Temporally restricted substrate interactions direct fate and specification of neural precursors derived from embryonic stem cells

Abstract

It was, until now, not entirely clear how the nervous system attains its cellular phenotypic diversity and wired complexity during development. Here we describe how environmental interactions alone can modify the development of neurogenic precursor cells. Upon evaluating distinct growth-permissive substrates in an embryonic stem cell-neurogenesis assay, we found that laminin, fibronectin, and gelatin instruct neural fate and alter the functional specification of neurons when applied at distinct stages of development. Changes in phenotypic, electrophysiological, and molecular characteristics could resemble cellular events and interactions in the early embryonic brain and may explain why these extracellular matrix components transiently demarcate certain developing brain structures.cell culture ͉ extracellular matrix ͉ fibronectin ͉ laminin ͉ sonic hedgehog T he extracellular matrix (ECM) assembles three-dimensional templates around functional units of developing brain and spinal cord before neurons complete their final positioning and alignment. These structures contain large numbers of extracellular molecules that disappear soon after synaptic stabilization (1, 2). It is believed that transient ECM molecules act as ''boundaries'' that instruct afferent fiber ingrowth through adhesive and repulsive cues and thus play a significant role during CNS pattern formation (3). The function of the ECM might extend beyond morphogenetic effects, because loss or disarrangement of boundaries is observed in CNS diseases and malformations (1, 4-6) and because the ECM is involved in regulating synaptic plasticity in the adult (7). However, early embryonic lethality in some animal knockout models (8, 9) and only minor phenotypic abnormalities in others (10) puzzles the study of individual matrix molecules and their receptors during critical periods of neural development in vivo. ES cells used as a model system for neurogenesis offer an alternative approach in vitro. Neural differentiation of pluripotent ES cells can be directed in culture mimicking the entire temporal sequence of CNS development (11-13). Series of defined media and growth factors yield highly enriched multipotent precursors, and, in subsequent culture steps, a variety of molecules and growth factors influence neural subtype specification. For instance, morphogenetic factors [e.g., sonic hedgehog (SHH), FGF8, FGF4, and retinoic acid] direct the differentiation of different rostral and caudal CNS neuron groups (14-16). The focus of the present study was on potential roles for ECM and cell-substrate interactions during fate specification and differentiation of ES cell-derived neural precursors (ESNPs). ResultsIn an attempt to optimize the efficacy of generating neurons and glia from ES cells in vitro, we compared distinct growth-permissive dish coatings in a controlled four-step neural induction protocol. In this paradigm, ES cells grow attached to culture dishes in step III (generation of neural precursors) and IV (maturation of neural phenotypes), thus permitting ...

The microenvironment of the embryonic neural stem cell: Lessons from adult niches?

“…As similar abnormalities of pial discontinuities and cortical ectopias are seen in the mice with deletion of the laminin ␥1 chain (so preventing laminin trimer formation) (Halfter et al, 2002), we can conclude that the interaction between the stem cell basal end foot and the pia is important for neuronal migration, as expected given that these cells use the basal process as a guidepost for migration (Rakic, 1995). However, the lack of a VZ abnormality is surprising given the levels of integrin expression in this region and recent evidence in ES cells showing that laminin 111 (comprising the ␣1, ␤1, and ␥1 chains) can efficiently facilitate transition of cells into neuronal precursors and neurons (Goetz et al, 2006), further suggesting a role for the integrin/extracellular matrix interaction in mediating cell proliferation and fate choices. However, given the lack of definitive in vivo evidence for such a regulatory role in the VZ, experimental approaches that specifically target the VZ while not perturbing the same interactions between the pial membrane and basal process may be required.…”

A two-step strategy for neuronal differentiation in vitro of human dental follicle cells

“…For our study surface modifications were used that previously facilitated neural differentiation of stem cells. Gelatin-coated wells, for example, were successfully used for neural and glial cell differentiation of embryonic stem cell-derived neural progenitor cells (Goetz et al, 2006;Zhang et al, 2006). In this study, gelatin does not improve neural differentiation of DFCs.…”

Electrophysiological evaluation of engrafted stem cell-derived neurons

“…(ii) New transplantation techniques are required to enable targeted, layer-or nucleus-specific introduction of solitary donor cells. (iii) A full grasp of the factors and protocols used to derive donor cells is required to generate ''custom'' neurons tailored to participate in the activities of specific local CNS networks 51 . …”