Micro- and nanoscale microenvironments for stem cell differentiation

Stem cells normally reside within specific extracellular microenvironments comprising a complex mixture of soluble and insoluble ECM signals, which regulate stem cell behaviors such as self-renewal, migration, and differentiation .  The project area focuses on developing methods to generate combinatorial extracellular matrix (ECM) arrays in a micro-/nano scale. One of the goals is to study the functions of several ECM molecules on hESC differentiation towards neurons focusing two important questions: i) what is the effect of ECM composition on the neurogenesis from human embryonic stem cells (hESCs)? and ii) what is the temporal and spatial effect of ECM materials with nanoscale features on hESC neurogenesis?  Trainees in the K. Uhrich (CCB) and K. Lee (CCB) labs will learn skills in biosurface chemistry and soft lithography (see Fig.4) and will generate the artificial microenvironment using various patterning tools, such as plasma-induced patterning and dip-pen nanolithography (DPN) (Fig. 4).  Current data by K. Uhrich (CCB) and M. Grumet (CDB) demonstrates that laminin-micropatterned surfaces promoted directional outgrowth of NL2.3; these neural stem cell clones overexpress the protein Notch to promote radial glial phenotype.  The next challenge for IGERT Trainees is to study cell differentiation behavior along 2-D bioactive gradients.  In a second effort, via collaboration with M. Schachner (Neuroscience), trainees in P. Moghe lab (CBE/BME) will provide organic nanoparticles functionalized with the neural cell adhesion molecule, L1, that promote ligand clustering and endocytosis (Fig. 5).  These ligand-nanoparticles will be (a) micropatterned using the microscale plasma-initialized patterning (mPIP) developed by the K. Uhrich (CCB); and (b) nanoimprinted using DPN by Trainees in the K. Lee lab (CCB) (Fig 4).

Fig. 4.  (A): Protein nanoarrays prepared via DPN generated affinity templates on gold. 3-D topographic image of a lysozyme nanoarray, consisting of a line grid and dots with intentionally varied feature dimensions.  (B): Protein nanoarrays prepared via direct-write DPN. (C): Topography image of the Retronectin protein array. (D): Immunostaining (actin and integrin) of cell adhesion on nano-pattern (C). DPN will be used as a tool to create nanoscale biofunctionalized substrates for stem cell adhesion and differentiation.

Figure 5.  Outgrowth of human ESCs from embryoid bodies was promoted on randomly organized ligand (L1)-presenting albumin nanoparticles compared to ligand-alone and nanoparticle controls. The IGERT project will micropattern or nanoimprint the ligand particles to control pluripotency vs. differentiation behaviors.