Developmentally coordinated extrinsic signals drive human pluripotent stem cell differentiation toward authentic DARPP-32+ medium-sized spiny neurons

Abstract

SUMMARYMedium-sized spiny neurons (MSNs) are the only neostriatum projection neurons, and their degeneration underlies some of the clinical features of Huntington's disease. Using knowledge of human developmental biology and exposure to key neurodevelopmental molecules, human pluripotent stem (hPS) cells were induced to differentiate into MSNs. In a feeder-free adherent culture, ventral telencephalic specification is induced by BMP/TGF inhibition and subsequent SHH/DKK1 treatment. + MSNs. Similar to mature MSNs, these neurons carry dopamine and A2a receptors, elicit a typical firing pattern and show inhibitory postsynaptic currents, as well as dopamine neuromodulation and synaptic integration ability in vivo. When transplanted into the striatum of quinolinic acid-lesioned rats, hPS-derived neurons survive and differentiate into DARPP-32 + neurons, leading to a restoration of apomorphine-induced rotation behavior. In summary, hPS cells can be efficiently driven to acquire a functional striatal fate using an ontogeny-recapitulating stepwise method that represents a platform for in vitro human developmental neurobiology studies and drug screening approaches.

Activin A directs striatal projection neuron differentiation of human pluripotent stem cells

“…Overgrowth of grafted cells, either as neural or teratoma-like tissue, appears to be a common feature in previous reports of hPSCderived striatal GABAergic neural transplantation (Aubry et al, 2008;Ma et al, 2012;Carri et al, 2013). By contrast, all the 16-week-old grafts in this study were of moderate size, free from proliferation and teratoma/neural overgrowth.…”

“…Despite a relatively high content of DARPP32 + cells in our grafts, the recipient animals did not show a difference in apomorphine-induced rotations compared with the pre-grafting baseline. This is intriguing, because we observed a hint of functional recovery in the Carri study (Carri et al, 2013) despite significantly lower numbers of DARPP32 + cells (the number of animals was too small to suggest a significant behavioural effect). Activin-treated cell-derived grafts were generally low in mass compared with those reported in the Ma and Carri studies (Ma et al, 2012;Carri et al, 2013), which might impact on the behavioural analysis.…”

“…Shh was first applied in an hESC differentiation protocol for generating DARPP32 + neurons (Aubry et al, 2008) and again recently with or without combinatorial inhibition of Wnt signalling (Ma et al, 2012;Carri et al, 2013;Nicoleau et al, 2013). The effect of Shh on the generation of ventral forebrain cellular phenotypes might be due to the derepression of GLI3, which is a key factor that dorsalises hESC-derived neural progenitors (Li et al, 2009).…”

“…To date, a small number of published protocols have reported the differentiation of MSN-like cells in vitro and post-transplantation in the rodent brain (Aubry et al, 2008;Ma et al, 2012;Carri et al, 2013). However, evidence is still limited as to the capacity of these cells to integrate into the host neural circuitry and receive dopaminergic inputs from the midbrain and glutamatergic inputs from the cortex while projecting fibres to the substantia nigra.…”

“…By contrast, activation of the canonical Wnt signalling pathway in the subpallium leads to repression of ventral telencephalic determinants including Nkx2.1, Gsx2, Dlx2 and Mash1. By applying this developmental knowledge, researchers have employed a defined dose of Shh, or Shh together with pharmacological inhibition of Wnt signalling, to obtain LGE-like neural progenitors from hESCs (Aubry et al, 2008;Li et al, 2009;Ma et al, 2012;Carri et al, 2013). However, genetic perturbation of the Shh-Gli pathway at the time of subpallial patterning in mouse 1376 RESEARCH ARTICLE Development (2015) 142, 1375-1386doi:10.1242 models does not affect LGE induction and subsequent striatal MSN neurogenesis (Rallu et al, 2002;Xu et al, 2010;Machold et al, 2003).…”