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Three-dimensional poly-(ε-caprolactone) nanofibrous scaffolds directly promote the cardiomyocyte differentiation of murine-induced pluripotent stem cells through Wnt/β-catenin signaling.

Chen Y, Zeng D, Ding L, Li XL, Liu XT, Li WJ, Wei T, Yan S, Xie JH, Wei L, Zheng QS - BMC Cell Biol. (2015)

Bottom Line: Furthermore, early inhibition of Wnt/β-catenin signaling by the selective antagonist Dickkopf-1 significantly reduced the activity of Wnt/β-catenin signaling and decreased the cardiomyocyte differentiation of miPSCs cultured on the 3D PCL nanofibrous scaffold, while the early activation of Wnt/β-catenin signaling by CHIR99021 further increased the cardiomyocyte differentiation of miPSCs.These results indicated that the electrospun 3D PCL nanofibrous scaffolds directly promoted the cardiomyocyte differentiation of miPSCs, which was mediated by the activation of the Wnt/β-catenin signaling during the early period of differentiation.These findings highlighted the biophysical role of 3D nanofibrous scaffolds during the cardiomyocyte differentiation of miPSCs and revealed its underlying mechanism involving Wnt/β-catenin signaling, which will be helpful in guiding future stem cell- and scaffold-based myocardium bioengineering.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Tangdu Hospital, Fourth Military Medical University, 1 Xinsi Road, Xi'an, 710038, China.

ABSTRACT

Background: Environmental factors are important for stem cell lineage specification, and increasing evidence indicates that the nanoscale geometry/topography of the extracellular matrix (ECM) directs stem cell fate. Recently, many three-dimensional (3D) biomimetic nanofibrous scaffolds resembling many characteristics of the native ECM have been used in stem cell-based myocardial tissue engineering. However, the biophysical role and underlying mechanism of 3D nanofibrous scaffolds in cardiomyocyte differentiation of induced pluripotent stem cells (iPSCs) remain unclear.

Results: Here, we fabricated a 3D poly-(ε-caprolactone) (PCL) nanofibrous scaffold using the electrospinning method and verified its nanotopography and porous structure by scanning electron microscopy. We seeded murine iPSCs (miPSCs) directly on the 3D PCL nanofibrous scaffold and initiated non-directed, spontaneous differentiation using the monolayer method. After the 3D PCL nanofibrous scaffold was gelatin coated, it was suitable for monolayer miPSC cultivation and cardiomyocyte differentiation. At day 15 of differentiation, miPSCs differentiated into functional cardiomyocytes on the 3D PCL nanofibrous scaffold as evidenced by positive immunostaining of cardiac-specific proteins including cardiac troponin T (cTnT) and myosin light chain 2a (MLC2a). In addition, flow cytometric analysis of cTnT-positive cells and cardiac-specific gene and protein expression of cTnT and sarcomeric alpha actinin (α-actinin) demonstrated that the cardiomyocyte differentiation of miPSCs was more efficient on the 3D PCL nanofibrous scaffold than on normal tissue culture plates (TCPs). Furthermore, early inhibition of Wnt/β-catenin signaling by the selective antagonist Dickkopf-1 significantly reduced the activity of Wnt/β-catenin signaling and decreased the cardiomyocyte differentiation of miPSCs cultured on the 3D PCL nanofibrous scaffold, while the early activation of Wnt/β-catenin signaling by CHIR99021 further increased the cardiomyocyte differentiation of miPSCs.

Conclusion: These results indicated that the electrospun 3D PCL nanofibrous scaffolds directly promoted the cardiomyocyte differentiation of miPSCs, which was mediated by the activation of the Wnt/β-catenin signaling during the early period of differentiation. These findings highlighted the biophysical role of 3D nanofibrous scaffolds during the cardiomyocyte differentiation of miPSCs and revealed its underlying mechanism involving Wnt/β-catenin signaling, which will be helpful in guiding future stem cell- and scaffold-based myocardium bioengineering.

No MeSH data available.


Spontaneous cardiac differentiation of monolayer cultured miPSCs on 3D PCL nanofibrous scaffolds. a Schematic of the protocol for the spontaneous CM differentiation of Oct4-GFP+ miPSC monolayers cultured without MEF feeder layers in vitro. b SEM images of the cell morphologies of purified miPSCs on gelatin-coated 3D PCL nanofibrous scaffolds (top) and in gelatin-coated TCPs (bottom) after 1 day of monolayer culture. 5000× magnification; scale bar, 10.0 μm. c Images of the spontaneous differentiation of miPSC monolayers cultured on 3D PCL nanofibrous scaffolds and in TCPs at different time points; scale bars, 100 μm. d RT-PCR analysis of gene expression of spontaneously differentiated miPSC monolayer cells over 15 days of differentiation on 3D PCL nanofibrous scaffolds. e Immunofluorescence images acquired on day 15 of spontaneous differentiation on 3D PCL nanofibrous scaffolds identifying both cTnT and MLC2a with DAPI for nuclei; scale bars, 100 μm
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Fig3: Spontaneous cardiac differentiation of monolayer cultured miPSCs on 3D PCL nanofibrous scaffolds. a Schematic of the protocol for the spontaneous CM differentiation of Oct4-GFP+ miPSC monolayers cultured without MEF feeder layers in vitro. b SEM images of the cell morphologies of purified miPSCs on gelatin-coated 3D PCL nanofibrous scaffolds (top) and in gelatin-coated TCPs (bottom) after 1 day of monolayer culture. 5000× magnification; scale bar, 10.0 μm. c Images of the spontaneous differentiation of miPSC monolayers cultured on 3D PCL nanofibrous scaffolds and in TCPs at different time points; scale bars, 100 μm. d RT-PCR analysis of gene expression of spontaneously differentiated miPSC monolayer cells over 15 days of differentiation on 3D PCL nanofibrous scaffolds. e Immunofluorescence images acquired on day 15 of spontaneous differentiation on 3D PCL nanofibrous scaffolds identifying both cTnT and MLC2a with DAPI for nuclei; scale bars, 100 μm

Mentions: To explore the influence of the nanofibrous topography and porous structure on the CM commitment of miPSCs, the purified Oct4-GFP+ miPSCs without MEFs were seeded on the 3D PCL nanofibrous scaffold and in TCPs for CM spontaneous differentiation using the monolayer culture method. The cell culture and differentiation protocol is presented in Fig. 3a. The miPSC morphology, as well as the interactions with the PCL nanofibers or with the surface of the TCPs, was captured by SEM, which revealed the morphological differences between the two groups (Fig. 3b). A more rounded and cilium-like structure was observed with the miPSCs cultured on 3D PCL nanofibrous scaffolds (Fig. 3b, top), while many pseudopodium-like structures with irregular cell edges were observed with the miPSCs cultured in TCPs (Fig. 3b Bottom). Furthermore, dynamic changes in GFP fluorescence and in cell morphology were observed during the spontaneous differentiation period. During differentiation, Oct4-GFP+ expression decreased gradually in both groups, which indicated the loss of pluripotency and the progression of differentiation (Fig. 3c). At day 10 of differentiation, floating cells and debris were also found in the media, as shown in images of D10 in Fig. 3c.Fig. 3


Three-dimensional poly-(ε-caprolactone) nanofibrous scaffolds directly promote the cardiomyocyte differentiation of murine-induced pluripotent stem cells through Wnt/β-catenin signaling.

Chen Y, Zeng D, Ding L, Li XL, Liu XT, Li WJ, Wei T, Yan S, Xie JH, Wei L, Zheng QS - BMC Cell Biol. (2015)

Spontaneous cardiac differentiation of monolayer cultured miPSCs on 3D PCL nanofibrous scaffolds. a Schematic of the protocol for the spontaneous CM differentiation of Oct4-GFP+ miPSC monolayers cultured without MEF feeder layers in vitro. b SEM images of the cell morphologies of purified miPSCs on gelatin-coated 3D PCL nanofibrous scaffolds (top) and in gelatin-coated TCPs (bottom) after 1 day of monolayer culture. 5000× magnification; scale bar, 10.0 μm. c Images of the spontaneous differentiation of miPSC monolayers cultured on 3D PCL nanofibrous scaffolds and in TCPs at different time points; scale bars, 100 μm. d RT-PCR analysis of gene expression of spontaneously differentiated miPSC monolayer cells over 15 days of differentiation on 3D PCL nanofibrous scaffolds. e Immunofluorescence images acquired on day 15 of spontaneous differentiation on 3D PCL nanofibrous scaffolds identifying both cTnT and MLC2a with DAPI for nuclei; scale bars, 100 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig3: Spontaneous cardiac differentiation of monolayer cultured miPSCs on 3D PCL nanofibrous scaffolds. a Schematic of the protocol for the spontaneous CM differentiation of Oct4-GFP+ miPSC monolayers cultured without MEF feeder layers in vitro. b SEM images of the cell morphologies of purified miPSCs on gelatin-coated 3D PCL nanofibrous scaffolds (top) and in gelatin-coated TCPs (bottom) after 1 day of monolayer culture. 5000× magnification; scale bar, 10.0 μm. c Images of the spontaneous differentiation of miPSC monolayers cultured on 3D PCL nanofibrous scaffolds and in TCPs at different time points; scale bars, 100 μm. d RT-PCR analysis of gene expression of spontaneously differentiated miPSC monolayer cells over 15 days of differentiation on 3D PCL nanofibrous scaffolds. e Immunofluorescence images acquired on day 15 of spontaneous differentiation on 3D PCL nanofibrous scaffolds identifying both cTnT and MLC2a with DAPI for nuclei; scale bars, 100 μm
Mentions: To explore the influence of the nanofibrous topography and porous structure on the CM commitment of miPSCs, the purified Oct4-GFP+ miPSCs without MEFs were seeded on the 3D PCL nanofibrous scaffold and in TCPs for CM spontaneous differentiation using the monolayer culture method. The cell culture and differentiation protocol is presented in Fig. 3a. The miPSC morphology, as well as the interactions with the PCL nanofibers or with the surface of the TCPs, was captured by SEM, which revealed the morphological differences between the two groups (Fig. 3b). A more rounded and cilium-like structure was observed with the miPSCs cultured on 3D PCL nanofibrous scaffolds (Fig. 3b, top), while many pseudopodium-like structures with irregular cell edges were observed with the miPSCs cultured in TCPs (Fig. 3b Bottom). Furthermore, dynamic changes in GFP fluorescence and in cell morphology were observed during the spontaneous differentiation period. During differentiation, Oct4-GFP+ expression decreased gradually in both groups, which indicated the loss of pluripotency and the progression of differentiation (Fig. 3c). At day 10 of differentiation, floating cells and debris were also found in the media, as shown in images of D10 in Fig. 3c.Fig. 3

Bottom Line: Furthermore, early inhibition of Wnt/β-catenin signaling by the selective antagonist Dickkopf-1 significantly reduced the activity of Wnt/β-catenin signaling and decreased the cardiomyocyte differentiation of miPSCs cultured on the 3D PCL nanofibrous scaffold, while the early activation of Wnt/β-catenin signaling by CHIR99021 further increased the cardiomyocyte differentiation of miPSCs.These results indicated that the electrospun 3D PCL nanofibrous scaffolds directly promoted the cardiomyocyte differentiation of miPSCs, which was mediated by the activation of the Wnt/β-catenin signaling during the early period of differentiation.These findings highlighted the biophysical role of 3D nanofibrous scaffolds during the cardiomyocyte differentiation of miPSCs and revealed its underlying mechanism involving Wnt/β-catenin signaling, which will be helpful in guiding future stem cell- and scaffold-based myocardium bioengineering.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Tangdu Hospital, Fourth Military Medical University, 1 Xinsi Road, Xi'an, 710038, China.

ABSTRACT

Background: Environmental factors are important for stem cell lineage specification, and increasing evidence indicates that the nanoscale geometry/topography of the extracellular matrix (ECM) directs stem cell fate. Recently, many three-dimensional (3D) biomimetic nanofibrous scaffolds resembling many characteristics of the native ECM have been used in stem cell-based myocardial tissue engineering. However, the biophysical role and underlying mechanism of 3D nanofibrous scaffolds in cardiomyocyte differentiation of induced pluripotent stem cells (iPSCs) remain unclear.

Results: Here, we fabricated a 3D poly-(ε-caprolactone) (PCL) nanofibrous scaffold using the electrospinning method and verified its nanotopography and porous structure by scanning electron microscopy. We seeded murine iPSCs (miPSCs) directly on the 3D PCL nanofibrous scaffold and initiated non-directed, spontaneous differentiation using the monolayer method. After the 3D PCL nanofibrous scaffold was gelatin coated, it was suitable for monolayer miPSC cultivation and cardiomyocyte differentiation. At day 15 of differentiation, miPSCs differentiated into functional cardiomyocytes on the 3D PCL nanofibrous scaffold as evidenced by positive immunostaining of cardiac-specific proteins including cardiac troponin T (cTnT) and myosin light chain 2a (MLC2a). In addition, flow cytometric analysis of cTnT-positive cells and cardiac-specific gene and protein expression of cTnT and sarcomeric alpha actinin (α-actinin) demonstrated that the cardiomyocyte differentiation of miPSCs was more efficient on the 3D PCL nanofibrous scaffold than on normal tissue culture plates (TCPs). Furthermore, early inhibition of Wnt/β-catenin signaling by the selective antagonist Dickkopf-1 significantly reduced the activity of Wnt/β-catenin signaling and decreased the cardiomyocyte differentiation of miPSCs cultured on the 3D PCL nanofibrous scaffold, while the early activation of Wnt/β-catenin signaling by CHIR99021 further increased the cardiomyocyte differentiation of miPSCs.

Conclusion: These results indicated that the electrospun 3D PCL nanofibrous scaffolds directly promoted the cardiomyocyte differentiation of miPSCs, which was mediated by the activation of the Wnt/β-catenin signaling during the early period of differentiation. These findings highlighted the biophysical role of 3D nanofibrous scaffolds during the cardiomyocyte differentiation of miPSCs and revealed its underlying mechanism involving Wnt/β-catenin signaling, which will be helpful in guiding future stem cell- and scaffold-based myocardium bioengineering.

No MeSH data available.