Elsevier

Brain Research Bulletin

Volume 156, March 2020, Pages 25-32
Brain Research Bulletin

Research report
Differential susceptibility of human neural progenitors and neurons to ischaemic injury

https://doi.org/10.1016/j.brainresbull.2019.12.005Get rights and content

Highlights

  • Structurally mature hESC-derived neurons are more sensitive to ischaemia.

  • hESC-derived neural progenitors are vulnerable to ischemia-reperfusion.

  • Changes in energy metabolism are not tightly linked to cell death.

Abstract

Background

Neuroprotection for stroke has shown great promise but has had little translational success. Developing drugs for humans logically requires human tissue evaluation. Human embryonic stem cell (hESC)-derived neuronal cultures at different developmental stages were subject to oxygen glucose deprivation (OGD) to determine how developing maturity altered response to ischemic injury.

Methods

H9 hESCs were induced by Noggin to generate neural progenitors (NPs) and highly arbourised structurally complex neurons. They were both subjected to OGD or OGD with reoxygenation (OGD-R) for 1−6 h.Outcome was assessed by measures of cell death, survival and morphology.

Results

NPs did not die after OGD but experienced progressive loss of metabolic activity. Highly arbourised neurons showed minimal cell death initially but 44 % and 78 % died after 4 and 6 h OGD. Metabolic dysfunction was greater in these more mature neurons (∼70 %) than in NPs and evident after 1 h OGD, before detection of neuronal death at 4 h. OGD-R salvaged metabolic activity but not cell death in mature neurons. In NPs there was little metabolic salvage and cell death was induced (50 % and 65 % at 4 and 6 h OGD-R, respectively).

Conclusions

Highly arbourised neurons are more sensitive to ischaemic injury than NPs which did however develop marked vulnerability to prolonged injury with reoxygenation. These observations imply that therapeutic potential may be highly dependent of the developmental state of the neurons we aim to protect.

Introduction

Translational neuroscience presents many challenges due to the complexity of the brain and its interdependence with all the other organs of the body. Even so, it has proven unexpectedly difficult to move from successful animal experiments to successful human clinical trials with reports of translational failure for diseases as diverse as stroke, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, traumatic brain injury and spinal cord injury (O’Collins et al., 2006; Watzlawick et al., 2014; Athauda and Foltynie, 2016; Pistollato et al., 2016; Tator et al., 2012; Antonic et al., 2014).

The breadth of this failure strongly suggests we lack essential knowledge specific to human brain responses to injury and disease but getting samples from the living human brain to address such questions is particularly difficult, and post-mortem tissues are limited in their potential to reveal the workings of the tissue during life. A range of immortal/immortalised cell lines has been used as surrogates for live human tissues. But for neurons, in particular, these provide a poor representation of the morphological complexity that exists in vivo, and the cell biology of continuously dividing cultures seems unlikely to provide a useful recapitulation of terminally differentiated non-dividing neurons (Schneider et al., 2011; Salvatore Amoroso et al., 1999).

Advances in human stem cell biology are beginning to provide solutions to these problems. It is now possible to generate an endless supply of cells that differentiate into neurons with the characteristic morphology and molecular biology of terminally differentiated human neurons.

Glial cells, endothelial cells, pericytes and invading and circulating inflammatory cells all influence neuronal responses to many stimuli after ischemic injury. Untangling these complex interactions is not the subject of this study. Understanding the complexity of such interactions is best served by first understanding the responses of individual cell types. In this study, we have used human embryonic stem cell (hESC)-derived neural progenitors and morphologically complex neurons to start this complex process in a proof of principle study of model human neuronal responses to ischaemic injury.

Section snippets

Cell Culture and induction of neural differentiation

The H9 (WA09) hESC line was supplied by the WiCell Research Institute and maintained in the Stem Cell Core Facility, Stem Cells Australia. Testing for mycoplasma contamination along with chromosome authentication by karyotyping was routinely performed.

hESC colonies were mechanically cut into small pieces and plated onto pre-prepared mitotically inactivated mouse embryonic fibroblasts (MEF). The outer edge of the colonies containing undifferentiated cells was sub-cultured in hES medium

Significant cell death in hESC-derived mature neurons but not NPs to OGD

The majority of MAP2+/arbour + cells in NP control cultures had an immature morphology with short branches and limited connectivity (Fig. 1A). A subpopulation of cells was MAP2+/arbour- (Fig. 1A, white arrows). After OGD, NP cultures displayed relatively little cell death but did exhibit focal bead-like swellings along the sparse MAP2+ neurites (Fig. 1B, B-1, white arrows, enlarged from Fig. 1B dotted frame). Even after 4 h OGD, cell death was not apparent, but MAP2+/arbour- cells were observed

Discussion

We have previously shown (Liu et al., 2019a) that 80 % ± 4.04 % (p < 0.001) of the cells present at 49DIV are MAP2 expressing neurons and that 80 % of these co-express NeuN. Quantitative PCR and immunohistochemistry indicated that the majority were either GABAergic or Glutamatergic with a small number of cholinergic and dopaminergic neurons and a small number of astrocytes and oligodendrocytes. This study found that susceptibility to ischaemia differed markedly between hESC derived neural

Conclusions

Our results demonstrate differences in the responses of human NPs and their arbourised structurally mature neuronal derivatives to two clinically relevant ischaemic injury conditions. These results imply we may be missing opportunities to maximise protection of the stroked brain during reperfusion but also that reperfusion therapies may pose additional risks to immature brains. Our data also suggest that hESC-derived neural cultures may provide an important test-bed for exploring human

Ethical approval

The use of H9 (WA09) hESC line was approved by University of Melbourne human ethics committee.

Author contributions

YL performed all experiments, assisted by AA. AEM maintained and provided the human embryonic stem cell lines. MD provided infrastructural support. YL and AA analysed the data. YL drafted the manuscript. EE and JMC and DWH revised the manuscript. DWH conceived the project and provided the funding.

Funding

This work was supported by the Australian National Health and Medical Research Council (NHMRC), grant number: 1013621(Improving Stroke Outcomes: Attenuating Progression and Recurrence) and 1037863(Biomarkers for assessing the effectiveness of hypothermia as a therapy in ischemic stroke patients).

Declaration of Competing Interest

The authors declare that they have no conflict of interest regarding research, authorship, and/or publication of this article.

References (23)

  • E.E. Franz Faul

    GPower3: a flexible statistical power analysis program for the social, behabioral, and biomedical sciences

    Behav. Res. Methods

    (2007)
  • Cited by (0)

    View full text