Spinal dI4 Interneuron Differentiation From Human Pluripotent Stem Cells

Abstract

Spinal interneurons (INs) form intricate local networks in the spinal cord and regulate not only the ascending and descending nerve transduction but also the central pattern generator function. They are therefore potential therapeutic targets in spinal cord injury and diseases. In this study, we devised a reproducible protocol to differentiate human pluripotent stem cells (hPSCs) from enriched spinal dI4 inhibitory GABAergic INs. The protocol is designed based on developmental principles and optimized by using small molecules to maximize its reproducibility. The protocol comprises induction of neuroepithelia, patterning of neuroepithelia to dorsal spinal progenitors, expansion of the progenitors in suspension, and finally differentiation into mature neurons. In particular, we employed both morphogen activators and inhibitors to restrict or "squeeze" the progenitor fate during the stage of neural patterning. We use retinoic acid (RA) which ventralizes cells up to the mid-dorsal region, with cyclopamine (CYC), an SHH inhibitor, to antagonize the ventralization effect of RA, yielding highly enriched dI4 progenitors (90% Ptf1a⁺, 90.7% Ascl1⁺). The ability to generate enriched spinal dI4 GABAergicINs will likely facilitate the study of human spinal IN development and regenerative therapies for traumatic injuries and diseases of the spinal cord.

Keywords: GABA; differentiate; human pluripotent stem cells; interneuron; spinal cord.

Figure 1

Summary of the dorsal domains and the transcription factors associated with the dI4 INs. This cartoon illustrates the dorsal domains and the corresponding cell types derived. On the left side, it depicts the dorsally derived BMP and WNT gradient. On the right side, the transcription factors are listed for dI4 progenitors and post-mitotic neurons, respectively. Adapted from Alaynick et al. (2011) and Lu et al. (2015).

Figure 2

In vitro differentiation protocol of spinal dI4 INs from hPSCs. (A) Cartoons showing the four stages of the differentiation and corresponding culture vessels, medium types, culture duration, and the morphological features under a phase contrast scope. (B–D) Characteristic antigenic expression in different stages of neural differentiation, Sox1 in neuroepithelia at day 7 (B), Ascl1 in the dorsal spinal dI4 progenitor (C) at day 15, and Ptf1a in maturing dI4 INs (D) at day 22. D, day; Scale bar, 50 μm.

Figure 3

Immunophenotyping of hPSC-derived dI4 INs. (A) Confocal images showing expression of Ascl1, Hoxb4, and Olig3 at D14; Ptf1a (B) at D22, Pax2 and Lhx1/5 (C) in dI4 neural progenitors at D28. (D) The percentage of positive cells in (A–C). (E) Immunostaining of differentiated dI4 mature neurons for GABA, NF-200, Pax2, Lhx1/5, NEUN, and MAP2 at D42. (F) The percentage of positive cells in (E). Scale bar, 50 μm. Data are presented as mean ± SEM of three independent experiments.

Figure 4

Immunophenotyping of 12-week human embryo spinal cord. (A) Immunostaining for Pax2 and Ptf1a in the spinal cord. Panel (B) is the magnified view on the inset in (A). (C) Immunostaining for Pax2 and Lhx1/5 in the spinal cord. Panel (D) is the magnified view on the inset in (C). Scale bar, 200 μm (A,C) and 20 μm (B,D).