Insulin-like growth factor binding protein-6 (IGFBP-6), the main regulator of insulin-like growth factor-2 (IGF-2), is a component of the stem cell niche in developing muscle cells. However, its role in muscle development has not been clearly defined. In this study, we investigated the role of IGFBP-6 in muscle commitment and differentiation of human mesenchymal stem cells derived from the placenta. We showed that placental mesenchymal stem cells (PMSCs) have the ability to differentiate into muscle cells when exposed to a specific culture medium by expressing muscle markers Pax3/7, MyoD, myogenin, and myosin heavy chain in a stage-dependent manner with the ultimate formation of multinucleated fibers and losing pluripotency-associated markers, OCT4 and SOX2. The addition of IGFBP-6 significantly increased pluripotency-associated markers as well as muscle differentiation markers at earlier time points, but the latter decreased with time. On the other hand, silencing IGFBP-6 decreased both pluripotent and differentiation markers at early time points. The levels of these markers increased as IGFBP-6 levels were restored. These findings indicate that IGFBP-6 influences MSC pluripotency and myogenic differentiation, with more prominent effects observed at the beginning of the differentiation process before muscle commitment.
Unlike embryonic stem cells which are derived from the early embryo, placental mesenchymal stem cells (PMSCs) are derived from human placentae that are usually discarded following delivery, and therefore a readily available and noncontroversial source of adult stem cells for possible use in tissue regenerative therapies in human patients [
Skeletal muscle development is a highly coordinated stepwise process utilizing a series of transcriptional factors, and structural and enzymatic proteins expressed to mark the different stages of skeletal muscle development. During myogenesis, committed progenitors differentiate into muscle lineage by upregulating the myogenic regulatory factors (MRFs) as well as muscle commitment transcription factors (Pax3 and Pax7), followed by the expression of early muscle cell markers (MyoD and myogenin) [
Mesenchymal stem cells isolated from bone marrow have the ability to differentiate into myocytes [
The insulin-like growth factor (IGF) family of peptides regulates cell growth, differentiation, and the maintenance of cell survival through several signal transduction pathways [
The IGF family has been shown previously to play a major role in muscle development. IGF-1R knockout mice die soon after birth due to breathing difficulties due to lack of functional respiratory muscles [
To our knowledge, the biological roles of IGFBP-6 in the differentiation of stem cells into the muscle lineage have not been reported. In this study, we determined if PMSCs can differentiate into skeletal muscle when exposed to muscle differentiation promoting conditions and then characterized the effects of IGFBP-6 on the differentiation of PMSCs into skeletal muscle.
PMSC isolation and experiments were conducted in accordance with the approval from the Health Sciences Research Ethics Board of Western University. Informed consent was obtained from healthy women undergoing therapeutic termination of pregnancy, and the PMSCs used in this study were isolated from 15 weeks preterm placental tissues. After surgery, chorionic villi were dissected, washed, minced with surgical scissors and forceps, and then subjected to enzymatic digestion with collagenase IV (369 IU/mg), hyaluronidase (999 IU/mg) (Sigma-Aldrich, Oakville, ON), and DNase I (2000 IU/mg) (Hoffmann-LaRoche, Mississauga, ON) for 10 minutes at room temperature, followed by 0.05% trypsin (Gibco/Invitrogen, Mississauga, ON) for 5 minutes at room temperature. The sample was then washed for 10 minutes with 10% FBS in DMEM/F12 medium, and the resulting single cell suspension was separated by density centrifugation over a Percoll (Sigma-Aldrich, Oakville, ON) discontinuous gradient using a modified protocol by Worton et al. [
Cells from Percoll gradient fractions number 3 and number 4 were plated on to T75 flasks, cultured, and maintained using DMEM/F12 media supplemented with 15% FBS serum (Gibco/Invitrogen, Mississauga, ON) and FGF-2 (50 ng/mL) (Sigma-Aldrich, Oakville, ON) containing 100 U/mL penicillin, 100
Cells were trypsinized for 10 minutes using recombinant trypsin (TrypLE EXpress, Gibco/Invitrogen, Mississauga, ON) diluted 1 : 1 in PBS, at 37°C. After the cells were detached from the flask, trypsin was neutralized with 10% FBS in DMEMF/12 medium, cells were washed and incubated for one hour with fluorochrome-labeled primary antibody against MSC markers. CD73 (number 550256) (BD Pharmingen, San Jose, CA), PE-conjugated CD105 (number 12-1057-73) (eBioscience, San Diego, CA), and CD-117/c-Kit (sc-13508) (Santa Cruz Biotechnology, Dallas, TX) were used (Supplementary Figure
Cells were plated in the presence of the muscle growth media (fetal bovine serum 0.05 mL/mL, fetuin 50
Recombinant Human IGFBP-6 (ProSpec, East Brunswick, NJ) was resuspended in sterile MilliQ-H2O and added to the media at a concentration of 375 ng/mL. IGFBP-6 was added every 3 days at the time of media change. IGFBP-6 concentration was determined by a dose-response experiment using PMSCs in muscle differentiation media (Supplementary Figure
To silence the endogenous IGFBP-6 expression, IGFBP6 siRNA (h) with a pool of 3 target-specific 19–25 nt siRNAs was used (Santa Cruz Biotechnology, Dallas, TX). 8
PMSCs were grown and differentiated on glass cover slips, stained with primary antibodies, and incubated at 4°C overnight. The primary antibodies were washed using 0.1% Tween-20 in PBS (3 times for 5 minutes), and cells were then incubated in the dark with the secondary antibody. The secondary antibody was washed 0.1% Tween-20 in PBS, and the nuclear stain was added for 7 minutes and then rinsed. The cover slips were mounted for 2 hours, and images were taken using a Zeiss confocal microscope. Each antibody was performed in triplicate.
Following experiment completion, each cell lysate containing 20
Human IGFBP-6 (RayBiotech®, Burlington, ON) and IGF-2 (ALPCO, Salem, NH) ELISA kits were used to measure the amount of IGFBP-6 and IGF-2 secreted into the media of different treatment conditions. Standards and samples were loaded into the wells and the immobilized antibody bound IGFBP-6 or IGF-2 present in the sample. The wells were washed, and biotinylated anti-human antibody was added. After washing, HRP-conjugated streptavidin was added; then a TMB substrate solution was used to develop a blue color in proportion to the amount of IGFBP-6 or IGF-2 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color was measured at 450 nm using Multiskan Ascent analysis software.
PMSC ALDH activity was assessed by flow cytometry. An Aldefluor™ kit (Stem Cell Technologies, Vancouver, BC) was used as per the manufacturer’s protocol. 5
For pluripotency markers, we used OCT4 antibody (N-19: sc-8628) (Santa Cruz Biotechnology, Dallas, TX), and SOX2 (2683-1) (Epitomics, Burlington, ON, CAN). For muscle differentiation markers, Pax3/7 (E-10:sc365613), MyoD (M-318: sc-760), myogenin (F5D: sc-12732), and myosin heavy chain (H-300: sc-20641) (Santa Cruz Biotechnology, Dallas, TX) were used. For loading control, pan-actin Ab-5 (Thermo Fisher Scientific, Fremont, CA) was used. For IGFBP-6, antibody (H-70: sc-13094) (Santa Cruz Biotechnology, Dallas, TX) was used. The secondary antibodies used for immunoblotting were goat anti-rabbit (number 170-6515) or anti-mouse (number 170-6516) HRP-conjugated antibodies (Bio-Rad, Hercules, CA), or donkey anti-goat antibody (Santa Cruz Biotechnology, Dallas, TX). The secondary antibodies used for immunocytochemistry were green-Alexa 488 or red-Alexa 568 (Invitrogen, Mississauga, ON).
All experiments were run in triplicates, and the specific protein levels were quantified and normalized for loading with the level of pan-actin in each lane. GraphPad Prism Software 5.0 was used to generate all graphs and analyses. A two-way ANOVA followed by a Bonferroni’s multiple comparison test or a one-way ANOVA followed by a Student
To determine if PMSCs can differentiate into skeletal muscle, PMSCs were grown under muscle differentiation conditions for up to 14 days. Compared to PMSCs grown in nondifferentiating conditions (10% FBS), differentiated PMSCs showed muscle morphology as early as day 1 postdifferentiation (compaction and elongated appearance) (Figures
PMSCs cultured under muscle differentiation conditions showed muscle morphology with lower OCT4 and higher MHC at 14 days postdifferentiation. (a, b) Compared to cells cultured under standard conditions in 10% FBS, PMSCs grown in muscle differentiation media showed skeletal muscle morphology as early as day 1 postdifferentiation (20x). (c, d) At 14 days postdifferentiation, PMSCs grown in muscle differentiation media showed increased skeletal muscle fiber compaction and the formation of multinucleated fibers (20x). 40x magnification is shown in the bottom right corner. Red arrows indicate multinucleated muscle cells. (e–h) Cells grown in muscle differentiation media showed less OCT4 (green-Alexa 488,
Under muscle differentiation conditions, PMSCs decreased pluripotency-associated protein levels of OCT4 and SOX2. OCT4 levels were reduced at day 1 compared to control with further decrease at 14 days postdifferentiation (Figure
PMSCs cultured under muscle differentiation conditions increased levels of muscle markers (Pax3/7, MyoD, MyoG, and MHC) and decreased OCT4 and SOX2 levels using Western blots. Compared to PMSCs cultured under control conditions, (a) OCT4 was decreased at 1 and 14 days under muscle differentiation conditions. (b) SOX2 levels were decreased at each time point under muscle differentiation conditions. (c) Pax3/7 was increased at day 7 and decreased by day 14 under muscle differentiation conditions. (d) MyoD, (e) MyoG, and (f) MHC were increased at each time point under muscle differentiation conditions. Protein levels were quantified by densitometry and normalized to
We used the Aldefluor assay to determine the frequency of primitive progenitor cells with high ALDH activity. In this context, high ALDH activity is a conserved characteristic of proliferative progenitor cells of multiple lineages [
PMSCs cultured under skeletal muscle differentiation conditions showed a decreased frequency of cells with high ALDH activity. Compared to PMSCs cultured under control conditions, PMSCs cultured under differentiated conditions showed significantly decreased frequency of cells with high ALDH activity. Even under control culture conditions, PMSCs showed diminished ALDH-activity over time. Data is presented as the mean ± SEM of 3 independent experiments. Two-way ANOVA with Bonferroni’s multiple comparison test was performed to determine
PMSCs under skeletal muscle differentiation conditions were investigated if they expressed IGFBP-6. Using immunocytochemistry, PMSCs cultured under differentiation conditions showed high intracellular IGFBP-6 immunoreactivity compared to PMSCs cultured under control conditions (Figures
PMSCs cultured under skeletal muscle differentiation conditions showed increased IGFBP-6 expression and secretion. (a, b) PMSCs cultured under muscle differentiation conditions showed higher IGFBP-6 staining (red-Alexa,
To test the effects of extracellular IGFBP-6 on developing muscle cells, recombinant human IGFBP-6 was added to the muscle differentiation media. Addition of extracellular IGFBP-6 into the culture media increased intracellular IGFBP-6 detection by Western blots, suggesting that recombinant human IGFBP-6 induced a positive feedback effect or was taken up by the differentiating cells (Figure
PMSCs treated with IGFBP-6 increased pluripotency-associated and muscle differentiation markers. IGFBP-6, OCT4, SOX2, Pax3/7, MyoD, MyoG, and MHC protein levels were quantified within PMSCs grown in muscle differentiation media with or without IGFBP-6 (375 ng/mL) supplementation using Western blots. (a) IGFBP-6 treatment increased IGFBP-6 levels as compared to PMSCs grown in muscle differentiation media only. IGFBP-6 treatment also increased pluripotency-associated markers (b) OCT4 and (c) SOX2 levels. (d) IGFBP-6 treatment increased muscle lineage commitment marker Pax3/7 at each time point. Muscle differentiation markers (e) MyoD, (f) MyoG, and (g) MHC levels increased with IGFBP-6 treatment at early time points (1–3 days) but showed reduced levels at later time points (7–14 days). Protein levels were quantified by densitometry and normalized to
Finally, IGFBP-6 treatment increased the levels of muscle-specific markers, MyoD, MyoG, and MHC, at the earlier time points with a decline over time in the prolonged presence of increased extracellular IGFBP-6 compared to unsupplemented muscle differentiation conditions (Figures
Due to the fact that both pluripotency-associated and differentiation markers increased by IGFBP-6 treatment in a time-dependent manner, we tested the cells for ALDH activity to determine the frequency of PMSCs that maintained high ALDH progenitor phenotype. PMSCs under muscle differentiation in the presence of IGFBP-6 increased ALDH activity compared to PMSCs under muscle differentiation alone at days 1 to 14 (Figure
IGFBP-6 treatment increased the frequency of PMSCs with high ALDH activity. At days 1, 3, and 7, PMSCs treated with IGFBP-6 showed increased frequency of cells with high ALDH-activity. Data is presented as the mean ± SEM of 3 independent experiments. Two-way ANOVA with Bonferroni’s multiple comparison test was performed to determine
PMSCs under muscle differentiation conditions treated with IGFBP-6 showed more muscle compaction with lower cell count and similar immunofluorescence for MHC compared to cells under muscle differentiation alone at 14 days. Compared to PMSCs cultured under muscle differentiation alone, PMSCs treated with IGFBP-6 showed high cell compaction at day 14 (10x) (a, b) and (c, d) 20x. (e, f) Using immunocytochemistry, PMSCs were immunoreactive for MHC (red-Alexa,
To evaluate the effects of IGFBP-6 silencing on pluripotency-associated and muscle differentiation markers in PMSCs, IGFBP-6 knockdown by siRNA was used during muscle differentiation over 7 days. As predicted, PMSC expression of IGFBP-6 was decreased for 1-2 days after IGFBP-6 knockdown compared to scrambled siRNA control. However, IGFBP-6 levels were equivalent to scrambled controls by day 3. Readministration of IGFBP-6 siRNA at day 3 prolonged IGFBP-6 reduction, but IGFBP-6 returned to control levels by day 6 (Figure
IGFBP-6 knockdown in PMSCs under muscle differentiation conditions reduced muscle markers protein levels. PMSCs were treated with siRNAs against IGFBP-6 or scrambled siRNA control every 3 days in muscle differentiation media. Using Western blots, (a) IGFBP-6 levels were significantly decreased at 1 and 2 days after siRNA treatment but recovered to control levels at day 3. When IGFBP-6 siRNA was reintroduced at day 3, there was a significant decrease for up to 5 days which returned to control levels at day 6. IGFBP-6 siRNA treatment decreased (b) OCT4 and (c) SOX2 levels at the early time points then returned to control levels at day 5. In contrast, muscle cell commitment marker (d) Pax3/7 was increased at days 1 and 2 when IGFBP-6 was knocked down. Muscle differentiation markers: (e) MyoD, (f) MyoG, and (g) MHC levels were decreased at early time points but recovered to control levels by day 5. Protein levels were quantified by densitometry and normalized to
As both pluripotency-associated and differentiation markers were decreased with IGFBP-6 silencing, ALDH activity was determined. Silencing of endogenous IGFBP-6 expression in PMSCs, decreased ALDH activity compared to control (scrambled siRNA) in a time-dependent manner (Figure
IGFBP-6 siRNA in PMSCs cultured under muscle differentiation conditions decreased the frequency of cells with high ALDH activity. PMSCs treated with IGFBP-6 siRNA showed significantly reduced frequency of cells with high ALDH activity. Data is presented as the mean ± SEM of 3 independent experiments. Two-way ANOVA with Bonferroni’s multiple comparison test was performed to determine
IGFBP-6 siRNA treatment maintained PMSCs cell morphology and inhibited IGFBP-6 secretion. PMSC skeletal muscle morphology was maintained for 7 days under muscle lineage differentiation conditions with (a) scrambled siRNA or (b) IGFBP-6 siRNA treatment. (c) Using ELISA, IGFBP-6 secretion was decreased at each time point with IGFBP-6 siRNA treatment that was applied every 3 days. Data is presented as the mean ± SEM of 3 independent experiments. Two-way ANOVA with Bonferroni’s multiple comparison test was performed to determine
Stem cell research has progressed in recent years, and the promise of using stem cells in tissue regeneration and cellular therapies are closer to becoming a reality in the clinics [
To be able to use stem cells to treat Duchenne muscular dystrophy and to be approved for clinical trials, cells need to be from a readily available source, maintain the muscle differentiated state, avoid immune rejection by the host, avoid tumorigenesis, and can be easily injected. Human placental mesenchymal stem cells achieve these criteria.
The human placenta is usually discarded tissue after birth and represents a rich source of adult mesenchymal stem cells for the development of tissue regeneration therapies [
The IGF family plays a central role in muscle development, differentiation, growth, and regeneration [
The aim of this study was to characterize the effects of IGFBP-6 on the early differentiation stage before PMSCs commit to the muscle lineage. As shown, when PMSCs were cultured under muscle differentiation-specific conditions, they showed the capacity to differentiate into multinucleated muscle fibers and commit to the muscle lineage. The biological effects of IGFBP-6 on this differentiation process, as determined by pluripotency-associated markers (OCT4 and SOX2), muscle commitment (Pax3/7), and differentiation (MyoD, MyoG, and MHC), were significantly changed at the earlier time points. Thus, IGFBP-6 induced muscle differentiation and could potentially be used to guide skeletal muscle regeneration using stem cell therapy.
IGFBP-6 was highly expressed in developing muscle cells [
The findings in this current study using PMSCs suggest that stem cells in the developing myotome or MSCs in a developed muscle tissue express IGFBP-6 in significant levels during differentiation, indicating IGFBP-6 as an integral protein during muscle development. In fact, as muscle differentiation progressed
When extracellular IGFBP-6 was supplemented into PMSC cultured under muscle differentiation conditions, the muscle commitment marker Pax3/7 was increased at all time points of study, while other muscle differentiation markers increased only at the earlier time points. As the differentiation progressed, IGFBP-6 treatment inhibited complete myogenic differentiation as demonstrated by decreased muscle differentiation markers MyoD, MyoG, and MHC. These findings together with the higher OCT4 and SOX2 levels indicate that IGFBP-6 promotes the commitment of PMSCs towards the muscle lineage, while the prolonged presence delays the differentiation process. Moreover, increased IGFBP-6 in the MSC microenvironment is expected to reduce the bioavailability of IGF-2 due to its high affinity for the peptide, confirmed by IGF-2 ELISA (Supplementary Figure
Knockdown of IGFBP-6 using siRNA decreased both intracellular and secreted IGFBP-6. This knockdown-mediated decrease in OCT4 and SOX2 supports the evidence that IGFBP-6 enhances pluripotency of PMSCs. In contrast, the significant early increase in Pax3/7 with IGFBP-6 silencing supports an earlier commitment towards the myogenic lineage. The increase in Pax3/7 could be due to the presence of a greater availability of extracellular IGF-2 (Supplementary Figure
Studies in various cell lines have shown mostly an inhibitory action of IGFBP-6 mainly via IGF-2-dependent actions. In L6A1 myoblast, IGFBP-6 inhibited muscle differentiation induced by IGF-2 but not IGF-1 [
Overall, we have demonstrated in this study that IGFBP-6 has both endogenous and exogenous actions that can promote or inhibit PMSC multipotency or differentiation. Exogenous IGFBP-6 exposure facilitates muscle lineage commitment while a prolonged exposure can inhibit late stage differentiation. Therefore, endogenous IGFBP-6 is required for maintaining multipotency and delaying commitment and enhancing late stage differentiation.
In conclusion, PMSCs are able to differentiate into skeletal muscle cells under appropriate environment or niche conditions and that this process is enhanced by the increase in extracellular IGFBP-6 and delayed by silencing the endogenous expression as evident by alterations in both pluripotent and muscle differentiation markers (Figure
Model of IGFBP-6 functions on PMSC differentiation into skeletal muscle. PMSCs under normal growth conditions (10% FBS) express high levels of pluripotency-associated markers OCT4 and SOX2. As these cells commit towards the skeletal muscle lineage, increased IGFBP-6 correlated with increased Pax3/7 that decreased as differentiation markers (MyoG, MyoD, and MHC) were increased. Both committed and differentiated muscle cells continued to express and secrete IGFBP-6. As IGFBP-6 increased, there was an increase in multipotency markers, as well as, an earlier commitment and differentiation towards the muscle lineage. Thus, IGFBP-6 was required for maintaining multipotency and enhancing muscle commitment and differentiation.
Some data in this paper has been presented as a poster at the Endocrine Society’s 96th Annual Meeting and Expo. Data was also submitted as part of a PhD thesis [
The authors declare that they have no conflicts of interest.
This work was supported by CIHR Grant 111024.
Supplementary Figure 1: PMSC Isolation from 15 weeks preterm placenta. (A) The dissected villous tissue was digested enzymatically and cells were separated using a discontinuous Percoll gradient. Five cell fractions were typically obtained corresponding to five different densities and cells were isolated from layers 3, 4, and 5. (B) Phase contrast images of the isolated PMSCs, from all three layers, grown in culture after 4 weeks. (C) PMSCs from passage 4 of all three layers were positive for CD73 and CD105 (>98%), and were negative for CD117 (<1%) (measured by flow cytometry). Flow cytometry histograms are representative of all 3 layers from 3 placental tissue as they showed the same results. Supplementary Figure 2: IGFBP-6 levels in response to IGFBP-6 supplementation in PMSCs under skeletal muscle differentiation conditions. (A) PMSCs cultured under muscle differentiation conditions showed increased IGFBP-6 protein levels, using western blots, in response to different doses of recombinant human IGFBP-6 protein supplementation with 375 ng/mL and 450 ng/mL having the highest band intensity. (B) IGFBP-6 secretion into the media was increased with the supplementation of recombinant human IGFBP-6 protein (375 ng/mL) that reduced by time and was lower compared to control at day 3. Data is presented as the mean ± SEM of 3 independent experiments. Two-way ANOVA with Bonferroni’s multiple comparison test was performed to determine