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Cell and Tissue Research

, Volume 373, Issue 2, pp 421–438 | Cite as

Neurospheres from neural stem/neural progenitor cells (NSPCs) of non-hydrocephalic HTx rats produce neurons, astrocytes and multiciliated ependyma: the cerebrospinal fluid of normal and hydrocephalic rats supports such a differentiation

  • Roberto Henzi
  • Montserrat Guerra
  • Karin Vío
  • César González
  • Cristian Herrera
  • Pat McAllister
  • Conrad Johanson
  • Esteban M. Rodríguez
Regular Article
  • 645 Downloads

Abstract

Fetal onset hydrocephalus and abnormal neurogenesis are two inseparable phenomena turned on by a cell junction pathology first affecting neural stem/progenitor cells (NSPCs) and later the multiciliated ependyma. The neurological impairment of children born with hydrocephalus is not reverted by derivative surgery. NSPCs and neurosphere (NE) grafting into the cerebrospinal fluid (CSF) of hydrocephalic fetuses thus appears as a promising therapeutic procedure. There is little information about the cell lineages actually forming the NE as they grow throughout their days in vitro (DIV). Furthermore, there is no information on how good a host the CSF is for grafted NE. Here, we use the HTx rat, a model with hereditary hydrocephalus, with the mutation expressed in about 30% of the litter (hyHTx), while the littermates develop normally (nHTx). The investigation was designed (i) to establish the nature of the cells forming 4 and 6-DIV NE grown from NSPCs collected from PN1/nHTx rats and (ii) to study the effects on these NEs of CSF collected from nHTx and hyHTx. Immunofluorescence analyses showed that 90% of cells forming 4-DIV NEs were non-committed multipotential NSPCs, while in 6-DIV NE, 40% of the NSPCs were already committed into neuronal, glial and ependymal lineages. Six-DIV NE further cultured for 3 weeks in the presence of fetal bovine serum, CSF from nHTx or CSF from hyHTx, differentiated into neurons, astrocytes and βIV-tubulin+ multiciliated ependymal cells that were joined together by adherent junctions and displayed synchronized cilia beating. This supports the possibility that ependymal cells are born from subpopulations of NSC with their own time table of differentiation. As a whole, the findings indicate that the CSF is a supportive medium to host NE and that NE grafted into the CSF have the potential to produce neurons, glia and ependyma.

Keywords

Congenital hydrocephalus Neural stem cells Neurospheres Ependymogenesis Cerebrospinal fluid 

Abbreviations

AJ

adherent junctions

AQP4

aquaporin 4

BrdU

bromodeoxyuridine

CSF

cerebrospinal fluid

DIV

days in vitro

EGF

epidermal growth factor

FBS

fetal bovine serum

FGF

fibroblast growth factor

GFAP

glial fibrillary acidic protein

GJ

gap junctions

hyHTx

hydrocephalic Texas rat

NSPCs

neural stem and progenitor cells

NE

neurospheres

nHTX

non-hydrocephalic Texas rat

PN

postnatal

SVZ

subventricular zone

VZ

ventricular zone

Notes

Acknowledgements

The authors wish to acknowledge the valuable technical support of Mrs Clara Jara, and Confocal and Electron Microscopy Core Facilities of Universidad Austral de Chile. Monoclonal antibodies against nestin and BrdU were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, Iowa.

Supplementary material

441_2018_2828_MOESM1_ESM.pptx (50.9 mb)
ESM 1 6DIV neurospheres cultured in the presence of FGF and then further cultured for 21 d in the presence of 5% FBS but no FGF. Phase contrast microscopy of an area similar to that shown in Fig. 6(d). The arrows point to cilia beating of ependymal cells generated in vitro. (PPTX 52118 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Roberto Henzi
    • 1
    • 2
  • Montserrat Guerra
    • 1
  • Karin Vío
    • 1
  • César González
    • 1
    • 3
  • Cristian Herrera
    • 1
  • Pat McAllister
    • 4
  • Conrad Johanson
    • 5
  • Esteban M. Rodríguez
    • 1
  1. 1.Instituto de Anatomía, Histología y Patología, Facultad de MedicinaUniversidad Austral de ChileValdiviaChile
  2. 2.Centro de Investigaciones Biomédicas, Facultad de MedicinaUniversidad de los AndesSantiagoChile
  3. 3.Facultad de MedicinaUniversidad San SebastiánPuerto MonttChile
  4. 4.Department of Neurosurgery Division of Pediatric NeurosurgeryWashington University and the Saint Louis Children’s HospitalSt. LouisUSA
  5. 5.Department of NeurosurgeryAlpert Medical School at Brown UniversityProvidenceUSA

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