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. 2021 Feb 3;2(1):100319.
doi: 10.1016/j.xpro.2021.100319. eCollection 2021 Mar 19.

Derivation of dorsal spinal sensory interneurons from human pluripotent stem cells

Sandeep Gupta  1 , Ken Yamauchi  1   2 , Bennett G Novitch  1   2   3 , Samantha J Butler  1   2   3
Affiliations

Derivation of dorsal spinal sensory interneurons from human pluripotent stem cells

Sandeep Gupta et al. STAR Protoc. .

Abstract

We describe two differentiation protocols to derive sensory spinal interneurons (INs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). In protocol 1, we use retinoic acid (RA) to induce pain, itch, and heat mediating dI4/dI6 interneurons, and in protocol 2, RA with bone morphogenetic protein 4 (RA+BMP4) is used to induce proprioceptive dI1s and mechanosensory dI3s in hPSC cultures. These protocols provide an important step toward developing therapies for regaining sensation in spinal cord injury patients. For complete details on the use and execution of this protocol, please refer to Gupta et al. (2018).

Keywords: Cell differentiation; Neuroscience; Stem cells.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Images of healthy and differentiated hESC/hiPSC colonies (A) Healthy hESC/hiPSC cultures should contain flat hESCs/hiPSC colonies with smooth edges. (B and C) In contrast, differentiated cultures have colonies that appear as clusters of scattered cells (B) or pigmented mounds (C). These colonies need to be removed before passaging. Scale bar (all panels), 500 μm.
Figure 2
Figure 2
Formation of embryoid bodies (A) By day 6 in SaND medium, the cells have formed a tight monolayer. (B) Embryoid bodies are formed after these cells are dissociated and seeded in ultra-low attachment plate as a single-cell suspension. (C) Large EBs should be observed in the culture, by day 3 after the seeding into the ultra-low attachment plate. Scale bar (all panels), 500 μm.
Figure 3
Figure 3
Immunostaining of D36 embryoid bodies to detect dI4-6, dI1, and dI3 neurons By day 36, EBs can be fixed, sectioned, and immunostained for dI specific markers. (A) The EBs derived from protocol 1 will contain Pax2/Lhx1/5+ dI4-dI6 neurons (A, A′, and A″) (B and C) In contrast, the EBs derived from protocol 2 contain a mixture of Lhx2+ dI1s (B) and Isl1+ Tlx3+ dI3s (C, C′, and C″). Scale bar (all panels), 100 μm.
Figure 4
Figure 4
Formation of embryoid bodies using the EZ passage tool (A) As an alternative method of making EBs, the EZ passage tool can be used to cut the monolayer of neuroectodermal cells at day 6, resulting in the square-shaped cell clusters (arrows). (B) Embryoid bodies are formed by day 7 after these clusters are transferred to an ultra-low attachment plate. Scale bar (all panels), 500 μm.
See this image and copyright information in PMC

References

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