Enhanced Differentiation of Human Embryonic Stem Cells Toward Definitive Endoderm on Ultrahigh Aspect Ratio Nanopillars

Abstract

Differentiation of human embryonic stem cells is widely studied as a potential unlimited source for cell replacement therapy to treat degenerative diseases such as diabetes. The directed differentiation of human embryonic stem cells relies mainly on soluble factors. Although, some studies have highlighted that the properties of the physical environment, such as substrate stiffness, affect cellular behavior. Here, mass‐produced, injection molded polycarbonate nanopillars are presented, where the surface mechanical properties, i.e., stiffness, can be controlled by the geometric design of the ultrahigh aspect ratio nanopillars (stiffness can be reduced by 25.0003). It is found that tall nanopillars, yielding softer surfaces, significantly enhance the induction of definitive endoderm cells from pluripotent human embryonic stem cells, resulting in more consistent differentiation of a pure population compared to planar control. By contrast, further differentiation toward the pancreatic ­endoderm is less successful on “soft” pillars when compared to “stiff” pillars or control, indicating differential cues during the different stages of differentiation. To accompany the mechanical properties of the nanopillars, the concept of surface shear modulus is introduced to describe the characteristics of engineered elastic surfaces through micro or nanopatterning. This provides a framework whereby comparisons can be drawn between such materials and bulk elastomeric materials.

High‐Aspect‐Ratio Nanostructured Surfaces as Biological Metamaterials

“…Although not normally considered a microfabrication technique, Stormonth‐Darling et al have shown injection molding can efficiently replicate 100 nm tip diameter polycarbonate nanopillars with very high aspect ratios (up to 20:1). Rasmussen et al showed how injection‐molded nanopillars could be used to study stem cell differentiation . Their work highlights how expensive electron‐beam‐patterned masters can be combined with high‐throughput manufacturing processes.…”

“…Physical cues also avoid the use of potential harmful chemicals in vivo, and are highly localized (unlike chemical cues which can diffuse into surrounding tissue) . Nanostructured surfaces have been proposed for: generating specific‐cell types in stem cell–based therapies; fabricating better in vitro models; and improving cell integration in tissue engineering …”

“…Multiple reports link differential fate to nanostructure geometry and density . Kong et al observed that the spacing of nanostructured surfaces influenced the regulation of differentiation‐related protein in human embryonic stem cells .…”

Injection Molding Micro‐ and Nanostructures in Thermoplastic Elastomers

“…Although applicable to many industries and applications, the interest in TPEs in this work stems primarily from the use of other elastomers like polydimethylsiloxane (PDMS) and other soft materials such as hydrogels in cell biology research. Flexible pillars offer a model for in vitro substrate rigidity that can be tuned by the height of the pillars and mechanical properties of the polymer . When cells adhere to such pillars they exert sufficient force to bend them and, by measuring the extent of the bending, it is possible to determine the magnitude of this force if the height and mechanical properties of the pillars are known.…”

“…If experiments such as these are to be up‐scaled it is desirable to use a high throughput technique like injection molding that can produce thousands of samples per day rather than PDMS casting which can take several hours to form a single replica. When the mechanical properties of TPEs are similar to these currently used materials (as in the work of Fu et al where the Young's Modulus of PDMS was 2.5 MPa) then injection molding may offer a route to high throughput production of samples that will enable this type of work to be done on a larger scale …”