Stem Cells Expanded from the Human Embryonic Hindbrain Stably Retain Regional Specification and High Neurogenic Potency

Abstract

Stem cell lines that faithfully maintain the regional identity and developmental potency of progenitors in the human brain would create new opportunities in developmental neurobiology and provide a resource for generating specialized human neurons. However, to date, neural progenitor cultures derived from the human brain have either been short-lived or exhibit restricted, predominantly glial, differentiation capacity. Pluripotent stem cells are an alternative source, but to ascertain definitively the identity and fidelity of cell types generated solely in vitro is problematic. Here, we show that hindbrain neuroepithelial stem (hbNES) cells can be derived and massively expanded from early human embryos (week 5-7, Carnegie stage 15-17). These cell lines are propagated in adherent culture in the presence of EGF and FGF2 and retain progenitor characteristics, including SOX1 expression, formation of rosette-like structures, and high neurogenic capacity. They generate GABAergic, glutamatergic and, at lower frequency, serotonergic neurons. Importantly, hbNES cells stably maintain hindbrain specification and generate upper rhombic lip derivatives on exposure to bone morphogenetic protein (BMP). When grafted into neonatal rat brain, they show potential for integration into cerebellar development and produce cerebellar granule-like cells, albeit at low frequency. hbNES cells offer a new system to study human cerebellar specification and development and to model diseases of the hindbrain. They also provide a benchmark for the production of similar long-term neuroepithelial-like stem cells (lt-NES) from pluripotent cell lines. To our knowledge, hbNES cells are the first demonstration of highly expandable neuroepithelial stem cells derived from the human embryo without genetic immortalization.

Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia

“…Different early human neural stem/progenitor cell types, including neuroepithelial-like cells, have been derived from hPSCs and the developing human brain (Conti and Cattaneo, 2010; Edri et al, 2015; Elkabetz et al, 2008; Sun et al, 2008, Tailor et al, 2013). However, to the best of our knowledge, stable cell lines of neuroepithelial cells have not been derived from the dorsal forebrain (prospective NCX) or the SC.…”

“…While recent work on hPSCs has expanded the range of neural cell types attainable (Conti and Cattaneo, 2010; Edri et al, 2015; Koch et al, 2009), and a population of NES cells has been derived from the hindbrain (Tailor et al, 2013), the NES cell system we described and characterized is, to our knowledge, the first neuropotent populations derived directly from the prospective NCX and spinal cord, respectively. As such, NES cells represent a substantial advance facilitating the study of early development and neuropathology in the human CNS.…”

Cytosine-5 RNA Methylation Regulates Neural Stem Cell Differentiation and Motility

“…To characterize the expression of NSUN2 during human neural differentiation, we used an NES cell line (Sai1) isolated from embryonic hindbrain (Carnegie stage 15) and neuroepithelial-like stem cells (AF22) derived from pluripotent cells (Falk et al., 2012, Tailor et al., 2013). In proliferating conditions, AF22 and Sai1 cells showed the characteristic “rosette structures” (Figure 1C) (Wilson and Stice, 2006).…”

How to make a midbrain dopaminergic neuron

“…5A-E), probably due to heterogeneity and/or late developmental stage of the cells and the protocols used. More recently, long-termneuroepithelial stem (lt-NES) cells have emerged as a more homogeneous early stage population that can be derived from either human fetal hindbrain tissue (Tailor et al, 2013) or PSCs, which can be differentiated into TH + neurons (Falk et al, 2012;Koch et al, 2009) (Fig. 5F).…”