Protocol for inducing dorsal spinal sensory interneurons from mouse embryonic stem cell-derived neuromesodermal progenitors

Abstract

Here, we present a protocol comprising two differentiation techniques to derive dorsal sensory spinal interneurons (dIs) from mouse embryonic stem cells (mESCs) through a neuromesodermal progenitor (NMP) precursor. First, we describe the steps for using retinoic acid (RA) to induce dI4s, dI5s, and dI6s, which mediate pain, itch, touch, and sensorimotor integration. Second, we detail the procedures for generating dI1s, dI2s, and dI3s, which mediate proprioception and mechanosensation, using RA together with bone morphogenetic protein (BMP) 4. For complete details on the use and execution of this protocol, please refer to Gupta et al.¹.

Keywords: Cell differentiation; Developmental biology; Neuroscience; Stem cells.

Graphical abstract

Figure 1

Images of cell morphologies from along the differentiation timeline (A–H) Phase contrast images of cells grown on either 0.1% gelatin, or Matrigel at day 1 (A), day 2 (B), day 3 (C), day 4 (D), day 5 (E), day 6 (F), day 8 (G) and day 9 (H). Note that mESCs initially form small clones on the gelatin at the beginning of the differentiation which rapidly expand when treated with FGF/CHIR (A–C). However, as differentiation progresses (day 4/5 onwards), the cells change shape becoming flatter, elongated and pyramidal in shape, and begin extending neuronal processes starting from day 7 in both the RA and RA+BMP4 conditions (arrowheads, G, H). The abundant small, bright round cells are either cells that have detached from the gelatin substrate, or floating dead cells and can be removed from the culture when passaging. Culturing the cells either on gelatin vs Matrigel, or in RA vs RA+BMP4 does not markedly change cellular morphology. Scale bar: 100μm.

Figure 2

NMP formation requires CHIR addition (A–D) Healthy mESC cultures have colonies with smooth edges. In contrast, cells cultured in N2/B27 medium with or without FGF/CHIR appear as nucleated, flat cell clones (B and C). NMPs are recognized by presence of small clones containing nucleated cells (D). (E–H) Day 3 mESC cultures lacking CHIR addition only express Sox2, similar to day 0 mESCs, demonstrating they have not progressed to the NMP state. In contrast, day 3 mESC cultures that have been treated with both bFGF and CHIR express both neural and mesodermal (Brachyury (T)/Cdx2) markers. Scale bar: 50μm.

Figure 3

Immunostaining to detect dorsal spinal progenitors and neurons (A) Schematic timeline of the RA±BMP4 mESC protocols. (B) Schematic depicting the location of the dorsal progenitor (dP) domains and the dIs, and some canonical nuclear markers. For a complete atlas of markers, see.^, (C and D) By day 7, both RA±BMP4 protocols are making Pax3⁺ Pax6⁺ dorsal spinal tissue. (E–H) By day 7, the RA±BMP4 protocols are both making Sox1⁺ neurons, that are extending Tuj1⁺ neural process. The Tuj1⁺ process become more extensive by day 9 (arrowheads, G, H). Scale bar: 50μm (C and D); 100μm (E, F, G).

Figure 4

Immunostaining to detect different dI populations (A and C) By day 9, the cultures derived from the RA protocol will contain a mixture of Pax2⁺ Lhx1/5⁺ dI4/dI6s and Lmx1b⁺ dI5s (not shown). (B and C) In contrast, the cells derived from the RA+BMP4 contain a mixture of Lhx2⁺ dI1s and Isl1⁺ dI3s (B and D). dI2s are present in these cultures but are most effectively detected by scRNA-Seq. Scale bar: 50μm.