Established monolayer differentiation of mouse embryonic stem cells generates heterogeneous neocortical‐like neurons stalled at a stage equivalent to midcorticogenesis

Abstract

Two existing and widely applied protocols of embryonic stem (ES) cell differentiation have been developed to enable in vitro generation of neurons resembling neocortical projection neurons in monolayer culture and from embryoid bodies. The monolayer approach offers advantages for detailed in vitro characterizations and potential mechanistic and therapeutic screening. We investigated whether mouse ES cells undergoing largely undirected neocortical differentiation in monolayer culture recapitulate progressive developmental programs of in vivo progenitor and postmitotic differentiation and whether they develop into specific neocortical subtypes. We find that ES-derived mitotic cells that have been dorsalized by the sonic hedgehog antagonist cyclopamine, and that express, as a total population, cardinal markers of telencephalic progenitors, are, in fact, molecularly heterogeneous. We next show that these progenitors subsequently generate small numbers of heterogeneous neocortical-like neurons that are “stalled” at an immature stage of differentiation, based on multiple developmental criteria. Although some aspects of neocortical development are recapitulated by existing protocols of ES cell differentiation, these data indicate that mouse ES-derived neocortical progenitors both are more heterogeneous than their in vivo counterparts and seemingly include many incorrectly specified progenitors. Furthermore, these ES-derived progenitors spontaneously differentiate into sparse, and incompletely and largely imprecisely differentiated, neocortical-like neurons that fail to adopt specific neuronal identities in vitro. These results provide both foundation and motivation for refining and enhancing directed differentiation of clinically important neocortical projection neuron subtypes.

Single-Cell Profiling of an In Vitro Model of Human Interneuron Development Reveals Temporal Dynamics of Cell Type Production and Maturation

“…A slow-down in gene expression dynamics has been observed in previous studies of human cortical development (Bakken et al, 2016; Colantuoni et al, 2011). It is possible that our in vitro observations reflect similar changes occurring at equivalent ages in vivo, or that some extrinsic element necessary for continued development of these cells is missing from our protocol, as previous work has suggested that development is comparatively slow in vitro (Sadegh and Macklis, 2014). …”

Organoid and Organ-on-a-Chip Systems: New Paradigms for Modeling Neurological and Gastrointestinal Disease

“…Utilizing iPSCs to generate neural tissue from patients with neurological diseases at scale can provide a better avenue with which to screen drugs targeting specific human diseases [75]. However, as mentioned above neurons produced in monolayers in the culture dish often lack developmental maturity [76, 77, 51]. Brain-chips taking advantage of unique co-culture geometries have successfully co-cultured human ES cell-derived neural progenitor cells, endothelial cells, mesenchymal stem cells, and microglia/macrophage precursors, allowing complex interactions reminiscent of the developing CNS [78].…”

“…Thus, the generation of CSMNs from familial ALS patient iPSCs may provide insight into the degeneration and preservation of these cells. It should be noted that upon careful examination, monolayer systems appear to exhibit stalled maturation of cortical subtypes in terms of inappropriate expression of laminar and postmitotic markers [51]. Impressively, the forebrain organoids generated by [25] harbor distinct cortical organization; and though an in-depth assessment regarding the existence of bona fide CSMNs in these organoids is yet to be reported, this culturing system provides promise towards the generation of this intricate cell type.…”

Fezf2transient expression via modRNA with concurrent SIRT1 inhibition enhances differentiation of cortical subcerebral / corticospinal neuron identity from mES cells

“…Midway through corticogenesis, post-mitotic SCPN identity is initially masked by transient co-expression of regulators of interhemispheric callosal projection neuron (CPN) development, including SATB2 (Alcamo et al, 2008;Britanova et al, 2008;Azim et al, 2009;Woodworth et al, 2012;Sadegh and Macklis, 2014;Leone et al, 2015). At later stages of maturation, SCPN discontinue expression of SATB2, and further resolve into diverse subpopulations of FEZF2-and CTIP2-expressing projection neurons with cortical area-and target-specific molecular identities, properties, and functional circuit Sadegh et al 4 connectivity (Woodworth et al, 2012;Custo Greig et al, 2013;Woodworth et al, 2016;Greig et al, 2016;Galazo et al, 2016).…”

“…ES/iPSC-based models of cortical differentiation are emerging as useful tools to investigate roles of chromatin modifications in cortical development (Tiberi et al, 2012;Juliandi et al, 2012). While protocols for directing cortical differentiation from ES/iPSC cells in systems ranging from monolayer cultures to organoids have succeeded in replicating some of the molecular characteristics of cortical development (Eiraku et al, 2008;Gaspard et al, 2008;Michelsen et al, 2015;Lancaster et al, 2013, Arlotta, 2018Pasca, 2018), the mature refinement of cortical subtypes is incomplete with these protocols; immature neurons become "stalled" at an mid-embryonic developmental stage (Sadegh and Macklis, 2014). These data suggest that ES-derived cortical cells are unlikely to have a sufficiently permissive molecular context for the precise refinement of SCPN identity.…”