Induction of Midbrain Dopaminergic Neurons from ES Cells by Stromal Cell–Derived Inducing Activity

Abstract

We have identified a stromal cell-derived inducing activity (SDIA) that promotes neural differentiation of mouse ES cells. SDIA accumulates on the surface of PA6 stromal cells and induces efficient neuronal differentiation of cocultured ES cells in serum-free conditions without use of either retinoic acid or embryoid bodies. BMP4, which acts as an antineuralizing morphogen in Xenopus, suppresses SDIA-induced neuralization and promotes epidermal differentiation. A high proportion of tyrosine hydroxylase-positive neurons producing dopamine are obtained from SDIA-treated ES cells. When transplanted, SDIA-induced dopaminergic neurons integrate into the mouse striatum and remain positive for tyrosine hydroxylase expression. Neural induction by SDIA provides a new powerful tool for both basic neuroscience research and therapeutic applications.

BMP-4 induces a Smad-dependent apoptotic cell death of mouse embryonic stem cell-derived neural precursors

“…27 The group of H Kawasaki has demonstrated that BMP-4 is able to redirect neuroectodermal precursors towards epidermal commitment. 6 However, the mechanism of BMP-4 neural inhibition remained unexplored so far. Here, we demonstrate that BMP-4, through the Smad signaling pathway, induces the apoptotic cell death of neuroectodermal precursor cells already engaged in the neural cell fate.…”

“…The absence of glial (Sox10, GFAP) markers confirmed the requirement of appropriate culture conditions to induce astrocytic (GFAP) and oligodendrocytic (Sox10) differentiation within this in vitro culture model. 6,19 Furthermore, no differentiation toward endodermal (AFP) and mesodermal (brachyury) lineages was observed ( Figure 1B).…”

“…6 Seeded on fixed fibroblastic feeder cells in the absence of serum, mouse ES cells differentiated efficiently within 7 days into neural precursors, Figure 1A (i)). As expected, virtually all colonies were immunoreactive to neural markers at day 6 (Table 1 and Figure S1), and at day 4 of differentiation, 80% of the colonies were already positive for the neural precursor marker Sox-1 (Table 1 and Figure S1).…”

“…A high level of neural differentiation has been obtained through aggregation of ES cells with retinoic acid treatment in the presence of serum, 5 or by the coculture of ES cells with the PA6 stromal cell line in the absence of serum. 6 In both cases, the addition of BMP-4 suppressed neural differentiation in a time-dependent manner, while it promoted mesodermal 5 or ectodermal engagement. 6 The attenuation of BMP signaling in ES cells appears to be essential but not sufficient for neural induction, since its specific blockade does not significantly promote neural differentiation, suggesting that some additional signals are required for mammalian neural induction.…”

“…6 In both cases, the addition of BMP-4 suppressed neural differentiation in a time-dependent manner, while it promoted mesodermal 5 or ectodermal engagement. 6 The attenuation of BMP signaling in ES cells appears to be essential but not sufficient for neural induction, since its specific blockade does not significantly promote neural differentiation, suggesting that some additional signals are required for mammalian neural induction. Recent data have shown that, in the absence of feeder-derived or serum-derived factors and at low cell densities, ES cells undergo a direct phenotypic change towards the neural fate, 7 a process that requires autocrine fibroblast growth factor (FGF).…”