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Differential susceptibility of human neural progenitors and neurons to ischaemic injury
Journal Pre-proof Differential susceptibility of human neural progenitors and neurons to ischaemic injury Ye Liu, Anna E. Michalska, Mirella Dottori, Emma Eaton, Jo-Maree Courtney, Ana Antonic, David W. Howells
PII:
S0361-9230(19)30484-8
DOI:
https://doi.org/10.1016/j.brainresbull.2019.12.005
Reference:
BRB 9823
To appear in:
Brain Research Bulletin
Received Date:
22 June 2019
Revised Date:
10 November 2019
Accepted Date:
10 December 2019
Please cite this article as: Liu Y, Michalska AE, Dottori M, Eaton E, Courtney J-Maree, Antonic A, Howells DW, Differential susceptibility of human neural progenitors and neurons to ischaemic injury, Brain Research Bulletin (2019), doi: https://doi.org/10.1016/j.brainresbull.2019.12.005
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Differential susceptibility of human neural progenitors and neurons to ischaemic injury
Ye Liua, Anna E. Michalskab, Mirella Dottoric, Emma Eatond, Jo-Maree Courtneyd, Ana Antonice, and David W. Howells*d
Department of Neurology, Huashan Hospital, Shanghai, 200040, China. State Key
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a
Laboratory of Medical Neurobiology, Fudan University, Shanghai, 200433, China. b
Stem Cell Core Facility, Stem Cells Australia, The University of Melbourne, Victoria 3010, Australia
Illawarra Health and Medical Research Institute, University of Wollongong, NSW, 2522
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c
Australia
School of Medicine, University of Tasmania, Hobart, Tasmania 7001, Australia
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Department of Neuroscience, Central Clinical School, Monash University, The Alfred
Corresponding author.
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*
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Centre, VIC 3004, Australia
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d
Email address: [email protected]
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Telephone numbers: +61 (3) 6226 4850
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.
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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 hours. Mild hypothermia (33°C) was used to assess neuroprotective potential in the structurally mature neurons
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after 1 or 4 hours OGD-R. 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 6h OGD. Metabolic dysfunction
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was greater in these more mature neurons (70%) than in NPs and evident after 1h OGD, before detection of neuronal death at 4h. OGD-R salvaged metabolic activity but not cell death in mature neurons. In NPs there was
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little metabolic salvage and cell death was induced (50% and 65% at 4 and 6h OGD-R, respectively).
Conclusions: Highly arbourised neurons are more sensitive to ischaemic injury than NPs which did however
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develop marked vulnerability to prolonged injury with reoxygenation. These observations imply that therapeutic
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potential may be highly dependent of the developmental state of the neurons we aim to protect.
hESC neuronal differentiation, OGD sensitivity, Stem cells
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Keywords:
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 [1-6].
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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 [7, 8].
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
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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
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Experimental procedures
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neuronal responses to ischaemic injury.
Cell Culture and induction of neural differentiation
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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.
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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 subcultured in hES medium containing 500ng/mL noggin (R&D system, 6057-NG-100) for 14 days to allow neural induction. After 14-days noggin treatment, the differentiated central parts of these new colonies were cut out and transferred to individual wells in a low attachment 96-well plate containing Neurobasal Medium/B27/N2
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supplemented with EGF and FGF to allow neurosphere (NS) formation.
Fourteen-day NSs were isolated, dissociated and allowed to continue their differentiation for a further 5 days in vitro (DIV) to provide neural progenitors. Highly arbourized neurons were also derived from 14-day NSs using methods developed in our lab [9], and maintained for up to 49DIV. Briefly, 14-day NSs were cut into smaller pieces and placed into individual wells of a 24-well plate pre-coated with poly-d-lysine (PDL) and laminin (10 µg/ml; Sigma, P6407 and Life Tech, 23017015, respectively). These pieces were cultured in Neurobasal Medium (NBM) made in our lab (supplement table2) without EGF and bFGF followed by re-collection and dissociation by accutase (Thermo Fisher, A1110501, supplied as ready-to-use cell dissociation reagent) at 28 days. Cell pellets were then re-suspended, filtered (40μm cell strainers, BD, 352340) and plated for immunostaining into 35mm2
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dishes containing coverslips (13mm round Menzel PK100, Grale Scientific, CS13100) and for MTT assay into 96-well plates. The seeding density for 35mm2 dishes with coverslips was 600,000 (NPs)/700,000 (49DIV neurons) and for 96-well plates was 20,000 (NPs)/30,000 (49DIV neurons) per well. The cells were then incubated at 37◦C in 5% CO2, 20% O2 with 95% humidity and ½ of the NBM was changed every second day until the experiments were terminated on day 49.
Oxygen and glucose deprivation and reoxygenation Plates with cultured NPs or 49DIV highly arbourized neurons were randomly allocated to the control or injury groups (4 time points: 1, 2, 4, and 6h) using the RAND function in Microsoft Excel. Glucose deprivation was achieved by replacing NBM with glucose-free NBM. The plates were then placed in a hypoxic chamber (STEMCELL, #27310), flushed with nitrogen at a rate of 20L/min for 10 minutes and placed in a 37°C incubator
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for 1, 2, 4 or 6h to induce the OGD injury. Cells in the OGD control groups were given fresh NBM and cultured at 37°C for 6h. Experiments in the OGD groups were terminated at the designated injury times and immediately processed for analysis. OGD-R groups including the control groups were given fresh NBM after the OGD injury to replace the deoxygenated medium and returned to the 37°C incubator until the total incubation time reached 24h in culture before the analysis. Cells in the OGD-R control group underwent the same procedure as OGD
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control followed by replenishment with fresh NBM and continued culture for another 18h.
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Detection of metabolic dysfunction by MTT assay
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide (MTT) is a yellow water-soluble tetrazolium salt, which is metabolised into a water-insoluble formazan in viable cells by the action of functional NAD+/NADP+
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dependent dehydrogenases. This assay is widely used as a surrogate of mitochondrial function. Briefly, 10l MTT (Sigma Cat#M2128, 5mg/mL in PBS) was added directly to each well following OGD or OGD-R and a vehicle control without cells but 100l NBM. The plates were then incubated at 37°C for 4h followed by the addition of
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100l DMSO. The absorbance was measured at 470nm using a plate reader (Bio-strategy, Spectra Max M3). Relative energy state was calculated by comparing the absorbance value in injured groups to those in the control groups.
Absorbance (Injury group) × 100% Absorbance (Control group)
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Relative energy state (%) =
These values were normalised to the measurements from uninjured controls. Cell death and morphology determined by staining A nuclear cell death and viability staining kit (ENZO-53004) containing a mixture of a blue fluorescent cellpermeable nucleic acid dye (excitation: 350nm, emission: 461nm) and a green fluorescent live-cell-impermeable nucleic acid dye (excitation: 503nm, emission: 524nm) was used to assess cell death after OGD and OGD-R injury. The dead cells appear cyan when combined with the nuclear stain, which is easily distinguished from either stain alone.
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Upon termination of the experiments, two coverslips were randomly selected, and placed into a new 24-well plate. 250l Blue/Green assay mixture (1:2000 in NBM) was added to each well and incubated at 37°C for 30 minutes. Cells were fixed with 2% PFA, and incubated with MAP2 antibody (Millipore, MAB5622, 1:200 dilution in 3% FCS and 0.3% Triton-X) at 4°C overnight. The next day, the cells were washed twice before addition of a donkey anti-rabbit (568) antibody (Life Tech, A10042, diluted 1:500 in 2% FCS) for 60 minutes at room temperature in the dark. Finally, 250µl of a 1:10,000 dilution of DAPI stock solution was incubated for 10 minutes followed by inverting and mounting coverslips on a standard glass microscope slide with a mounting medium containing a fluorescence anti-fade reagent (Molecular Probes, P36930).
Statistical analysis For the cell death assay and immunocytochemical staining of MAP2, the sample size was determined by power
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analysis using the software G*power [10]. To detect an effect size of 0.35 using one-way analysis of variance (ANOVA) with a fixed effects omnibus model with error probability of 0.05, power (1- error probability) 0.95 for five groups, giving a total number of 160 for samples that need to be analysed.
Each experiment was repeated on three different occasions. Within each experiment, five visual fields (centre,
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top, bottom, left, right) were selected from each of at least two samples (coverslips) for each time point. All images were taken at 200X magnification using a Zeiss Axio Observer 7 inverted fluorescence microscope. Images were then re-labelled and randomised by another researcher to blind the counting of positively stained (dead) cells using
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the Cell Counter plugin of Image J (NIH, 1.50i). MAP2+/arbour+ cells have MAP2 staining of both the cell soma and processes. MAP2+/arbour- cells did not have process staining. Dead cell number was expressed as a % of
background cell death.
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the total number of DAPI positive cells. Normalisation against uninjured controls was used to correct for
The same sampling regime and power assumptions were used for the quantitation of the MTT assay (which was
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replicated twice) as the effect size was expected to be larger than the cell death assay.
All values are presented as mean ± SEM, and all graphics are drafted using PRISM7. One-way and two-way ANOVA with Bonferroni post-hoc error correction was used to identify significant differences within and
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between the groups at the different experimental times.
Results
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
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MAP2+ neurites (Fig 1B, 1B-1, white arrows, enlarged from Fig 1B dotted frame). Even after 4h OGD, cell death was not apparent, but MAP2+/arbour- cells were observed to aggregate in the culture (Fig 1C, white box).
Counting cells permeable to the nuclear stain did not detect significant changes in cell death with increasing OGD duration (24.851.9% and 32.133.17% for 1h OGD and 6h OGD, respectively). Similarly, the number of the MAP2+/arbour+ cells did not alter significantly as OGD duration increased (Fig 1D). However, the mitochondrial activity assessed by MTT assay was significantly suppressed to 66.783.72% (p<0.001) of control activity within 1h OGD and continued to decline as OGD duration increased. After 6h OGD, only 35.242.87% (p<0.001) of MTT detectable mitochondrial activity remained (Fig 1D). The mature neurons cultured for 49DIV exhibited complex network structures, with long and arbourised processes
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making frequent contacts with each other in the control culture. MAP2+/arbour+ neurons were the predominant cellular type, comprising 601.68% of the total cell number (Fig 2A, D). Process disruption (neuritic breakage) on MAP2+/arbour+ neurons was detected after 1h OGD (Fig 2B-1, white arrows, enlarged from Fig 2B, dotted frame). However, increased cell death only became apparent after 4h OGD (44.172.7%, p<0.001), and increased further by 6h OGD (78.113.72%, p<0.001) (Fig 2D). Loss of MAP2+/arbour+ neurons mirrored total cell death
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with significant differences detected at 4h (42.842.07%, p<0.001) and 6h OGD (16.462.67%, p<0.001) (Fig 2D).
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The metabolic activity of the highly arbourised neurons exhibited extreme sensitivity to OGD. Only 30.69±1% (p<0.001) of the cells maintained normal function after 1h OGD, however, there was no further deterioration with
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increased OGD duration (2h: 31.63±1.05%, 4h: 31.15±0.95%, 6h: 29.37±0.91%) (Fig 2D).
Significant cell death in hESC derived NPs and highly arbourised neurons after OGD-R
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Neuritic beading showing damage to NP projections was also detected early after OGD-R (Fig 3B, white dotted box and Fig 3B-1). However, while NPs survived well after OGD, marked cell death was detected after OGD-R and the cultures acquired essentially the same cell death profile as seen in more mature arbourised neurons in response to OGD. 49.784.95% and 65.335.37% (p<0.001) of NPs died after 4 and 6h OGD-R, respectively (Fig 3D). While the number of MAP2+/arbour+ neurons changed little after OGD-R, the number of
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MAP2+/arbour- cells fell as cell death became apparent with very few left in the cultures by 6h (41.23%, p<0.001). Metabolic activity in NPs after OGD-R was less severely affected than after OGD alone, but was significantly reduced at all time points (1h 84.111.62%, p<0.001; 2h 78.83.22%, p<0.001; 4h 72.212.37%, p<0.001 and 6h 36.65%, p<0.00) (Fig 3D). Highly arbourised more mature neurons suffered marked damage to neuronal processes after 1h OGD-R (Fig 4B1, white arrows, enlarged from Fig 4B, dotted frame) although substantial neuronal death was not detected. After 4h OGD-R, neurites became sparser (Fig 4C) and neuronal death continued to increase (65.433.85%, p<0.001) until 6h (85.132.82%, p<0.001) (Fig 4D). Loss of MAP2+/arbour+ neurons mirrored cell death with a reduction to 28.773.07%, p<0.001 and 13.382.45, p<0.001 after 4 and 6h OGD-R respectively (Fig 4D). Unlike the
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response of arbourised mature neurons to OGD, metabolic activity was maintained after OGD-R and only became significantly depleted after 6h (76.426.44%, p<0.001) (Fig 4D).
Comparison of cell death and metabolic activity after OGD and OGD-R injury responses To permit a clearer comparison across experiments, the quantitative data were plotted by outcome measure in Figure 5 where 2-way ANOVA has been used to examine differences in the response of the two cell types in the presence of OGD or OGD-R.
The MTT assay of metabolic activity showed marked differences between NPs and more mature arbourised neurons and between OGD and OGD-R. For NPs, the steady decline in mitochondrial activity detected by MTT in OGD was attenuated but not prevented by OGD-R. At 1, 2 and 4h, suppression of mitochondrial activity was
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17.33% (p<0.01), 16.26% (p<0.01) and 21.6% (p<0.001) less in OGD-R than in OGD. However, by 6h of injury, the degree of compromise was similar in both groups. While arbourised neurons showed a dramatic and early loss of mitochondrial function after OGD, this loss was markedly suppressed after OGD-R with 60.29% (p<0.001), 55.15% (p<0.001), 55.31% (p<0.001) and 47.05% (p<0.001) less loss at 1, 2, 4 and 6h respectively (Fig 5A).
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In NP cultures, the profile of cell death after OGD-R was similar to that for OGD for the first 2h of the experiment with few cells dying. However, the profiles of death diverged thereafter, with 22.093.43% (p<0.001) and 33.24.95% (p<0.001) more death after OGD-R at 4 and 6h respectively. This later profile was very similar to
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that seen for more mature arbourised neurons under both OGD and OGD-R conditions (Fig 5B).
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In NP cultures, MAP2+/arbour+ neurons accounted for approximately 40% of the total cell population, and this proportion did not alter significantly despite the induction of OGD or OGD-R injury. In contrast, in mature neuron cultures, the number of MAP2+/arbour+ neurons was initially higher (~60%) and started to decline dramatically after 4h in both OGD and OGD-R injury models (Fig 5C). It is not clear whether the change in cell death (Fig 5B)
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and MAP2+/arbour+ neuron number (Fig 5C) at 1h after OGD-R in mature neuron cultures represents a biologically important change or a statistical aberration.
A MAP2+/arbour- subpopulation of cells existed only in the NP cultures. While they were not affected by OGD, they exhibited marked vulnerability to OGD-R. Little difference was evident until 4h (OGD: 39.14% vs OGD-R: 10.7%, p<0.001) and 6h (OGD: 33.3% vs OGD-R: 4.03%) when most of these cells had been lost in OGD-R
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compared to OGD (Fig 5D).
Discussion We have previously shown [9] 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
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markedly between hESC derived neural progenitors and the highly arbourised neuronal products of their differentiation.
While NP appeared insensitive to up to six hours of OGD, morphologically complex neuronal cultures, which we have previously shown to contain ~80% NeuN+ neurons and to express the neurochemical machinery required for neurotransmission [9], died rapidly with OGD durations of more than two hours. This neuronal death, detected by staining dependent on loss of nuclear integrity, was mirrored by the loss of the MAP2+/arbour+ neurons. Despite the lack of cell death detected by the nuclear stain, NPs did show signs of damage. Beading of MAP2+ projections on NPs was identified within one hour of OGD and the MTT assay detected a steady loss of metabolic activity as OGD progressed. However, while morphologically complex neurons reached maximum suppression of this activity within one hour of OGD, NPs took six hours to reach the same level. These data are consistent
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with a higher resistance to hypoxic depolarisation in mitochondria from human new-born brains than those from adult brains [11], suggesting that this might be because neural stem/progenitor cells have a lower requirement for oxidative metabolism than mature neurons [12].
These data indicate that it is inappropriate to use morphologically simple cells in attempts to model stroke, the results will clearly be misleading. Whether we can directly extrapolate the behaviour of these highly arborized
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human neurons in culture to those that might inhabit the brain of an elderly subject before a stroke remains unclear. Nevertheless, understanding the principle that arborisation increases sensitivity to injury seems likely to be
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important.
In the stroke research field were no neuroprotective strategies have successfully translated from either animal or
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animal tissue models to the clinic, understanding that the state of the cells used to model disease is critical for the outcomes measures is important. Unfortunately, until candidates have been screened prospectively as suggested here and progressed to completed clinical trial, we cannot know for certain. However, since we have previously
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shown that using arborized human neurons allows us to distinguish between NXY-059[13] which failed in clinical trial[14] and hypothermia[6] which does protect the brain in neonates with hypoxic ischemic encephalopathy (HIE) and during surgery requiring cardiac arrest[16], it seems likely that testing in human cells as described here will become an important part of the stroke drug development armamentarium. Moreover, whilst our laboratory’s focus is stroke, these findings might have more immediate impact in screening for drugs to better treat HIE where currently untestable arguments about how well these cells might mimic the responses possible in the aged brain
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are redundant. Importantly, understanding the molecular differences that underpin these markedly different responses will not only advance our knowledge of how ischaemic injury accrues but also point to important therapeutic targets.
We found that reperfusion provided no protection to mature neurons in terms of cell death even though energy metabolism was dramatically restored. This indicates that after the initial trigger, components of the cell death response are irrevocable, and they are not tied entirely to reduced energy metabolism. Indeed, out data would seem to suggest the metabolic activity of the surviving neurons dramatically increases. This is graphically illustrated in Supplementary Figure 1 which shows the MTT metabolic activity data normalised for surviving
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neuron number. Sanderson and colleagues propose an explanation for this phenomenon, dephosphorylation primed hyperactivity of oxidative phosphorylation upon reperfusion[17]. The phenomenon of reperfusion injury is well recognised in the literature [18, 19]. These observations emphasize that while reperfusion of the ischemiaaffected brain is essential, doing so may not prevent neuronal death that has already been initiated. Understanding what the point of no-return is for different cells under different circumstances is critical for delivery of the most effective therapy across a range of human diseases experiencing ischaemia of different degrees at different stages of development. Our data emphasises that the earliest possible intervention has the greatest potential for benefit.
In NP cultures, reperfusion was less effective at protecting metabolic activity and appeared to increase cell death, especially of the MAP2- cells. This indicates that active management of reperfusion injury may be particularly critical for the neonatal and still-developing juvenile brain exposed to HIE, stroke or asphyxiation. In the adult
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brain, endogenous neural stem cell populations such as those believed to be important for gliogenesis or neuroplasticity may be particularly at risk of reperfusion injury[20-22]. Moreover, if stem-cell based therapeutic interventions for brain injuries become a reality, the implanted cells would be expected to be at risk.
The disparity between the results of the assays of cell death and energy metabolism, which are widely used and often interpreted as assays of cell viability, is important. This is consistent with similar observations from
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systematic review and meta-analysis studies of OGD in SH-SY5Y cells, the most widely used surrogate for human neurons [23]. This indicates that it is inappropriate to use the morphologically simple cells typically used to model
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stroke in vitro as the results will be misleading.
In the NPs, the majority of cells, even after six hours OGD, have compromised mitochondrial function (58%),
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but few are actually dead (7%) suggesting a potential for salvage of approximately half of the population by some form of neuroprotection. For the mature neurons, extrapolation suggests the window for significant neuroprotection may be closing between four and six hours after injury of OGD (Table 1). This is entirely
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consistent with the window available for thrombolytic therapy in the clinic [24].
One limitation of this proof of principle study is that a single hESC line has been used, the particularly well studied H9 line. Verification of our findings in other lines will be important but selecting from amongst the 400 hESC lines listed as commercially available by NIH will present a challenge beyond the scope of funding and ethics approval available for this study. It would seem logical to also verify this data in primary adult human neurons.
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However, human neurons excised during epilepsy or tumour surgery and then cultured may not be truly representative of basal activity in the intact brain.
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
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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 ischaemic biology and screening of candidate therapies.
Conflict of Interest.
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
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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
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(Biomarkers for assessing the effectiveness of hypothermia as a therapy in ischemic stroke patients).
Declaration of conflicting of interests
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The authors declare that they have no conflict of interest regarding research, authorship, and/or publication of
Ethical Approval
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this article.
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The use of H9 (WA09) hESC line was approved by University of Melbourne human ethics committee.
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[20] G. Yadirgi, S. Marino, Adult neural stem cells and their role in brain pathology, The Journal of Pathology, 217 (2009) 242-253. [21] M.A. Curtis, M. Kam, R.L.M. Faull, Neurogenesis in humans, European Journal of Neuroscience, 33 (2011) 1170-1174. [22] G.L. Ming, H. Song, Adult neurogenesis in the mammalian brain: significant answers and significant questions, Neuron, 70 (2011) 687-702. [23] Y. Liu, E.D. Eaton, T.E. Wills, S.K. McCann, A. Antonic, D.W. Howells, Human Ischaemic Cascade Studies Using SH-SY5Y Cells: a Systematic Review and Meta-Analysis, Translational Stroke Research, DOI 10.1007/s12975-018-0620-4(2018). [24] E.B. Kennedy R Lees, Rüdiger von Kummer, Thomas G Brott, Danilo Toni, James C Grotta, Gregory W Albers, Markku Kaste, John R Marler, Scott A Hamilton, Barbara C Tilley, Stephen M Davis, Geoff rey A Donnan, Werner Hacke, for the ECASS, ATLANTIS, NINDS, and, E.r.-P.S.G. Investigators*, Time to treatment with intravenous alteplase and outcome in stroke- an updated pooled analysis of ECASS, ATLANTIS, NINDS, and EPITHET trials, The Lancet, 375 (2010) 1695-1703. [25] K. Kimura, Y. Sakamoto, J. Aoki, Y. Iguchi, K. Shibazaki, T. Inoue, Clinical and MRI predictors of no early recanalization within 1 hour after tissue-type plasminogen activator administration, Stroke, 42 (2011) 3150-3155. [26] P. Lyden, T. Hemmen, J. Grotta, K. Rapp, K. Ernstrom, T. Rzesiewicz, S. Parker, M. Concha, S. Hussain, S. Agarwal, B. Meyer, J. Jurf, I. Altafullah, R. Raman, Collaborators, Results of the ICTuS 2 Trial (Intravascular Cooling in the Treatment of Stroke 2), Stroke, 47 (2016) 2888-2895. [27] World Stroke Congress Abstracts, 2018, International Journal of Stroke, 13 (2018) 3217. [28] S. Shankaran, A.R. Laptook, R.A. Ehrenkranz, J.E. Tyson, S.A. McDonald, E.F. Donovan, A.A. Fanaroff, W.K. Poole, L.L. Wright, R.D. Higgins, N.N. Finer, W.A. Carlo, S. Duara, W. Oh, C.M. Cotten, D.K. Stevenson, B.J. Stoll, J.A. Lemons, R. Guillet, A.H. Jobe, H. National Institute of Child, N. Human Development Neonatal Research, Whole-body hypothermia for neonates with hypoxic-ischemic encephalopathy, The New England journal of medicine, 353 (2005) 1574-1584. [29] Y.H. Wan, C. Nie, H.L. Wang, C.Y. Huang, Therapeutic hypothermia (different depths, durations, and rewarming speeds) for acute ischemic stroke: a meta-analysis, Journal of stroke and cerebrovascular diseases : the official journal of National Stroke Association, 23 (2014) 2736-2747. [30] J.W. Lampe, L.B. Becker, State of the art in therapeutic hypothermia, Annual review of medicine, 62 (2011) 79-93.
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Figure 5
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Table 1: Summary of the differences between the assays of cell death and energy function.
Critical functional change
Irreversible cell death Chromatin
Metabolic dysfunction (MTT)
condensation (ICC cell death assay)
OGD 1h
30%
3%
OGD 2h
33%
4%
OGD 4h
43%
6%
OGD 6h
58%
7%
NPs
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Mature neurons 69%
0.3%
OGD 2h
68%
1%
OGD 4h
69%
19%
OGD 6h
71%
53%
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OGD 1h
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PII:
S0361-9230(19)30484-8
DOI:
https://doi.org/10.1016/j.brainresbull.2019.12.005
Reference:
BRB 9823
To appear in:
Brain Research Bulletin
Received Date:
22 June 2019
Revised Date:
10 November 2019
Accepted Date:
10 December 2019
Please cite this article as: Liu Y, Michalska AE, Dottori M, Eaton E, Courtney J-Maree, Antonic A, Howells DW, Differential susceptibility of human neural progenitors and neurons to ischaemic injury, Brain Research Bulletin (2019), doi: https://doi.org/10.1016/j.brainresbull.2019.12.005
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Differential susceptibility of human neural progenitors and neurons to ischaemic injury
Ye Liua, Anna E. Michalskab, Mirella Dottoric, Emma Eatond, Jo-Maree Courtneyd, Ana Antonice, and David W. Howells*d
Department of Neurology, Huashan Hospital, Shanghai, 200040, China. State Key
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a
Laboratory of Medical Neurobiology, Fudan University, Shanghai, 200433, China. b
Stem Cell Core Facility, Stem Cells Australia, The University of Melbourne, Victoria 3010, Australia
Illawarra Health and Medical Research Institute, University of Wollongong, NSW, 2522
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c
Australia
School of Medicine, University of Tasmania, Hobart, Tasmania 7001, Australia
e
Department of Neuroscience, Central Clinical School, Monash University, The Alfred
Corresponding author.
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*
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Centre, VIC 3004, Australia
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d
Email address: [email protected]
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Telephone numbers: +61 (3) 6226 4850
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.
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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 hours. Mild hypothermia (33°C) was used to assess neuroprotective potential in the structurally mature neurons
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after 1 or 4 hours OGD-R. 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 6h OGD. Metabolic dysfunction
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was greater in these more mature neurons (70%) than in NPs and evident after 1h OGD, before detection of neuronal death at 4h. OGD-R salvaged metabolic activity but not cell death in mature neurons. In NPs there was
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little metabolic salvage and cell death was induced (50% and 65% at 4 and 6h OGD-R, respectively).
Conclusions: Highly arbourised neurons are more sensitive to ischaemic injury than NPs which did however
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develop marked vulnerability to prolonged injury with reoxygenation. These observations imply that therapeutic
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potential may be highly dependent of the developmental state of the neurons we aim to protect.
hESC neuronal differentiation, OGD sensitivity, Stem cells
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Keywords:
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 [1-6].
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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 [7, 8].
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
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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
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Experimental procedures
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neuronal responses to ischaemic injury.
Cell Culture and induction of neural differentiation
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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.
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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 subcultured in hES medium containing 500ng/mL noggin (R&D system, 6057-NG-100) for 14 days to allow neural induction. After 14-days noggin treatment, the differentiated central parts of these new colonies were cut out and transferred to individual wells in a low attachment 96-well plate containing Neurobasal Medium/B27/N2
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supplemented with EGF and FGF to allow neurosphere (NS) formation.
Fourteen-day NSs were isolated, dissociated and allowed to continue their differentiation for a further 5 days in vitro (DIV) to provide neural progenitors. Highly arbourized neurons were also derived from 14-day NSs using methods developed in our lab [9], and maintained for up to 49DIV. Briefly, 14-day NSs were cut into smaller pieces and placed into individual wells of a 24-well plate pre-coated with poly-d-lysine (PDL) and laminin (10 µg/ml; Sigma, P6407 and Life Tech, 23017015, respectively). These pieces were cultured in Neurobasal Medium (NBM) made in our lab (supplement table2) without EGF and bFGF followed by re-collection and dissociation by accutase (Thermo Fisher, A1110501, supplied as ready-to-use cell dissociation reagent) at 28 days. Cell pellets were then re-suspended, filtered (40μm cell strainers, BD, 352340) and plated for immunostaining into 35mm2
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dishes containing coverslips (13mm round Menzel PK100, Grale Scientific, CS13100) and for MTT assay into 96-well plates. The seeding density for 35mm2 dishes with coverslips was 600,000 (NPs)/700,000 (49DIV neurons) and for 96-well plates was 20,000 (NPs)/30,000 (49DIV neurons) per well. The cells were then incubated at 37◦C in 5% CO2, 20% O2 with 95% humidity and ½ of the NBM was changed every second day until the experiments were terminated on day 49.
Oxygen and glucose deprivation and reoxygenation Plates with cultured NPs or 49DIV highly arbourized neurons were randomly allocated to the control or injury groups (4 time points: 1, 2, 4, and 6h) using the RAND function in Microsoft Excel. Glucose deprivation was achieved by replacing NBM with glucose-free NBM. The plates were then placed in a hypoxic chamber (STEMCELL, #27310), flushed with nitrogen at a rate of 20L/min for 10 minutes and placed in a 37°C incubator
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for 1, 2, 4 or 6h to induce the OGD injury. Cells in the OGD control groups were given fresh NBM and cultured at 37°C for 6h. Experiments in the OGD groups were terminated at the designated injury times and immediately processed for analysis. OGD-R groups including the control groups were given fresh NBM after the OGD injury to replace the deoxygenated medium and returned to the 37°C incubator until the total incubation time reached 24h in culture before the analysis. Cells in the OGD-R control group underwent the same procedure as OGD
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control followed by replenishment with fresh NBM and continued culture for another 18h.
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Detection of metabolic dysfunction by MTT assay
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide (MTT) is a yellow water-soluble tetrazolium salt, which is metabolised into a water-insoluble formazan in viable cells by the action of functional NAD+/NADP+
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dependent dehydrogenases. This assay is widely used as a surrogate of mitochondrial function. Briefly, 10l MTT (Sigma Cat#M2128, 5mg/mL in PBS) was added directly to each well following OGD or OGD-R and a vehicle control without cells but 100l NBM. The plates were then incubated at 37°C for 4h followed by the addition of
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100l DMSO. The absorbance was measured at 470nm using a plate reader (Bio-strategy, Spectra Max M3). Relative energy state was calculated by comparing the absorbance value in injured groups to those in the control groups.
Absorbance (Injury group) × 100% Absorbance (Control group)
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Relative energy state (%) =
These values were normalised to the measurements from uninjured controls. Cell death and morphology determined by staining A nuclear cell death and viability staining kit (ENZO-53004) containing a mixture of a blue fluorescent cellpermeable nucleic acid dye (excitation: 350nm, emission: 461nm) and a green fluorescent live-cell-impermeable nucleic acid dye (excitation: 503nm, emission: 524nm) was used to assess cell death after OGD and OGD-R injury. The dead cells appear cyan when combined with the nuclear stain, which is easily distinguished from either stain alone.
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Upon termination of the experiments, two coverslips were randomly selected, and placed into a new 24-well plate. 250l Blue/Green assay mixture (1:2000 in NBM) was added to each well and incubated at 37°C for 30 minutes. Cells were fixed with 2% PFA, and incubated with MAP2 antibody (Millipore, MAB5622, 1:200 dilution in 3% FCS and 0.3% Triton-X) at 4°C overnight. The next day, the cells were washed twice before addition of a donkey anti-rabbit (568) antibody (Life Tech, A10042, diluted 1:500 in 2% FCS) for 60 minutes at room temperature in the dark. Finally, 250µl of a 1:10,000 dilution of DAPI stock solution was incubated for 10 minutes followed by inverting and mounting coverslips on a standard glass microscope slide with a mounting medium containing a fluorescence anti-fade reagent (Molecular Probes, P36930).
Statistical analysis For the cell death assay and immunocytochemical staining of MAP2, the sample size was determined by power
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analysis using the software G*power [10]. To detect an effect size of 0.35 using one-way analysis of variance (ANOVA) with a fixed effects omnibus model with error probability of 0.05, power (1- error probability) 0.95 for five groups, giving a total number of 160 for samples that need to be analysed.
Each experiment was repeated on three different occasions. Within each experiment, five visual fields (centre,
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top, bottom, left, right) were selected from each of at least two samples (coverslips) for each time point. All images were taken at 200X magnification using a Zeiss Axio Observer 7 inverted fluorescence microscope. Images were then re-labelled and randomised by another researcher to blind the counting of positively stained (dead) cells using
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the Cell Counter plugin of Image J (NIH, 1.50i). MAP2+/arbour+ cells have MAP2 staining of both the cell soma and processes. MAP2+/arbour- cells did not have process staining. Dead cell number was expressed as a % of
background cell death.
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the total number of DAPI positive cells. Normalisation against uninjured controls was used to correct for
The same sampling regime and power assumptions were used for the quantitation of the MTT assay (which was
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replicated twice) as the effect size was expected to be larger than the cell death assay.
All values are presented as mean ± SEM, and all graphics are drafted using PRISM7. One-way and two-way ANOVA with Bonferroni post-hoc error correction was used to identify significant differences within and
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between the groups at the different experimental times.
Results
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
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MAP2+ neurites (Fig 1B, 1B-1, white arrows, enlarged from Fig 1B dotted frame). Even after 4h OGD, cell death was not apparent, but MAP2+/arbour- cells were observed to aggregate in the culture (Fig 1C, white box).
Counting cells permeable to the nuclear stain did not detect significant changes in cell death with increasing OGD duration (24.851.9% and 32.133.17% for 1h OGD and 6h OGD, respectively). Similarly, the number of the MAP2+/arbour+ cells did not alter significantly as OGD duration increased (Fig 1D). However, the mitochondrial activity assessed by MTT assay was significantly suppressed to 66.783.72% (p<0.001) of control activity within 1h OGD and continued to decline as OGD duration increased. After 6h OGD, only 35.242.87% (p<0.001) of MTT detectable mitochondrial activity remained (Fig 1D). The mature neurons cultured for 49DIV exhibited complex network structures, with long and arbourised processes
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making frequent contacts with each other in the control culture. MAP2+/arbour+ neurons were the predominant cellular type, comprising 601.68% of the total cell number (Fig 2A, D). Process disruption (neuritic breakage) on MAP2+/arbour+ neurons was detected after 1h OGD (Fig 2B-1, white arrows, enlarged from Fig 2B, dotted frame). However, increased cell death only became apparent after 4h OGD (44.172.7%, p<0.001), and increased further by 6h OGD (78.113.72%, p<0.001) (Fig 2D). Loss of MAP2+/arbour+ neurons mirrored total cell death
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with significant differences detected at 4h (42.842.07%, p<0.001) and 6h OGD (16.462.67%, p<0.001) (Fig 2D).
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The metabolic activity of the highly arbourised neurons exhibited extreme sensitivity to OGD. Only 30.69±1% (p<0.001) of the cells maintained normal function after 1h OGD, however, there was no further deterioration with
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increased OGD duration (2h: 31.63±1.05%, 4h: 31.15±0.95%, 6h: 29.37±0.91%) (Fig 2D).
Significant cell death in hESC derived NPs and highly arbourised neurons after OGD-R
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Neuritic beading showing damage to NP projections was also detected early after OGD-R (Fig 3B, white dotted box and Fig 3B-1). However, while NPs survived well after OGD, marked cell death was detected after OGD-R and the cultures acquired essentially the same cell death profile as seen in more mature arbourised neurons in response to OGD. 49.784.95% and 65.335.37% (p<0.001) of NPs died after 4 and 6h OGD-R, respectively (Fig 3D). While the number of MAP2+/arbour+ neurons changed little after OGD-R, the number of
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MAP2+/arbour- cells fell as cell death became apparent with very few left in the cultures by 6h (41.23%, p<0.001). Metabolic activity in NPs after OGD-R was less severely affected than after OGD alone, but was significantly reduced at all time points (1h 84.111.62%, p<0.001; 2h 78.83.22%, p<0.001; 4h 72.212.37%, p<0.001 and 6h 36.65%, p<0.00) (Fig 3D). Highly arbourised more mature neurons suffered marked damage to neuronal processes after 1h OGD-R (Fig 4B1, white arrows, enlarged from Fig 4B, dotted frame) although substantial neuronal death was not detected. After 4h OGD-R, neurites became sparser (Fig 4C) and neuronal death continued to increase (65.433.85%, p<0.001) until 6h (85.132.82%, p<0.001) (Fig 4D). Loss of MAP2+/arbour+ neurons mirrored cell death with a reduction to 28.773.07%, p<0.001 and 13.382.45, p<0.001 after 4 and 6h OGD-R respectively (Fig 4D). Unlike the
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response of arbourised mature neurons to OGD, metabolic activity was maintained after OGD-R and only became significantly depleted after 6h (76.426.44%, p<0.001) (Fig 4D).
Comparison of cell death and metabolic activity after OGD and OGD-R injury responses To permit a clearer comparison across experiments, the quantitative data were plotted by outcome measure in Figure 5 where 2-way ANOVA has been used to examine differences in the response of the two cell types in the presence of OGD or OGD-R.
The MTT assay of metabolic activity showed marked differences between NPs and more mature arbourised neurons and between OGD and OGD-R. For NPs, the steady decline in mitochondrial activity detected by MTT in OGD was attenuated but not prevented by OGD-R. At 1, 2 and 4h, suppression of mitochondrial activity was
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17.33% (p<0.01), 16.26% (p<0.01) and 21.6% (p<0.001) less in OGD-R than in OGD. However, by 6h of injury, the degree of compromise was similar in both groups. While arbourised neurons showed a dramatic and early loss of mitochondrial function after OGD, this loss was markedly suppressed after OGD-R with 60.29% (p<0.001), 55.15% (p<0.001), 55.31% (p<0.001) and 47.05% (p<0.001) less loss at 1, 2, 4 and 6h respectively (Fig 5A).
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In NP cultures, the profile of cell death after OGD-R was similar to that for OGD for the first 2h of the experiment with few cells dying. However, the profiles of death diverged thereafter, with 22.093.43% (p<0.001) and 33.24.95% (p<0.001) more death after OGD-R at 4 and 6h respectively. This later profile was very similar to
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that seen for more mature arbourised neurons under both OGD and OGD-R conditions (Fig 5B).
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In NP cultures, MAP2+/arbour+ neurons accounted for approximately 40% of the total cell population, and this proportion did not alter significantly despite the induction of OGD or OGD-R injury. In contrast, in mature neuron cultures, the number of MAP2+/arbour+ neurons was initially higher (~60%) and started to decline dramatically after 4h in both OGD and OGD-R injury models (Fig 5C). It is not clear whether the change in cell death (Fig 5B)
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and MAP2+/arbour+ neuron number (Fig 5C) at 1h after OGD-R in mature neuron cultures represents a biologically important change or a statistical aberration.
A MAP2+/arbour- subpopulation of cells existed only in the NP cultures. While they were not affected by OGD, they exhibited marked vulnerability to OGD-R. Little difference was evident until 4h (OGD: 39.14% vs OGD-R: 10.7%, p<0.001) and 6h (OGD: 33.3% vs OGD-R: 4.03%) when most of these cells had been lost in OGD-R
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compared to OGD (Fig 5D).
Discussion We have previously shown [9] 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
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markedly between hESC derived neural progenitors and the highly arbourised neuronal products of their differentiation.
While NP appeared insensitive to up to six hours of OGD, morphologically complex neuronal cultures, which we have previously shown to contain ~80% NeuN+ neurons and to express the neurochemical machinery required for neurotransmission [9], died rapidly with OGD durations of more than two hours. This neuronal death, detected by staining dependent on loss of nuclear integrity, was mirrored by the loss of the MAP2+/arbour+ neurons. Despite the lack of cell death detected by the nuclear stain, NPs did show signs of damage. Beading of MAP2+ projections on NPs was identified within one hour of OGD and the MTT assay detected a steady loss of metabolic activity as OGD progressed. However, while morphologically complex neurons reached maximum suppression of this activity within one hour of OGD, NPs took six hours to reach the same level. These data are consistent
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with a higher resistance to hypoxic depolarisation in mitochondria from human new-born brains than those from adult brains [11], suggesting that this might be because neural stem/progenitor cells have a lower requirement for oxidative metabolism than mature neurons [12].
These data indicate that it is inappropriate to use morphologically simple cells in attempts to model stroke, the results will clearly be misleading. Whether we can directly extrapolate the behaviour of these highly arborized
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human neurons in culture to those that might inhabit the brain of an elderly subject before a stroke remains unclear. Nevertheless, understanding the principle that arborisation increases sensitivity to injury seems likely to be
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important.
In the stroke research field were no neuroprotective strategies have successfully translated from either animal or
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animal tissue models to the clinic, understanding that the state of the cells used to model disease is critical for the outcomes measures is important. Unfortunately, until candidates have been screened prospectively as suggested here and progressed to completed clinical trial, we cannot know for certain. However, since we have previously
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shown that using arborized human neurons allows us to distinguish between NXY-059[13] which failed in clinical trial[14] and hypothermia[6] which does protect the brain in neonates with hypoxic ischemic encephalopathy (HIE) and during surgery requiring cardiac arrest[16], it seems likely that testing in human cells as described here will become an important part of the stroke drug development armamentarium. Moreover, whilst our laboratory’s focus is stroke, these findings might have more immediate impact in screening for drugs to better treat HIE where currently untestable arguments about how well these cells might mimic the responses possible in the aged brain
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are redundant. Importantly, understanding the molecular differences that underpin these markedly different responses will not only advance our knowledge of how ischaemic injury accrues but also point to important therapeutic targets.
We found that reperfusion provided no protection to mature neurons in terms of cell death even though energy metabolism was dramatically restored. This indicates that after the initial trigger, components of the cell death response are irrevocable, and they are not tied entirely to reduced energy metabolism. Indeed, out data would seem to suggest the metabolic activity of the surviving neurons dramatically increases. This is graphically illustrated in Supplementary Figure 1 which shows the MTT metabolic activity data normalised for surviving
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neuron number. Sanderson and colleagues propose an explanation for this phenomenon, dephosphorylation primed hyperactivity of oxidative phosphorylation upon reperfusion[17]. The phenomenon of reperfusion injury is well recognised in the literature [18, 19]. These observations emphasize that while reperfusion of the ischemiaaffected brain is essential, doing so may not prevent neuronal death that has already been initiated. Understanding what the point of no-return is for different cells under different circumstances is critical for delivery of the most effective therapy across a range of human diseases experiencing ischaemia of different degrees at different stages of development. Our data emphasises that the earliest possible intervention has the greatest potential for benefit.
In NP cultures, reperfusion was less effective at protecting metabolic activity and appeared to increase cell death, especially of the MAP2- cells. This indicates that active management of reperfusion injury may be particularly critical for the neonatal and still-developing juvenile brain exposed to HIE, stroke or asphyxiation. In the adult
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brain, endogenous neural stem cell populations such as those believed to be important for gliogenesis or neuroplasticity may be particularly at risk of reperfusion injury[20-22]. Moreover, if stem-cell based therapeutic interventions for brain injuries become a reality, the implanted cells would be expected to be at risk.
The disparity between the results of the assays of cell death and energy metabolism, which are widely used and often interpreted as assays of cell viability, is important. This is consistent with similar observations from
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systematic review and meta-analysis studies of OGD in SH-SY5Y cells, the most widely used surrogate for human neurons [23]. This indicates that it is inappropriate to use the morphologically simple cells typically used to model
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stroke in vitro as the results will be misleading.
In the NPs, the majority of cells, even after six hours OGD, have compromised mitochondrial function (58%),
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but few are actually dead (7%) suggesting a potential for salvage of approximately half of the population by some form of neuroprotection. For the mature neurons, extrapolation suggests the window for significant neuroprotection may be closing between four and six hours after injury of OGD (Table 1). This is entirely
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consistent with the window available for thrombolytic therapy in the clinic [24].
One limitation of this proof of principle study is that a single hESC line has been used, the particularly well studied H9 line. Verification of our findings in other lines will be important but selecting from amongst the 400 hESC lines listed as commercially available by NIH will present a challenge beyond the scope of funding and ethics approval available for this study. It would seem logical to also verify this data in primary adult human neurons.
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However, human neurons excised during epilepsy or tumour surgery and then cultured may not be truly representative of basal activity in the intact brain.
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
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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 ischaemic biology and screening of candidate therapies.
Conflict of Interest.
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
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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
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(Biomarkers for assessing the effectiveness of hypothermia as a therapy in ischemic stroke patients).
Declaration of conflicting of interests
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The authors declare that they have no conflict of interest regarding research, authorship, and/or publication of
Ethical Approval
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this article.
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The use of H9 (WA09) hESC line was approved by University of Melbourne human ethics committee.
References
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Table 1: Summary of the differences between the assays of cell death and energy function.
Critical functional change
Irreversible cell death Chromatin
Metabolic dysfunction (MTT)
condensation (ICC cell death assay)
OGD 1h
30%
3%
OGD 2h
33%
4%
OGD 4h
43%
6%
OGD 6h
58%
7%
NPs
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Mature neurons 69%
0.3%
OGD 2h
68%
1%
OGD 4h
69%
19%
OGD 6h
71%
53%
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OGD 1h
18