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Early differentiation patterning of mouse embryonic stem cells in response to variations in alginate substrate stiffness.

Candiello J, Singh SS, Task K, Kumta PN, Banerjee I - J Biol Eng (2013)

Bottom Line: Upon generation of functionally mature ESC derived islet-like cells, they need to be implanted into diabetic patients to restore the loss of islet activity.Encapsulation in alginate microcapsules is a promising route of implantation, which can protect the cells from the recipient's immune system.The insight into these biophysical phenomena found in this study can be used along with other cues to enhance the differentiation of embryonic stem cells toward a specific lineage fate.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA. pkumta@pitt.edu.

ABSTRACT

Background: Embryonic stem cells (ESCs) have been implicated to have tremendous impact in regenerative therapeutics of various diseases, including Type 1 Diabetes. Upon generation of functionally mature ESC derived islet-like cells, they need to be implanted into diabetic patients to restore the loss of islet activity. Encapsulation in alginate microcapsules is a promising route of implantation, which can protect the cells from the recipient's immune system. While there has been a significant investigation into islet encapsulation over the past decade, the feasibility of encapsulation and differentiation of ESCs has been less explored. Research over the past few years has identified the cellular mechanical microenvironment to play a central role in phenotype commitment of stem cells. Therefore it will be important to design the encapsulation material to be supportive to cellular functionality and maturation.

Results: This work investigated the effect of stiffness of alginate substrate on initial differentiation and phenotype commitment of murine ESCs. ESCs grown on alginate substrates tuned to similar biomechanical properties of native pancreatic tissue elicited both an enhanced and incrementally responsive differentiation towards endodermal lineage traits.

Conclusions: The insight into these biophysical phenomena found in this study can be used along with other cues to enhance the differentiation of embryonic stem cells toward a specific lineage fate.

No MeSH data available.


Related in: MedlinePlus

Change in pluripotent (A), mesoderm (B), ectoderm (C), and endoderm (D) gene expression during spontaneous differentiation of ESD3 embryonic stem cells due to changes in substrate stiffness. For pluripotent markers (Rex1, Oct4, and Sox2) there was either down regulation or no upregulation of gene expression after 5 days of differentiation. FGF8 (B, red line) was the only mesoderm marker that demonstrated any level of upregulation. The primitive ectoderm marker FGF5 (C, red line) was dramatically upregulated when compared to undifferentiated cells, however, this increase was notable for all substrate stiffnesses. The endoderm genes (D) CXCR4 and AFP demonstrated a strong upregulation in relation to the alginate substrates, while SOX17 and FOXA2 did not.
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Figure 4: Change in pluripotent (A), mesoderm (B), ectoderm (C), and endoderm (D) gene expression during spontaneous differentiation of ESD3 embryonic stem cells due to changes in substrate stiffness. For pluripotent markers (Rex1, Oct4, and Sox2) there was either down regulation or no upregulation of gene expression after 5 days of differentiation. FGF8 (B, red line) was the only mesoderm marker that demonstrated any level of upregulation. The primitive ectoderm marker FGF5 (C, red line) was dramatically upregulated when compared to undifferentiated cells, however, this increase was notable for all substrate stiffnesses. The endoderm genes (D) CXCR4 and AFP demonstrated a strong upregulation in relation to the alginate substrates, while SOX17 and FOXA2 did not.

Mentions: After allowing the ES-D3 cells to spontaneously differentiate on the various alginate substrates for 5 days in experimental media free of any additional soluble signaling factors, we analyzed the differentiated cell population for its phenotypic commitment. Our aim was to determine which germ layers, if any, were sensitive to the changes in mechanical properties of the underlying substrate. To accomplish this we chose representative markers for pluripotency and early markers for the three germ layers and analyzed it through real time RT-PCR. The pluripotency markers used for this study were REX1, OCT4, and SOX2. Figure 4A compares the relative change in these pluripotency markers across the nine alginate gels of varying Young’s modulus. For the most part we noted a downregulation of the pluripotency marker genes, and specifically very little sensitivity to the changes in gel stiffness. None of the markers demonstrated any significant upregulation as is expected with differentiation.


Early differentiation patterning of mouse embryonic stem cells in response to variations in alginate substrate stiffness.

Candiello J, Singh SS, Task K, Kumta PN, Banerjee I - J Biol Eng (2013)

Change in pluripotent (A), mesoderm (B), ectoderm (C), and endoderm (D) gene expression during spontaneous differentiation of ESD3 embryonic stem cells due to changes in substrate stiffness. For pluripotent markers (Rex1, Oct4, and Sox2) there was either down regulation or no upregulation of gene expression after 5 days of differentiation. FGF8 (B, red line) was the only mesoderm marker that demonstrated any level of upregulation. The primitive ectoderm marker FGF5 (C, red line) was dramatically upregulated when compared to undifferentiated cells, however, this increase was notable for all substrate stiffnesses. The endoderm genes (D) CXCR4 and AFP demonstrated a strong upregulation in relation to the alginate substrates, while SOX17 and FOXA2 did not.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643844&req=5

Figure 4: Change in pluripotent (A), mesoderm (B), ectoderm (C), and endoderm (D) gene expression during spontaneous differentiation of ESD3 embryonic stem cells due to changes in substrate stiffness. For pluripotent markers (Rex1, Oct4, and Sox2) there was either down regulation or no upregulation of gene expression after 5 days of differentiation. FGF8 (B, red line) was the only mesoderm marker that demonstrated any level of upregulation. The primitive ectoderm marker FGF5 (C, red line) was dramatically upregulated when compared to undifferentiated cells, however, this increase was notable for all substrate stiffnesses. The endoderm genes (D) CXCR4 and AFP demonstrated a strong upregulation in relation to the alginate substrates, while SOX17 and FOXA2 did not.
Mentions: After allowing the ES-D3 cells to spontaneously differentiate on the various alginate substrates for 5 days in experimental media free of any additional soluble signaling factors, we analyzed the differentiated cell population for its phenotypic commitment. Our aim was to determine which germ layers, if any, were sensitive to the changes in mechanical properties of the underlying substrate. To accomplish this we chose representative markers for pluripotency and early markers for the three germ layers and analyzed it through real time RT-PCR. The pluripotency markers used for this study were REX1, OCT4, and SOX2. Figure 4A compares the relative change in these pluripotency markers across the nine alginate gels of varying Young’s modulus. For the most part we noted a downregulation of the pluripotency marker genes, and specifically very little sensitivity to the changes in gel stiffness. None of the markers demonstrated any significant upregulation as is expected with differentiation.

Bottom Line: Upon generation of functionally mature ESC derived islet-like cells, they need to be implanted into diabetic patients to restore the loss of islet activity.Encapsulation in alginate microcapsules is a promising route of implantation, which can protect the cells from the recipient's immune system.The insight into these biophysical phenomena found in this study can be used along with other cues to enhance the differentiation of embryonic stem cells toward a specific lineage fate.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA. pkumta@pitt.edu.

ABSTRACT

Background: Embryonic stem cells (ESCs) have been implicated to have tremendous impact in regenerative therapeutics of various diseases, including Type 1 Diabetes. Upon generation of functionally mature ESC derived islet-like cells, they need to be implanted into diabetic patients to restore the loss of islet activity. Encapsulation in alginate microcapsules is a promising route of implantation, which can protect the cells from the recipient's immune system. While there has been a significant investigation into islet encapsulation over the past decade, the feasibility of encapsulation and differentiation of ESCs has been less explored. Research over the past few years has identified the cellular mechanical microenvironment to play a central role in phenotype commitment of stem cells. Therefore it will be important to design the encapsulation material to be supportive to cellular functionality and maturation.

Results: This work investigated the effect of stiffness of alginate substrate on initial differentiation and phenotype commitment of murine ESCs. ESCs grown on alginate substrates tuned to similar biomechanical properties of native pancreatic tissue elicited both an enhanced and incrementally responsive differentiation towards endodermal lineage traits.

Conclusions: The insight into these biophysical phenomena found in this study can be used along with other cues to enhance the differentiation of embryonic stem cells toward a specific lineage fate.

No MeSH data available.


Related in: MedlinePlus