Derivation of Engrafting Skeletal Muscle Precursors from Human Embryonic Stem Cells Using Serum-Free Methods

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Published Date: May 22, 2015

Derivation of Engrafting Skeletal Muscle Precursors from Human Embryonic Stem Cells Using Serum-Free Methods

Karlijn J. Wilschut1,4*, Wenhui Gong1, Peter E. Oishi1,3* and Harold S. Bernstein1-3,5

1 Department of Pediatrics, University of California San Francisco, San Francisco, CA 94143 USA

2 Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA 94143 USA

3 Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA 94143 USA

4 Department of Surgery, University of California San Francisco, San Francisco, CA 94143 USA

5 The Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029 USA

*Corresponding authors: Karlijn J. Wilschut, University of California San Francisco, 513 Parnassus Avenue, Box 0932, San Francisco, CA 94143-1346, Tel: (+1) 415-476-9369; Email:

Peter E. Oishi, University of California San Francisco, 513 Parnassus Avenue, Box 1346, San Francisco, CA 94143-1346, Tel: (+1) 415-476-1043; Fax: (+1) 415-514-0235; Email:,


Citation: Wilschut KJ, Gong W, Oishi PE, Bernstein HS (2015) Derivation of Engrafting Skeletal Muscle Precursors from Human Embryonic Stem Cells Using Serum-Free Methods. J Stem Trans Bio 1(1): 106. Doi:




Degenerative muscle diseases affect muscle tissue integrity and function. Human embryonic stem cells (hESC) are an attractive source of cells to use in regenerative therapies due to their unlimited capacity to divide and ability to specialize into a wide variety of cell types. A practical way to derive therapeutic myogenic stem cells from hESC is lacking. In this study, we demonstrate the development of two serum-free conditions to direct the differentiation of hESC towards a myogenic precursor state. Using TGFß and PI3Kinase inhibitors in combination with bFGF we showed that one week of differentiation is sufficient for hESC to specialize into PAX3+/PAX7+ myogenic precursor cells. These cells also possess the capacity to further differentiate in vitro into more specialized myogenic cells that express MYOD, Myogenin, Desmin and MYHC, and showed engraftment in vivo upon transplantation in immunodeficient mice. Ex vivo myomechanical studies of dystrophic mouse hindlimb muscle showed functional improvement one month post-transplantation. In summary, this study describes a promising system to derive engrafting muscle precursor cells solely using chemical substances in serum-free conditions and without genetic manipulation.

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Muscular dystrophies are debilitating degenerative muscle diseases caused by defective muscle proteins that disrupt normal homeostasis [1,2]. During embryogenesis Pax3 and Pax7-expressing cells drive muscle formation, growth and repair [3-8]. In the postnatal period, these cells are termed satellite cells –stem cells that remain quiescent beneath the basal lamina of muscle fibers under normal conditions. Tissue damage triggers these cells to proliferate and differentiate into myoblasts, leading to the formation of multinucleated myotubes and fibers [9-12]. However, dystrophic tissue induces repeated activation of satellite cells, ultimately leading to stem cell exhaustion, limiting the ability for further restorative myogenesis. Given their role in muscle formation, growth and repair, satellite cells, or other myogenic precursors with similar characteristics, would be attractive candidates for cell-based therapies for muscular dystrophies. Successful cell therapy would require myogenic precursor cells to: i. repopulate damaged muscle, ii. contribute to the formation of muscle fibers, and iii. lead to expression of normal muscle proteins in host tissue [13]. In addition, an important therapeutic benefit would be repopulation of the muscle stem cell niche to allow for ongoing homeostasis.

Embryonic stem cells (ESC) have the capacity to expand indefinitely, while maintaining the ability to differentiate into a wide variety of cell types. As such ESC may be useful for cell transplantation therapy or tissue engineering. However, a non-genetics method to obtain hESC derivatives with muscle regenerative abilities is lacking. Promising attempts to derive human myogenic precursors from pluripotent stem cells have relied on viral–mediated MYOD-expression [14-18], serum-containing media [14,15], use of demethylating agents [15], or selective multi-step cell culture approaches and cell sorting procedures that may be difficult to implement on a large-scale [16]. The most efficient methods described to date used genetic manipulation of human ESC (hESC) to overexpress the myogenic transcription factors, PAX3 and PAX7 [19]. Importantly, the use of genetically manipulated or virally induced cells, as well as cells exposed to serum that contains non-human components, may be difficult to use in human trials due to safety concerns.

 Therefore, in order to establish alternative ways of driving early myogenic specification, we leveraged previous studies describing chemical compounds that stimulate and facilitate hESC differentiation toward paraxial mesoderm, the lineage responsible for limb and limb-associated skeletal muscle development. hESC can be chemically stimulated by activation of the bone morphogenetic protein (BMP) pathway, which is a key inducer of mesoderm formation during embryogenesis [14,20-24]. Treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor, Ly294002 hydrochloride (Ly), facilitates the differentiation of hESC by reducing self-renewal ability [25,26]. Additionally, paraxial mesoderm, the lineage responsible for limb muscle development, can be formed by brief stimulation with BMP and inhibition of the TGFb/Activin pathway using the small molecule, SB431542 (SB) [27]. This compound specifically inhibits the Activin pathway without affecting BMP signaling. It has been shown that inhibition of the TGFb/Activin pathway using SB up-regulates myogenic genes during hESC differentiation in defined culture conditions [14]. Another important factor in mesoderm formation is fibroblast growth factor (FGF), which also determines myogenic fate during development and plays a role in satellite cell activation [28-31]. Therefore, it is plausible that these components could be used to drive myogenesis in hESC cultures.

Based on these observations, we hypothesized that engrafting myogenic precursors could be derived from hESC by the sequential and coordinated manipulation of the BMP, TGFß and FGF pathways. Importantly, we sought to prove that myogenic specification of hESC could be achieved without the use of allogeneic components in serum or genomic alteration. To this end, we tested whether myogenesis could be induced through embryoid body (EB) differentiation in medium containing basic fibroblast growth factor (bFGF), Ly and low levels of FGF, in combination with short exposure to BMP4 (referred to as KFLy(B)S medium). Separately, a combination of FGF and SB alone was tested for the ability to drive myogenesis (referred to as KFS medium). We determined the capacity of hESC-derived myogenic precursors to engraft in skeletal muscle upon transplantation in hindlimb muscles of SCID mice. In addition, we determined functional changes in transplanted muscle of mdx mice using ex vivo myomechanical analysis. The novel differentiation approach described in this study circumvents many of the limitations encountered in the transition of pluripotent stem cells into myogenic precursor cells, while enhancing our understanding of the mechanisms that govern stem cell differentiation and skeletal myoge

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Materials and Methods 


Human ESC culture and myogenic differentiation     

All work with hESC was done with the approval of UCSF Stem Cell Research Oversight Committee. The H9 hESC (WA09; WiCell) were maintained on irradiated CF1 mouse embryonic fibroblasts (MEF; Millipore, Temecula, CA) in KSR medium [Knockout Dulbecco’s Modified Eagle Medium containing 20% Knockout Serum Replacement, 2mM glutamine, 0.1 mM nonessential amino acids, 0.1 mM β-mercaptoethanol (all from Invitrogen, Carlsbad, CA)] supplemented with 12.5 ng/ml recombinant human fibroblast growth factor basic (bFGF; R&D Systems, Minneapolis, MN) as previously described [32].

hESC colonies were detached after brief exposure to Collagenase IV (1 mg/ml; Sigma-Aldrich, St. Lois, MO), washed and collected by settlement. Differentiation was initiated by embryoid body (EB) formation in pre-differentiation medium KFLy(B)S: KSR medium freshly supplemented with 20 ng/ml bFGF, 10 μM Ly294,002 hydrochloride (Ly; Sigma-Aldrich), 10 μM SB431542 hydrate (SB; Sigma-Aldrich), and for the first 36 hours supplementing with recombinant human bone morphogenetic protein-4 (BMP-4) (10 ng/ml; HumanZyme, Chicago, IL), or pre-differentiation medium KFS: KSR medium freshly supplemented with 40 ng/ml bFGF and 10 μM SB. Both pre-differentiation media were supplemented one time with 10 μM Rho-associated protein kinase inhibitor (ROCKi) at the day of EB formation. SB and Ly were reconstituted in dimethylsulfoxide (DMSO) at 20 mM, and diluted to the final concentration in pre-differentiation medium. Human EBs were kept in suspension in ultra-low cluster 6-well plates (Costar, Corning, NY) and placed onto an orbital shaker (Daigger, Vernon Hills, IL) at a speed of 90 rpm. After 36 hours, medium was refreshed, which was repeated every other day until day six.

To assess the myocyte-forming potential of EB-derived myogenic precursor cells derived in KFLy(B)S and KFS (differentiation day 6) media, EBs were plated onto 12.5 μg/cm2 fibronectin (from human plasma; Sigma-Aldrich) in 0.1% gelatin-coated culture surfaces in pre-differentiation medium to stimulate cell outgrowth. After two days, medium was replaced with muscle differentiation medium [DMEM-high glucose, 10% fetal bovine serum (FBS; Hyclone, Thermo Scientific, South Logan, UT), 5% horse serum (HS; also from Hyclone) supplemented with 5 ng/ml bFGF] and cultured for an additional 4 days.

Quantitative real-time PCR

RNA was extracted from cells using the RNeasy Mini Kit including DNase treatment (Qiagen, Valencia, CA) according to the manufacturer's instruction. RNA concentration was measured using a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies) to convert equal quantities of mRNA into cDNA using the Superscript™ III First-Strand Synthesis System (Invitrogen). Relative gene expression was determined using TaqMan Assay (Applied Biosytems) on an ABI 7300 Real-Time PCR system with the following human primer pairs (ABI): OCT4 (Hs00742896_s1); Brachyury (T) (HS00610080_m1); PAX3 (Hs00240950_m1); PAX7 (Hs00242962m1); MYOD (Hs00159528_m1); Desmin (Hs01090875_m1); Myogenin (Hs002311167_m1); MYHC (Hs00430042_m1) and GAPDH (4326317E). Cycle threshold (Ct) value for detection of gene of interest was normalized against Ct value of the housekeeping gene, human GAPDH, and relative changes were calculated according the ΔΔCt method. Results of relative fold expression were analyzed using GraphPad prism software in a two-way ANOVA with Bonferroni post-test.


Immunofluorescence staining was performed on EB-outgrowths cultured in of KFLy(B)S or KFS with mouse anti-human Pax7 (undiluted), Myogenin (F5D; undiluted) and MyHC (MF20; 1:20) antibodies (all three supernatants from Developmental Studies Hybridoma Bank); mouse anti-human MyoD (5.8A; 20 μg/ml; R&D systems); or mouse anti-human Desmin (D33; 10 μg/ml; Dako, Glostrup, Denmark). Cells were fixed in 4% paraformaldehyde (PFA; EMS, Fort Washington, PA) for 15 min. and permeabilized in 0.4% Triton-X100 for 10 min. After blocking for 30 min in blocking solution [2% normal goat serum (NGS; Dako), 1% bovine serum albumin (BSA; Sigma-Aldrich), 0.1% cold fish gelatin (Sigma-Aldrich), 0.1% Triton-X100, 0.05% Tween-20 in phosphate buffered saline (PBS)], cells were subsequently incubated with the primary antibodies diluted in blocking solution for 1 hr. After washing (three times; 5 min in PBS with 0.05% Tween-20), primary antibodies were detected using Alexa Fluor® 488-labeled goat anti-mouse (1:200; Life Technologies, Grand Island, NY) for 30 min. All steps were performed at room temperature (RT). Fluorescence microscopy was performed using a Nikon Eclipse TE300 microscope and Zeiss AxioScope Imager Z.2 with Apotome (Zeiss, Inc, Thornwood, NY).

Intramuscular cell transplantation

All experiments involving animals were done with the approval of the UCSF Institutional Animal Care and Use Committee. Hindlimbs of CB17.B6-PrkdcscidLyst bg/Crl (8-12 wk-old) and C57BL/10ScSn-Dmdmdx/J (X-linked muscular dystrophy; aged 6-8 weeks) mice (Jackson Laboratories, Bar Harbor, ME) were exposed to gamma radiation (5 Gy and 18 Gy, respectively) three days before cell transplantation to inhibit muscle regeneration by host muscle cells. Mice cages were supplied with a water bottle containing an antibacterial oral suspension (200 mg sulfamethoxazole-40 mg trimethoprim/ 200 ml water) from the day of irradiation through euthanasia. For intramuscular injection, EB-derived in KFLy(B)S or KFS were trypsinized and filtered through 40 μm nylon cell strainer (Gibco) to obtain a single cell suspension. One million cells cultured in KFLy(B)S or KFS were suspended in 50 μl PBS (supplemented with 10 μM ROCKi) and injected into the tibialis anterior (TA) muscle at two sites. As control, the contra-lateral muscle was injected with 50 μl PBS with 10 μM ROCKi. TA muscles were analyzed by immunohistochemistry one month post-transplantation, as described above.


TA muscles were embedded in OCT, snap-frozen in liquid nitrogen and stored at -80°C. Cryosections (5 mm) were fixed in acetone (ice cold, 10 min) and blocked in blocking solution [5% NGS, 5% Normal Donkey Serum (Dako), 5% BSA, 0.1% cold fish gelatin, 0.1% Triton-X100, 0.05% Tween-20 in PBS]. Sections were incubated with rabbit anti-human Dystrophin (1:2500; Novus Biologicals, Littleton, CO), chicken anti-mouse Laminin (2 mg/ml; Abcam), mouse anti-human Desmin (10 μg/ml; D33; Dako), or mouse anti-human HLA-1 (W6/32; antigen determinant common to HLA-A, B and C; 4 mg/ml; provided by Frances Brodsky, Dept. of Biopharmaceutical Sciences, UCSF) in blocking buffer for 1 hour. Alexa488/594-conjugated goat anti-mouse IgG, Alexa594-Donkey anti-Rabbit IgG, or Alexa488-Goat anti-Chicken IgG (1:250; all from Life Technologies) were used to detect primary antibodies. Sections were then washed and dehydrated in ethanol (70%, 90%, 100%; 5 min each), air-dried, and mounted in Vectashield with DAPI (Vector, Burlingame, CA). For immunohistochemical detection, sections were additionally blocked for endogenous peroxidase (0.3% H2O2 in PBS, 20min). Primary antibody HLA-1 was detected by peroxidase labeled goat anti-mouse. Visualization was performed by a 3-amino-9-ethyl-carbazole (AEC; Sigma) substrate incubation for 4 minutes. Nuclei were counterstained with Mayer’s haematoxyline (2 min, Fluka, Buchs, Zwitserland). All steps were performed at RT. Fluorescence microscopy was performed using a Nikon Eclipse TE300 microscope and Zeiss AxioScope Imager Z.2 with Apotome (Zeiss, Inc, Thornwood, NY).

Ex vivo myomechanical analysis of transplanted EDL muscle in mdx mouse model

Ex vivo myography was performed to determine the increase in force production and to determine muscle fatigue level after cell transplantation into the extensor digitorum longus (EDL) muscle of mdx mouse. Myomechanical analysis was performed as previously described [33,34]. Force-frequency fatigue was measured by exposing muscle to a supramaximal stimulus train of 3-5 pulses (300 msec duration separated by 3 sec) at successive frequencies (30, 60, 100 and 140 Hz), with 5 minute intervals between stimulations. Low-frequency time-fatigue was measured by supramaximal tetanic muscle stimulation at low frequency (60 Hz) for duration of 300 msec, repeated every 3 seconds for a period of 10 minutes. For myo-mechanical analysis, the left and right leg variables of an animal were averaged in each group and muscle mass, cross-sectional area, and length was measured. Significant differences were present between the means of each group compared to the PBS group. Multiple t-tests were performed with the number of comparisons adjusted by Bonferroni correction. A value of p <0.05 was considered significant.

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Inhibition of TGFβ pathway directs myogenic commitment

To compare previously described, serum-containing methods for their capacity to induce the formation of myogenic precursors, we differentiated hESC by a selective culture approach using serum-containing (10% FBS) medium to enrich for CD73+ mesodermal stem cells followed by a second cell sort for expression of neural cell adhesion molecule (NCAM), a molecule also expressed by myogenic cells [16]. Furthermore, we differentiated hESC-EB in low serum conditions (2% HS) [35-37] and high serum conditions (10% FBS) during 2-4 weeks in culture [15]. We observed expression of PAX7 in cells derived through the selective multi-step methods (preCD73/NCAM sort) and when exposed to low and high serum conditions (Figure 1A). Addition of SB, a compound that inhibits the TGFb pathway, to cells differentiated in 10% FBS culture conditions resulted in markedly increased PAX7 levels (Figure 1A) *p<0.01. This indicated the myogenic stimulatory effect of TGFb inhibition in hESC. Next, we transiently stimulated the BMP pathway with BMP4 during EB differentiation combined with Ly and high levels of bFGF in serum-free conditions (KFLy(B)). This resulted in high levels of PAX7 expression (Figure 1A) p<0.001. This was also seen when EB were exposed solely to high concentrations of FGF (KF) (Figure 1A) p<0.001. These step-wise differentiation schemes are illustrated in Supplementary Figure S1.

Given the observed PAX7 expression in differentiated hESC in the presence of SB, we examined the expression of other genes involved in myogenesis [38-41] in cells differentiated under these conditions. We also examined the requirement for SB in inducing muscle gene expression. Human ESC differentiated in serum-free KFLy(B) medium supplemented with SB (KFLy(B)S) showed an increase in PAX3 expression, marking early myogenesis, and MYOD, a transcription factor involved in the initiation of myogenesis (Figure 1B) p<0.001 compared to culture in KFly(B) in the absence of SB. Here, SB supplementation was not beneficial with respect to PAX7 expression. Human ESC differentiated in KF medium supplemented with SB (KFS) showed a strong increase in expression of PAX3, MYOD and PAX7, compared to culture in KF in the absence of SB (Figure 1B) right, p<0.05. These data indicate the stimulatory effect of SB on myogenic commitment in hESC differentiated in serum-free conditions.


Figure 1: Differentiation of human embryonic stem cells along the myogenic lineageRelative expression levels of transcripts normalized to GAPDH were determined by qRT-PCR. (A): Expression levels of PAX7 in myogenic progenitor cells derived from hESC using serum-containing methods (low= 2%HS; high= 10%FBS), hESC treated with SB in high serum, KFLy(B), and KF, relative to undifferentiated hESC. Data shown are mean±S.E.M. (N=3). *, p<0.01; **, p<0.001. (B): Expression levels of PAX3, PAX7 and MYOD in KFly(B)S and KFS compared to media without SB are shown. Data shown are mean±S.E.M. (N=3). *, p<0.05. CM, conditioned medium; FBS, fetal bovine serum; hESC; human embryonic stem cells; HS, horse serum; NCAM, neural cell adhesion molecule; SB, TGFb inhibitor SB431542.


Figure S1: Schematic overview of differentiation methods. bFGF, basic fibroblast growth factor; EB, embryoid body; hESC, human embryonic stem cells.


Myogenic differentiation in EB culture

We hypothesized that myogenic precursor cells would be more likely to engraft in host muscle, and have a greater chance of repopulating the stem cell niche, than fully differentiated myoblasts. To monitor the stages of myogenic differentiation, we cultured hESC-EB in serum-free KFLy(B)S or KFS and examined myogenic gene expression by qRT-PCR at days 2, 4, 6 and 8, relative to undifferentiated hESC (day 0). OCT4 expression was used as an indicator of residual pluripotency after initiation of hESC differentiation. In both differentiation conditions, OCT4 expression fell dramatically by day 2 and further diminished through day 6 of differentiation (Figure 2) p<0.001. To assess mesoderm lineage commitment, Brachyury expression was evaluated. Brachyury expression was maximal at days 2 and 4 in KFLy(B)S, then decreased by day 6. This drop in expression indicates that the cells can adopt a different tissue fate. The early Brachyury expression peak during differentiation follows a similar pattern shown in other studies related to mesoderm differentiation [20,24]. In here they observed that mesoderm commitment decreases after day 2 and is absent following day 6 of differentiation. EB differentiated in KFS did not show elevated levels of Brachyury expression during EB differentiation, which could suggest that cells passed through early stages of mesoderm differentiation, undetected by the sampling times chosen for these experiments. Myogenic commitment was evaluated based on PAX3, PAX7, and MYOD expression. Expression levels of the early myogenic factor, PAX3, peaked for both differentiation conditions at day 6 (p<0.001). At this time point, PAX7 expression also was at its maximum (p<0.001), however, PAX7 expression fell as differentiation proceeded, as indicated by an increase in MYOD expression (KFLy(B)S, p<0.05; KFS, p<0.001). MYOD expression levels continued to increase over the following days, whereas PAX3 and PAX7 expression declined suggesting myoblast differentiation. Taken together, these data suggested that 6 days of differentiation in KFLy(B)S or KFS was sufficient to produce myogenic precursors capable of undergoing myogenesis.

Figure 2: Myogenic commitment during 8 days of EB differentiation. Relative expression levels of transcripts were determined by qRT-PCR after differentiation day 2, 4, 6 and 8 of differentiation. Expression of indicated transcripts in hESC-derived myogenic precursors cultured in KFLy(B)S and KFS are shown relative to undifferentiated hESC. Data shown are mean±S.E.M. (N=3). **, p<0.01; *, p<0.05. hESC, human embryonic stem cells; ns, non-significant.


KFLy(B)S- and KFS-derived precursors differentiate into myoblasts in vitro

Directed differentiation of stem cells can be stimulated with exposure to specific extracellular matrix (ECM) components they would likely encounter in the stem cell niche [42]. This has specifically been shown for stem cell differentiation toward the myogenic lineage [43-45]. EB differentiated for 6 days in KFLy(B)S or KFS were plated in dishes coated with Matrigel, laminin or fibronectin to stimulate myogenic cell outgrowth. We observed that attachment to fibronectin-coated dishes, compared to Matrigel and laminin, more effectively promoted myogenic differentiation (data not shown). Therefore, fibronectin coating was used to evaluate the myogenic potential of EB-outgrowth cells (EB-O) during the subsequent 6 days in muscle differentiation medium.

Cells pre-differentiated in KFLy(B)S showed expression of PAX7, MYOD, Myogenin, MYHC and Desmin based on immunofluorescence staining (Figure 3). This was supported by upregulation of MYOD, Myogenin, and MYHC expression levels determined by qRT-PCR at EB-O day 2 and day 6 (Figure 3A) p<0.001. At this stage levels of PAX3 and PAX7 remained similar suggesting that besides terminal myogenic differentiation PAX7+ cells were likely able to continue proliferation. In KFS-differentiated EB-O day 6, large areas of PAX7+ cells were observed, with detection of Myogenin-, MYHC- and Desmin-expressing cells (Figure 3B). These PAX7+ areas were only observed in KFS-differentiated EB-O. MYOD + cells were not observed indicating that downregulation of MYOD led to terminal differentiation.

Figure 3: Determination of in vitro myogenic potential of hEB-derived myogenic precursor cellsEB-derived cells were plated onto fibronectin to stimulate myogenic outgrowth in differentiation medium. Indicated muscle-specific proteins were detected by immunofluorescence staining. Nuclei were counterstained with DAPI (blue). (A): Adherent KFLy(B)S-derived precursors display outgrowth of cells expressing PAX7, MYOD, Myogenin, Desmin and MYHC (green). Corresponding qRT-PCR data shows myogenic differentiation towards myoblasts based on elevated expression of MYOD, Myogenin and MYHC. (B): Outgrowth of KFS-derived EB showed large patches of PAX7 expressing cells. Myogenin, MYHC and Desmin were also detected, while MYOD was not detected. Corresponding qRT-PCR data supported the observation of further differentiation after EB plating and outgrowth, as expression of MYHC doubled. Data shown are mean±S.E.M.. (N=3). *, p<.0001. Scale bar= 50 mm.


hESC-derived myogenic precursors contribute to new myofiber formation in vivo

To determine the in vivo myogenic potential of hESC-derived myogenic precursors, one million single cells from dissociated EB were injected into the irradiated TA muscle of immunodeficient SCID mice. KFLy(B)S- and KFS-derived transplanted cells were detected in host muscle using a human-specific HLA-1 antibody one month after transplantation [46,47] (Figure 4). To determine whether transplanted precursor cells differentiated in situ into muscle, we performed immunofluorescence staining of serial sections with an antibody directed against Desmin, a marker of muscle intermediate filaments (Figure 4). Desmin staining confirmed that the transplanted cells underwent terminal differentiation. This was supported by observation of centrally located nuclei in the positive fibers characteristic of newly formed myofibers (Figure 4A, B). Furthermore, the newly formed myofibers were also identified by the detection of Dystrophin co-staining with HLA-1 (Figure 4C). To anatomically localize the transplanted cells, immunohistochemical analysis was performed on transverse and longitudinal cryosections of TA muscle with anti-HLA-1. Staining demonstrated that transplanted cells resided adjacent to the myofiber (Figure 5A). In sham-injected tissue, no HLA-1-positive cells were seen. Myofibers co-stained with anti-laminin to visualize the basal lamina demonstrated the juxtaposition of these cells beneath the basal lamina (Figure 5B) arrows. This finding suggests that the hESC-derived myogenic precursors take up residence within the anatomical niche that is associated with resident muscle stem cells (e.g., satellite cells).

Figure 4: Engraftment of hESC-derived myogenic precursors in transplanted TA muscle of SCID micePresence of human cells in host muscle tissue was observed at one month by immunohistochemistry using a human-specific HLA-1 antibody. (A): Detection of transplanted cells treated with KFL(B)S (HLA-1, green) and KFS (HLA-1, green; red, Laminin). (B) KFLy(B)S-treated, HLA-1+ cells are visualized in red. Muscle tissue is visualized using an anti-Desmin antibody (green). Donor-derived fiber formation shows expression of Desmin in corresponding areas (white dotted line) in serial sections indicating formation of newly formed fibers within the host muscle. Magnification of these areas identifies newly formed fibers by centrally located nuclei (arrow) within myofibers (right panel). Basal lamina was identified by anti-laminim staining (green). (C): KFL(B)S- and KFS- treated cells were visualized with HLA-1 (green). Areas containing human donor cells show expression of Desmin in corresponding areas (white dotted line). Dystrophin was visualized in red. (D): Newly formed tissue visualized by HLA-1 staining (green) showed expression of Dystrophin (red). Arrow indicated the central located nuclei in regenerative fibers. Nuclei are stained with DAPI (blue). Scale bar= 25 mm.


Figure 5: Seeding of host muscle with hESC-derived myogenic precursors after transplantation. Detection of human cells in TA muscle by immunohistochemistry using anti-HLA-1 antibody 1 month after transplantation. (A): Location of donor cells on transverse and longitudinal sections within close proximity of muscle fiber (HLA-1, red). No positive staining was observed in sections of uninjected TA muscle (right panel). Scale bar= 50 mm. (B): Localization of human donor cells (red; arrow) enclosed in basal lamina of muscle in a co-staining with LAMININ (green). Left picture is KFLy(B)S- derived myogenic precursor cells and right picture represents KFS- derived myogenic precursor cells. Nuclei are stained with DAPI (blue). Scale bar= 25 mm.


Ex vivo myomechanical analysis demonstrates functional improvement with transplantation of hESC-derived myogenic precursors into mdx hindlimb muscle

In order to evaluate changes in muscle function with transplantation of hESC-derived myogenic precursors, we performed myomechanical analysis on harvested EDL muscle from mdx mice previously injected with PBS or cells derived in KFLy(B)S or KFS media. The analysis is summarized in Table 1. There were no differences in muscle mass or muscle cross-sectional area between groups. Analysis of muscle twitch demonstrated that KFLy(B)S and KFS mice (KFLy(B)S 125±13 mN, KFS 112 ± 11 mN) generated higher maximal tension (Pt) than PBS and uninjected mice (PBS 102 ± 8 mN and 105±10, p<0.05). There were no differences in specific maximal twitch tension (sPt), contraction times (CT) or half-relaxation time (HRT) between groups. Analysis of tetanus revealed greater maximal tetanic tension (Po) in KFLy(B)S (205±65 mN) vs PBS (148.8±28 mN, p<0.05) mice. Otherwise, there were no differences in maximal tetanic tension (Po), specific maximal tetanic tension (sPo), maximal rate of rise of tetanus (MRRT), or the Pt/Po ratio between groups.

Likewise, force-frequency analysis showed greater force generation at 140Hz in EDL injected with KFLy(B)S-derived cells compared to KFS-derived cells, PBS and uninjected EDL p<0.05, (Figure 6A). However, with the low-frequency fatigue protocol, EDL injected with KFS-derived cells maintained greater force generation beginning at 6 minutes that was sustained throughout the 10 minute fatigue period compared to EDL injected with KFLy(B)S-derived cells, PBS and uninjected EDL p<0.05 (Figure 6A).

Figure 6: Ex vivo myomechanical analysis of EDL muscle from mdx mice one month after transplantation. Dissected EDL muscle was mounted on a force transducer (see Methods section). Muscle tension is expressed as mean percent maximum force (N=13 PBS; N=7 KFLy(B)S; N=8 KFS; N=5 uninjected). (A) - EDL muscles injected with hESC-derived myogenic precursors cultured in KFLy(B)S generate a significantly higher force at 140 Hz compared to EDL injected with PBS and uninjected EDL (*p<0.05). (B): Mice transplanted with hESC-derived myogenic precursors cultured in KFS showed a significantly greater maximum contraction (less fatigue) beginning at 6 min and continuing throughout the 10 min fatigue protocol compared to EDL injected with PBS and uninjected EDL (*p<0.05).


PAX3 and PAX7 are critical factors in embryonic skeletal myogenesis and participate after birth in muscle homeostasis. They are, therefore, important indicators of a myogenic precursor stage that harbors regenerative capacity. Several methods have been published describing the myogenic differentiation of hESC, however, limitations of published methods include the use of non-human serum and genetic manipulation that may hamper therapeutic application due to safety concerns [48,49]. This study underscores that myogenesis can be achieved in hESC cultures in a short time frame when exposed to appropriate developmental cues. We have demonstrated two approaches to derive PAX3- and PAX7-expressing myogenic precursors from hESC using a novel sequence of chemical compounds that direct a myogenic cell fate. Upon transplantation, these muscle precursor cells engrafted into muscle, formed new fibers, and improved muscle strength.

In the course of this study a publication revealed the success of a chemically defined culture system in transgenic zebrafish that directs skeletal myogenesis of human induced pluripotent stem cells [50]. Therefore, the combination of pathway inhibitors and stimulators described in this study provide an effective platform to advances approaches to muscle differentiation from hESC. Among the benefits are the avoidance of the use of serum and genetic alteration to promote gene expression. Furthermore, we showed that hESC entered the myogenic program in less then one-week making this approach faster than other published methods applying hESC differentiation during 2-4 weeks. Additionally, lacking selective cell culture approaches contributed to a method that is costs effective and easy.

In these studies, we show evidence of myogenic potential by detection of muscle-specific expression in vitro and engraftment and differentiation in vivo. PAX7 positive cells were easily detected in cultures demonstrating the capacity to maintain muscle precursor features. Based on this analysis, KFLy(B)S-derived cells appear to be more prone to terminal differentiation, while KFS-derived cells show characteristics of cells at a precursor stage. Aside from the relatively few cells that go on to express MHC, it could be that PAX7-positive cells incapable of differentiating are continuing to proliferate, as evidenced by large areas of PAX7-expressing cells in adherent cell cultures. Fusion of the myogenic hESC-derived myogenic precursor into myotubes in culture was not observed, however, this may be due to insufficient cell-cell contact among suitably differentiated cells. After transplantation directly into muscle, the cells localized beneath the basal lamina of newly formed and existing muscle fibers, characteristic of endogenous satellite cells. Interestingly, the transplanted cells were able to contribute to Desmin-expressing new fibers surrounded by Dystrophin, indicating mature myofiber structures.

In mdx mice, transplantation with hESC-derived precursors derived using these methods resulted in improved mechanical force generation and decreased fatigue. Both muscles injected with KFLy(B)S and KFS-derived cells demonstrated higher maximal tension than muscle injected with PBS or EDL that was not injected with cells, indicating improved functional capacity. Interestingly, EDL from mice injected with KFLy(B)S-derived cells demonstrated greater maximal tetanic tension, compared to both EDL from mice injected with PBS and KFS-derived cells and EDL that was not injected. However, force-frequency analysis demonstrated decreased fatigue in muscle from mice injected with KFS-derived cells compared to the other groups. The mechanisms that might account for these differences are unclear, but it is intriguing that the ESC-derived precursors could alter functional capacity by affecting different muscle properties (e.g strength vs endurance). Most importantly, however, the findings of the myomechanical analysis indicate preliminary proof-of-concept for functional myogenic potential for these hESC-derived precursor cells.

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We have shown that a short exposure of hESC to chemically defined media promotes differentiation of hESC into engrafting myogenic precursor cells that can contribute to the formation of new fibers in vivo and improve muscle strength as analyzed ex vivo. These techniques are less laborious than previously published methods, and the avoidance of xenobiotic agents and serum may mitigate safety concerns. Further studies to assess durability of transplanted cells and newly formed fibers, as well as their contribution to muscle homeostasis over time are warranted.

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Copyright: © 2015 Karlijn J. Wilschut, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.