM), a tissue that contributes tothe limbs and body wall mesenchyme and to the long bones of the limbs but which doesnot form skeletal muscle. 

Q6. What are the advantages of the protocol described here?

Key advantages of the differentiation protocol reported here are the ease ofimplementation of a simple and robust 2D-culture system, higher yields, the productionof both myogenic progenitors and mature fiber cell types, and a faster experimentaltimeline over existing protocols16,19,46-48. 

Q7. What is the key population for muscle regeneration in vivo?

Elegant mouse genetics experiments have shown thatsatellite cells represent the key population allowing muscle regeneration in vivo 116-118. 

Q8. What are the ways to assess the hPSCstatus?

Complementary ways to assess the hPSCstatus include evaluation of colony growth, immunohistochemistry or Fluorescence-activated flow cytometry (FACS) analysis for stem cell markers expression, embryoidbodies formation and histological analysis of hPSCs-derived teratoma60. 

Q9. How long can myogenic subcultures be maintained in SkGM?

The authors found that confluent subcultures can be maintained in SkGM media foran extended period of time without losing their myogenic potential when furtherpassaged. 

Q10. What are the biomedical applications of hPSCs differentiated in vitro?

the authors expect that thefirst biomedical applications of PSCs differentiated in vitro will be in the domain of highthroughput screening, in vitro disease modeling and cell toxicity assays65,76,166, for whichsuch approval is not required. 

Q11. What is the key to the development of paraxial mesoderm?

At the heart of the protocol presented here lies the use of dual modulation of the Wnt and BMP pathways to efficiently induce paraxial mesoderm from hPSCs. 

Q12. How can The authorkeep myogenic cells in SkGM?

Cells can be further subcultured, over 100 days for downstream applications or kept in SkGM at high density without losing myogenic competency. 

Q13. How many days can a culture be expanded?

After 3 days, culture can be expanded again following steps 28-42END BOX● TIMING Steps 1–4, pluripotent cells preparation: 5-7 d Steps 5–19, predifferentiation setup: 2 d Steps 20–25, directed differentiation: 30-40d+ 

Q14. What is the effect of the dissociation of the primary culture?

The dissociation of the dense primary culture will eliminate dead cells, debris, ECMdeposit, unwanted derivatives but also existing (postmitotic) myofibers (Figure 3d). 

Q15. What is the use of hPSC-derived myogenic progenitors?

Production of hPSC-derived myogenic progenitors may also be used as a more relevant and standardizedsource of cells for bioengineering approaches that have traditionally relied on cell linesand biopsies (Figure 5). 

Q16. How long does it take to mature a muscle fiber?

This canbe achieved within a week, although muscle fibers continue to mature for 2 weeks, asevidenced by fiber size and myofibrills.