ENGINEERING ARTIFICIAL STEM CELL NICHES - Biologically-Responsive Hybrid Biomaterials - page 283

Protein tethering was achieved by attaching a heterofunctional PEG linker
to a protein of interest and then crosslinking this conjugate into the forming
gel network. To ensure site-selectivity in protein immobilization, engineered
Fc-chimeric proteins were linked on the gel surface via binding to an
intermediate auxiliary protein, ProteinA, that contains four high-affinity binding
sites (K a = 10 8 per mole) for the Fc-region of human, mouse and rabbit
immunoglobulins ( Fig. 4a ). To specifically functionalize gels and immobilize
proteins only at the bottom of microwells, rather than homogeneously
distributing proteins across the entire array (bulk modification), the commonly
used micromolding process was augmented by adding a protein microcontact
printing step ( Fig. 4b ): PEG-functionalized ProteinA was adsorbed onto the
posts of the PDMS stamp (step 1,2) and the hydrogel polymerized onto the
ProteinA/PDMS (step 3,4), transferring both the topographic pattern and protein
pattern onto the gel surface. Immunofluorescence microscopy revealed that
microcontact printed proteins, such as a BSA-FITC model protein were localized
at the bottom of the microwells ( Fig. 4c ). When ProteinA was used, Fc-chimeric
proteins such as N-Cadherin (N-Cad) were shown via immunostaining to be
effectively tethered ( Fig. 4d ). Indeed, selective modification of microwells with
adhesion proteins such as fibronectin or Fc-chimeric Vascular Cell Adhesion
Molecule-1 (VCAM-1) ensured efficient confinement and tracking of HSCs over
long culture periods, whereas the cells escaped from the microwells within a few
hours when the bulk of the surface was modified. Using this platform, individual
HSCs were exposed to selected putative protein components and their growth
tracked by time-lapse microscopy. It was demonstrated that single HSCs can
undergo self-renewal divisions in vitro in response to selected immobilized
proteins in these artificial niches. Notably, a reduction in proliferation kinetics or
an increase in asynchronous division of single HSCs in microwells in response to
the protein Wnt3a or N-Cadherin correlated well with subsequent serial long-
term blood reconstitution in mice in vivo. These results validate the protein-
tethered hydrogel microwell platform as a broadly applicable paradigm for
dissecting the regulatory role of specific signals within a complex stem cell
niche.
Ochsner and colleagues recently demonstrated that it is possible to form
microwells having dimensions comparable to that of single cells in order to
achieve complete cell confinement in protein-functionalized microwells. 73 If it
were possible to pattern the microwell surfaces with different types of proteins
(or protein mixtures) on these arrays, a true mimicry of a single stem cell niche
with spatially well-defined polarization of protein cues could become possible.