Cell Surface Proteins for Enrichment and In Vitro Characterization of Human Pluripotent Stem Cell-Derived Myogenic Progenitors

Abstract

Human myogenic progenitors can be derived from pluripotent stem cells (PSCs) for use in modeling natural and pathological myogenesis, as well as treating muscle diseases. Transgene-free methods of deriving myogenic progenitors from different PSC lines often produce mixed populations that are heterogeneous in myogenic differentiation potential, yet detailed and accurate characterization of human PSC-derived myogenic progenitors remains elusive in the field. The isolation and purification of human PSC-derived myogenic progenitors is thus an important methodological consideration when we investigate the properties and behaviors of these cells in culture. We previously reported a transgene-free, serum-free floating sphere culture method for the derivation of myogenic progenitors from human PSCs. In this study, we first performed comprehensive cell surface protein profiling of the sphere culture cells through the screening of 255 antibodies. Next, we used magnetic activated cell sorting and enriched the cells according to the expression of specific surface markers. The ability of muscle differentiation in the resulting cells was characterized by immunofluorescent labeling and quantification of positively stained cells. Our results revealed that myotube-forming cells resided in the differentiated cultures of CD29⁺, CD56⁺, CD271⁺, and CD15^- fractions, while thick and multinucleated myotubes were identified in the differentiated cultures from CD9⁺ and CD146⁺ fractions. We found that PAX7 localization to the nucleus correlates with myotube-forming ability in these sorted populations. We also demonstrated that cells in unsorted, CD271⁺, and CD15^- fractions responded differently to cryopreservation and prolonged culture expansion. Lastly, we showed that CD271 expression is essential for terminal differentiation of human PSC-derived myogenic progenitors. Taken together, these cell surface proteins are not only useful markers to identify unique cellular populations in human PSC-derived myogenic progenitors but also functionally important molecules that can provide valuable insight into human myogenesis.

Figure 1

Graphical abstract of antibody screening for surface marker profiling of human PSC-derived myogenic progenitors. FGF-2-treated and nontreated cells derived from human PSCs were stained with 255 antibodies against surface markers using the BD Lyoplate™ Human Cell Surface Marker Screening Panel.

Figure 2

Selection of potential surface markers to enrich human PSC-derived myogenic progenitors. (a) 33 antibodies among the 89 antibodies stained positive in FGF-2-treated cells showed noticeable different expression levels in nontreated cells: 10 showed higher expression in FGF-2-treated cells and 23 showed lower expression in FGF-2-treated cells. (b) FGF-2-treated cells stained positive for CD29 and CD56 but negative for CD6 and CD83. Pink = positive for the surface marker indicated; blue = all nuclei. Scale bar = 100 μm; enlarged image scale bar = 50 μm. (c) Higher CD271 expression was seen in FGF-2 treated cells, whereas CD15 expression was higher in nontreated cells. Scale bar = 100 μm; enlarged image scale bar = 20 μm. (d) CD9 and CD15 fit our criteria of selection that 30-70% of myogenic progenitor culture was stained positive with high intensity and clear morphology; CD61, CD49a, and CD95 did not fit the criteria. Top panel scale bar = 100 μm; bottom panel scale bar = 50 μm.

Figure 3

Myotube-forming efficiency of human iPSC-derived myogenic progenitors enriched using identified markers. (a–c) Representative ICC images of MyHC expression (stained green) in differentiated cultures of human iPSC-derived myogenic progenitors sorted using MACS. Scale bar = 50 μm. (d) Means of % MyHC⁺ cells in differentiated cultures of unsorted, positive, and negative fractions. Error bars represent SEM from three independent experiments. Statistical significance was calculated by one-way ANOVA followed by Fisher's LSD post hoc test. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. (e) Means of % MyHC⁺ cells of sorted fractions normalized by the % MyHC⁺ cells of parallel unsorted fractions. Error bars represent SEM from three independent experiments. Statistical significance was calculated by one-way ANOVA followed by Fisher's LSD post hoc test. ^∗p < 0.05, significantly different compared to unsorted fractions.

Figure 4

Characterization of myotubes formed by human iPSC-derived myogenic progenitors enriched using CD9 and CD146. (a) Representative ICC images of MyHC expression (stained green) in differentiated cultures of sorted human iPSC-derived myogenic progenitors. Scale bar = 50 μm. (b–d) Properties of myotubes formed by differentiated cultures of unsorted cells and CD9⁺ and CD9^– human iPSC-derived myogenic progenitors: means of myotube width in microns, means of % myotube with ≥3 nuclei, and means of % myotube with 1 to ≥6 nuclei. (e–g) Properties of myotubes formed by differentiated cultures of unsorted and CD146⁺ and CD146^– human iPSC-derived myogenic progenitors: means of myotube width in microns, means of % myotube with ≥3 nuclei, and means of % myotube with 1 to ≥6 nuclei. Error bars represent SEM from three independent experiments. Statistical significance was calculated by one-way ANOVA followed by Fisher's LSD post hoc test. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 5

PAX expression level and distribution pattern of human iPSC-derived myogenic progenitors enriched using identified markers. (a) Representative ICC images of PAX7 expression (stained green) in undifferentiated cells of sorted human iPSC-derived EZ spheres. Long yellow arrows indicate nuclear localization of PAX7. Pink arrowheads indicate cytoplasmic distribution of PAX7. Scale bar = 20 μm. (b) Means of % PAX7⁺ cells in undifferentiated cultures of sorted fractions. Error bars represent SEM from two independent experiments. No statistical significance as determined by one-way ANOVA. (c) Means of % cells with PAX7 expression localized in the nuclei and % cells with PAX7 expression scattered across the cytoplasm among PAX7⁺ cells in undifferentiated cultures of sorted fractions. Error bars represent SEM from two independent experiments. Statistical significance was calculated by one-way ANOVA followed by Bonferroni's post hoc test for pairwise comparisons. ^∗∗p < 0.01, ^∗∗∗p < 0.001. (d) Means of % nuclear PAX7⁺ cells in undifferentiated cultures of sorted fractions. Error bars represent SEM from two independent experiments. Statistical significance was calculated by one-way ANOVA followed by Bonferroni's post hoc test for pairwise comparisons. ^∗p < 0.05, ^∗∗p < 0.01. (e) Adapted from data in Figure 3(d), means of % MyHC⁺ cells in differentiated cultures of sorted fractions. Error bars represent SEM from three independent experiments. Statistical significance for pairwise comparisons was calculated by unpaired two-tailed Student's t-test. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 6

Purification of human iPSC-derived myogenic progenitors altered culture resilience to cryopreservation and expansion. (a) Measurements and formulas to calculate volume of each sphere. (b) Growth rate of sphere cultures prepared from cells in unsorted, CD271⁺, and CD15^– fractions after cryopreservation and culture expansion, presented as relative change in volume over 9 days in expansion medium. Error bars represent SEM from three independent experiments. Values on day 9 were analyzed by one-way ANOVA followed by Dunnett's post hoc test. ^∗∗∗p < 0.001. (c) Means of % MyHC⁺ cells in differentiated cultures prepared from unsorted, CD271⁺, and CD15^– spheres after cryopreservation and expansion. Error bars represent SEM from three independent experiments. No statistical significance as calculated by one-way ANOVA. (d) Representative ICC images of MyHC expression (stained green) corresponding to (c). Top panel scale bar = 100 μm; bottom panel scale bar = 50 μm.

Figure 7

Knockdown of CD271 in human PSC-derived myogenic progenitors reduced and impaired myotube-forming ability. (a) Western blot analysis showed successful knockdown of CD271 levels after treatment with CD271 siRNA #1 and #2 compared to scrambled as a negative control. This antibody clone might recognize another posttranslational fragment, hence the faint bands in the CD271 blotting. Beta-actin served as the loading control. (b) Means of % MyHC⁺ cells in differentiated cultures prepared from cells treated with CD271 siRNA scrambled (negative control) #1 or #2. Error bars represent SEM from 6 analyzed images. Statistical significance was calculated by one-way ANOVA followed by Dunnett's post hoc test. ^∗∗∗p < 0.001. (c) Representative immunocytochemistry images of MyHC expression (stained green) corresponding to (b). Scale bar = 20 μm.