ENGINEERING ARTIFICIAL STEM CELL NICHES - Biologically-Responsive Hybrid Biomaterials - page 285

2.3. Niche protein microarrays
An outstanding challenge in stem cell biology is to unravel the complexity of
biomolecular signaling that controls stem cell behavior. Conventional
experimental paradigms, i.e. the testing of one signal at a time, fail to deal with
this complexity. Several groups have started to address this challenge by
screening the effects of combinations of multiple putative microenvironmental
signals on stem cell fate. 74,75,76,77 Mixtures of protein signaling cues such as
ECM components, morphogens, and other signaling proteins were microarrayed
on flat glass substrates using robotic spotting technology. Stem cells can be
exposed to such multicomponent artificial niches, and their response quantified
at the single cell level via multi-parameter analyses involving, for example,
immunocytochemistry-based read-outs (i.e. staining of cell surface markers).
These high-throughput analyses of signaling networks have begun to characterize
the effects of combinations of stem cell regulatory proteins on self-renewal and
differentiation. In one seminal study by Soen et al. , 75 primary human neural
precursor cells were cultured on printed protein arrays to explore the extent and
direction of differentiation into neurons and glias ( Fig. 5 ). Co-stimulation with
two specific developmental morphogens (Wnt and Notch ligands) maintained
stem cells in an undifferentiated-like state suggesting that this combination of
signals may be found in the native niche, in contrast to bone morphogenetic
protein-4, which led to the expression of differentiation markers. In another
study by LaBarge, Bissell and co-authors, protein microarrays were utilized
to dissect the instructive function of the microenvironment in human mammary
progenitor cell regulation. 77 In combination with organotypic 3D culture
models and micropatterned substrates, previously unrecognized combinatorial
microenvironments of cell-extrinsic mammary gland proteins (including the
Notch ligand Jagged-1, P- and E-Cadherin) as well as ECM molecules (including
Laminin-1) were identified. Bhatia and colleagues, who pioneered this approach,
recently expanded the breadth of these platforms by compartmentalizing protein
arrays using gaskets to produce a multi-well plate. This advance allowed them to
simultaneously probe the interactions of ECM components and soluble growth
factors on stem cell fate. 78 The application of robotic spotting technologies to
hydrogels, and also to microwell arrays in order to probe nonadherent stem cell
populations, should allow the effects of combinatorial signaling to be studied in
stem cells grown under physicochemical conditions, more closely resembling
niches.
While the above studies have resulted in important discoveries with regards
to extrinsic stem cell regulation, they have invariably been conducted on flat