Spatiotemporal analysis of putative notochordal cell markers reveals CD24 and keratins 8, 18, and 19 as notochord-specific markers during early human intervertebral disc development

Abstract

In humans, the nucleus pulposus (NP) is composed of large vacuolated notochordal cells in the fetus but, soon after birth, becomes populated by smaller, chondrocyte-like cells. Although animal studies indicate that notochord-derived cells persist in the adult NP, the ontogeny of the adult human NP cell population is still unclear. As such, identification of unique notochordal markers is required. This study was conducted to determine the spatiotemporal expression of putative human notochordal markers to aid in the elucidation of the ontogeny of adult human NP cells. Human embryos and fetuses (3.5-18 weeks post-conception (WPC)) were microdissected to isolate the spine anlagens (notochord and somites/sclerotome). Morphology of the developing IVD was assessed using hematoxylin and eosin. Expression of keratin (KRT) 8, KRT18, KRT19, CD24, GAL3, CD55, BASP1, CTGF, T, CD90, Tie2, and E-cadherin was assessed using immunohistochemistry. KRT8, KRT18, KRT19 were uniquely expressed by notochordal cells at all spine levels at all stages studied; CD24 was expressed at all stages except 3.5 WPC. While GAL3, CD55, BASP1, CTGF, and T were expressed by notochordal cells at specific stages, they were also co-expressed by sclerotomal cells. CD90, Tie2, and E-cadherin expression was not detectable in developing human spine cells at any stage. This study has identified, for the first time, the consistent expression of KRT8, KRT18, KRT19, and CD24 as human notochord-specific markers during early IVD development. Thus, we propose that these markers can be used to help ascertain the ontogeny of adult human NP cells. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 34:1327-1340, 2016.

Keywords: intervertebral disc degeneration; notochordal cells; nucleus pulposus; ontogeny; phenotype.

Figure 1

H&E staining of a cohort of developing spines. The notochord develops as a rod‐like centrally located structure formed by large and vacuolated notochordal cells (arrows) and surrounded by somites which will later become sclerotomal cells. During the analyzed stages, the notochord involutes to become localized to the central IVD region (NP anlagen) and sclerotomal cells adopt a segmented pattern; densely organized sclerotomal regions will form the developing AF. Sclerotomal cells in the adjacent regions have a round morphology and will later form the VB. (A) CS10, (B) CS16, (C) 7 WPC, (D) 8 WPC, (E) 9 WPC, (F) 10 WPC, (G) 12 WPC, (H) 14 WPC, (I) 17 WPC, (J) 18 WPC.

Figure 2

KRT18 immunostaining of a cohort of developing spines showing notochord‐specific expression of this marker. No KRT18 expression was seen in the surrounding sclerotomal AF and VB cells. (A) CS16, (B) 7 WPC, (C) 10 WPC, (D) 18 WPC. For stages 10 and 18 WPC, two higher magnifications are highlighted (squares), one that is centered to the developing notochordal NP (C2 and D2) and the other that is centered to the developing sclerotomal AF (C3 and D3).

Figure 3

CD24 immunostaining of a cohort of fetal spines showing notochord‐specific expression of this marker between CS16 and 18 WPC. No CD24 expression was seen in the surrounding sclerotomal cells in the developing AF and VB in all analyzed stage. (A) CS16, (B) 8 WPC, (C) 10 WPC, (D) 18 WPC. For stages 10 and 18 WPC, two higher magnifications are highlighted (squares), one that is centered to the developing notochordal NP (C2 and D2) and the other that is centered to the developing sclerotomal AF (C3 and D3).

Figure 4

GAL3 immunostaining of a cohort of fetal spines. GAL‐3 was notochord‐specific between 7 and 9 WCP after which it became co‐expressed by sclerotomal VB cells. (A) CS16, (B) 7.5 WPC, (C) 11 WPC, (D) 18 WPC. For stages 11 and 18 WPC, three higher magnifications are highlighted (squares), one that is centered to the developing notochordal NP (C2 and D2), other that is centered to the developing sclerotomal AF (C3 and D3) and another that is centered to the developing VB (C4 and D4).

Figure 5

CD55 immunostaining of a cohort of developing spines. CD55 was notochord‐specific between CS16 and 10 WPC, after which it became co‐expressed by sclerotomal cells in the developing AF. (A) CS16, (B) 7.5 WPC, (C) 12 WPC, (D) 18 WPC. For stages 12 and 18 WPC, two higher magnifications are highlighted (squares), one that is centered to the developing notochordal NP (C2 and D2) and the other that is centered to the developing sclerotomal AF (C3 and D3).

Figure 6

T immunostaining of a cohort of developing spines showing notochord and sclerotomal (VB anlagen) co‐expression between CS16 and 18 WPC. (A) CS16, (B) 7 WPC, (C) 10 WPC, (D) 18 WPC. For stages 10 and 18 WPC, three higher magnifications are highlighted (squares), one that is centered to the developing notochordal NP (C2 and D2), other that is centered to the developing sclerotomal AF (C3 and D3) and another that is centered to the developing VB (C4 and D4).

Figure 7

CTGF immunostaining of a cohort of fetal spines showing notochord and VB co‐expression between CS16 and 18 WPC. (A) CS16, (B) 7 WPC; (C) 11 WPC, (D) 18 WPC. For stages 11 and 18 WPC, three higher magnifications are highlighted (squares), one that is centered to the developing notochordal NP (C2 and D2), other that is centered to the developing sclerotomal AF (C3 and D3) and another that is centered to the developing VB (C4 and D4).

Figure 8

BASP1 immunostaining of a cohort of developing spines. BASP1 was localized to all notochordal and somite/sclerotomal cells at all stages analyzed, except at CS16, where no developing spine anlagen staining was found. (A) CS16 WPC, (B) 7.5 WPC, (C) 11 WPC, (D) 18 WPC. For stages 11 and 18 WPC, three higher magnifications are highlighted (squares), one that is centered to the developing notochordal NP (C2 and D2), other that is centered to the developing sclerotomal AF (C3 and D3) and another that is centered to the developing VB (C4 and D4).

Figure 9

Negative developing spine markers. CD90, Tie2, and E‐Cad immunostaining was not seen in any developing spine anlagen at any of the stages analyzed. (A) CD90 immunostaining in specimens A1: CS16 and A2: 18 WPC, A3 depicts CD90 staining of kidney (positive control). (B) Tie2 immunostaining in specimens B1: CS16 and B2: 18 WPC. B3 depicts Tie2 staining of placenta (positive control). (C) E‐Cad immunostaining in specimens C1: CS16 and C2: 18 WPC. C3 depicts E‐Cad staining of kidney (positive control).

Figure 10

Schematic representation of the protein staining in the developing human spine. KRT8, KRT18, and KRT19 were notochord‐specific markers at all stages analyzed and CD24 was notochord‐specific between 5.5 and 18 WPC. The expression of GAL3, CD55, CTGF, BASP1, and T varied with development stage. CD90, Tie2, and E‐Cad were not expressed by any developing spine cell anlagen.