Time Course of Spinal Doublecortin Expression in Developing Rat and Porcine Spinal Cord: Implication in In Vivo Neural Precursor Grafting Studies

Abstract

Expression of doublecortin (DCX), a 43-53 kDa microtubule binding protein, is frequently used as (i) an early neuronal marker to identify the stage of neuronal maturation of in vivo grafted neuronal precursors (NSCs), and (ii) a neuronal fate marker transiently expressed by immature neurons during development. Reliable identification of the origin of DCX-immunoreactive cells (i.e., host vs. graft) requires detailed spatial and temporal mapping of endogenous DCX expression at graft-targeted brain or spinal cord regions. Accordingly, in the present study, we analyzed (i) the time course of DCX expression in pre- and postnatal rat and porcine spinal cord, and (ii) the DCX expression in spinally grafted porcine-induced pluripotent stem cells (iPS)-derived NSCs and human embryonic stem cell (ES)-derived NSCs. In addition, complementary temporospatial GFAP expression study in porcine spinal cord was also performed. In 21-day-old rat fetuses, an intense DCX immunoreactivity distributed between the dorsal horn (DH) and ventral horn was seen and was still present in the DH neurons on postnatal day 20. In animals older than 8 weeks, no DCX immunoreactivity was seen at any spinal cord laminae. In contrast to rat, in porcine spinal cord (gestational period 113-114 days), DCX was only expressed during the pre-natal period (up to 100 days) but was no longer present in newborn piglets or in adult animals. Immunohistochemical analysis was confirmed with a comparable expression profile by western blot analysis. Contrary, the expression of porcine GFAP started within 70-80 days of the pre-natal period. Spinally grafted porcine iPS-NSCs and human ES-NSCs showed clear DCX expression at 3-4 weeks postgrafting. These data indicate that in spinal grafting studies which employ postnatal or adult porcine models, the expression of DCX can be used as a reliable marker of grafted neurons. In contrast, if grafted neurons are to be analyzed during the first 4 postnatal weeks in the rat spinal cord, additional markers or grafted cell-specific labeling techniques need to be employed to reliably identify grafted early postmitotic neurons and to differentiate the DCX expression from the neurons of the host.

In vivo reprogramming of NG2 glia enables adult neurogenesis and functional recovery following spinal cord injury

“…We first examined cellular responses to SCI by staining for DCX, a well-established molecular marker for immature neurons during adult neurogenesis (Couillard-Despres et al, 2005;Kempermann et al, 2004;Knoth et al, 2010). Consistent with previous reports (Juhasova et al, 2015;Su et al, 2014) ). The thoracic T7-T9 level of the spinal cord was then injured by crushing.…”

“…DCX is normally expressed in neuroblasts and immature neurons and can serve as a reliable marker for adult neurogenesis (Couillard-Despres et al, 2005;Kempermann et al, 2004;Knoth et al, 2010). It is highly expressed in the developing spinal cord but completely turned off in the adult (Juhasova et al, 2015). Although DCX + cells have been observed previously surrounding the lesion site in the adult spinal cord (Ziv et al, 2006), their origin and final fate is not clear.…”

Chromatin establishes an immature version of neuronal protocadherin selection during the naive-to-primed conversion of pluripotent stem cells

“…20). In this dataset, PCDHGC4 [37][38][39] and DCX 43 reach the lowest expression after 1-2 years of age, whereas PCDHGC5 and synaptic and glial components (CAMK2A, SCN1B, PLP1, and GFAP 42 ) reach the highest expression after this age ( Fig. 6b, profiles).…”

Establishment of Transgene‐Free Porcine Induced Pluripotent Stem Cells

“…The potential benefits of non-integrative (transgene-free) reprogramming were already well established, as integrative methods can suffer from persistent transgene expression or re-activation, which can influence differentiation (Yu et al, 2007) and raise the risk of tumorigenesis (Okita et al, 2007) or immunogenicity (Zhao et al, 2011). However, despite efforts over the following years (Aravalli et al, 2012;Bui et al, 2012;Congras et al, 2016;Juhasova et al, 2015;Park et al, 2013;Strnadel et al, 2018), most reprogramming reports continued to be performed using integrative strategies, such as retrovirus-(as in Wu et al, 2009), lentivirus-(as in Ezashi et al, 2009), or transposon-mediated (as in Kues et al, 2013) reprogramming, rather than episomal plasmids. The lingering challenges of low reprogramming efficiency and episomal plasmid retention (Okita et al, 2008;Du et al, 2015;Telugu et al, 2010) remained persistent obstacles.…”