High yield extraction of pure spinal motor neurons, astrocytes and microglia from single embryo and adult mouse spinal cord

Abstract

Extraction of mouse spinal motor neurons from transgenic mouse embryos recapitulating some aspects of neurodegenerative diseases like amyotrophic lateral sclerosis has met with limited success. Furthermore, extraction and long-term culture of adult mouse spinal motor neurons and glia remain also challenging. We present here a protocol designed to extract and purify high yields of motor neurons and glia from individual spinal cords collected on embryos and adult (5-month-old) normal or transgenic mice. This method is based on mild digestion of tissue followed by gradient density separation allowing to obtain two millions motor neurons over 92% pure from one E14.5 single embryo and more than 30,000 from an adult mouse. These cells can be cultured more than 14 days in vitro at a density of 100,000 cells/cm2 to maintain optimal viability. Functional astrocytes and microglia and small gamma motor neurons can be purified at the same time. This protocol will be a powerful and reliable method to obtain motor neurons and glia to better understand mechanisms underlying spinal cord diseases.

Transplanted enteric neural stem cells integrate within the developing chick spinal cord: implications for spinal cord repair

“…Their study revealed that transplanted iPSCMN localised to spinal cord white matter, with projections extending into the PNS, similar to the results presented here. Such localisation was also observed by Belkind-Gerson et al (2016). Those authors demonstrated that tail vein delivery of ENSC into mice resulted in homing of ENSC-derived cells to an injury site and white matter within the brain.…”

“…Future work will be aimed at examining the extent to which transplanted ENSCs contribute to the donor neural population in the host spinal cord post-transplant, including specific neural subtypes. Notably, Belkind-Gerson et al (2016) reported neuronal differentiation of ENSCs following transplantation into the brain, and previous studies within our lab showed neurogenesis following ENSC transplant into the gut using labelling with BrdU (Cooper et al 2016;McCann et al 2017), making ENSC a promising source of replacement neurons following SCI.…”

“…Although preclinical animal studies of ENSC have to date focused almost exclusively on ENS repair (Burns et al 2016;Stamp, 2017) the ENS shares several features with the SC that strongly support the use of ENSC as a stem cell-based therapy for SCI. Immunofluorescence analysis of cultured murine ENSC has revealed TuJ1 + neurons, S100 + glia and SOX10 + /S100-labelled stem cells (Metzger et al 2009b;Natarajan et al 2014), all of which are markers that are also expressed in central nervous system (CNS) tissues (Barnabe-Heider et al 2010;Beaudet et al 2015). Thus, there is a rationale for using ENSC to replace cells lost through SCI.…”

ALS-linked SOD1 Mutants Enhance Outgrowth, Branching, and the Formation of Actin-Based Structures in Adult Motor Neurons

“…These mice develop symptoms more rapidly, with hind-limb paralysis seen as early as 5 months of age (Gurney, 1994). Using a well-characterized protocol for the high yield extraction of spinal motor neurons from adult mice (Beaudet et al, 2015), we established cultures of adult motor neurons from mutant SOD1 and non-transgenic mice (referred to as NTg). We then performed a large-scale quantitative analysis of these cells' ability to extend new processes.…”

“…Embryo spinal cords were obtained from timed pregnant G93A-DL and C57BL/6J mice at embryonic day 14 as previously described in detail (Beaudet et al, 2015). Once embryos were removed from the uterus, spinal cords were extracted under sterile conditions in a laminar flow hood with the aid of a dissecting microscope (SMZ800, NIKON INSTRUMENTS INC.) and small forceps and placed into cold Leibovitz's L-15 medium (Life Technologies, Grand Island, NY) supplemented with 25 μg ml −1 penicillinstreptomycin (Life Technologies).…”