Deriving excitatory neurons of the neocortex from pluripotent stem cells

Abstract

The human cerebral cortex is an immensely complex structure that subserves critical functions that can be disrupted in developmental and degenerative disorders. Recent innovations in cellular reprogramming and differentiation techniques have provided new ways to study the cellular components of the cerebral cortex. Here, we discuss approaches to generate specific subtypes of excitatory cortical neurons from pluripotent stem cells. We review spatial and temporal aspects of cortical neuron specification that can guide efforts to produce excitatory neuron subtypes with increased resolution. Finally, we discuss distinguishing features of human cortical development and their translational ramifications for cortical stem cell technologies.

Figure 1. Pathways for generating cortical excitatory neurons from pluripotent cells in vivo and in vitro

a) Pluripotent cells of the inner cell mass in the embryonic blastocyst are thought to differentiate into cells of the anterior neuroectoderm in the absence of any instructive signals through a series of default fate decisions. Shown in red are morphogens that promote alternative differentiation fates. Shown in green are factors that inhibit those morphogens and facilitate the default pathway. b) The anterior (rostral) neuroectoderm gives rise to the telencephalon upon closure of the neural tube. Dorsal-ventral (D–V) patterning is driven by dorsally expressed Wnts and ventrally expressed SHH, which converge on the activity of Gli3 to determine whether FGF signaling will be restrained or promote ventralization. c) The division of the adult cortex into specialized functional areas develops in response to embryonic patterning signals, produced at discrete locations, that induce gradients of expression for key transcription factors. The combinatorial expression levels of these transcription factors – Emx2, Coup-TF1, Sp8, and Pax6 – define a cell's position in relation to the rostral-caudal and dorsomedial-ventrolateral (dm-vl) axes of the embryonic cortex. The cross-repressive and co-stimulatory interactions depicted on the left have been defined genetically, and in some cases biochemically. d) Through a series of asymmetric, self-renewing divisions, radial glial cells (RG) give rise to all the subtypes of cortical excitatory neurons in a defined temporal sequence. This neurogenesis is typically indirect, since the daughter cell is often an intermediate progenitor cell (IP) that divides again to produce two neurons (N). The markers depicted represent some subclasses of glutamatergic neurons: Cajal-Retzius cells (layer I, Reelin); corticothalamic neurons (layer VI, Tbr1); subcerebral projection neurons (layer V, Ctip2); and callosal projection neurons (layers II–III, Cux1). After neurogenesis, RG convert to gliogenic cells that give rise to astrocytes (A). e) Producing cortical excitatory neurons in vitro from ES or iPS cells occurs in phases that resemble the natural developmental sequence. i) ES cells differentiate into anterior neural cells through default mechanisms that are promoted using biological or chemical inhibitors of the Smad and Wnt pathways. *Although exogenous FGFs promote caudal CNS fates, FGF inhibitors should not be used to favor telencephalization since proliferation of primitive neural cells requires autogenous FGF signaling (Smukler et al., 2006; Zeng et al., 2010). ii) Dorsal fates can be induced in ES-derived telencephalic cells by Wnt stimulation and/or SHH inhibition, or sometimes through intrinsic mechanisms. iii) ES-derived dorsal telencephalic cells respond to the intrapallial patterning signals present in the embryonic telencephalon. Low-density cultures intrinsically adopt a caudal cortex identity. iv) ES-derived cortical progenitor cells produce excitatory neuron subtypes in the same sequence that occurs during cortical neurogenesis in the embryo, followed by glial cell production. DAPT, a chemical inhibitor of Notch, can be used to prevent stem cell self-renewal and promote synchronous differentiation of neural precursor cells.