Acta Biomaterialia 101 (2020) 102–116

Contents lists available at ScienceDirect

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: neil.cameron@monash.edu (N.R. Cameron),

Carmel.O’Brien@csiro.au (C.M. O’Brien).

1

(

d

m

y

a

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

t

t

s

t

l

s

t

a

s

i

s

b

o

d

n

c

i

s

r

s

t

a

o

m

s

r

r

t

b

a

p

s

n

t

b

t

i

n

n

c

[

a

s

w

s

f

m

t

m

a

t

H

t

p

i

v

t

r

o

p

c

e

s

1

a

a

o

p

n

t

d

c

t

c

s

c

t

c

s

m

t

c

l

2

2

o

s

A

t

L

a

m

N

1

a

u

s

c

t

d

t

c

a

5

i

w

e

p

t

a

i

fi

p

2

t

a

d

t

v

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

2

s

b

i

e

w

w

w

4

f

w

t

S

S

r

r

O

(

a

(

v

2

[

(

p

fi

t

a

S

i

s

2

c

p

c

p

s

t

g

i

d

1

r

c

a

i

n

h

N

1

N

a

t

2

6

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

l

w

(

P

m

a

v

9

a

i

s

2

s

p

9

e

F

M

t

t

m

a

a

t

β

I

I

p

g

1

I

F

a

w

d

w

a

s

M

2

c

2

R

s

A

s

p

t

d

u

–

m

F

2

2

u

n

R

n

r

L

t

A

1

c

2

R

T

M

w

(

f

1

a

i

C

c

R

S

I

s

(

G

(

2

f

(

t

c

2

t

P

b

4

s

r

N

1

u

o

1

i

(

l

A

f

u

a

f

a

o

g

t

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).

m

c

T

i

H

H

(

t

m

s

P

l

d

b

l

N

i

(

n

m

t

5

t

n

h

t

n

b

c

c

s

i

i

t

d

i

f

t

m

c

(

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).

i

e

2

2

b

(

n

n

d

t

s

r

s

r

c

i

H

w

t

t

3

p

s

i

d

5

p

c

b

p

c

c

s

o

m

P

t

s

c

c

m

s

t

s

(

P

a

s

r

a

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).

f

c

w

l

fl

s

o

w

(

3

p

3

w

B

m

s

t

s

p

B

5

a

i

t

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).

[

d

w

o

i

o

h

p

fl

d

t

f

f

t

o

c

b

d

h

d

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

f

p

s

H

H

r

b

d

m

l

t

s

e

a

c

s

5

5

s

2

2

s

T

f

h

w

s

n

a

2

s

c

t

5

p

a

t

n

i

c

h

h

n

w

T

M

m

d

w

i

2

p

u

h

h

d

T

t

l

H

S

5

H

r

c

( 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).

H

t

h

m

h

n

p

r

3

d

f

d

i

n

n

t

5

t

p

i

C

p

c

t

a

(

a

t

t

w

g

p

t

f

d

w

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

3

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.

a

fl

p

w

d

l

t

e

o

i

f

s

w

f

i

a

e

m

f

d

c

a

d

r

(

c

f

o

n

t

(

b

d

n

w

m

d

c

P

c

a

c

S

p

s

d

t

o

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

a

d

a

f

u

b

c

t

n

f

b

i

t

d

w

m

h

i

t

d

n

G

t

T

s

o

d

u

d

o

d

e

7

d

b

A

t

c

w

d

F

t

c

a

i

e

g

a

w

c

r

t

s

M

o

5

s

w

o

c

v

n

p

n

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

a

g

d

p

r

c

t

d

e

c

n

c

m

i

d

s

t

t

e

t

c

s

f

H

i

c

s

d

t

s

t

a

5

c

a

p

g

r

N

H

N

o

m

c

w

N

p

t

s

y

r

t

w

t

s

w

t

t

t

c

D

F

a

A

s

a

S

f

R

[

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 .

eferences

[1] E. Carletti , A. Motta , C. Migliaresi , Scaffolds for tissue engineering and 3D cellculture, in: J.W. Haycock (Ed.), 3D Cell Culture, Humana Press, 2011, pp. 17–39 .

[2] I.W.Y. Mak , N. Evaniew , M. Ghert , Lost in translation: animal models and clin-ical trials in cancer treatment, Am. J. Transl. Res. 6 (2) (2014) 114–118 .

[3] X.-h. Xu , Z. Zhong , Disease modeling and drug screening for neurological dis-eases using human induced pluripotent stem cells, Acta Pharmacol. Sin. 34 (6)

(2013) 755–764 . [4] A .R. Murphy , A . Laslett , C.M. O’Brien , N.R. Cameron , Scaffolds f or 3D in vitro

culture of neural lineage cells, Acta Biomater. 54 (2017) 1–20 .

[5] B. Dhandayuthapani , Y. Yoshida , T. Maekawa , D.S. Kumar , Polymeric scaffoldsin tissue engineering application: a review, Int. J. Polym. Sci. 2011 (2011) .

[6] M.K. Włodarczyk-Biegun , K. Farbod , M.W.T. Werten , C.J. Slingerland , F.A. deWolf , J.J.J.P. van den Beucken , S.C.G. Leeuwenburgh , M.A. Cohen Stuart ,

M. Kamperman , Fibrous hydrogels for cell encapsulation: a modular andsupramolecular approach, PLoS One 11 (5) (2016) e0155625 .

[7] J. Malda , T.J. Klein , Z. Upton , The roles of hypoxia in the in vitro engineering of

tissues, Tissue Eng. 13 (9) (2007) 2153–2162 . [8] X. Yin , B.E. Mead , H. Safaee , R. Langer , J.M. Karp , O. Levy , Stem cell organoid

engineering, Cell Stem Cell 18 (1) (2016) 25–38 . [9] A.R. Murphy , I. Ghobrial , P. Jamshidi , A. Laslett , C.M. O’Brien , N.R. Cameron ,

Tailored emulsion-templated porous polymer scaffolds for iPSC-derived humanneural precursor cell culture, Polym. Chem. 8 (43) (2017) 6617–6627 .

[10] Y. Tang , P. Yu , L. Cheng , Current progress in the derivation and therapeutic

application of neural stem cells, Cell Death Dis. 8 (10) (2017) e3108 . [11] S. Weiss , C. Dunne , J. Hewson , C. Wohl , M. Wheatley , A.C. Peterson ,

B.A. Reynolds , Multipotent CNS stem cells are present in the adult mammalianspinal cord and ventricular neuroaxis, J. Neurosci. 16 (23) (1996) 7599–7609 .

[12] D. Ferrari , E. Binda , L.D. Filippis , A.L. Vescovi , Isolation of neural stem cellsfrom neural tissues using the neurosphere technique, Curr. Protoc. Stem Cell

Biol. 15 (1) (2010) 1–18 .

[13] A. Mothe , C.H. Tator , Isolation of neural stem/progenitor cells from theperiventricular region of the adult rat and human spinal cord, J. Vis. Exp. 99

(99) (2015) e52732 JoVE . [14] S.F. Pagano , F. Impagnatiello , M. Girelli , L. Cova , E. Grioni , M. Onofri , M. Cav-

allaro , S. Etteri , F. Vitello , S. Giombini , Isolation and characterization of neu-ral stem cells from the adult human olfactory bulb, Stem Cells 18 (4) (20 0 0)

295–300 .

[15] B. Reynolds , S. Weiss , Generation of neurons and astrocytes from isolated cellsof the adult mammalian central nervous system, Science 255 (5052) (1992)

1707–1710 . [16] B.A. Reynolds , W. Tetzlaff, S. Weiss , A multipotent EGF-responsive striatal em-

bryonic progenitor cell produces neurons and astrocytes, J. Neurosci. 12 (11)(1992) 4565–4574 .

[17] S.-C. Zhang , M. Wernig , I.D. Duncan , O. Brüstle , J.A. Thomson , In vitro differen-

tiation of transplantable neural precursors from human embryonic stem cells,Nat. Biotechnol. 19 (12) (2001) 1129–1133 .

[18] E. Banda , L. Grabel , in: Directed Differentiation of Human Embryonic StemCells Into Neural progenitors, Human Embryonic Stem Cell Protocols, Springer,

2014, pp. 289–298 . [19] Y. Wen , S. Jin , Production of neural stem cells from human pluripotent stem

cells, J. Biotechnol. 188 (2014) 122–129 . 20] J. Kim , J.A. Efe , S. Zhu , M. Talantova , X. Yuan , S. Wang , S.A. Lipton , K. Zhang ,

S. Ding , Direct reprogramming of mouse fibroblasts to neural progenitors, Proc.

Natl. Acad. Sci. 108 (19) (2011) 7838–7843 . [21] E. Shahbazi , S. Moradi , S. Nemati , L. Satarian , M. Basiri , H. Gourabi , N.Z. Mehr-

jardi , P. Günther , A. Lampert , K. Händler , Conversion of human fibroblasts tostably self-renewing neural stem cells with a single zinc-finger transcription

factor, Stem Cell Rep. 6 (4) (2016) 539–551 .

116 A.R. Murphy, J.M. Haynes and A.L. Laslett et al. / Acta Biomaterialia 101 (2020) 102–116

[22] S. Zhu , R. Ambasudhan , W. Sun , H.J. Kim , M. Talantova , X. Wang , M. Zhang ,Y. Zhang , T. Laurent , J. Parker , Small molecules enable OCT4-mediated direct

reprogramming into expandable human neural stem cells, Cell Res. 24 (1)(2014) 126 .

[23] L. Li , J. Chao , Y. Shi , Modeling neurological diseases using iPSC-derived neuralcells, Cell Tissue Res. 371 (1) (2018) 1–9 .

[24] K.M. Haston , S. Finkbeiner , Clinical trials in a dish: The potential of pluripo-tent stem cells to develop therapies for neurodegenerative diseases, Annu. Rev.

Pharmacol. Toxicol. 56 (1) (2016) 489–510 .

[25] V. Sundaramoorthy, Modelling lyssavirus infections in stem cell-derived hu-man neuronal cultures (in preparation), (2019).

[26] J.C. Jones , K. Sabatini , X. Liao , H.T. Tran , C.L. Lynch , R.E. Morey , V. Glenn-Pratola ,F.S. Boscolo , Q. Yang , M.M. Parast , Y. Liu , S.E. Peterson , L.C. Laurent , J.F. Loring ,

Y.C. Wang , Melanocytes derived from transgene-free human induced pluripo-tent stem cells, J. Invest. Dermatol. 133 (8) (2013) 2104–2108 .

[27] J.A. Thomson , J. Itskovitz-Eldor , S.S. Shapiro , M.A. Waknitz , J.J. Swiergiel ,

V.S. Marshall , J.M. Jones , Embryonic stem cell lines derived from human blas-tocysts, Science 282 (5391) (1998) 1145–1147 .

[28] C.A. Schneider , W.S. Rasband , K.W. Eliceiri , NIH image to ImageJ: 25 years ofimage analysis, Nat. Methods 9 (7) (2012) 671–675 .

[29] Reinnervate Ltd., Alvetex® Scaffold Protocols, 2016. http://reinnervate.com/alvetex/alvetex-documentation/protocols/ . 2018).

[30] J. Nichols , B. Zevnik , K. Anastassiadis , H. Niwa , D. Klewe-Nebenius , I. Cham-

bers , H. Schöler , A. Smith , Formation of pluripotent stem cells in the mam-malian embryo depends on the POU transcription factor Oct4, Cell 95 (3)

(1998) 379–391 . [31] X. Zhang , C.T. Huang , J. Chen , M.T. Pankratz , J. Xi , J. Li , Y. Yang , T.M. LaVaute ,

X.-J. Li , M. Ayala , Pax6 is a human neuroectoderm cell fate determinant, CellStem Cell 7 (1) (2010) 90–100 .

[32] L. Kan , N. Israsena , Z. Zhang , M. Hu , L.-R. Zhao , A. Jalali , V. Sahni , J.A. Kessler ,

Sox1 acts through multiple independent pathways to promote neurogenesis,Dev. Biol. 269 (2) (2004) 580–594 .

[33] S. Zhao , J. Nichols , A.G. Smith , M. Li , SoxB transcription factors specify neuroec-todermal lineage choice in ES cells, Mol. Cell. Neurosci. 27 (3) (2004) 332–342 .

[34] J. Menezes , M. Luskin , Expression of neuron-specific tubulin defines a novelpopulation in the proliferative layers of the developing telencephalon, J. Neu-

rosci. 14 (9) (1994) 5399–5416 .

[35] M.K. Lee , J.B. Tuttle , L.I. Rebhun , D.W. Cleveland , A. Frankfurter , The expres-sion and posttranslational modification of a neuron-specific β-tubulin isotype

during chick embryogenesis, Cell Motil. Cytoskelet. 17 (2) (1990) 118–132 . [36] K. De Gendt , E. Denolet , A. Willems , V.W. Daniels , L. Clinckemalie , S. Denayer ,

M.F. Wilkinson , F. Claessens , J.V. Swinnen , G. Verhoeven , Expression of tubb3, abeta-tubulin isotype, is regulated by androgens in mouse and rat sertoli cells,

Biol. Reprod. 85 (5) (2011) 934–945 .

[37] C. Liu , Y. Zhong , A. Apostolou , S. Fang , Neural differentiation of human embry-onic stem cells as an in vitro tool for the study of the expression patterns of

the neuronal cytoskeleton during neurogenesis, Biochem. Biophys. Res. Com-mun. 439 (1) (2013) 154–159 .

[38] L.F. Eng , R.S. Ghirnikar , Y.L. Lee , Glial fibrillary acidic protein: GFAP-thirty-oneyears (1969–20 0 0), Neurochem. Res. 25 (9-10) (20 0 0) 1439–1451 .

[39] C. Eliasson , C. Sahlgren , C.-H. Berthold , J. Stakeberg , J.E. Celis , C. Betsholtz ,J.E. Eriksson , M. Pekny , Intermediate filament protein partnership in astrocytes,

J. Biol. Chem. 274 (34) (1999) 23996–24006 .

[40] U. Lendahl , L.B. Zimmerman , R.D.G. McKay , CNS stem cells express a new classof intermediate filament protein, Cell 60 (4) (1990) 585–595 .

[41] J. Zhang , J. Jiao , Molecular biomarkers for embryonic and adult neural stemcell and neurogenesis, BioMed Res. Int. (2015) 2015 .

[42] D.L. Clarke , C.B. Johansson , J. Wilbertz , B. Veress , E. Nilsson , H. Karlström ,U. Lendahl , J. Frisen , Generalized potential of adult neural stem cells, Science

288 (5471) (20 0 0) 1660–1663 .

[43] R. Galli , U. Borello , A. Gritti , M.G. Minasi , C. Bjornson , M. Coletta , M. Mora ,M.G.C. De Angelis , R. Fiocco , G. Cossu , Skeletal myogenic potential of human

and mouse neural stem cells, Nat. Neurosci. 3 (10) (20 0 0) 986–991 . [44] M. Archana , T. Yogesh , K. Kumaraswamy , Various methods available for detec-

tion of apoptotic cells-A review, Indian J. Cancer 50 (3) (2013) 274–283 . [45] B.-Y. Hu , J.P. Weick , J. Yu , L.-X. Ma , X.-Q. Zhang , J.A. Thomson , S.-C. Zhang ,

Neural differentiation of human induced pluripotent stem cells follows devel-

opmental principles but with variable potency, Proc. Natl. Acad. Sci. 107 (9)(2010) 4335–4340 .

[46] B. Wiedenmann , W.W. Franke , C. Kuhn , R. Moll , V.E. Gould , Synaptophysin: amarker protein for neuroendocrine cells and neoplasms, PNAS 83 (10) (1986)

3500–3504 . [47] L. Tarsa , Y. Goda , Synaptophysin regulates activity-dependent synapse forma-

tion in cultured hippocampal neurons, PNAS 99 (2) (2002) 1012–1016 .

[48] K.K. Kim , R.S. Adelstein , S. Kawamoto , Identification of neuronal nuclei (NeuN)as fox-3, a new member of the fox-1 gene family of splicing factors, J. Biol.

Chem. 284 (45) (2009) 31052–31061 . [49] V. Gusel’Nikova , D. Korzhevskiy , NeuN as a neuronal nuclear antigen and neu-

ron differentiation marker, Acta Naturae 7 (2 (25)) (2015) . [50] R.J. Mullen , C.R. Buck , A.M. Smith , NeuN, a neuronal specific nuclear protein in

vertebrates, Development 116 (1) (1992) 201–211 .

[51] S.M. O’Connor , D.A. Stenger , K.M. Shaffer , D. Maric , J.L. Barker , W. Ma , Primaryneural precursor cell expansion, differentiation and cytosolic Ca2 + response in

three-dimensional collagen gel, J. Neurosci. Methods 102 (2) (20 0 0) 187–195 .

[52] W. Ma , W. Fitzgerald , Q.Y. Liu , T.J. O’Shaughnessy , D. Maric , H.J. Lin , D.L. Alkon ,J.L. Barker , CNS stem and progenitor cell differentiation into functional neu-

ronal circuits in three-dimensional collagen gels, Exp. Neurol. 190 (2) (2004)276–288 .

[53] A.M. Cruz Gaitán , N.M. Torres-Ruíz , N.G. Carri , Embryonic neural stem cells ina 3D bioassay for trophic stimulation studies, Brain Res. Bull. 115 (2015) 37–44 .

[54] S. Tasneem , K. Farrell , M.-Y. Lee , C.R. Kothapalli , Sensitivity of neural stem cellsurvival, differentiation and neurite outgrowth within 3D hydrogels to envi-

ronmental heavy metals, Toxicol. Lett. 242 (2016) 9–22 .

[55] Y.H. Kim , S.H. Choi , C. D’Avanzo , M. Hebisch , C. Sliwinski , E. Bylykbashi ,K.J. Washicosky , J.B. Klee , O. Brustle , R.E. Tanzi , D.Y. Kim , A 3D human neu-

ral cell culture system for modeling Alzheimer’s disease, Nat. Protocols 10 (7)(2015) 985–1006 .

[56] Z.X. Zhang , Q.X. Zheng , Y.C. Wu , D.J. Hao , Compatibility of neural stem cellswith functionalized self-assembling peptide scaffold in vitro , Biotechnol. Bio-

process Eng. 15 (4) (2010) 545–551 .

[57] H. Aligholi , S.M. Rezayat , H. Azari , S. Ejtemaei Mehr , M. Akbari , S.M. Modar-res Mousavi , F. Attari , F. Alipour , G. Hassanzadeh , A. Gorji , Preparing neural

stem/progenitor cells in puramatrix hydrogel for transplantation after braininjury in rats: A comparative methodological study, Brain Res. 1642 (2016)

197–208 . [58] S. Hong , K. Yang , B. Kang , C. Lee , I.T. Song , E. Byun , K.I. Park , S.-W. Cho , H. Lee ,

Hyaluronic acid catechol: A biopolymer exhibiting a pH-Dependent adhesive or

cohesive property for human neural stem cell engineering, Adv. Funct. Mater.23 (14) (2013) 1774–1780 .

[59] Z.-N. Zhang , B.C. Freitas , H. Qian , J. Lux , A. Acab , C.A. Trujillo , R.H. Herai ,V.A.N. Huu , J.H. Wen , S. Joshi-Barr , Layered hydrogels accelerate iPSC-derived

neuronal maturation and reveal migration defects caused by mecp2 dysfunc-tion, Proc. Natl. Acad. Sci. 113 (12) (2016) 3185–3190 .

[60] R.S. Ashton , A. Banerjee , S. Punyani , D.V. Schaffer , R.S. Kane , Scaffolds based on

degradable alginate hydrogels and poly (lactide-co-glycolide) microspheres forstem cell culture, Biomaterials 28 (36) (2007) 5518–5525 .

[61] S.C. Owen , M.S. Shoichet , Design of three-dimensional biomimetic scaffolds, J.Biomed. Mater. Res. Part A 94 (4) (2010) 1321–1331 .

[62] S. Ali , I.B. Wall , C. Mason , A.E. Pelling , F.S. Veraitch , The effect of young’s mod-ulus on the neuronal differentiation of mouse embryonic stem cells, Acta Bio-

mater. 25 (2015) 253–267 .

[63] A. Banerjee , M. Arha , S. Choudhary , R.S. Ashton , S.R. Bhatia , D.V. Schaffer ,R.S. Kane , The influence of hydrogel modulus on the proliferation and dif-

ferentiation of encapsulated neural stem cells, Biomaterials 30 (27) (2009)4695–4699 .

[64] N.D. Leipzig , M.S. Shoichet , The effect of substrate stiffness on adult neuralstem cell behavior, Biomaterials 30 (36) (2009) 6 867–6 878 .

[65] K. Saha , A.J. Keung , E.F. Irwin , Y. Li , L. Little , D.V. Schaffer , K.E. Healy , Sub-

strate modulus directs neural stem cell behavior, Biophys. J. 95 (9) (2008)4 426–4 438 .

[66] D.-N. Kim , J. Park , W.-G. Koh , Control of cell adhesion on poly(ethylene gly-col) hydrogel surfaces using photochemical modification and micropatterning

techniques, J. Ind. Eng. Chem. 15 (1) (2009) 124–128 . [67] X. Li , X. Liu , B. Josey , C.J. Chou , Y. Tan , N. Zhang , X. Wen , Short Laminin peptide

for improved neural stem cell growth, Stem Cells Transl. Med. 3 (5) (2014)662–670 .

[68] G.J. Her , H.-C. Wu , M.-H. Chen , M.-Y. Chen , S.-C. Chang , T.-W. Wang , Control

of three-dimensional substrate stiffness to manipulate mesenchymal stem cellfate toward neuronal or glial lineages, Acta Biomater. 9 (2) (2013) 5170–5180 .

[69] A. Banerjee , M. Arha , S. Choudhary , R.S. Ashton , S.R. Bhatia , D.V. Schaffer ,R.S. Kane , The influence of hydrogel modulus on the proliferation and dif-

ferentiation of encapsulated neural stem cells, Biomaterials 30 (27) (2009)4695–4699 .

[70] K. Saha , A.J. Keung , E.F. Irwin , Y. Li , L. Little , D.V. Schaffer , K.E. Healy , Sub-

strate modulus directs neural stem cell behavior, Biophys. J. 95 (9) (2008)4 426–4 438 .

[71] I. Smith , V. Silveirinha , J.L. Stein , L. de la Torre-Ubieta , J.A. Farrimond ,E.M. Williamson , B.J. Whalley , Human neural stem cell-derived cultures in

three-dimensional substrates form spontaneously functional neuronal net-works, J. Tissue Eng. Regener. Med. (2015) n/a-n/a .

[72] A.B. MacDermott , V. Henzi , D.B. Reichling , 15 - Characterization of glutamate

receptor function using calcium photometry and imaging, in: T. Narahashi(Ed.), Methods in Neurosciences, Academic Press, 1994, pp. 283–303 .

[73] H.J. Luhmann , A. Sinning , J.-W. Yang , V. Reyes-Puerta , M.C. Stüttgen ,S. Kirischuk , W. Kilb , Spontaneous neuronal activity in developing neocortical

networks: from single cells to large-scale interactions, Front. Neural Circuits 10(2016) 40 .

[74] T. Gorba , O. Klostermann , P. Wahle , Development of neuronal activity and

activity-dependent expression of brain-derived neurotrophic factor mRNA inorganotypic cultures of rat visual cortex, Cereb. Cortex 9 (8) (1999) 864–877 .

[75] R.E. Baker , M.A. Corner , J. van Pelt , Spontaneous neuronal discharge patterns indeveloping organotypic mega-co-cultures of neonatal rat cerebral cortex, Brain

Res. 1101 (1) (2006) 29–35 . [76] J. Kuijlaars , T. Oyelami , A. Diels , J. Rohrbacher , S. Versweyveld , G. Meneghello ,

M. Tuefferd , P. Verstraelen , J.R. Detrez , M. Verschuuren , Sustained synchronized

neuronal network activity in a human astrocyte co-culture system, Sci. Rep. 6(2016) 36529 .