IADR Abstract Archives
Ultrasound/Tensile Forces Induce Differentiation and Proliferation in Human Pluripotent Stem Cells
Objectives: To determine the extent to which tensile forces generated by Acoustic Tweezing Cytometry (ATC) promote human pluripotent stem cell (hPSC) differentiation and proliferation.
Methods: 45,000 undifferentiated hPSCs (H1 and H9 cell lines) were seeded on matrigel-coated 60 mm tissue culture plates overnight. Next, microbubbles, functionalized with RGD peptides, were linked to integrin on the surface of hPSCs. Subsequently, hPSCs were treated with ATC, an ultrasound-based subcellular mechanical stimulation technique. Ultrasound pulse (1 MHz center frequency, 50 ms pulse duration, and 5% duty cycle) with 0.05 MPa amplitude was applied to the cells at a 45 degree angle beneath the petri dish for 30 minutes. Confocal microscopy and immunoblot techniques were used to assess cell proliferation and differentiation.
Results: hPSCs with ATC applied resulted in enhanced cell differentiation and proliferation. hPSCs treated with ATC-mediated forces induced shuttling of pluripotent transcription factors, Oct3/4 and Nanog, from the nucleus to the cytoplasm in a time-dependent manner, while Sox2 was not affected. Additionally, immunoblot analysis demonstrated that ATC-treated hPSCs resulted in significantly decreased Oct3/4 and Nanog. Further, hPSCs treated with ATC resulted in an increase in the cell proliferation marker, Ki-67, via immunoblot. Thus, these data demonstrate through ATC treatment of hPSCs that the pluripotent transcription factors are mechano-sensitive proteins and rapidly respond to tensile forces.
Conclusions: Mechanical stresses applied to hPSCs induce the shuttling of pluripotent transcription factors from the nucleus to the cytoplasm to promptly direct cell differentiation. These findings will better our understanding of how mechanical forces affect the developing embryo and ATC treatment of hPSCs may allow us to rapidly generate large numbers of cell progenitor for regenerative medicine.
Methods: 45,000 undifferentiated hPSCs (H1 and H9 cell lines) were seeded on matrigel-coated 60 mm tissue culture plates overnight. Next, microbubbles, functionalized with RGD peptides, were linked to integrin on the surface of hPSCs. Subsequently, hPSCs were treated with ATC, an ultrasound-based subcellular mechanical stimulation technique. Ultrasound pulse (1 MHz center frequency, 50 ms pulse duration, and 5% duty cycle) with 0.05 MPa amplitude was applied to the cells at a 45 degree angle beneath the petri dish for 30 minutes. Confocal microscopy and immunoblot techniques were used to assess cell proliferation and differentiation.
Results: hPSCs with ATC applied resulted in enhanced cell differentiation and proliferation. hPSCs treated with ATC-mediated forces induced shuttling of pluripotent transcription factors, Oct3/4 and Nanog, from the nucleus to the cytoplasm in a time-dependent manner, while Sox2 was not affected. Additionally, immunoblot analysis demonstrated that ATC-treated hPSCs resulted in significantly decreased Oct3/4 and Nanog. Further, hPSCs treated with ATC resulted in an increase in the cell proliferation marker, Ki-67, via immunoblot. Thus, these data demonstrate through ATC treatment of hPSCs that the pluripotent transcription factors are mechano-sensitive proteins and rapidly respond to tensile forces.
Conclusions: Mechanical stresses applied to hPSCs induce the shuttling of pluripotent transcription factors from the nucleus to the cytoplasm to promptly direct cell differentiation. These findings will better our understanding of how mechanical forces affect the developing embryo and ATC treatment of hPSCs may allow us to rapidly generate large numbers of cell progenitor for regenerative medicine.
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