Three-dimensional differentiation of human pluripotent ...
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Acta Biomaterialia 101 (2020) 102–116
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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actbio
Full length article
Three-dimensional differentiation of human pluripotent stem
cell-derived neural precursor cells using tailored porous polymer
scaffolds
Ashley R. Murphy
a , b , John M. Haynes c , Andrew L. Laslett b , d , Neil R. Cameron a , e , ∗, Carmel M. O’Brien
b , d , ∗∗
a Department of Materials Science and Engineering, Monash University, 22 Alliance Lane, Clayton, VIC 3800, Australia b CSIRO Manufacturing, Research Way, Clayton, VIC 3168, Australia c Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia d Australian Regenerative Medicine Institute, Science, Technology, Research and Innovation Precinct (STRIP), Monash University, Clayton Campus, Wellington
Road, Clayton, VIC 3800, Australia e School of Engineering, University of Warwick, Coventry CV4 7AL, UK
a r t i c l e i n f o
Article history:
Received 23 July 2019
Revised 30 September 2019
Accepted 7 October 2019
Available online 11 October 2019
Keywords:
Porous polymer
3D scaffold
Neural stem cell
Neuron
Neural differentiation
Tissue engineering
3D cell culture
a b s t r a c t
This study investigates the utility of a tailored poly(ethylene glycol) diacrylate-crosslinked porous poly-
meric tissue engineering scaffold, with mechanical properties specifically optimised to be comparable to
that of mammalian brain tissue for 3D human neural cell culture. Results obtained here demonstrate the
attachment, proliferation and terminal differentiation of both human induced pluripotent stem cell- and
embryonic stem cell-derived neural precursor cells (hPSC-NPCs) throughout the interconnected porous
network within laminin-coated scaffolds. Phenotypic data and functional analyses are presented demon-
strating that this material supports terminal in vitro neural differentiation of hPSC-NPCs to a mixed pop-
ulation of viable neuronal and glial cells for periods of up to 49 days. This is evidenced by the upregula-
tion of TUBB3, MAP2, SYP and GFAP gene expression, as well as the presence of the proteins βIII-TUBULIN,
NEUN, MAP2 and GFAP. Functional maturity of neural cells following 49 days 3D differentiation culture
was tested via measurement of intracellular calcium. These analyses revealed spontaneously active, syn-
chronous and rhythmic calcium flux, as well as response to the neurotransmitter glutamate. This tailored
construct has potential application as an improved in vitro human neurogenesis model with utility in
platform drug discovery programs.
Statement of significance
The interconnected porosity of polyHIPE scaffolds exhibits the ability to support three-dimensional neural
cell network formation due to limited resistance to cellular migration and re-organisation. The previously
developed scaffold material displays mechanical properties similar to that of the mammalian brain. This
research also employs the utility of pluripotent stem cell-derived neural cells which are of greater clinical
relevance than primary neural cell lines. This scaffold material has future potential in better mimicking
three-dimensional neural networks found in the human brain and may result in improved in vitro models
for disease modelling and drug screening applications.
© 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
∗ Corresponding author at: Department of Materials Science and Engineering,
Monash University, 22 Alliance Lane, Clayton, VIC 3800, Australia. ∗∗ Corresponding author at: CSIRO Manufacturing, Research Way, Clayton, VIC
3168, Australia.
E-mail addresses: [email protected] (N.R. Cameron),
Carmel.O’[email protected] (C.M. O’Brien).
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https://doi.org/10.1016/j.actbio.2019.10.017
1742-7061/© 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
. Introduction
Human somatic cells cultured in flat, stiff, two-dimensional
2D) environments typically display an irregular morphology and
evelop unnatural cell-cell interactions [1] . While conventional
onolayer cell cultures are simple and convenient to anal-
se, tissue-specific architecture, mechanical and biochemical cues,
nd cell-cell communication can all be lost to various degrees
A.R. Murphy, J.M. Haynes and A.L. Laslett et al. / Acta Biomaterialia 101 (2020) 102–116 103
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hroughout the process. This leads to physiological inaccuracies
hat can be problematic for disease modelling and pre-clinical drug
creening. It has also been found that animal model studies of-
en do not result in successful translation to human trials due to
imited similarity to human physiology [2] . Both these pre-clinical
creening techniques can have considerable detrimental impacts on
he progression of new drug candidate research to clinical trials,
nd can be particularly evident when modelling complex disease
tates such as those found in the central nervous system (CNS) [3] .
The brain is the least understood organ in the human body. It
s difficult to access, highly susceptible to damage and complex in
tructure and function. The limited understanding of the human
rain is reflected in the paucity of effective treatments for vari-
us neurological disorders such as Parkinson’s disease, Alzheimer’s
isease and motor neuron disorders. To address this research gap,
ew methods for the culture of human neural (neuronal and glial)
ells, particularly in vitro three-dimensional (3D) culture, are be-
ng developed to more accurately reconstruct the complex in vivo
tructure and function of the human brain and provide more
ealistic in vitro models for disease interrogation and treatment
tudies.
Engineering neural tissue that is more closely representative of
hat found in the human brain and central nervous system requires
scaffold or matrix to recreate the 3D in vivo microenvironment
r niche. Various materials (natural and synthetic) in different for-
ats (gels, porous solids and fibres) can be used as scaffolds to as-
ist the 3D culture of replicating and terminally differentiated neu-
al cells [4] . To aid neural tissue growth, and increase physiological
elevance, scaffolds can be topographically modified, mechanically
uned or chemically/biologically functionalised, all of which have
een shown to aid neural precursor cell attachment, proliferation
nd differentiation [4] .
Porous polymeric materials, such as polymerised high internal
hase emulsion (polyHIPE) materials, possess great potential as
caffolds f or 3D cell culture and f or applications in tissue engi-
eering due to their high and fully interconnected porosity, long-
erm stability and ease of manufacture [5] . Unlike electrospun fi-
rous mats and some 3D printed lattice structures, polyHIPE ma-
erials are consistently porous in all three dimensions, facilitat-
ng a more realistic capacity for 3D cellular growth. The intercon-
ected porosity of polyHIPE scaffolds potentially allows for more
atural cell migration, as opposed to hydrogels which can in some
ases retard cell movement throughout viscous polymer networks
6] . Solid porous scaffolds offer the ability to control the macro-
rrangement of cells into structures that are not limited by diffu-
ion of nutrients and waste [5 , 7] , a common problem associated
ith scaffold-free aggregate cultures such as organoids and neuro-
pheres [8] .
PolyHIPE scaffolds synthesised using thiol-ene ‘click’ chemistry
rom poly(ethylene glycol) diacrylate and tri-functionalised thiol
onomers (PEGDA polyHIPE) were previously developed to mimic
he bulk mechanical properties of mammalian brain tissue [9] . This
aterial was demonstrated to exhibit greater cell culture media
bsorption capacity and improved optical transparency compared
o previously developed polyHIPE scaffold materials. PEGDA poly-
IPE scaffolds have also been demonstrated to support the at-
achment, infiltration and short-term expansion of human induced
luripotent stem cell-derived neural precursor cells (hiPSC-NPCs)
n vitro [9] .
The study of live, mature, human neuronal and glial cells in
itro is substantially limited by a lack of safe and ethical harvesting
echniques, as well as their limited proliferative capacity [10] . Neu-
al precursor cells (NPCs) are a valuable research tool for the study
f mature neural cells in vitro , given their proliferative capacity and
otential to differentiate to all neural cell types. Neural precursor
ells can be directly isolated from primary tissue [11–16] , differ-
ntiated from pluripotent stem cells (PSCs, including embryonic
tem cells (ESCs) and induced pluripotent stem cells (iPSCs)) [17–
9] and transdifferentiated from somatic cells [20–22] . Differenti-
tion of hPSCs to NPCs currently provides the most safe, renew-
ble and technically reliable source of hNPCs for the production
f mature neural cells for in vitro research purposes [10] . Induced
luripotent stem cell (iPSC) technology also presents an opportu-
ity for the derivation of mature neural cells with diseased pheno-
ypes [23] . A variety of protocols have already been developed to
irect the differentiation of PSC-derived NPCs (PSC-NPCs) to spe-
ific neuronal and glial sub-types [24] . However, 2D NPC differen-
iation techniques fail to produce a population of cells that realisti-
ally mimic the natural, 3D, networked nature of the human brain.
This study investigates the ability of a tailored PEGDA polyHIPE
caffold to support the 3D expansion and long term differentiation
ulture of hPSC-NPCs. In order to investigate any potential advan-
ages of the low modulus PEGDA polyHIPE scaffold for 3D neural
ell culture applications, this scaffold is compared to a polyHIPE
caffold (TMPTA) of similar morphology but elastic and storage
oduli greater than the reported ranges for mammalian brain
issue. This study has employed a physiologically relevant cell
ulture system using two hPSC-NPC (hESC-NPC and hiPSC-NPC)
ines generated by noggin-induced BMP inhibition of hPSCs.
. Materials and methods
.1. PolyHIPE synthesis
The polyHIPE synthesis procedure was replicated from the work
f Murphy et al. [9] . Briefly, a hydrophobic phase mixture con-
isting of: trimethylolpropane tris(3-mercaptopropionate) (Sigma-
ldrich); poly(ethylene glycol) diacrylate (M n = 700) (PEGDA) or
rimethylolpropane triacrylate (TMPTA), HYPERMER
TM B-246SF-
Q-(AP) (surfactant, Croda) (3% w/w of hydrophobic phase); and
diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide/2-hydroxy-2-
ethylpropiophenone blend (photoinitiator, Sigma-Aldrich, Cat
o: 405,663) (5% w/w of hydrophobic phase) was dissolved in
,2-dichloroethane (Sigma-Aldrich). The mixture was added to
two-neck 250 mL round-bottom flask and stirred at 350 rpm
sing a D-shaped polytetrafluoroethylene (PTFE) overhead paddle
tirrer (Sigma-Aldrich). A hydrophilic phase of Milli-Q water,
omprising of 80% v/v of the final emulsion volume, was added to
he hydrophobic phase mixture drop-wise, at approximately 0.5–1
rops per second, until all water was added. The mixture was
hen stirred for a further 2 h. The HIPE mixture was poured into a
ylindrical PTFE mould between two glass plates and passed under
high intensity ultraviolet (UV) light irradiator at a power flux of
W/cm
2 . The resulting polyHIPE was then washed by immersion
n acetone (Merck) overnight, in order to exchange water trapped
ithin the structure. The polyHIPE was further washed by Soxhlet
xtraction using dichloromethane (Merck) at 77 °C for 48 h. The
olyHIPE was air-dried overnight to prevent any sudden contrac-
ion and damage to the materials, then vacuum-dried for 24 h
t room temperature. PolyHIPE micro-structure was confirmed by
maging using a Nova NanoSEM 450 FEGSEM (FEI Company). A
nal polyHIPE porosity of 80% was previously confirmed by helium
ycnometry [9] .
.2. Rheological measurements
Dynamic shear tests were performed using a Physica rheome-
er (Anton Paar) with a plate-plate configuration comprising of
quartz lower plate and a stainless steel upper plate of 12 mm
iameter. The lower plate was heated using a digital tempera-
ure controller (PolyScience®) and data was analysed via RheoPlus
3.62 rheometer software (Anton Paar). PolyHIPE samples were cut
104 A.R. Murphy, J.M. Haynes and A.L. Laslett et al. / Acta Biomaterialia 101 (2020) 102–116
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into 200 μm thick disks of 15 mm diameter, pre-wet with PBS and
placed onto the quartz lower plate heated to 37 °C. The upper plate
was lowered to a gap size of 200 μm or until a force was de-
tected, indicative of the upper plate touching the polyHIPE sample.
Frequency, strain and time sweep experiments were performed in
triplicate.
2.3. Cell culture
All work using hPSCs, and derivative hPSC-NPCs, was carried
out in accordance with Australia’s National Health and Medical Re-
search Council (NHMRC) ‘National Statement on Ethical Conduct in
Human Research’ (2007, updated 2018), the ‘Australian Code for
the Responsible Conduct of Research’ (2007, updated 2018) and
with approvals from Monash University and the Commonwealth
Scientific and Industrial Research Organization (CSIRO) Human Re-
search Ethics Committees.
Renewing human neural precursor cell lines were previ-
ously derived and characterised as described by Sundaramoor-
thy [25] from parental HDF51i-509-iPSCs [26] and WA09(H9)-ESCs
[27] . All 2D hNPC maintenance and differentiation culture methods
were performed as previously described [25] .
Parental HDF51i-509-iPSCs and WA09(H9)-ESCs were cultured
on Geltrex TM LDEV-Free, hESC-qualified, reduced growth factor
basement membrane matrix (Thermo Fisher Scientific) coated cul-
tureware in Essential 8 TM cell culture medium (Thermo Fisher Sci-
entific), with daily medium changes for the purpose of providing
control RNA samples in qPCR studies.
2.4. Three-dimensional hNPC polyHIPE maintenance and neural
differentiation culture
2.4.1. Preparation, assembly and sterilisation
Circular disk scaffolds of 15 mm diameter and 200 μm thick-
ness were cut from a cylinder polyHIPE monolith by sectioning
using a VT10 0 0 S vibrating-blade microtome (vibratome, Thermo
Fisher Scientific). Samples that were too soft to section with a
vibratome ( e.g. PEGDA polyHIPE) were embedded in Tissue-Tek®
optimum cutting temperature compound (VWR International) in
Peel-A-Way® Disposable Histology Moulds (Sigma-Aldrich) and
frozen at –20 °C. Cryo-embedded polyHIPE monoliths were then
sectioned into 200 μm thick disks using a CM3050-S Cryostat (Le-
ica). Disks were then assembled into Alvetex® Scaffold 12-well in-
serts (Bio-Scientific Pty Ltd), with the Alvetex® scaffolds removed,
and placed in a Falcon® 12-well Clear Flat Bottom Plate (12-well
plate, Bio-Strategy), (3.8 cm
2 /well). Scaffolds were sterilised by im-
mersion in 5 mL of 70% w/v ethanol (Merck) in water per well for
30 min under the UV sterilisation light of the biosafety cabinet.
Scaffolds were then washed twice with 3.5 mL of 1:1 DMEM/Hams
F12 (DMEM/F12), (Thermo Fisher Scientific) per well for 5 min
each wash.
2.4.2. Human NPC maintenance within polyHIPE scaffolds
Scaffolds were coated in 3.5 mL (12-well plate) of 10 μg/mL
Laminin from Englebreth-Holm-Swarm murine sarcoma basement
membrane (laminin, Sigma-Aldrich) in DMEM/F12 per well for
2 h at room temperature. The laminin solution was aspirated and
1 × 10 6 hNPCs in 150 μL of STEMdiffTM Neural Progenitor Medium
(NPM), (STEMCELL Technologies) was placed on top of the scaffold.
Scaffolds were incubated at 37 °C in an atmosphere of 5% CO 2 in
air for 3 h to allow cells to settle and attach to the material, af-
ter which an additional 3.5 mL of STEMdiffTM NPM was carefully
added so as not to disturb cell attachment. Human NPCs were al-
lowed to settle for a further 48 h and subsequently maintained in
STEMdiffTM NPM with medium changes every 1–2 days.
.4.3. Human NPC neural differentiation culture within polyHIPE
caffolds
Assembled and sterilised scaffolds in 12-well plates were incu-
ated in 3.5 mL of 15 μg/mL poly-L-ornithine (PLO, Sigma-Aldrich)
n Dulbecco’s Phosphate-Buffered Saline (DPBS, Thermo Fisher Sci-
ntific) per well for 2 h at room temperature, then washed twice
ith 3.5 mL of DPBS per well for 5 min each wash. Scaffolds
ere then coated in 3.5 mL of 10 μg/mL laminin in DMEM/F12 per
ell overnight with the well plate parafilm sealed and stored at
°C. Human NPCs were seeded on to PLO/laminin-coated scaf-
olds and cultured for 14 days in STEMdiffTM NPM (as above), after
hich differentiation was commenced with 50% v/v media changes
o BrainPhys TM neural medium supplemented with NeuroCult TM
M1 Neuronal Supplement 200 × (STEMCELL Technologies), N2
upplement-A 100 × (STEMCELL Technologies), 20 ng/mL human
ecombinant BDNF (STEMCELL Technologies), 20 ng/mL human
ecombinant GDNF (STEMCELL Technologies), 1 mM N
6 , 2 ′ --dibutyryladenosine 3 ′ , 5 ′ -cyclic monophosphate sodium salt
Sigma-Aldrich), 200 nM ascorbic acid (STEMCELL Technologies)
nd 50 U/mL penicillin-streptomycin (Thermo Fisher Scientific),
complete BrainPhys TM neural medium). Medium changes of 50%
/v were performed each 2 and 3 days for up to 50 days.
.5. Histology and staining
Scaffold cultures were fixed and stained as previously described
9] . Briefly, scaffolds were fixed in neutral buffered formalin (pH 7)
Sigma-Aldrich) and processed to remove water and infiltrate with
araffin wax. Processed scaffolds were then embedded in paraf-
n wax and sectioned in to 10 μm sections using a Rotary Micro-
ome CUT 4060 (microTEC). Mounted sections were then dewaxed
nd stained in Harris’s Haematoxylin and Eosin for 5 min each.
tained sections were imaged with an Olympus BX51 microscope
n brightfield mode using a × 20 objective and Nuance FX Multi-
pectral Imaging System.
.6. Double strand DNA quantification assay
After washing with PBS to remove any unbound cells, scaffold
ultures were placed directly into 1 mL lysis buffer [10 mM Tris
H 8, 1 mM EDTA and 0.2% v/v Triton X-100] within 1.7 mL mi-
rotubes. Samples were vortexed for 10 s every 5 min, for a total
eriod of 30 min, and kept on ice between mixes. At this point,
amples were either placed in –80 °C for storage or processed fur-
her. Samples were then homogenised 10–15 times using a 21-
auge needle to produce cell lysate. Lysate was then diluted 1:10
n 1 × TE buffer (Thermo Fisher Scientific). Quant-iT TM PicoGreen®
sDNA reagent (Thermo Fisher Scientific) was diluted 1:200 in
× TE buffer. A 1:1 mixture of lysate solution and 1 × PicoGreen®
eagent was placed in an OptiPlate-96, black opaque 96-well mi-
roplate (PerkinElmer). Fluorescence was measured at excitation
nd emission wavelengths of 460 nm and 540 nm, respectively, us-
ng an EnSpire TM 2300 Multilabel Plate Reader (Perkin Elmer) run-
ing EnSpire 3.0 software (Perkin Elmer).
Results were compared to a standard curve for 1–10 × 10 6
PSC
–NPCs analysed as above for dsDNA amount. Human PSC-
PCs cultured on laminin-coated 2D TCPS were harvested via
0 min incubation in a solution of 500 μL Accutase. Human PSC-
PCs were quantified using the trypan blue exclusion method with
0.4% w/v solution of trypan blue and counted using a hemocy-
ometer.
.7. Cell viability assay
Human PSC-NPCs were maintained in STEMdiffTM NPM for 2,
, 10 and 14 days on laminin-coated TCPS cultureware and within
A.R. Murphy, J.M. Haynes and A.L. Laslett et al. / Acta Biomaterialia 101 (2020) 102–116 105
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aminin-coated polyHIPE scaffolds. Scaffolds were removed from
ell inserts and placed in a Falcon® 6-well Clear Flat Bottom Plate
Bio-Strategy) with 1 mL of STEMdiffTM NPM. A volume of 100 μL
restoBlue® Cell Viability Reagent (Thermo Fisher Scientific) was
ixed into the 1 mL of STEMdiffTM NPM and incubated at 37 °C in
humidified atmosphere of 5% CO 2 in air for 35 min. A 200 μL
olume of the mixture was placed in an OptiPlate-96 black opaque
6-well microplate. Fluorescence was then measured at excitation
nd emission wavelengths of 560 nm and 590 nm, respectively, us-
ng an EnSpire TM 2300 Multilabel Plate Reader running EnSpire 3.0
oftware.
.8. Immunocytochemistry
Immunocytochemical staining was performed as previously de-
cribed [9] . Briefly fixed 10 μm sections of scaffold cultures pre-
ared by histology underwent heat induced epitope retrieval at
8 °C for 30 min. Neural progenitor cell maintenance and differ-
ntiation cultures on coated 8-well glass chamber slides (Thermo
isher Scientific) were fixed using 4% paraformaldehyde (Electron
icroscopy Sciences) in PBS and permeabilised with 0.1% v/v Tri-
on X-100 (Sigma-Aldrich) and did not undergo the epitope re-
rieval process. Fixed samples were incubated with 10% v/v nor-
al goat serum in PBS (blocking buffer) for 1 h at room temper-
ture. Samples were then incubated with the following primary
ntibodies diluted in blocking buffer for 1 h at room tempera-
ure: anti-SOX1 IgG, anti-VIMENTIN IgM, anti-NESTIN IgG 1 , anti-
III-TUBULIN IgG 2a , anti-NEUN IgG 1 , anti-MAP2 IgY and anti-GFAP
gG, as well as the isotype control antibodies: rabbit IgG, mouse
gM, mouse IgG 1 , mouse IgG 2a and chicken IgY ( Table S1 ). Sam-
les were washed with PBS then incubated with the following
oat secondary antibodies all diluted 1:500 in blocking buffer for
h at room temperature: anti-IgG ( H + L ) Alexa Fluor® 568, anti-
gM Alexa Fluor® 568, anti-IgG 1 Alexa Fluor® 568, anti-IgG 2a Alexa
luor® 488, anti-IgG 1 Alexa Fluor® 568, anti-IgY Alexa Fluor® 568,
nti-IgG Alexa Fluor® 647 ( Table S2 ). Samples were finally washed
ith PBS, counter-stained with 4 ′ ,6-diamido-2-phenylindole dihy-
rochloride (DAPI), (1:500, Thermo Fisher Scientific) and mounted
ith Fluoromount TM Aqueous Mounting Medium (Sigma-Aldrich)
coverslip. Samples were imaged with an Olympus BX51 micro-
cope in fluorescence mode using a × 20 objective and Nuance FX
ultispectral Imaging System.
.9. RNA analysis by quantitative reverse transcriptase-polymerase
hain reaction
.9.1. RNA isolation and quality assessment
RNA was harvested and prepared from cellular materials using
Neasy Mini kit (Qiagen) in accordance with manufacturer’s in-
tructions. In brief, cells were harvested from 2D cultures using
ccutase®, resuspended in RLT buffer and passed through a QIA
hredder homogenisation column (Qiagen). Scaffold cultures were
laced directly in RLT buffer (without Accustase® harvest) and vor-
ex mixed for 1 min before being eluted through a QIA shred-
er homogenisation column. RNA was isolated and concentrated
sing an RNeasy Mini Kit (Qiagen). RNA samples were stored at
80 °C until further required. RNA concentration and purity were
easured using a Nanodrop ND-10 0 0 spectrophotometer (Thermo
isher Scientific) to confirm an absorbance ratio A 260 /A 280 of
.0–2.1.
.9.2. Complementary DNA synthesis
Complementary DNA (cDNA) was produced from isolated RNA
sing a High-Capacity cDNA Reverse Transcription Kit (Life Tech-
ologies) following manufacturer’s instructions. In brief, 1 μg of
NA sample was placed in a reaction mixture of random primers,
ucleotides and reverse transcriptase without RNase inhibitor. The
eaction mixture was placed in a T100 TM Thermal Cycler (Bio-Rad
aboratories) for 10 min annealing at 25 °C, 120 min polymerisa-
ion at 37 °C, 5 min deactivation at 85 °C and finally held at 4 °C.
fter reaction completion, newly synthesised cDNA was diluted
:12 in RNase-free water and stored at –80 °C. An RNA to cDNA
onversion efficiency of 100% was assumed.
.9.3. Reverse transcriptase PCR
As per the ’TaqMan® Universal PCR Master Mix’ User Guide,
ev. E (Applied Biosystems), a 20 μL reaction volume mixture of
aqMan® Universal PCR MasterMix (Thermo Fisher Scientific), Taq-
an® Gene Expression Assay ∗ (Thermo Fisher Scientific) and cDNA
as prepared in MicroAmp
TM Optical 96-Well Reaction Plates
Thermo Fisher Scientific). Reactions were carried out utilising the
ollowing thermal cycling conditions: 2 min incubation at 50 °C;
0 min activation at 95 °C; and 40 cycles of 15 s denaturation
t 95 °C followed by 1 min annealing and extending at 60 °C, us-
ng a 7500 Real-Time PCR System (Applied Biosystems). Reaction
t values were normalised to the housekeeping gene GAPDH and
alibrated to the control parental HDF51i-509-iPSC and H9-ESC
NA using 7500 Fast System SDS v1.4 software (Thermo Fisher
cientific). ∗TaqMan® Gene Expression Assay’s for genes: POU5F1 (Assay
D: Hs04260367_gH), PAX6 (Assay ID: Hs01088114_m1), SOX1 (As-
ay ID: Hs10157642_s1), TUBB3 (Assay ID: Hs00801390_s1), MAP2
Assay ID: Hs0 025890 0_m1), SYP (Assay ID: Hs0 030 0531_m1),
FAP (Assay ID: Hs00909233_m1) and house-keeping gene GAPDH
Hs02786624_g1) were all used.
.10. Statistical analysis
Statistical analyses of minimum triplicate cultures were per-
ormed by either a one-way or two-way analysis of variance
ANOVA) using either Dunnett’s or Tukey’s multiple comparison
ests in Prism 7.01 software (GraphPad). Statistical significance was
onsidered as p < 0.05.
.11. Calcium imaging
Calcium imaging was performed on day 49 neural differentia-
ion cultures derived from both HDF51i-509- and H9-hNPCs within
EGDA polyHIPE scaffolds and on 2D TCPS. Cultures were incu-
ated in complete BrainPhys TM neural medium with 10 μM fluo-
AM (Thermo Fisher Scientific) for 30 min at 37 °C in an atmo-
phere of 5% CO 2 in air. Media was removed from cultures and
eplaced with HEPES buffered salt solution (consisting of 145 mM
aCl, 5 mM KCl, 1 mM MgSO 4 , 10 mM HEPES, 2 mM CaCl 2 and
0 mM glucose at pH 7.4) and imaged at atmospheric conditions
sing an A1R confocal microscope (Nikon) equipped with a × 20
bjective, (excitation 488 nm; emission 525–550 nm). Following a
0-minute equilibration period, and 3 and 4 min of baseline activ-
ty imaging, the agonist L-glutamic acid (glutamate, Sigma-Aldrich),
1, 10 or 100 μM) was added. Cultures were then allowed to equi-
ibrate for 3–4 min, before the addition of 100 mM KCl (Sigma-
ldrich). Cultures were given a further 2 min to re-equilibrate be-
ore recording was stopped. Only cells that responded to KCl were
sed for further analysis.
Live cell calcium imaging recordings were analysed using Im-
geJ Image Processing and Analysis software [28] . Video files (.nd2
ormat) obtained from the Nikon A1R confocal microscope were
nalysed in ImageJ using the ‘Nikon ND2 Reader’ plugin. The soma
f cells morphologically identified as neurons were selected as re-
ions of interest (ROI) using the ‘ROI Manager’ and measured using
he ‘Multi Measure’ function.
106 A.R. Murphy, J.M. Haynes and A.L. Laslett et al. / Acta Biomaterialia 101 (2020) 102–116
Fig. 1. Visualization of hiPSC-NPC migration and expansion within PEGDA and TMPTA polyHIPE scaffolds : HDF51i-509-hNPCs (passage 14) were seeded at 1 × 10 6 cells onto
laminin-coated ( A ) PEGDA and ( B ) TMPTA polyHIPE scaffolds. After ( i ) 2, ( ii ) 6, ( iii ) 10 and ( iv ) 14 days culture in NPC maintenance medium, scaffolds were fixed, sectioned
at 10 μm thickness, stained with hematoxylin and eosin and assessed by brightfield microscopy (scale bars = 100 μm).
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Synchronicity of neuronal firing was quantified using a Pear-
son correlation to determine the Pearson correlation coefficient,
a value between –1.0 and 1.0 that represents the in-phase (posi-
tive) and out-of-phase (negative) correlation between curves. The
Pearson correlation coefficient (R) between two ROI data sets
x(t) = {x 1 ,…,x n } and y(t) = {y 1 ,…,y n } is represented by the follow-
ing equation ( Eq. (1) ):
R ( x, y ) =
∑ n i =1 ( x i − x ) ( y i − y ) √ ∑ n
i =1 ( x i − x ) 2 √ ∑ n
i =1 ( y i − y ) 2
(1)
The synchronicity score (S) was then determined by calculating the
mean of all elements of the correlation coefficient matrix (R ij ).
3. Results
3.1. Three-dimensional maintenance of hPSC-NPCs within polyHIPE
scaffolds
3.1.1. Expansion and viability of hiPSC-NPCs within polyHIPE scaffolds
HDF51i-509-hNPCs were seeded onto both PEGDA and TMPTA
polyHIPE scaffold materials coated with laminin and maintained in
STEMdiff NPM (NPC maintenance medium) for 14 days. Transverse
histological sections of PEGDA and TMPTA scaffold cultures visu-
ally demonstrated an increase in HDF51i-509-hNPC number over
a 14-day culture period ( Fig. 1 . A.i–iv ). There were observable dif-
ferences in the methods of attachment, infiltration and migration
of HDF51i-509-hNPCs throughout the PEGDA scaffold (G’ = 1.4 kPa,
Fig. S1 ) when compared to the TMPTA scaffold (G’ = 36 kPa,
Fig. S2 ). HDF51i-509-hNPCs within the PEGDA scaffold material
were observed to first settle randomly throughout the depth of
the scaffold ( Fig. 1 .A.i ). Small HDF51i-509-hNPC clusters were then
observed to radially expand to form larger 3D cellular aggregates
( Fig. 1 . A.ii ). These aggregates then appeared to spread and homo-
geneously fill the scaffold voids ( Fig. 1 .A.iii-iv ). Conversely, within
the TMPTA scaffold material, HDF51i-509-hNPCs were initially
observed to evenly distribute themselves towards the top (seeded)
surface of the material ( Fig. 1 .B.i ). HDF51i-509-hNPCs then ap-
peared to migrate downwards throughout the 200 μm depth of the
TMPTA scaffold ( Fig. 1 . B.ii–iv ).
Complete recovery of cells from structurally similar 3D cell cul-
ture scaffolds has previously been proven difficult, demonstrated
by the modest 58% recovery of HepG2 cells from Alvetex® cell cul-
ture scaffolds [29] . It was hypothesised that the recovery of intra-
cellular components of cells within polyHIPE scaffolds via cell lysis
ay be a more efficient method of quantifying cell number when
ompared to attempts to extract whole cells out of the scaffolds.
he amount of dsDNA harvested by cell lysis was therefore used to
ndirectly quantify the number of HDF51i-509-hNPCs inside poly-
IPE scaffolds.
As determined by dsDNA quantification, significant increases in
DF51i-509-hNPC number were observed within both the PEGDA
p < 0.001) and TMPTA scaffolds ( p < 0.001), as well as in the con-
rol 2D TCPS ( p < 0.001) from days 2 to 14 of culture in NPC
aintenance medium post-seeding ( Fig. 2 .A ), indicating cell expan-
ion in all systems. The number of HDF51i-509-hNPCs within the
EGDA scaffolds 2 days post-seeding was found to be significantly
ess ( p < 0.05) than that observed on control 2D TCPS, possibly in-
icating a lower attachment efficiency. The number of cells within
oth the PEGDA and TMPTA scaffolds was shown to be significantly
ess ( p < 0.05) than that on the 2D TCPS at days 6, 10 and 14 in
PC maintenance medium, which is potentially a consequence of
nitial low cell attachment efficiency onto the polyHIPE scaffolds
Fig. 2 .A ).
HDF51i-509-hNPCs cultured on 2D TCPS reached a maximum
umber of 10.5 × 10 6 cells (SD = 0.91) after 6 days culture in NPC
aintenance medium, after which cell number was observed not
o significantly change ( p > 0.05) ( Fig. 2 .A ). The number of HDF51i-
09-hNPCs within the TMPTA scaffold continued to increase up
o 5.5 × 10 6 (SD = 0.54) at day 10, after which it ceased to sig-
ificantly change ( p > 0.05) ( Fig. 2 .A ). The number of HDF51i-509-
NPCs within the PEGDA scaffold significantly increased ( p < 0.001)
o 4.0 × 10 6 cells (SD = 0.57) after 14 days culture in NPC mainte-
ance medium ( Fig. 2 .A ).
Viability of 2D TCPS HDF51i-509-hNPC cultures, as determined
y PrestoBlue® Cell Viability Reagent, was found to be in con-
ordance with cell number trends determined by dsDNA quantifi-
ation ( Fig. 2 .A and B ). The viability of both PEGDA and TMPTA
caffold cultures steadily increased from days 2 to 14 culture
n NPC maintenance medium ( Fig. 2 .B ). Interestingly, the viabil-
ty of the PEGDA HDF51i-509-hNPC scaffold cultures was found
o be significantly greater than the TMPTA scaffold cultures at
ays 10 ( p < 0.05) and 14 ( p < 0.01) ( Fig. 2 .B ), despite there be-
ng significantly fewer ( p < 0.001) cells within the PEGDA scaf-
old at the same time points as determined by dsDNA quan-
ification ( Fig. 2 .A ). After 6 days culture in NPC maintenance
edium, the viability of the 2D TCPS culture ceased to signifi-
antly change ( p > 0.05), in concordance with cell number results
Fig. 2 .A and B ).
A.R. Murphy, J.M. Haynes and A.L. Laslett et al. / Acta Biomaterialia 101 (2020) 102–116 107
Fig. 2. Quantification of hiPSC-NPC expansion and viability within PEGDA and TMPTA polyHIPE scaffolds for 14 days culture : ( A ) Number of HDF51i-509-hNPCs cultured within
laminin-coated PEGDA and TMPTA scaffolds and on 2D TCPS, as determined by dsDNA quantification at 2, 6, 10 and 14 days culture in NPC maintenance medium (mean ±standard deviation, N = 3); ( B ) viability of HDF51i-509-hNPC cultured within laminin-coated PEGDA scaffolds, TMPTA scaffolds and on 2D TCPS at 2, 6, 10 and 14 days culture
in NPC maintenance medium (mean ± standard deviation, N = 3); ( C ) overall viability of HDF51i-509-hNPC cultures grown within laminin-coated PEGDA scaffolds, TMPTA
scaffolds and on 2D TCPS normalised to cell number at 2, 6, 10 and 14 days culture in NPC maintenance medium (mean ± standard deviation, N = 3). Statistical analysis was
performed by a Two-Way ANOVA utilising a Dunnett’s multiple comparison test (mean ± standard deviation, N = 3, n.s p > 0.05, 0.01 <
∗p < 0.05, 0.001 <
∗∗p < 0.01, ∗∗∗p <
0.001).
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To better evaluate the average viability per cell, culture viabil-
ty data was normalised to cell number data to determine the av-
rage viability on a per cell basis ( Fig. 2 .C ). PEGDA, TMPTA and
D TCPS cultures displayed maximum normalised viability at day
post-seeding, most likely due to a minimum number of cells
eing present and reduced competition for cell culture nutrients
Fig. 2 .C ). As HDF51i-509-hNPC numbers increased over time, the
ormalised viability of PEGDA, TMPTA and 2D TCPS cultures sig-
ificantly decreased between days 2 and 6 culture ( p < 0.001) and
id not significantly change ( p > 0.05) from day 6 to day 14 cul-
ure ( Fig. 2 .C ). The normalised viability of 2D TCPS and PEGDA
caffold cultures were similar over the entire 14-day culture pe-
iod ( Fig. 2 . C ). Conversely, the normalised viability of the TMPTA
caffold cultures were lower over the entire 14-day culture pe-
iod when compared to both the PEGDA scaffold and 2D TCPS
ultures ( Fig. 2 .C ), possibly a result of HDF51i-509-hNPCs exit-
ng the stem cell state and undergoing apoptosis. Nevertheless,
DF51i-509-hNPCs cultured within the PEGDA polyHIPE scaffold
ere found to be more viable on a per cell basis than those cul-
ured within the TMPTA polyHIPE scaffold and also comparable to
hose on 2D TCPS.
.1.2. Protein and mRNA expression of hPSC-NPCs maintained within
olyHIPE scaffolds
Having established HDF51i-509-hNPC attachment to and expan-
ion within both TMPTA and PEGDA polyHIPE scaffolds, we next
nvestigated the cellular phenotype of cultures after long-term (14
ays) maintenance within these materials. The status of HDF51i-
09-hNPCs and H9-hNPCs cultured within both PEGDA and TMPTA
olyHIPE scaffolds after 14 days in NPC maintenance medium was
ompared to routinely maintained 2D TCPS cultures and assessed
y quantitative reverse transcriptase-PCR (qRT-PCR) for mRNA ex-
ression of the genes: POU5F1 ( OCT-3/4 ), a transcription factor
ritical in embryonic development and the maintenance of stem
ell pluripotency [30] ; PAX6 , a marker of neuroectodermal lineage
pecification in human embryos as well as neural differentiation
f hESCs [31] ; SOX1, an early transcribed neuroectodermal linage
arker and transcription factor present in the nucleus of the hN-
Cs [32 , 33] ; βIII-Tubulin ( TUBB3 ), a class III member of the β-
ubulin protein family that forms cellular microtubule networks
pecifically present in immature neurons [34 , 35] and also in testis
ells [36] , as well as being weakly expressed in neural progenitor
ells [37] ; and GFAP , an intermediate filament protein present in
ature astrocytes of the CNS [38] , integral in maintaining glia cell
hape and structure [38] ( Fig. 3 .A-E ).
After 14 days culture in NPC maintenance medium, as expected,
he expression of POU5F1 mRNA remained downregulated in 3D
caffold cultures of both HDF51i509-hNPC and H9-hNPC cultures
Fig. 3 .A ). Expression of the early neuroectodermal lineage markers
AX6 and SOX1 were significantly downregulated in both PEGDA
nd TMPTA cultures of both cell lines when compared to corre-
ponding 2D cultures ( Fig. 3 .B and C ). The expression of TUBB3
emained constant in both PEGDA and TMPTA HDF51i-509-hNPC
nd H9-hNPC cultures when compared to 2D HDF51i-509-hNPC
108 A.R. Murphy, J.M. Haynes and A.L. Laslett et al. / Acta Biomaterialia 101 (2020) 102–116
Fig. 3. Relative qRT-PCR study of hPSC-NPCs maintained within PEGDA and TMPTA polyHIPE scaffolds : HDF51i-509- and H9-hNPCs were cultured within PEGDA and TMPTA
polyHIPE scaffolds f or 14 days in NPC maintenance medium. RNA was extracted and analysed for the expression of: ( A ) POU5F1 , ( B ) PAX6 , ( C ) SOX1 , ( D ) TUBB3 and ( E )
GFAP . Expression data was controlled using the house-keeping gene GAPDH, normalised to parental HDF51i-509-iPSC and H9-ESC expression data and analysed for statistical
difference to 2D TCPS hNPC maintenance cultures (black). Statistical analysis performed by a One-Way ANOVA utilising a Tukey multiple comparison test (mean ± standard
deviation, N = 3, 0.01 <
∗p < 0.05, 0.001 <
∗∗p < 0.01, ∗∗∗p < 0.001).
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and H9-hNPC cultures ( Fig. 3 .D ). Interestingly, the expression of
GFAP was significantly upregulated ( p < 0.001) in both PEGDA and
TMPTA HDF51i-509-hNPC and H9-hNPC cultures when compared
to 2D HDF51i-509-hNPC and H9-hNPC cultures ( Fig. 3 .E ). The
downregulation of early neuroectodermal lineage markers com-
bined with the upregulation of a glial cell marker possibly in-
dicates the initiation of spontaneous glial-lineage differentiation
of PSC-hNPCs after 14 days culture in NPC maintenance medium,
within both PEGDA and TMPTA polyHIPE scaffolds.
Following 14 days culture in NPC maintenance medium within
both TMPTA and PEGDA polyHIPE scaffolds, HDF51i-509-hNPCs
were investigated via in-situ immunocytochemistry for the pres-
ence of the proteins: SOX1; VIMENTIN, a cytoskeletal type III in-
termediate filament protein, expressed in radial glia cells, a type of
migrating neural progenitor in the developing CNS [39] ; NESTIN,
an intermediate filament protein (type VI) expressed transiently
in NPCs, which downregulates upon differentiation of these cells
[40 , 41] ; and βIII-TUBULIN to examine any phenotypic changes oc-
curring within the polyHIPE scaffold environments ( Fig. 4 ). After 14
days culture in NPC maintenance medium, immunocytochemistry
revealed that HDF51i-509-hNPCs grown within both PEGDA and
TMPTA scaffold materials expressed the proteins NESTIN ( Fig. 4 .A.i
and B.i ), VIMENTIN ( Fig. 4 .A.ii and B.ii ) SOX1 ( Fig. 4 .A.iii and
B.iii ) and βIII-TUBULIN ( Fig. 4 .A.iv and B.iv ). This indicates that
such cultures retain an early neural phenotype, comparable to
maintenance control cultures on 2D TCPS ( Fig. 4 .C ). The autoflu-
orescent emission wavelength of the PEGDA scaffold was distinct
rom the other fluorescent molecules within immunocytochemi-
ally stained sections, which allowed the assignment of its own
avelength channel using spectral un-mixing software. This al-
owed the scaffold to be labelled its own colour (white) in the
uorescent images shown ( Fig. 4 .A ). The autofluorescent emis-
ion wavelength of the TMPTA scaffold could not be assigned its
wn colour in the fluorescent images as it was too similar in
avelength maximum to the DAPI emission fluorescence spectra
Fig. 4 . B ).
.2. Three-dimensional neural differentiation of hPSC-NPCs within
olyHIPE scaffolds
.2.1. Organisation and morphology of differentiated hiPSC-NPCs
ithin polyHIPE scaffolds
To initiate neural differentiation, a switch to complete
rainPhys TM neural medium (neural differentiation medium) was
ade after 14 days culture of HDF51i-509-hNPCs within polyHIPE
caffolds in NPC maintenance medium ( Fig. 5 .A.i and B.i ). Hema-
oxylin and eosin stained sections of PEGDA and TMPTA polyHIPE
caffold cultures showed no visible change in the amount of cells
resent from days 0 to 10 neural differentiation ( Fig. 5 .A.ii and
.ii ). Phase contrast microscopy revealed an increase in HDF51i-
09-hNPC death and/or detachment from viable 2D TCPS compar-
tive cultures at day 10 neural differentiation ( Fig. 5 .C.ii ), which
s typical of cells exiting from an NSC state and failing to viably
ransition to neural and glial progenitor lineage restricted cells
A.R. Murphy, J.M. Haynes and A.L. Laslett et al. / Acta Biomaterialia 101 (2020) 102–116 109
Fig. 4. Immunocytochemical detection of early neural lineage protein markers in hiPSC-NPCs maintained within PEGDA and TMPTA polyHIPE scaffolds : HDF51i-509-hNPCs (passage
13) were seeded onto ( A ) PEGDA polyHIPE scaffolds, ( B ) TMPTA polyHIPE scaffolds and ( C ) control 2D culture-glass slides, and cultured for 14 days in NPC maintenance
medium. All cultures were fixed, with scaffold cultures sectioned, and immunocytochemically stained for the detection of the proteins; ( i ) SOX1 detected with AF568 (red),
( ii ) VIMENTIN detected with AF568 (red), ( iii ) NESTIN detected with AF568 (red) and ( iv ) βIII-TUBULIN detected with AF488 (green). Respective isotype (left) and secondary
antibody only (right) controls are shown in the top right inset panels. The nuclear counter-stain DAPI (blue, ii-iv ) and PEGDA scaffold (white, A ) can be visualised (scale
bars = 100 μm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Morphological analysis of hiPSC-NPCs differentiated within PEGDA and TMPTA polyHIPE scaffolds : HDF51i-509-hNPCs (passage 13) were seeded onto ( A ) PEGDA, ( B )
TMPTA scaffolds and ( C ) control 2D TCPS and cultured for ( i ) 0, ( ii ) 10, ( iii ) 19, ( iv ) 28 and ( v ) 45 days in neural differentiation medium. Scaffold cultures were fixed in
10% neutral buffered formalin, sectioned, stained with hematoxylin and eosin and imaged by brightfield microscopy (scale bars = 100 μm). Images of viable 2D TCPS cultures
were captured via phase contrast microscopy (scale bars = 200 μm).
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42 , 43] , with the change to differentiation culture conditions. At
ays 19 and 28 differentiation, a noticeably lower number of cells
ere visually observed in hematoxylin and eosin stained sections
f PEGDA and TMPTA scaffold cultures when compared to day 0
nitiation of differentiation ( Fig. 5 .A.iii–iv and B.iii–iv ). Some cells
bserved inside both PEGDA and TMPTA scaffolds display a dense
ematoxylin stained nucleus with condensed eosin stained cyto-
lasm, a morphological characteristic of cell apoptosis [44] . Con-
uency of cells on 2D TCPS was shown to be maintained between
ays 19 and 28 neural differentiation ( Fig. 5 .C.iii and C.iv ). The
ime period for neural differentiation was extended to 45 days
or both PEGDA scaffold cultures and 2D TCPS cultures to support
unctional neuronal in vitro maturation. At day 45 differentiation,
he arrangement of cells within the PEGDA scaffold revealed areas
f large aggregated cellular migration extending beyond the oc-
upancy of the scaffold voids ( Fig. 5 .A.v ). Here cells appeared to
e completely surrounding the PEGDA scaffold material. At day 45
ifferentiation on 2D TCPS, cell bodies were visually observed to
ave aggregated and formed rounded cluster structures with ra-
ially extended neurites that were networking individual clusters
110 A.R. Murphy, J.M. Haynes and A.L. Laslett et al. / Acta Biomaterialia 101 (2020) 102–116
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( Fig. 5 .C.v ), typical of maturing iPSC- and ESC-derived neuronal
monolayer cultures [45] .
Unlike biological tissue, for which histological techniques are
typically used, cells grown throughout these scaffolds do not have
an established, continuous ECM to support and protect cells from
abrasive damage. Some cells in sectioned scaffolds appear well
outside the area of the scaffold when visualised via brightfield mi-
croscopy ( Fig. 5 .A.iv ), suggesting that these cells have most likely
been displaced from their original position on or within the scaf-
fold section. It should therefore be taken into consideration that
such images may not always provide an entirely accurate repre-
sentation of the structure present during culture, particularly when
fragile cells such as neurons begin to form.
3.2.2. Regulation of neural lineage mRNA markers throughout neural
differentiation
To assess the gene expression profiles of both HDF51i-509-
and H9-hNPCs differentiated terminally within PEGDA and TMPTA
polyHIPE scaffolds, cultures were analysed via qRT-PCR for the
expression of mRNA encoded by the following genes: POU5F1;
PAX6; SOX1; TUBB3 ; Microtubule-associated protein 2 ( MAP2 ), a
neuron specific protein that stabilises the microtubule network
in the dendrites of post-mitotic neurons, whose expression has
been demonstrated shortly after that of βIII-Tubulin in mice [34] ;
Synaptophysin ( SYP ), a presynaptic vesicular membrane glycopro-
tein [46] present in neuronal synapses [47] ; and GFAP . The ex-
pression of mRNA was normalised to parental pluripotent HDF51i-
509-iPSC and H9-ESC lines and compared for statistically signifi-
cant changes relative to maintenance cultures of HDF51i-509- and
H9-hNPCs on 2D TCPS.
As expected, the expression of the pluripotency marker POU5F1
was observed to be downregulated when compared to parental
hPSC mRNA, and did not change in PEGDA, TMPTA and control
2D TCPS HDF51i-509-hNPC differentiation cultures ( Fig. 6 . A.i ) and
H9-hNPC neural differentiation cultures ( Fig. 6 .A.ii ). No significant
change in the expression of PAX6 mRNA was detected at days
10, 19, 28 and 45 in PEGDA HDF51i-509-hNPC differentiation cul-
tures when compared to 2D HDF51i-509-hNPC maintenance cul-
tures ( Fig. 6 .B.i ). PAX6 mRNA in TMPTA HDF51i-509-hNPC differen-
tiation cultures was observed to be significantly down regulated,
when compared to 2D HDF51i-509-hNPC maintenance cultures, at
days 10, 19 and 28 ( Fig. 6 .B.i ). PAX6 expression was found to be
significantly upregulated in 2D TCPS HDF51i-509-hNPC differentia-
tion cultures at days 10, 19 and 28 when compared to 2D HDF51i-
509-hNPC maintenance cultures ( Fig. 6 .B.i ). Similar to HDF51i-509-
hNPC differentiation cultures, no significant change was detected
in the expression of PAX6 mRNA at days 10, 19, 28 and 45 in
PEGDA H9-hNPC differentiation cultures when compared to 2D H9-
hNPC maintenance cultures ( Fig. 6 .B.ii ). TMPTA H9-hNPC differen-
tiation cultures also showed no significant change in the expres-
sion of PAX6 mRNA at days 10, 19 and 28 when compared to
2D H9-hNPC maintenance cultures ( Fig. 6 .B.ii ). Similar to 2D TCPS
HDF51i-509-hNPC differentiation cultures, 2D TCPS H9-hNPC dif-
ferentiation cultures showed upregulation of PAX6 mRNA at days
19 and 28 when compared to 2D H9-hNPC maintenance cultures
( Fig. 6 .B.ii ). Significant downregulation of SOX1 mRNA was de-
tected at day 28 and day 45 in PEGDA HDF51i-509-hNPC differ-
entiation cultures when compared to 2D HDF51i-509-hNPC main-
tenance cultures ( Fig. 6 .C.i ). Significant downregulation of SOX1
mRNA was observed at days 10, 19 and 28 in TMPTA HDF51i-509
differentiation cultures when compared to 2D HDF51i-509-hNPC
maintenance cultures ( Fig. 6 .C.i ). No significant change in SOX1
mRNA was detected at days 10, 19 and 28 in 2D TCPS HDF51i-509-
hNPC differentiation cultures when compared to 2D HDF51i-509-
hNPC maintenance cultures ( Fig. 6 .C.i ). No significant change in
SOX1 mRNA was detected in either PEGDA or TMPTA H9-hNPC dif-
erentiation cultures at any differentiation time points when com-
ared to 2D H9-hNPC maintenance cultures ( Fig. 6 .C.ii ). A small
ignificant upregulation in SOX1 mRNA was detected in 2D TCPS
9-hNPC differentiation cultures at day 19 when compared to 2D
9-hNPC maintenance cultures, however, this upregulation did not
emain significant after 28 days ( Fig. 6 .C.ii ).
Expression of the neuronal marker TUBB3 mRNA was found to
e significantly upregulated at day 10 in PEGDA HDF51i-509-hNPC
ifferentiation cultures when compared to 2D HDF51i-509-hNPC
aintenance cultures, however, was observed to decrease back to a
evel comparable to that of 2D HDF51i-509-hNPC maintenance cul-
ures after days 28 and 45 ( Fig. 6 .D.i ). Expression of TUBB3 was ob-
erved to consistently decrease in TMPTA HDF51i-509-hNPC differ-
ntiation cultures and was seen to be significantly downregulated
t day 28 when compared to 2D HDF51i-509-hNPC maintenance
ultures ( Fig. 6 .D.i ). Expression of TUBB3 mRNA was observed to be
ignificantly upregulated at days 10, 19 and 28 in 2D TCPS HDF51i-
09-hNPCs differentiation cultures when compared to 2D HDF51i-
09-hNPC maintenance cultures ( Fig. 6 .D.i ). TUBB3 mRNA expres-
ion was observed not to significantly change at days 10, 19 and
8 in PEGDA H9-hNPC differentiation cultures when compared to
D H9-hNPC maintenance cultures, however, was observed to be
ignificantly down regulated at day 45 ( Fig. 6 .D.ii ). Expression of
UBB3 mRNA did not significantly change in TMPTA H9-hNPC dif-
erentiation cultures at day 10, 19 or 28 when compared to 2D H9-
NPC maintenance cultures ( Fig. 6 .D.ii ). 2D TCPS H9-hNPC cultures
ere found to have significantly upregulated TUBB3 mRNA expres-
ion at days 10 and 19 when compared to 2D H9-hNPC mainte-
ance cultures, however, this was observed to decrease back to
level comparable to 2D H9-hNPC maintenance cultures at day
8 ( Fig. 6 .D.ii ). PEGDA HDF51i-509 differentiation cultures demon-
trated a significant upregulation of MAP2 mRNA at day 19 when
ompared to 2D HDF51i-509-hNPC maintenance cultures, however
his decreased back to a level comparable to that of 2D HDF51i-
09-hNPC maintenance cultures at days 28 and 45 ( Fig. 6 .E.i ). Ex-
ression of MAP2 mRNA was observed not to significantly change
fter 28 days differentiation in TMPTA HDF51i-509-hNPC differen-
iation cultures when compared to 2D HDF51i-509-hNPC mainte-
ance cultures ( Fig. 6 .E.i ). Expression of MAP2 mRNA was signif-
cantly upregulated in 2D TCPS HDF51i-509-hNPC differentiation
ultures at days 10, 19 and 28 when compared to 2D HDF51i-509-
NPC maintenance cultures ( Fig. 6 .E.ii ). Unlike the HDF51i-509-
NPC line, PEGDA H9-hNPC differentiation cultures exhibited sig-
ificant upregulation of MAP2 mRNA at days 10, 19, 28, and 45
hen compared to 2D H9-hNPC maintenance cultures ( Fig. 6 .E.ii ).
MPTA H9-hNPC differentiation cultures exhibited upregulation of
AP2 mRNA at day 19 when compared to 2D HDF51i-509-hNPC
aintenance cultures, however, this decreased to an insignificantly
ifferent level at day 28 ( Fig. 6 .E.ii ). Expression of MAP2 mRNA
as found to be significantly upregulated at days 10, 19 and 28
n 2D TCPS H9-hNPC differentiation cultures when compared to
D H9-hNPC maintenance cultures ( Fig. 6 .E.ii ). Expression of the
resynaptic marker SYP mRNA was observed to be significantly
pregulated at days 10, 19, 28 and 45 in PEGDA HDF51i-509-
NPC differentiation cultures when compared to 2D HDF51i-509-
NPC maintenance cultures ( Fig. 6 .F.i ). Expression of SYP mRNA
id not significantly change at any differentiation time point in
MPTA HDF51i-509-hNPC differentiation cultures when compared
o 2D HDF51i-509-hNPC maintenance cultures ( Fig. 6 .F.i ). Simi-
ar to PEGDA HDF51i-509-hNPC differentiation cultures, 2D TCPS
DF51i-509 differentiation cultures displayed an upregulation of
YP mRNA at days 10, 19 and 28 when compared to 2D HDF51i-
09-hNPC maintenance cultures ( Fig. 6 .F.i ). PEGDA and 2D TCPS
9-hNPC differentiation cultures both displayed a significant up-
egulation of SYP mRNA at all differentiation time points when
ompared to 2D H9-hNPC maintenance cultures ( Fig. 6 .F.ii ). TMPTA
A.R. Murphy, J.M. Haynes and A.L. Laslett et al. / Acta Biomaterialia 101 (2020) 102–116 111
Fig. 6. Relative qRT-PCR study of neural lineage markers of hPSC-NPCs differentiated within PEGDA and TMPTA polyHIPE scaffolds : ( i ) HDF51i-509-hNPCs and ( ii ) H9-hNPCs
cultured within PEGDA and TMPTA scaffolds for 10, 19, 28 and 45 days in neural differentiation media. RNA was extracted and analysed for the expression of: ( A ) POU5F1 ,
( B ) PAX6, ( C ) SOX1, ( D ) TUBB3 , ( E ) MAP2 , ( F ) SYP and ( G ) GFAP markers. Expression data was controlled using the house-keeping gene GAPDH , normalised to parental HDF51i-
509-iPSC and H9-ESC RNA and analysed for statistical difference to 2D TCPS hNPC maintenance cultures (black). Statistical analysis was performed via a Two-Way ANOVA
utilising a Dunnett’s multiple comparison test (mean ± standard error measurement, N = 3, 0.01 <
∗p < 0.05, 0.001 <
∗∗p < 0.01, ∗∗∗p < 0.001).
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9-hNPCs differentiation cultures displayed a significant upregula-
ion of SYP mRNA at day 19 and 28 when compared to 2D H9-
NPC maintenance cultures ( Fig. 6 .F.ii ). Expression of the glial cell
arker GFAP mRNA in PEGDA, TMPTA and 2D TCPS HDF51i-509-
NPC and H9-hNPC differentiation cultures was observed to be sig-
ificantly upregulated at all differentiation time points when com-
ared to 2D HDF51i-509-hNPC and H9-hNPC maintenance cultures,
espectively ( Fig. 6 .G.i and 6.G.ii ).
.2.3. Expression of neural-lineage protein markers throughout
ifferentiation within polyHIPE scaffolds
The differentiation of HDF51i-509-hNPCs within PEGDA scaf-
olds, TMPTA scaffolds and on control 2D substrates in neural
ifferentiation medium was also assessed via immunocytochem-
cal detection of the following proteins: βIII-TUBULIN; MAP2;
euronal nuclear protein (NEUN), an RNA binding protein for
euron-specific splicing regulation [48] , typically expressed in
he nucleus of post-mitotic neurons [49 , 50] ; and GFAP. HDF51i-
09-hNPCs differentiated within PEGDA scaffolds were observed
o express the cytoskeletal protein βIII-TUBULIN and the nuclear
rotein NEUN from day 10 differentiation to day 45 ( Fig. 7 .A.i–iv )
n cells cultured throughout the scaffold’s entire 200 μm depth.
ells positive for detection of βIII-TUBULIN and NEUN also ap-
eared at day 10 differentiation and throughout to day 28 on
ontrol 2D substrates ( Fig. S3.A ). The number of cells positive for
he expression of βIII-TUBULIN within TMPTA scaffolds visually
ppeared to decrease between day 10 and day 28 differentiation
Fig. 7 .B.i–iii ), and appeared to be less in number than those seen
t day 14 NPC maintenance cultures (day 0 differentiation) from
he same initial seeding ( Fig. 4 .B.iv ), however this was not quan-
ified. Cells positive for the NEUN protein were few and sparse
ithin TMPTA scaffolds at all differentiation time points investi-
ated ( Fig. 7 .B.i–iii ). Cells positive for detected expression of the
rotein MAP2 were observed within PEGDA scaffolds from day 10
o day 45 differentiation ( Fig. 7 .C.i–iv ). Interestingly, cells positive
or MAP2 expression did not appear on control 2D substrates until
ay 19 differentiation ( Fig. S3.B ), later than detected expression
ithin PEGDA scaffolds. Cells positive for MAP2 within TMPTA
112 A.R. Murphy, J.M. Haynes and A.L. Laslett et al. / Acta Biomaterialia 101 (2020) 102–116
Fig. 7. Immunocytochemical detection of the neural lineage proteins in hiPSC-NPCs differentiated within PEGDA and TMPTA polyHIPE scaffolds : HDF51i-509-hNPCs (passage 13)
were seeded onto ( A, C, E ) PEGDA and ( B, D, F ) TMPTA polyHIPE scaffolds, and cultured f or ( i ) 10, ( ii ) 19, ( iii ) 28 and ( iv ) 45 days (shown f or PEGDA) in neural differentiation
medium. All cultures were fixed, sectioned, and immunocytochemically stained for the detection of the proteins ( A, B ) βIII-TUBULIN using AF488 (green) and NEUN using
AF568 (red), ( C, D ) MAP2 using AF568 (red) and ( E, F ) GFAP using AF647 (magenta). The nuclear counter-stain DAPI (blue) and PEGDA scaffold (white) can be visualised
(scale bars = 100 μm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
s
s
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p
scaffolds were observed between days 10 and 28 differentiation
and mainly resided at the top and bottom surfaces of the scaffold
( Fig. 7 .D.i–iii ). GFAP expressing cells were first detected within
PEGDA scaffolds from day 19 differentiation and throughout to
day 45 differentiation ( Fig. 7 .E.i–iv ). GFAP expressing cells were
also seen within TMPTA scaffolds ( Fig. 7 .F.i–iii ) and on control 2D
substrates ( Fig. S3.C ) at days 19 and 28 differentiation. Similar re-
sults to control 2D HDF51i-509-hNPC differentiation cultures were n
een with control 2D H9-hNPC differentiation cultures (data not
hown).
.3. Calcium imaging of hPSC-neuronal cell cultures within PEGDA
olyHIPE scaffolds
HDF51i-509- and H9-hNPCs were differentiated for 49 days in
eural differentiation medium within PEGDA polyHIPE scaffolds
A.R. Murphy, J.M. Haynes and A.L. Laslett et al. / Acta Biomaterialia 101 (2020) 102–116 113
Fig. 8. Calcium imaging response curves of hPSC-NPCs differentiated for 49 days within PEGDA polyHIPE scaffolds : ( A ) HDF51i-509-hNPCs and ( B ) H9-hNPCs were differentiated
within PEGDA polyHIPE scaffolds for 49 days and imaged using the fluo-4 AM calcium binding dye. Cells exhibiting a small, compact cell soma with long thin axonal
extensions, morphologically characteristic of neurons, were identified as regions of interest (ROI) for intensity measurements. Large, rounded star-shaped cells morphologically
characteristic of glial cells were avoided as ROI selections. ( i ) Representative spontaneous calcium oscillations and ( ii ) elevations of intracellular calcium in response to ( A.ii )
100 μM glutamate and ( B.ii ) increasing concentrations of 1, 10 and 100 μM glutamate. Arrows illustrate the time point at which glutamate was added.
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nd on control 2D TCPS surfaces, then imaged for activity using
uo-4, a fluorescent, calcium-sensitive binding dye. The PEGDA
olyHIPE scaffold was observed to emit light in the 525–550 nm
avelength, similar to the fluo-4 molecule, making it difficult to
istinguish between the PEGDA scaffold material and intracellu-
ar calcium fluorescent signals. The scaffold material also emit-
ed light in the 550–649 nm detector channel, without the 561 nm
mission laser on, which we then used to distinguish fluo-4 flu-
rescence from scaffold fluorescence. The focal plane from which
mages were captured was adjusted to be approximately 100 μm
rom the top and bottom surfaces of the scaffold, in order to en-
ure data was captured from cells cultured, three-dimensionally
ithin the scaffold and not two-dimensionally on the outer sur-
aces of the scaffold. Where cultures displayed spontaneous activ-
ty synchronicity was measured, where they did not glutamate was
dded. Beyond day 30 differentiation, H9-hNPC 2D control differ-
ntiation cultures displayed significant cell clustering and detach-
ent (data not shown). To combat this difficulty, H9-hNPC 2D dif-
erentiation cultures were carefully dissociated (using Accutase) at
ay 30 differentiation and replated at a density of 1 × 10 5 cells per
m
2 .
Spontaneous and synchronous calcium activity was observed
cross a field of view of cells displaying neuronal morphology,
erived from HDF51i-509-hNPCs, after 49 days culture in neu-
al differentiation medium within the PEGDA polyHIPE scaffold
Fig. 8 .A.i, Video S1 ). This synchronous spike activity, with a syn-
hronicity score of S = 0.83 (out of 1), was observed to occur at a
requency of 1.2 oscillations per minute, suggesting the formation
f a network between cells grown throughout the scaffold. Spont a-
eous calcium activity was also observed in H9-hNPC-derived cul-
ures, terminally differentiated for 49 days within PEGDA scaffolds
Fig. 8 .B.i ). Spike activity appeared to be rhythmic in some cells,
ut was not seen to be synchronous across the culture ( S = 0.01),
issimilar to the observations for the HDF51i-509-hNPC-derived
euronal cultures. HDF51i-509-hNPCs differentiated for 49 days
ithin the PEGDA scaffold were also shown to respond to gluta-
ate with rapid elevations of intracellular calcium ( Fig. 8 .A.ii ), in-
icating the presence of functional glutamate receptors on these
ells. H9-hNPC neural differentiation cultures (day 49) within the
EGDA polyHIPE scaffold also showed elevations in intracellular
alcium in response to glutamate ( Fig. 8 .B.ii ).
Calcium imaging of HDF51i-509-hNPCs terminally differenti-
ted for 49 days on control 2D TCPS displayed a spike in calcium
oncentration in response to glutamate (10 μM) ( Fig. S4.A ).
ome spontaneous waves were also observed in these cultures
re-agonist addition, however did not display any rhythmic or
ynchronous behaviour as seen in 3D cultures ( Fig. S4.A ). After 49
ays differentiation (replated at day 30) H9-hNPC neural differen-
iation cultures responded to glutamate (10 μM) with an elevation
f intracellular calcium ( Fig. S4.B ).
114 A.R. Murphy, J.M. Haynes and A.L. Laslett et al. / Acta Biomaterialia 101 (2020) 102–116
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4. Discussion
The combination of biomaterial cell culture scaffolds and in
vitro renewable neural stem cell systems shows great promise for
the development of new therapeutic tools that allow for the inter-
rogation and eventual treatment of human neurological disorders.
Evaluating the influence of different 3D scaffolds on the attach-
ment, proliferation and differentiation of hNPCs is an important
step in the development of new 3D neurological models. A vari-
ety of hydrogel systems have been employed for the maintenance
and neural lineage differentiation of hNPCs such as animal-derived
collagen hydrogels [51-54] , Matrigel TM [55] , PuraMatrix TM hydro-
gels [56 , 57] , hyaluronic acid-modified hydrogels [58 , 59] and PLGA
hydrogels [60] . Due to their typically high elastic modulus com-
pared to hydrogels, solid porous scaffolds are rarely utilised in 3D
neural cell culture applications [4] . Solid macroporous cell culture
scaffolds offer larger void volumes (compared to hydrogels), allow-
ing for potentially greater nutrient and waste diffusion [61] and
more natural 3D cellular migration. The solid macroporous PEGDA
polyHIPE cell culture scaffold was developed to mimic the me-
chanical properties of mammalian brain tissue, whilst simultane-
ously addressing certain impracticalities such as transparency and
hydrophobicity [9] .
This study investigated the ability of a PEGDA polyHIPE cell
culture scaffold to support the long-term expansion and differ-
entiation of both ESC- and iPSC-derived hNPCs. PEGDA polyHIPE
scaffolds were developed with mechanical properties compa-
rable to that of mammalian brain tissue, which has previously
been shown to be an important characteristic in supporting
and inducing neural cell differentiation [62-65] . This study also
compared the performance of the PEGDA polyHIPE scaffold to
the mechanically stiffer, more hydrophobic and opaque TMPTA
polyHIPE scaffold f or the purpose of investigating whether this
new tailored polyHIPE scaffold influences any phenotypic changes
during long-term expansion and in vitro neural differentiation of
HDF51i-509-hNPCs and H9-hNPCs.
PEGDA polyHIPE scaffolds were shown to support the vi-
able, three-dimensional expansion of HDF51i-509-hNPCs for a
period of 14 days in NPC maintenance medium as determined
both qualitatively, by hematoxylin and eosin staining, as well
as quantitatively, by dsDNA quantification and PrestoBlue® cell
viability assay. HDF51i-509-hNPCs displayed a 13-fold increase in
cell number from day 2 to 14 post-seeding, compared to a 4-fold
increase within the stiffer TMPTA polyHIPE scaffold, and a 5-fold
increase on control 2D TCPS, over the same 14-day culture period.
However, initial hNPC attachment efficiency on the PEGDA poly-
HIPE material was found to be the poorest of the three systems
investigated, revealing only 31% of the 1 × 10 6 cells initially seeded
onto the material, at 2 days post-seeding. This relative measure of
hNPC attachment efficiency was significantly lower than the 132%
observed for the TMPTA scaffold and 201% seen for the control
2D TCPS cultures at 2 days post-seeding. Reduced attachment
of HDF51i-509-hNPCs onto the PEGDA polyHIPE scaffold may
reflect the hydrophilic properties of the PEGDA material induced
by its PEG-based co-monomer [66] , which could result in poor
physical adhesion of laminin coating and/or HDF51i-509-hNPCs.
Attachment efficiency could potentially be improved by chemical
functionalisation of the PEGDA polyHIPE material with cell binding
motifs, such as the IKVAV peptide sequence, which has previously
been shown to enhance primary hNPC attachment and migration
on PEG hydrogels [67] . Interestingly, the PEGDA HDF51i-509-hNPC
scaffold maintenance cultures consistently revealed greater via-
bility than the TMPTA cultures, despite consistently displaying a
lower overall number of cells over the 14-day culture period. This
could reflect a preference for the chemistry and/or mechanical
properties of the PEGDA polyHIPE scaffold material. In addition,
phenotypic change in both HDF51i-509- and H9-hNPCs was
etected by qRT-PCR after 14 days culture within both PEGDA
nd TMPTA polyHIPE scaffolds. Downregulation in gene expression
or the early neuroectodermal markers PAX6 and SOX1 as well as
pregulation of the glial cell marker GFAP was observed across
oth hESC- and hiPSC-NPC lines, which could possibly reflect the
hange in cell culture dimensionality or an adaptive response
o the foreign scaffold material environment. However, the early
eural lineage protein markers SOX1, NESTIN and VIMENTIN were
ound to remain expressed in HDF51i-509-hNPCs cultured within
oth PEGDA and TMPTA polyHIPE scaffolds after 14 days culture
n NPC maintenance medium. HDF51i-509-hNPCs were observed
o homogenously fill the voids of the scaffold material after 14
ays culture in NPC maintenance medium. Consequently, this
as chosen as the time point to switch from NPC maintenance
edium to neural differentiation medium in order to initiate
PSC-NPC neural lineage differentiation.
Phenotypes indicative of neuronal and glial cells were evident
n both HDF51i-509- and H9-hNPC lines differentiated within
he PEGDA polyHIPE scaffold as early as day 10 culture in neural
ifferentiation media, evident by the upregulated expression of
euronal genes TUBB3 and MAP2 , as well as the glial cell marker
FAP , and the synapse marker SYP , then further confirmed by
he detection of proteins βIII-TUBULIN, MAP2, NEUN and GFAP.
his data suggests that the PEGDA polyHIPE scaffold is capable of
upporting the differentiation of hPSC-NPCs to a mixed population
f neuronal and glial cell types. Interestingly, HDF51i-509-hNPCs
ifferentiated within the stiffer TMPTA scaffold exhibited downreg-
lation of TUBB3 and MAP2 , and upregulation of GFAP and scarcely
etectable NEUN protein, indicative of preferential differentiation
f HDF51i-509-hNPCs to glial cells rather than neurons. Similar
ifferentiation phenomena have been observed on hydrogels with
lastic moduli greater than that of mammalian brain tissue [68-
0] . However, this result was not replicated with the H9-hNPC line
ifferentiated within the TMPTA scaffold demonstrating inherent
iological variation in the application of human stem cell sources.
fter 45 days terminal differentiation, some cells were observed
o have formed large aggregates growing within and beyond the
onstraints of the scaffold voids, a phenomenon not seen in similar
ork using stiffer Alvetex® emulsion-templated scaffolds for the
ifferentiation of primary human neural progenitor cells [71] .
ollowing 45 days differentiation, PEGDA polyHIPE scaffold cul-
ures exhibited markers characteristic of mature neuronal and glial
ell formation by immunocytochemical detection and qRT-PCR
nalyses.
To further validate this phenotype, and demonstrate the abil-
ty of the PEGDA polyHIPE scaffold to support 3D neural differ-
ntiation, PEGDA polyHIPE hPSC-NPCs cultures that had under-
one long-term neural differentiation were assessed for function-
lity by calcium imaging. HDF51i-509- and H9-derived neurons
ithin PEGDA polyHIPE scaffolds displayed an increase in intra-
ellular calcium concentration in response to the excitatory neu-
otransmitter glutamate. This result indicates the presence of func-
ional glutamatergic receptors on cells within the PEGDA polyHIPE
caffold, typical of most neurons found in the human brain [72] .
ore interesting, however, was the spontaneous calcium activity
bserved within PEGDA polyHIPE differentiation cultures. HDF51i-
09-derived neurons within PEGDA polyHIPE scaffolds were ob-
erved to spontaneously fire without agonist addition, in unison
ith surrounding neurons ( S = 0.83) and at a rhythmic frequency
f 1.2 oscillation per minute. Repetitive and synchronised burst dis-
harges across large fractions of cultures have been identified in
itro in neocortical rat neuron cultures [73] as well as organotypic
eocortical in vitro rat slice cultures [74 , 75] , and is typical of early
ostnatal development. Burst frequency of hiPSC-derived cortical
eurons has been studied in 2D cultures and shown to occur at
A.R. Murphy, J.M. Haynes and A.L. Laslett et al. / Acta Biomaterialia 101 (2020) 102–116 115
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[
frequency of roughly of 1.5 oscillations per minute [76] , slightly
reater than what was observed in PEGDA polyHIPE HDF51i-509-
erived neurons. Spontaneous activity was also observed in PEGDA
olyHIPE H9-derived neurons at a similar frequency to those de-
ived from HDF51i-509-hNPCs, although with less observed syn-
hronicity ( S = 0.01), possibly due to reduced network formation in
he H9-derived 3D neural cultures or the enriched generation of
ifferent neuronal phenotypes ( e.g. GABAergic neurons).
When differentiated on TCPS, both HDF51i-509- and H9-hNPCs
xhibited significant cell clustering by day 49 differentiation. These
ell clusters would detached easily, which made identification of
eurons practically challenging for functional analysis. Replating of
ells during the differentiation protocol was required to conserve a
onolayer of cells for the purpose of allowing the morphological
dentification of neurons during functional analysis. Clustering and
etachment of cells, however, was not found to be a significant is-
ue for PEGDA polyHIPE differentiation cultures, which permitted
he uninterrupted maturation of cultures and possibly contributed
o the synchronous spontaneous activity observed. This is consid-
red an advantage of the 3D scaffold system.
Despite having some limitations in live cell imaging ability, due
o the optical properties of the scaffold, and extraction of live
ells out of the scaffold, the PEGDA polyHIPE material is demon-
trated to be capable of supporting the viable expansion and dif-
erentiation of hPSC-NPCs to neurons and glia. The PEGDA poly-
IPE scaffold is unique compared to hydrogel scaffold materials
n that it utilises natural cellular migration, as opposed to me-
hanical dispersion of cells throughout a hydrogel. The PEGDA
caffold is more suited to long term cultures compared to hy-
rogel scaffolds, such as those comprised of animal collagen, due
o its demonstrated structural stability. Further optimisation this
ynthetic biomaterial scaffold could rendered 3D neural cell cul-
ures more reproducible for future high throughput drug screening
pplications.
. Conclusion
This study explored the ability of tailored PEGDA polyHIPE cell
ulture scaffolds to support the three-dimensional maintenance
nd differentiation of both HDF51i-509- and H9-hNPCs. PEGDA
olyHIPE scaffolds were shown to support the attachment, mi-
ration and expansion of HDF51i-509-hNPCs over a 14-day pe-
iod in NPC maintenance culture conditions. Culture viability and
PC number were also shown to increase over this 14-day period.
DF51i-509-hNPCs cultured within PEGDA scaffolds for 14 days in
PC maintenance medium were demonstrated to express markers
f early neuroectodermal-lineage, similar to 2D HDF51i-509-hNPC
aintenance cultures. Following culture in neural differentiation
onditions for 45 days, upregulation of MAP2, SYP and GFAP mRNA
as detected from PEGDA polyHIPE cultures compared to 2D hPSC-
PC maintenance cultures as well as the detected expression of
roteins MAP2, NEUN and GFAP. HDF51i-509- and H9-hNPCs cul-
ured in neural differentiation medium within PEGDA polyHIPE
caffolds f or 49 days were shown by fluo-4 calcium imaging anal-
ses to transiently increase intracellular calcium concentration in
esponse to 100 μM glutamate. Both cell lines also displayed spon-
aneous transient increases in intracellular calcium concentration,
ithout the addition of chemical agonist in long term differentia-
ion cultures. HDF51i-509-neural differentiation cultures displayed
pontaneous calcium activity in a synchronous, rhythmic fashion
hile H9-neural differentiation cultures displayed spontaneous ac-
ivity, however, not in a comparable synchronous fashion. This ma-
erial provides an alternative to tradition hydrogel materials used
o support 3D neural cell cultures and potentially has future appli-
ation in high throughput drug screening.
eclaration of Competing Interest
None.
unding sources
This research did not receive any specific grant from funding
gencies in the public, commercial, or not-for-profit sectors.
cknowledgements
The authors acknowledge the use of facilities and technical as-
istance of Monash Histology Platform, Department of Anatomy
nd Developmental Biology, Monash University, Victoria, Australia.
upplementary materials
Supplementary material associated with this article can be
ound, in the online version, at doi: 10.1016/j.actbio.2019.10.017 .
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