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, 35 (19), 5098-109

Carbon Nanotube-Based Substrates for Modulation of Human Pluripotent Stem Cell Fate

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Carbon Nanotube-Based Substrates for Modulation of Human Pluripotent Stem Cell Fate

Marina V Pryzhkova et al. Biomaterials.

Abstract

We investigated the biological response of human pluripotent stem cells (hPSCs) cultured on a carbon nanotube (CNT) array-based substrate with the long term goal to direct hPSC germ layer specification for a wide variety of tissue engineering applications. CNT arrays were fabricated using a chemical vapor deposition system allowing for control over surface roughness and mechanical stiffness. Our results demonstrated that hPSCs readily attach to hydrophilized and extracellular matrix coated CNT arrays. hPSCs cultured as colonies in conditions supporting self-renewal demonstrated the morphology and marker expression of undifferentiated hPSCs. Conditions inducing spontaneous differentiation lead to hPSC commitment to all three embryonic germ layers as assessed by immunostaining and RT-PCR analysis. Strikingly, the physical characteristics of CNT arrays favored mesodermal specification of hPSCs. This is contradictory to the behavior of hPSCs on traditional tissue culture plastic which promotes the development of ectoderm. Altogether, these results demonstrate the potential of CNT arrays to be used in the generation of new platforms that allow for precise control of hPSC differentiation by tuning the characteristics of their physical microenvironment.

Keywords: Cell adhesion; Cytoskeleton; Differentiation; Human pluripotent stem cells; Multi-walled carbon nanotubes; Surface roughness.

Figures

Fig. 1
Fig. 1
SEM images of CNT arrays on silicon wafer. (A) Entire array structure from side view. (B, C) Incorporation of ECM proteins (Geltrex) onto the surface of UV/ozone treated CNT arrays (magnification 4000 and 10,000 correspondingly). (D) Hydrophobic CNT arrays on the silicon wafers before UV/ozone treatment and the same CNT arrays became hydrophilic after oxidation as seen with water drop assay.
Fig. 2
Fig. 2
AFM analysis of surface roughness. Representative images of cell culture surface topography: CNT – carbon nanotubes, TCP – tissue culture plastic, Ra – the average deviation from mean.
Fig. 3
Fig. 3
hPSC colonies grown on CNT arrays demonstrate typical morphology and express pluripotency-associated markers characteristic for undifferentiated cells. (A) SEM images of hPSC colonies on CNT array (magnification 300, 1000, 500 and 5000). (B) Expression of transcription factor OCT4 (red) in hPSC colonies (scale bar 50 μm and 100 mm). (C) Expression of trans-membrane protein TRA-1-81 (green) (scale bar 100 μm and 50 μm correspondingly). Cell nuclei are stained with DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
Behavior of hPSCs on CNT arrays. (A) hPSCs lose pluripotency marker expression in the middle of colonies first (Day 8 of differentiation) (scale bar 100 μm). (B) By Day 24 of differentiation cells lose cell–cell contacts, migrate out of colonies, and demonstrate multiple focal adhesion sites (scale bar 50 μm).
Fig. 5
Fig. 5
hPSC from a monolayer culture seeded on CNT arrays as a homogenous single cell suspension, in contrast to cells on TCP, underwent apoptosis within 24 h if ROCK inhibitor (Y-27632) was not added. However the addition of 10 μm ROCK inhibitor allowed single hPSCs to migrate on CNT arrays and reestablish cell–cell contacts similar to cells on TCP.
Fig. 6
Fig. 6
Schematic of hPSC differentiation process in colonies (Col) and embryoid bodies (EB).
Fig. 7
Fig. 7
Spontaneous differentiation of hESC H9 colonies on CNT arrays and tissue culture plastic analyzed on Day 16 (A) and 24 (B) (scale bar 50 μm).
Fig. 8
Fig. 8
Spontaneous differentiation of hiPSC BM1M colonies on CNT arrays and tissue culture plastic analyzed on Day 16 (A) and 24 (B) (scale bar 50 μm).
Fig. 9
Fig. 9
Spontaneous differentiation of hESC H9 embryoid bodies on CNT arrays and tissue culture plastic analyzed on Day 16 (scale bar 50 μm).
Fig. 10
Fig. 10
Spontaneous differentiation of hESC H9 embryoid bodies on CNT arrays and tissue culture plastic analyzed on Day 24 (scale bar 50 μm).
Fig. 11
Fig. 11
Spontaneous differentiation of hiPSC BM1M embryoid bodies on CNT arrays and tissue culture plastic analyzed on Day 16 (A) and Day 24 (B) (scale bar 50 μm).
Fig. 12
Fig. 12
RT-PCR analysis of gene expression of undifferentiated H9 hESC and Day 16 and Day 24 cells differentiated on CNT arrays and TCP from colonies and embryoid bodies.

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