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Modulating human mesenchymal stem cell plasticity using micropatterning technique.

Tijore A, Wen F, Lam CR, Tay CY, Tan LP - PLoS ONE (2014)

Bottom Line: Altogether, we designed four groups of experiments; 1) Patterned hMSCs cultured in normal growth medium serving as a positive control; 2) Patterned hMSCs cultured in normal growth medium for 14 days followed by osteogenic and adipogenic media for next 7 days (to study the robustness of the effect of micropatterning); 3) Patterned hMSCs (initially grown in normal growth medium for 14 days) trypsinized and recultured in different induction media for next 7 days (to study the robustness of the effect of micropatterning without any shape constrain) and 4) Patterned hMSCs cultured in osteogenic and adipogenic media for 14 days (to study the effects of biochemical cues versus biophysical cues).It was found that hMSCs that were primed to commit to myocardial lineage (Groups 2 and 3) were able to maintain myocardial lineage commitment despite subsequent culturing in osteogenic and adipogenic media.However, for hMSCs that were not primed (Group 4), the biochemical cues seem to dominate over the biophysical cue in modulating hMSCs differentiation.

View Article: PubMed Central - PubMed

Affiliation: Division of Materials Technology, School of Materials Science and Engineering, Nanyang Technological University, Singapore, Singapore.

ABSTRACT
In our previous work, we have reported that enforced elongation of human mesenchymal stem cells (hMSCs) through micropatterning promoted their myocardial lineage commitment. However, whether this approach is robust enough to retain the commitment when subsequently subjected to different conditions remains unsolved. This de-differentiation, if any, would have significant implication on the application of these myocardial-like hMSCs either as tissue engineered product or in stem cell therapy. Herein, we investigated the robustness of micropatterning induced differentiation by evaluating the retention of myocardial differentiation in patterned hMSCs when challenged with non-myocardial differentiation cues. Altogether, we designed four groups of experiments; 1) Patterned hMSCs cultured in normal growth medium serving as a positive control; 2) Patterned hMSCs cultured in normal growth medium for 14 days followed by osteogenic and adipogenic media for next 7 days (to study the robustness of the effect of micropatterning); 3) Patterned hMSCs (initially grown in normal growth medium for 14 days) trypsinized and recultured in different induction media for next 7 days (to study the robustness of the effect of micropatterning without any shape constrain) and 4) Patterned hMSCs cultured in osteogenic and adipogenic media for 14 days (to study the effects of biochemical cues versus biophysical cues). It was found that hMSCs that were primed to commit to myocardial lineage (Groups 2 and 3) were able to maintain myocardial lineage commitment despite subsequent culturing in osteogenic and adipogenic media. However, for hMSCs that were not primed (Group 4), the biochemical cues seem to dominate over the biophysical cue in modulating hMSCs differentiation. It demonstrates that cell shape modulation is not only capable of inducing stem cell differentiation but also ensuring the permanent lineage commitment.

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Investigation of tissue-lineage specificity of trypsinized and re-cultured hMSCs (re-cultured in osteo- and adipogenic induction medium).Trypsinized hMSCs (group 3) were re-cultured in osteogenic (a, b, e, f) and adipogenic medium (c, d, g, h) for 7 days and lineage commitment was validated by detecting the tissue-lineage specific markers; β-MHC, osteocalcin along with oil red O dye. Immunostaining results proved that trypsinized and re-cultured patterned cells exhibited cardiac MHC expression (a, c) in osteo- and adipogenic medium respectively, whereas these re-cultured patterned cells failed to express osteogenic marker (b) or to produce oil globules (d). Trypsinized and re-cultured unpatterned cells exhibited no traces of cardiac marker expression (e, g) but showed osteocalcin expression (f) and tiny oil globules formation (h). The scale bar is 100 µm.
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pone-0113043-g006: Investigation of tissue-lineage specificity of trypsinized and re-cultured hMSCs (re-cultured in osteo- and adipogenic induction medium).Trypsinized hMSCs (group 3) were re-cultured in osteogenic (a, b, e, f) and adipogenic medium (c, d, g, h) for 7 days and lineage commitment was validated by detecting the tissue-lineage specific markers; β-MHC, osteocalcin along with oil red O dye. Immunostaining results proved that trypsinized and re-cultured patterned cells exhibited cardiac MHC expression (a, c) in osteo- and adipogenic medium respectively, whereas these re-cultured patterned cells failed to express osteogenic marker (b) or to produce oil globules (d). Trypsinized and re-cultured unpatterned cells exhibited no traces of cardiac marker expression (e, g) but showed osteocalcin expression (f) and tiny oil globules formation (h). The scale bar is 100 µm.

Mentions: In the third group of experiments, we would like to answer the question of whether the effect of micropatterning on the lineage commitment is robust enough to be retained even after the cells were trypsinized and re-plated onto unpatterned surfaces. To do that, committed hMSCs grown in normal growth medium (for 14 days) were trypsinized and reseeded onto conventional fibronectin coated cover glasses for further 7 days in the presence of normal growth medium, osteogenic medium and adipogenic medium respectively. Morphology of trypsinized cells (especially trypsinized patterned cells) was taken into consideration to see whether there are any morphological changes after they were exposed to various culture media and are shown in Figure S5 in File S1. Trypsinized patterned cells exhibited an elongated morphology in all three culture media suggesting that the cells were able to ‘remember’ previously assumed shape by maintaining their muscle-like phenotype. In contrast, unpatterned cells acquired various random shapes. It clearly indicated that trypsinized patterned cells tend to retain elongated cell morphology which might be crucial for the maintenance of myocardial lineage commitment. Our observations were supported by a recent study showing that hMSCs retained mechanical memory of its former growth environment. In addition, it was shown that the transcriptional coactivators YAP and TAZ played a deterministic role in storing this information from the mechanical environment which can critically shape the stem cell’s future fate [29]. Thus, trypsinized and recultured cell samples from both patterned and unpatterned groups were assessed for β-MHC, osteocalcin expression and oil globules formation respectively to determine the lineage commitment. Remarkably, immunostaining data illustrated that trypsinized and recultured patterned cells grown in normal growth medium sustained prominent cardiac MHC expression and very weak osteocalcin expression, emphasizing the maintenance of patterned cell’s myocardial lineage commitment (Figure 5a & 5b). Yet again, unpatterned cells did not show any significant expression of cardiac MHC, but visible osteocalcin expression was observed, indicative of osteogenesis (Figure 5c & 5d). Similarly, trypsinized and recultured patterned cells grown respectively in osteogenic and adipogenic media for 7 days stained positively for cardiac MHC (Figure 6a & 6c), while no traces of osteocalcin expression and oil globules were observed in those recultured patterned cells (Figure 6b & 6d). Myocardial lineage commitment of trypsinized and recultured patterned cells grown in different media was reconfirmed with cTnT immunostaining results (Figure S6 in File S1). Trypsinized and recultured unpatterned cells grown in osteogenic and adipogenic medium respectively did not express cardiac MHC (Figure 6e & 6 g). However, these recultured unpatterned cells showed distinct osteocalcin expression and oil globule formation in osteogenic and adipogenic medium respectively, indicating that osteogenic and adipogenic lineage commitment is dependent on induction medium used (Figure 6f & 6 h).


Modulating human mesenchymal stem cell plasticity using micropatterning technique.

Tijore A, Wen F, Lam CR, Tay CY, Tan LP - PLoS ONE (2014)

Investigation of tissue-lineage specificity of trypsinized and re-cultured hMSCs (re-cultured in osteo- and adipogenic induction medium).Trypsinized hMSCs (group 3) were re-cultured in osteogenic (a, b, e, f) and adipogenic medium (c, d, g, h) for 7 days and lineage commitment was validated by detecting the tissue-lineage specific markers; β-MHC, osteocalcin along with oil red O dye. Immunostaining results proved that trypsinized and re-cultured patterned cells exhibited cardiac MHC expression (a, c) in osteo- and adipogenic medium respectively, whereas these re-cultured patterned cells failed to express osteogenic marker (b) or to produce oil globules (d). Trypsinized and re-cultured unpatterned cells exhibited no traces of cardiac marker expression (e, g) but showed osteocalcin expression (f) and tiny oil globules formation (h). The scale bar is 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4234627&req=5

pone-0113043-g006: Investigation of tissue-lineage specificity of trypsinized and re-cultured hMSCs (re-cultured in osteo- and adipogenic induction medium).Trypsinized hMSCs (group 3) were re-cultured in osteogenic (a, b, e, f) and adipogenic medium (c, d, g, h) for 7 days and lineage commitment was validated by detecting the tissue-lineage specific markers; β-MHC, osteocalcin along with oil red O dye. Immunostaining results proved that trypsinized and re-cultured patterned cells exhibited cardiac MHC expression (a, c) in osteo- and adipogenic medium respectively, whereas these re-cultured patterned cells failed to express osteogenic marker (b) or to produce oil globules (d). Trypsinized and re-cultured unpatterned cells exhibited no traces of cardiac marker expression (e, g) but showed osteocalcin expression (f) and tiny oil globules formation (h). The scale bar is 100 µm.
Mentions: In the third group of experiments, we would like to answer the question of whether the effect of micropatterning on the lineage commitment is robust enough to be retained even after the cells were trypsinized and re-plated onto unpatterned surfaces. To do that, committed hMSCs grown in normal growth medium (for 14 days) were trypsinized and reseeded onto conventional fibronectin coated cover glasses for further 7 days in the presence of normal growth medium, osteogenic medium and adipogenic medium respectively. Morphology of trypsinized cells (especially trypsinized patterned cells) was taken into consideration to see whether there are any morphological changes after they were exposed to various culture media and are shown in Figure S5 in File S1. Trypsinized patterned cells exhibited an elongated morphology in all three culture media suggesting that the cells were able to ‘remember’ previously assumed shape by maintaining their muscle-like phenotype. In contrast, unpatterned cells acquired various random shapes. It clearly indicated that trypsinized patterned cells tend to retain elongated cell morphology which might be crucial for the maintenance of myocardial lineage commitment. Our observations were supported by a recent study showing that hMSCs retained mechanical memory of its former growth environment. In addition, it was shown that the transcriptional coactivators YAP and TAZ played a deterministic role in storing this information from the mechanical environment which can critically shape the stem cell’s future fate [29]. Thus, trypsinized and recultured cell samples from both patterned and unpatterned groups were assessed for β-MHC, osteocalcin expression and oil globules formation respectively to determine the lineage commitment. Remarkably, immunostaining data illustrated that trypsinized and recultured patterned cells grown in normal growth medium sustained prominent cardiac MHC expression and very weak osteocalcin expression, emphasizing the maintenance of patterned cell’s myocardial lineage commitment (Figure 5a & 5b). Yet again, unpatterned cells did not show any significant expression of cardiac MHC, but visible osteocalcin expression was observed, indicative of osteogenesis (Figure 5c & 5d). Similarly, trypsinized and recultured patterned cells grown respectively in osteogenic and adipogenic media for 7 days stained positively for cardiac MHC (Figure 6a & 6c), while no traces of osteocalcin expression and oil globules were observed in those recultured patterned cells (Figure 6b & 6d). Myocardial lineage commitment of trypsinized and recultured patterned cells grown in different media was reconfirmed with cTnT immunostaining results (Figure S6 in File S1). Trypsinized and recultured unpatterned cells grown in osteogenic and adipogenic medium respectively did not express cardiac MHC (Figure 6e & 6 g). However, these recultured unpatterned cells showed distinct osteocalcin expression and oil globule formation in osteogenic and adipogenic medium respectively, indicating that osteogenic and adipogenic lineage commitment is dependent on induction medium used (Figure 6f & 6 h).

Bottom Line: Altogether, we designed four groups of experiments; 1) Patterned hMSCs cultured in normal growth medium serving as a positive control; 2) Patterned hMSCs cultured in normal growth medium for 14 days followed by osteogenic and adipogenic media for next 7 days (to study the robustness of the effect of micropatterning); 3) Patterned hMSCs (initially grown in normal growth medium for 14 days) trypsinized and recultured in different induction media for next 7 days (to study the robustness of the effect of micropatterning without any shape constrain) and 4) Patterned hMSCs cultured in osteogenic and adipogenic media for 14 days (to study the effects of biochemical cues versus biophysical cues).It was found that hMSCs that were primed to commit to myocardial lineage (Groups 2 and 3) were able to maintain myocardial lineage commitment despite subsequent culturing in osteogenic and adipogenic media.However, for hMSCs that were not primed (Group 4), the biochemical cues seem to dominate over the biophysical cue in modulating hMSCs differentiation.

View Article: PubMed Central - PubMed

Affiliation: Division of Materials Technology, School of Materials Science and Engineering, Nanyang Technological University, Singapore, Singapore.

ABSTRACT
In our previous work, we have reported that enforced elongation of human mesenchymal stem cells (hMSCs) through micropatterning promoted their myocardial lineage commitment. However, whether this approach is robust enough to retain the commitment when subsequently subjected to different conditions remains unsolved. This de-differentiation, if any, would have significant implication on the application of these myocardial-like hMSCs either as tissue engineered product or in stem cell therapy. Herein, we investigated the robustness of micropatterning induced differentiation by evaluating the retention of myocardial differentiation in patterned hMSCs when challenged with non-myocardial differentiation cues. Altogether, we designed four groups of experiments; 1) Patterned hMSCs cultured in normal growth medium serving as a positive control; 2) Patterned hMSCs cultured in normal growth medium for 14 days followed by osteogenic and adipogenic media for next 7 days (to study the robustness of the effect of micropatterning); 3) Patterned hMSCs (initially grown in normal growth medium for 14 days) trypsinized and recultured in different induction media for next 7 days (to study the robustness of the effect of micropatterning without any shape constrain) and 4) Patterned hMSCs cultured in osteogenic and adipogenic media for 14 days (to study the effects of biochemical cues versus biophysical cues). It was found that hMSCs that were primed to commit to myocardial lineage (Groups 2 and 3) were able to maintain myocardial lineage commitment despite subsequent culturing in osteogenic and adipogenic media. However, for hMSCs that were not primed (Group 4), the biochemical cues seem to dominate over the biophysical cue in modulating hMSCs differentiation. It demonstrates that cell shape modulation is not only capable of inducing stem cell differentiation but also ensuring the permanent lineage commitment.

Show MeSH
Related in: MedlinePlus