UC Berkeley

The native microenvironment in which a cell resides greatly affects its function. The biochemical signals presented to the cell, the three-dimensional (3D) spatial organization or manner in which they are presented, and even the mechanical properties of the surrounding tissue all play an integral role in regulating its innate function. For example, neural stem cells (NSCs) - cells that are able to continuously replicate without losing their intrinsic properties (self-renew) as well as differentiate into all mature neural cell types (multipotent) - and pluripotent cells in the blastocyst - which gives rise to human embryonic stem cells (hESCs) in culture - naturally reside within a local microenvironment that carefully regulates their proliferation and subsequent differentiation. However, in general, numerous downstream applications involving stem cells require cell isolation and extensive culture outside the body, and when these cells are extracted from their natural environments, it is exceedingly difficult to control their behavior. To be able to expand these stem cells and their progeny one needs to understand and emulate their natural environments, or niches.

Within the stem cell niche, numerous cellular signaling systems involve the presentation of multivalent ligands, which upon binding to their cognate receptor induce a process of receptor clustering that is apparently critical for signal transduction. In our investigation of factors that naturally form higher-order signaling complexes, we discovered that the transmembrane protein ephrin-B2 significantly enhances neuronal differentiation of NSCs upon ligand multimerization. By inducing signaling through its receptor EphB4, ephrin-B2 upregulates transcription factors which have been identified as contributors to neuronal differentiation. Through in vivo immunostaining and short hairpin RNA (shRNA) interference, we have also identified astrocytes as the endogenous source of ephrin-B2 in the adult rat brain. These findings indicate the first example of an Eph-family protein in regulating stem cell lineage commitment in the adult nervous system and the first example of a cell membrane-bound factor that contributes to adult hippocampal neurogenesis through multivalent signaling.

Multivalent binding of ligands can often initiate cellular signal transduction, either secreted or cell-surface tethered, to target cell receptors, leading to receptor clustering. Since multivalent ligands can be significantly more potent than corresponding monovalent interaction, engineering synthetic multivalent ligands to organize receptors into nanoscale clusters is an attractive approach to elicit desired downstream cellular responses. We create for the first time multivalent ligands that influence stem cell fate, both in vitro and in vivo. The ectodomain of ephrin-B2, normally an integral membrane protein ligand, was conjugated to a soluble biopolymer to yield multivalent conjugates that potently induced signaling within and neuronal differentiation of neural stem cells, in culture and within the brain. Furthermore, synthetic multivalent conjugates of ephrin-B1 strongly enhanced human embryonic and induced pluripotent stem cell differentiation into functional dopaminergic neurons. Multivalent bioconjugates thus represent a powerful tool for controlling stem cell fate in vitro and in vivo.

Currently, the technological capacity to restore neuronal function in degenerating or injured regions of the adult brain is severely limited. Only in two regions of the adult mammalian brain - the subventricular zone (SVZ) and hippocampus - are NSCs capable of continuously generating new neurons, enabled by a complex repertoire of factors that precisely regulate the activation, proliferation, differentiation, and integration of the newborn cells. A growing number of studies also report low level neurogenesis in regions of the adult brain outside these established neurogenic niches - potentially via NSC recruitment or activation of local, quiescent NSCs - under perturbations such as ischemia, cell death, or viral gene delivery of proneural growth factors. We have explored whether implantation of engineered biomaterials can stimulate neurogenesis in normally quiescent regions of the brain. Specifically, recombinant versions of factors found within the NSC microenvironment, Sonic hedgehog and ephrin-B2, were conjugated to long polymers, thereby creating highly bioactive, multivalent ligands that begin to emulate components of the neurogenic niche. In this engineered biomaterial microenvironment, new neuron formation was observed in normally non-neurogenic regions of the brain, the striatum and cortex, and combining these multivalent biomaterials with SDF-1α increased neuronal commitment of newly divided cells 7- to 8-fold in these regions. Additionally, the decreased hippocampal neurogenesis of geriatric rodents was partially rescued toward levels of young animals. We thus demonstrate for the first time de novo neurogenesis in both the cortex and striatum of adult rodents stimulated solely by delivery of synthetic biomaterial forms of proteins naturally found within adult neurogenic niches, offering the potential to replace neurons lost in neurodegenerative disease or injury as an alternative to cell implantation.