Modeling central nervous system development in vitro using pluripotent stem cells
The directed differentiation of pluripotent stem cells to neurons for the treatment of neuromuscular diseases has received considerable attention during the last decade. Several studies have described the efficient generation of anterior neurons of the cortex and midbrain such as motor neurons and dopaminergic neurons. However, until recently, the generation of posterior spinal cord neurons had proved challenging. To generate specific type of neurons along the anterior-posterior (AP) axis we developed a system that allows timely expression of specific Hox genes. Hox genes are expressed in distinct domains along the AP axis and provide cells with positional identity. Strikingly, timely activation of a Hoxb1 transgene resulted in the generation of neurons with specific hindbrain identity (Figure 1) (Gouti & Gavalas, 2008).
Figure 1: Directed differentiation of mouse pluripotent stem cells to hindbrain neural progenitor cells. Induction of Hoxb1 transgene at a specific time window during differentiation results in the downregulation of anterior identity (otx2) and activation of a hindbrain specific genetic program (Hoxb1).
However, induction of more posterior 5' Hox genes failed to generate specific spinal cord neurons. This suggested that the cellular context is very important for the induction of specific neuronal cell types and that the anterior neural progenitors were unable to support the generation of more posterior cell types. An additional advantage of this in vitro approach is that it can provide insights into the function of specific transcription factors during development (Gouti et al, 2011; Bami et al 2011).
Several lines of evidence indicate that the cells contributing to the spinal cord originate from a different source to those forming more anterior regions such as the brain and hindbrain. Striking evidence came from retrospective clonal lineage analysis experiments in the mouse embryo (Tzouanacou et al, 2009) where a single labelled cell found in the posterior part of the elongating embryo can give descendants to both the spinal cord and paraxial mesoderm. These cells called neuromesodermal progenitors (NMPs) reside in the caudal lateral epiblast region and are important for axis elongation but have been largely ignored in the stem cell field. We have recently described the generation of the NMP population in vitro from mouse and human pluripotent stem cells, which results in the efficient generation of spinal cord neurons (Figure 2), (Gouti et al, 2014). Our results illustrate the importance of mimicking normal embryonic development for the efficient generation of specific cell types from pluripotent stem cells.
Figure 2: Directed differentiation of mouse pluripotent stem cells to hindbrain neural progenitor cells expressing Hoxb4. Induction of NMP identity results in the efficient generation of neurons with brachial (Hoxc6) and thoracic spinal cord identity (Hoxc9).
In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity.
Gouti M, Tsakiridis A, Wymeersch FJ, Huang Y, Kleinjung J, Wilson V, Briscoe J.
PLoS Biol. 2014 Aug 26;12(8)
Directed neural differentiation of mouse embryonic stem cells is a sensitive system for the identification of novel Hox gene effectors.
Bami M, Episkopou V, Gavalas A, Gouti M.
PLoS One. 2011;6(5):e20197
Anterior Hox genes interact with components of the neural crest specification network to induce neural crest fates.
Gouti M, Briscoe J, Gavalas A.
Stem Cells. 2011 May;29(5):858-70
Hoxb1 controls cell fate specification and proliferative capacity of neural stem and progenitor cells.
Gouti M, Gavalas A.
Stem Cells. 2008 Aug;26(8):1985-97