Laminin α5 subs a es p omo e su i al, ne wo k o ma ion and
unc ional de elopmen o human plu ipo en s em cell-de i ed neu ons
in i o
Anu Hyysalo
a,
⁎, Me i Ris ola
a
, Mee i E.-L. Mäkinen
a
, Se gei Häy ynen
b
, Ma i Nyk e
b
, Susanna Na kilah i
a
a
Neu oG oup, BioMediTech, Uni e si y o Tampe e, Lääkä inka u 1, 33520 Tampe e, Finland
b
Compu a ional Biology, BioMediTech, Uni e si y o Tampe e, Lääkä inka u 1, 33520 Tampe e, Finland
abs ac a icle in o
A icle his o y:
Recei ed 7 Decembe 2016
Recei ed in e ised o m 28 Augus 2017
Accep ed 7 Sep embe 2017
A ailable online 12 Sep embe 2017
Laminins a e one o he majo p o ein g oups in he ex acellula ma ix (ECM) and specific laminin iso o ms a e
c ucial o neu onal unc ions in he cen al ne ous sys em in i o. In he p esen s udy, we compa ed ecombi-
nan human laminin iso o ms (LN211, LN332, LN411, LN511, and LN521) and laminin iso o m agmen (LN511-
E8) in in i o cul u es o human plu ipo en s em cell (hPSC)-de i ed neu ons. We showed ha laminin sub-
s a es con aining he α5-chain a e impo an o neu onal a achmen , iabili y and ne wo k o ma ion, as de-
ec ed by phase con as imaging, iabili y s aining, and immunocy ochemis y. Gene exp ession analysis
showed ha he molecula mechanisms in ol ed in he p e e ence o hPSC-de i ed neu ons o specificlaminin
iso o ms could be ela ed o ECM emodeling and cell adhesion. Impo an ly, he mic oelec ode a ay analysis
e ealed he wides dis ibu ion o elec ophysiologically ac i e neu ons on laminin α5 subs a es, indica ing
mos e ficien de elopmen o neu onal ne wo k unc ionali y. This s udy shows ha specific laminin α5sub-
s a es p o ide a con olled in i o cul u e en i onmen o hPSC-de i ed neu ons. These subs a es can be u i-
lized no only o enhance he p oduc ion o unc ional hPSC-de i ed neu ons o in i o applica ions like disease
modeling, oxicological s udies, and d ug disco e y, bu also o he p oduc ion o clinical g ade hPSC-de i ed
cells o egene a i e medicine applica ions.
© 2017 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://
c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
Keywo ds:
Alpha5 laminin subs a e
Human plu ipo en s em cell-de i ed neu on
In i o cul u ing
Mic oelec ode a ay
Neu onal ne wo ks
1. In oduc ion
Laminins, one o he majo p o ein g oups in he ex acellula ma ix
(ECM), play an impo an ole in he cen al ne ous sys em (CNS)
(Ba os e al., 2011). Laminins a e la ge he e o ime ic glycop o eins
consis ing o α,βand γchains, which assemble in o c oss-shaped mol-
ecules. To da e, 5 α,3β, and 3 γchains ha e been iden ified and associ-
a e o o m a leas 15 di e en laminin iso o ms (Indyk e al., 2003).
Laminins a e in ol ed in many aspec s o CNS physiology and neu onal
unc ions, bu hei exac biological oles in he o ma ion, de elopmen ,
and unc ion o neu onal ne wo ks emain la gely unknown
(Domoga skaya e al., 2012).
In o ma ion ega ding exp ession and unc ion o specific laminin
chains o iso o ms in di e en CNS egions has mainly been collec ed
om gene exp ession and inac i a ion s udies in animal models and
s udies o human laminin diseases. Ex ensi e mRNA sequencing has
e ealed he exp ession o se e al laminin chains (α1, α3, α4, α5, β1,
β2, γ1) in he e al human and emb yonic mouse en icula zone,
sub en icula zone and co ical pla e, bu he biological significance
o hese chains was no e alua ed (Fie z e al., 2012). Fu he mo e, he
unc ional ole o laminins con aining he α5chainhasbeenshownin
neu al ube o ma ion and neu al c es cell mig a ion du ing mouse em-
b yogenesis (Coles e al., 2006;Mine e al., 1998). La e in de elop-
men , laminin (LN) 511 (α5, β1, γ1) has been iden ified as he majo
neu onal laminin in he basemen memb ane o mouse hippocampus
(Indyk e al., 2003). In addi ion, co ical his ogenesis, de elopmen
and pial basemen memb ane o ma ion a e dis u bed by mu a ions in
he laminin β2, γ1, and γ3chains(Ba ak e al., 2011;Hal e e al.,
2002;Radne e al., 2013). LN411 o LN511 and LN111 o LN211,
which a e p oduced by endo helial cells and as ocy es, espec i ely,
pa icipa e in he o ma ion o he blood-b ain ba ie (Six e al.,
2001), and laminins con aining he α2 chain a e equi ed o oligoden-
d ocy e ma u a ion and CNS myelina ion in mice (Chun e al., 2003).
Thus, al hough se e al laminin chains o iso o ms ha e been iden ified
in he CNS, knowledge ega ding he o e all exp ession and unc ions
o laminins is no comp ehensi e.
S udying laminin iso o ms in i o is challenging due o he o e lap-
ping unc ions o laminin chains and iso o ms (Simon-Assmann e al.,
S em Cell Resea ch 24 (2017) 118–127
Abb e ia ions: hPSC, human plu ipo en s em cell; LN, laminin; MEA, mic oelec ode
a ay.
⁎Co esponding au ho a : Ins i u e o Biosciences and Medical Technology
(BioMediTech), Uni e si y o Tampe e, Lääkä inka u 1, 33520 Tampe e, Finland.
E-mail add ess: anu.hyysalo@s a .u a.fi(A. Hyysalo).
h p://dx.doi.o g/10.1016/j.sc .2017.09.002
1873-5061/© 2017 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
Con en s lis s a ailable a ScienceDi ec
S em Cell Resea ch
jou nal homepage: www.else ie .com/loca e/sc
2011). Howe e , he p oduc ion o eliably pu ified and comme cially
a ailable laminin iso o ms has enabled mo e de ailed in i o expe i-
men s o be pe o med o examine he oles o specific laminin iso o ms.
Chemically defined, xeno- ee, ecombinan laminin iso o ms ha a e
sui able o clinical use ha e been ma ke ed o a ew yea s. In i o s ud-
ies wi h mouse do sal oo ganglion neu ons ha e shown ha LN111
and LN511 p o ide supe io suppo o neu i e ou g ow h compa ed
o LN211 and LN411 (Plan man e al., 2008). LN511 also ema kably
p omo es he elonga ion o axons and dend i es o a hippocampal
neu ons compa ed o LN111, LN211 and LN411 (Fusaoka-Nishioka e
al., 2011). Fu he mo e, he E8 agmen o LN511 p omo es axon and
dend i e ou g ow h and inc eases he numbe o dend i es in a hippo-
campal neu ons (Fusaoka-Nishioka e al., 2011). Howe e , he esul s
ob ained wi h mu ine neu ons canno be di ec ly ansla ed o human
neu ons.
Human plu ipo en s em cells (hPSCs) a e conside ed as an excellen
ool o esea ch and clinical pu poses. P e iously, LN111, LN332,
LN511, LN521, and he E8 agmen o LN511 ha e been shown o e fi-
cien ly suppo he sel - enewal o hPSCs (Lu e al., 2014;Miyazaki e
al., 2008;Miyazaki e al., 2012a;Rodin e al., 2010). Cu en ly, only a
ew s udies add essing he use o specific laminin iso o ms o agmen s
and di e en ia ing hPSC-de i ed neu ons ha e been published. Dopa-
mine gic neu ons we e p oduced om human induced plu ipo en
s em cells (hIPSCs) unde xeno- ee condi ions using LN521 o he E8
agmen o LN511 (Doi e al., 2014;Lu e al., 2014;Nakagawa e al.,
2014).
In addi ion o he mo phological and biochemical cha ac e iza ion, i
is c ucial o assess he unc ionali y o neu onal ne wo ks in i o. De-
spi e he impo ance o his aspec , i has ba ely been add essed in p e-
ious laminin s udies. Neu onal ne wo k unc ionali y and
de elopmen in i o can be exclusi ely e alua ed using mic oelec ode
a ay (MEA) echnology, as p e iously shown o hPSC-de i ed neu ons
(Heikkilä e al., 2009;Yla-Ou inen e al., 2010). In his s udy, we com-
pa ed he ecombinan human laminin iso o ms LN211, LN411, LN332,
LN511, and LN521, as well as he LN511-E8 agmen as subs a es o
hPSC-de i ed neu onal cul u es. To ou knowledge, his is he fi s pub-
lica ion o epo he e ec s o di e en laminin iso o ms as in i o coa -
ing subs a es on neu onal ne wo k ac i i y.
2. Ma e ials and me hods
2.1. Neu onal di e en ia ion and laminins
The human emb yonic s em cell (hESC) line Regea 08/023, de i ed
and cha ac e ized a he Ins i u e o Biosciences and Medical Technolo-
gy (BioMediTech, Uni e si y o Tampe e, Finland) (Sko man, 2010),
was used. An addi ional hESC line (Regea 11/013; (Sko man, 2010))
and hIPSC line (04511WTs; (Ojala e al., 2016)) we e s udied wi h less
ex ensi e analyses o confi m he ob ained esul s. Neu ons we e di e -
en ia ed in suspension cul u es, as p e iously desc ibed (Lappalainen e
al., 2010). Fo neu onal ma u a ion and compa ison o he cell beha io s
on di e en laminin subs a es, p edi e en ia ed cells we e pla ed
on polys y ene o MEA (Axion Biosys ems, A lan a, GA, USA) coa ed
wi h he human ecombinan laminins LN211, LN332, LN411, LN511,
LN521 (2 μg/cm
2
, BioLamina, Sundbybe g, Sweden), o he LN511-E8
agmen (iMa ix-511, 1 μg/cm
2
, Clon ech, Taka a Bio Inc., Shiga,
Japan). Chain composi ions o he laminins used in his s udy a e
lis ed in Supplemen a y Table 1. Laminin om Engelb e h-Holm-
Swa m mu ine sa coma basemen memb anes (2 μg/cm
2
, Sigma-Al-
d ich, S . Louis, MO, USA) and laminin om human placen a (2 μg/
cm
2
, Sigma Ald ich) we e used as con ol coa ing subs a es, as we
ha e ou inely used hese subs ances o hPSC-de i ed neu ons
(Lappalainen e al., 2010;Toi onen e al., 2013). Mo phology o he
cells on di e en laminin subs a es was e alua ed using phase con-
as imaging.
2.2. Cell iabili y and sp eading analysis
Viabili y and sp eading o neu ons cul u ed on laminin subs a es o
one week we e in es iga ed using an au oma ed cell coun e (Coun -
ess®, The mo Fishe Scien ific) o a LIVE/DEAD® Viabili y/Cy o oxici y
Ki o mammalian cells (The mo Fishe Scien ific) as p e iously de-
sc ibed (Ylä-Ou inen e al., 2014).
2.3. Immunocy ochemis y
Immunocy ochemical cha ac e iza ion waspe o med o in es iga e
neu onal p o ein exp ession, as p e iously desc ibed (Lappalainen e al.,
2010). Neu ons cul u ed on laminin subs a es o one week we e fixed
and s ained wi h mouse an i-SRY (sex de e mining egion Y)-box 2
(SOX2; 1:200, R&D sys ems Inc., Minneapolis, MN, USA), abbi an i-mi-
c o ubule-associa ed p o ein (MAP-2, 1:400; Me ck Millipo e), abbi
an i-β- ubulin iso ype III (1:2000; GenSc ip , Pisca away, NJ, USA),
and chicken an i-glial fib illa y acidic p o ein (GFAP; 1:4000, Abcam,
Camb idge, UK). Fo seconda y an ibody labeling, Alexa Fluo 488-
(1:400) and Alexa Fluo 568-conjuga ed an ibodies (1:400) (bo h
om The mo Fishe Scien ific) we e used.
2.4. Gene exp ession analysis
To al RNA was ex ac ed om neu ons cul u ed o one week on
laminin iso o ms using a NucleoSpin RNA XS ki (Mache ey-Nagel,
Dü en, Ge many). The concen a ion and quali y o he RNA we e spec-
oscopically moni o ed. RNA was e e se ansc ibed in o cDNA using
he High Capaci y cDNA Re e se T ansc ip ion ki (Applied Biosys ems,
The mo Fishe Scien ific). Eigh y- ou genes o in e es we e analyzed
using a comme cial TaqMan® A ay o Human Ex acellula Ma ix &
Adhesion Molecules (Applied Biosys ems, The mo Fishe Scien ific).
Fu he mo e, a subse o hese genes we e also analyzed using
TaqMan® Gene Exp ession Assays.
2.5. Mic oelec ode a ay
The spon aneous elec ical ac i i y o he neu onal ne wo ks gene -
a ed on di e en laminin subs a es was measu ed using s anda d 12-
well pla e MEAs, he Maes o MEA sys em, and AxIS so wa e (all
om Axion Biosys ems). Ten minu e eco dings we e pe o med once
a week o a minimum o h ee weeks. Spike coun s and bu s analyses
we e pe o med using a cus om-made MATLAB sc ip (Kapucu e al.,
2012), wi h u he modifica ions.
Mo e de ailed desc ip ion o he Ma e ials and me hods a e p o id-
ed in he Suppo ing in o ma ion.
3. Resul s
3.1. LN511 e ficien ly suppo s ne wo k o ma ion and iabili y o hPSC-de-
i ed neu ons
In his s udy, hPSC -de i ed neu ons we e cul u ed on he ecombi-
nan human laminin iso o ms LN211, LN332, LN411 and LN511. P e i-
ously, he exp ession o specific chains om all hese iso o ms in he
CNS has been epo ed (Fie z e al., 2012). Cell mo phology and ne wo k
o ma ion was isually assessed om phase con as images. The hPSC-
de i ed neu ons a ached mos e ficien ly and o med ne wo ks wi h
he highes densi y and mos e en dis ibu ion on LN511 (Fig. 1A).
The beha io o cells on LN511 was isually simila o cells on mouse
laminin. Fewe cells a ached o LN411 compa ed o he o he laminin
iso o ms, and ins ead o o ming a ne wo k, he iable cells agg ega ed.
The beha io o cells cul u ed on LN411 esembled he cul u es on
human laminin. Cell a achmen and neu onal ne wo k o ma ion on
he LN211 and LN332 iso o ms we e mo e e ficien han on LN411 bu
lesse han in cul u es on LN511 and mouse laminin.
119A. Hyysalo e al. / S em Cell Resea ch 24 (2017) 118–127
Cell iabili y and he amoun s o li e and dead cells we e quan ified
wi h au oma ed cell coun e . In addi ion, sp eading o he iable cells in
he cul u es was de e mined wi h li e/dead s aining and quan ified as
he pe cen age o cul u e a ea co e ed wi h cells posi i e o li e o
dead s aining. The highes cell iabili y (median [mdn] 94%, p ≤0.045)
and li e cell coun (mdn 1.42 × 10
6
cells/cm
2
) we e de ec ed on
LN511 (Fig. 1B–C). Simila esul was ob ained om analysis o li e
cell co e age, as li e cell co e age was significan ly lowe in cul u es
on he o he laminin iso o ms compa ed o cul u es on LN511
(mdn
LN511
61%, all p b0.01) and mouse laminin (mdn
mouse laminin
50%,
all p b0.03). Cell iabili y on o he subs a es, anged om 78%
(LN411 and human laminin) o 88% (LN332). The amoun s o li e cells
and li e cell co e ages, howe e , demons a ed di e ences be ween
he cul u es mo e clea ly. The li e cell coun s de ec ed on LN211 and
LN332 we e 0.50 and 0.68 × 10
6
cells/cm
2
, espec i ely, whe eas he
li e cell co e age on bo h subs a es was 34%. The lowes li e cell coun s
(mdn 0.39 and 0.31 × 10
6
cells/cm
2
) and li e cell co e ages (mdn 21%
and 17%) we e obse ed in cul u es on LN411 and human laminin. Sim-
ila le els o dead cell co e age and dead cell coun s we e de ec ed on
e e y subs a e.
3.2. The la ges numbe o hPSC-de i ed neu ons is de ec ed on LN511
To al cell coun s and he numbe o cells posi i e o he neu onal
ma ke s MAP-2 and β- ubulin
III
we e quan ified o confi m he neu o-
nal iden i y o he di e en ia ed cells on he laminin iso o ms. The ma-
jo i y o he cells in all cul u es we e s ained wi h MAP-2 and β-
ubulin
III
and ew GFAP-posi i e as ocy es we e ound, ega dless o
he coa ing subs a e. Neu al p ecu so cells in he cul u es we e de ec -
ed wi h SOX2. Rep esen a i e images o cells s ained o MAP-2, β-
ubulin
III
, and GFAP a e p esen ed in Fig. 2A.
The highes o al cell (mdn 1610 cells/image) and neu on (mdn
1173 neu ons/image) coun s we e de ec ed in cul u es on LN511 (Fig.
2B–C). The o al cell coun was only significan ly inc eased on LN511
(p b0.01) compa ed o mouse laminin (mdn 1112 cells/image) (Fig.
2B). The neu on coun on LN511 was also highe bu no significan ly
di e en (p = 0.08) om mouse laminin (mdn 799 neu ons/image)
(Fig. 2C). Bo h cell coun s we e significan ly inc eased on LN511 com-
pa ed o all o he laminin iso o ms ( o al cell coun , all; p b0.01) (neu-
on coun , LN211 [p b0.01], LN332 [p = 0.05], LN411 [p b0.01]) (Fig.
2B–C). The neu on pe cen age (neu on coun / o al cell coun ) in he
cul u es a ied be ween 59% (LN411) and 80% (LN211). Mos o he e-
maining popula ion exp essed SOX2, while some SOX2-posi i e cells
also exp essed MAP-2 and β- ubulin
III
(Supplemen al Fig. 1).
The hESC line Regea 11/013 and hIPSC line 04511WTs we e used
and analyzed in e ms o cell a achmen and mo phology, and p o ein
exp ession o confi m ha he esul s ob ained wi h hESC line Regea
08/023 a e independen o he used cell line. Simila di e ences be-
ween he laminin iso o ms we e de ec ed wi h bo h cell lines,
suppo ing ou esul s wi h Regea 08/023 cells (Supplemen al Fig. 2).
Taken oge he , he immunocy ochemical analysis confi med ha he
LN511 iso o m mos e ficien ly suppo ed he o ma ion o dense and
Fig. 1. Cell mo phology and li e/dead analysis o hPSC-de i ed neu ons on di e en
laminin iso o ms. The hPSC-de i ed neu ons we e cul u ed on LN211, LN332, LN411,
LN511, mouse laminin and human laminin o one week. A) Rep esen a i e phase
con as images o he neu onal ne wo ks o med on di e en laminin iso o ms. Scale
ba : 100 μm. B) Rep esen a i e images o li e/dead-s ained cells on di e en laminin
iso o ms p esen ed wi h median cell iabili y pe cen age on each cul u e subs a e.
Scale ba : 100 μm. C) Quan ifica ion o cell sp eading (li e/dead cell co e age) and li e
and dead cell coun s in he cul u es. The cell sp eading quan ifica ion da a om h ee
biological expe imen s a e p esen ed as Tukey boxplo s on he le y-axis. Mann-
Whi ney U- es (*p ≤0.05 compa ed o LN511 and mouse laminin; #p ≤0.05 compa ed
o mouse laminin). All iso o ms and human laminin we e fi s compa ed pai wise wi h
mouse laminin. Fu he mo e, LN211, LN332 and LN411 we e hen compa ed pai wise
wi h LN511. The co esponding li e and dead cell coun s o each cul u e subs a e a e
p esen ed as median alues on he igh y-axis.
120 A. Hyysalo e al. / S em Cell Resea ch 24 (2017) 118–127
iable neu onal ne wo ks, whe eas LN411 and human laminin p o ided
he leas suppo .
3.3. Gene exp ession p ofiles o ECM-associa ed molecules in neu ons cul-
u ed on di e en laminin iso o ms
The exp ession p ofiles o 84 genes in neu ons cul u ed on di e en
laminin iso o ms we e compa ed. Fou een o hese genes we e exclud-
ed om he analyses due o hei conside ably low o unde ec ed ex-
p ession. In gene al, he exp ession o se e al genes ela ed o human
ECM and cell adhesion we e up egula ed compa ed o suspension sam-
ples (Fig. 3A). The majo i y o he s udied genes we e exp essed a
lowe le els in neu ons cul u ed on LN411 han on he o he laminin
iso o ms and mouse laminin. The gene exp ession p ofile o cells cul-
u ed on LN411 mainly esembled cells cul u ed on human laminin.
Based on he esul s om his s udy showing ha neu on a ach-
men , iabili y and ne wo k o ma ion a e he mos e ficien on LN511
and leas success ul on LN411, he gene exp ession p ofiles be ween
neu ons cul u ed on hese subs a es we e compa ed in mo e de ail. Fi -
een genes wi h ei he a minimum o a h ee- old di e ence in ela i e
exp ession o s a is ically significan di e ences be ween cul u es on
LN511 and LN411 we e iden ified (Fig. 3B). Se en o hese genes
encoded ECM p o eins (COL1A1,COL8A1,COL11A1,COL15A1,LAMB3,
ECM1, and FN1). Se e al α1 chains om di e en collagens (COL1A1
3.5-, COL8A1 10.6-, COL11A1 2.4- [p = 0.04], COL15A1 3.3- old, espec-
i ely) we e conside ably up egula ed in cells cul u ed on LN511 com-
pa ed o he cul u es on LN411. Laminin β3-chain (LAMB3)was
up egula ed 11.1- old in he cul u es on LN511. Addi ional ECM p o-
eins ha a e no included in he laminin o collagen amilies, ex acel-
lula ma ix p o ein 1 (ECM1)andfib onec in (FN1), we e up egula ed
4.6- and 4.4- old, espec i ely, in cells cul u ed on LN511.
Conside able di e ences in he ela i e exp ession le els o se e al
cell su ace adhesion molecules we e also de ec ed be ween cells cul-
u ed on LN511 and LN411 (Fig. 3B). Th ee in eg in subuni s, α1, αV,
and β4(ITGA1,ITGAV,andITGB4) we e up egula ed 3.3-, 1.7- (p =
0.04), and 3.1- old, espec i ely, and in e cellula adhesion molecule 1
(ICAM1) was significan ly up egula ed in cells cul u ed on LN511
(12.7- old, p = 0.01). Two di e en p o eases, ma ix me allop o einase
10 (MMP10) and a disin eg in and me allop o einase wi h a
h ombospondin ype 1 mo i , membe 13 (ADAMTS13), we e up egu-
la ed 4.6- old and down egula ed 1.2- old (p = 0.004), espec i ely, in
cul u es on LN511. Connec i e issue g ow h ac o (CTGF)and
ans o ming g ow h ac o , be a-induced ECM p o ein (TGFBI) we e
bo h significan ly up egula ed in cells cul u ed on LN511 (3.0- old (p
= 0.045) and 8.0- old (p = 0.04), espec i ely).
Simila end in gene exp ession le els be ween cells cul u ed on
LN511 and LN411 was obse ed wi h Regea 11/013 and 04511WTs –de-
i ed cells. The mos dis inc i e di e ences be ween LN511 and LN411
cul u es wi hin all s udied cell lines we e de ec ed as up egula ion o
ICAM1 and TGFBI in cells cul u ed on LN511 (Supplemen al Fig. 3). In
summa y, conside able di e ences we e disco e ed in he exp ession
p ofiles o genes encoding ECM p o eins, cell su ace adhesion mole-
cules, p o eases, and g ow h ac o s, and majo i y o hese genes we e
up egula ed in cul u es on LN511 compa ed o cul u es on LN411.
3.4. Wides dis ibu ion o elec ophysiologically ac i e neu ons is de ec ed
on LN511
MEA echnology was used o e alua e he unc ionali y o he neu o-
nal ne wo ks gene a ed on di e en laminin iso o ms. Elec ophysio-
logical ac i i y was assessed using he ollowing pa ame e s:
p opo ion o spike-de ec ing elec odes; spon aneous spiking equen-
cy in ac i e elec odes; p opo ion o bu s -de ec ing elec odes o all
elec odes; and o al bu s coun s in he bu s -de ec ing elec odes.
The neu onal ne wo k densi y and cell mo phology we e e alua ed
du ing he MEA expe imen s using phase con as mic oscopy. Cell
a achmen , mo phology, and ne wo k o ma ion on MEAs coa ed
wi h di e en laminin iso o ms we e simila o he esul s desc ibed
abo e, de ec ed on polys y ene. The a ached cells p oli e a ed on all
coa ing subs a es, bu he ini ial di e ences in ne wo k densi y also
emained a la e ime poin s (Supplemen al Fig. 4).
Cells o med spon aneously ac i e neu onal ne wo ks on all es ed
subs a es and showed bo h spike and bu s ac i i y (Fig. 4A–B). The
highes pe cen ages o spike- and bu s -de ec ing elec odes we e ob-
se ed on LN511 a e e y ime poin . The pe cen ages o spike- and
bu s -de ec ing elec odes on LN511 inc eased om 14% o 33% and
om 5% o 11%, espec i ely, o e ime. The ac i e elec ode le els
we e significan ly educed in cul u es on LN211 compa ed o LN511 a
one and wo weeks, bu a h ee weeks, no significan di e ences
we e de ec ed (Fig. 4A–B). On LN211, he ac i e elec ode le els we e
simila o he cul u es on mouse laminin. Cul u es on LN332 and
LN411 showed he lowes le els o spike- and bu s -de ec ing
Fig. 2. Immunocy ochemical cha ac e iza ion o hPSC-de i ed neu ons on di e en
laminin iso o ms. P o ein exp ession o he neu onal ma ke s MAP-2 and β- ubulin
III
we e de e mined in hPSC-de i ed neu ons cul u ed on LN211, LN332, LN411, LN511,
mouse laminin and human laminin o one week. A) Rep esen a i e images o MAP-2/β-
ubulin
III
/GFAP-s ained cells on di e en laminin iso o ms. Scale ba : 100 μm. B) To al
cell coun s and C) neu on coun s we e quan ified in cul u es on LN211, LN332, LN411,
LN511, mouse laminin and human laminin a e one week. Pa ial cell de achmen was
de ec ed on LN411, LN332, and human laminin du ing immunocy ochemis y, which
may ha e a ec ed he esul s o he quan ifica ion. The da a om h ee biological
expe imen s p esen ed as Tukey boxplo s. Mann-Whi ney U es (*p ≤0.05; **p ≤0.01;
***p ≤0.005). All iso o ms and human laminin we e fi s compa ed pai wise wi h
mouse laminin. Fu he mo e, LN211, LN332 and LN411 we e hen compa ed pai wise
wi h LN511.
121A. Hyysalo e al. / S em Cell Resea ch 24 (2017) 118–127
elec odes ( ange LN332[spike] 1–10%; LN332[bu s ] 0–3%;
LN411[spike] 2–6%; and LN411[bu s ] 0–1%). Compa ed o he cul u es
on LN511, he ac i e elec ode coun s we e significan ly educed on
bo h hese subs a es a e e y ime poin , excluding he bu s -de ec ing
elec ode le el on LN411 a one week (Fig. 4A–B). The ac i e elec ode
le els on LN332 and LN411 we e simila o cul u es on human laminin.
In gene al, he ela i e amoun s o spike- and bu s -de ec ing elec odes
de eloped simila ends in all cul u es, and he highes ac i i y was de-
ec ed a he h ee week ime poin .
Inc eased spiking equency and bu s ing ac i i y a e conside ed as a
sign o unc ional ma u a ion in cul u ed neu onal ne wo ks (Wagenaa
e al., 2006). A sligh inc easing end in spon aneous spiking equency
was de ec ed in all cul u es o e ime (Fig. 4C). Fo example, cells g own
on mouse laminin p esen ed median spiking equencies o 0.10, 0.12,
and 0.13 Hz a one, wo, and h ee weeks, espec i ely. When he spik-
ing equencies in cul u es on each laminin iso o m we e compa ed o
cul u es on LN511 and mouse laminin, ew significan di e ences
we e obse ed a one and wo weeks, bu no di e ences we e obse ed
a he h ee week ime poin . Cells on LN332 showed a significan ly
lowe spiking equency (0.06 Hz) han on LN511 (0.10 Hz) o mouse
laminin (0.10 Hz) a one week (p[LN511] b0.01, p[mouse LN] =
0.01). The spiking equency o cells cul u ed on LN411 (0.10 Hz) was
significan ly educed a wo weeks compa ed o cells cul u ed on
LN511 (0.21 Hz) (p b0.01). The o al bu s coun s in bu s -de ec ing
elec odes we e no a ec ed by he coa ing subs a e o ime (Fig. 4D).
The only significan di e ence was obse ed be ween cells cul u ed
on LN211 (2.9 bu s s/min) and mouse laminin (1.5 bu s s/min) a he
h ee week ime poin .
In conclusion, he neu onal ne wo ks gene a ed on LN511 con ain
he wides dis ibu ion o unc ional neu ons. Spike o bu s equencies
in ac i e elec odes we e clea ly no a ec ed by he subs a e.
3.5. All laminin α5 subs a es suppo a achmen and ne wo k o ma ion
o hPSC-de i ed neu ons
In his s udy, we ha e shown ha LN511 e ficien ly suppo s cell i-
abili y, ne wo k o ma ion, and he g ow h o hPSC-de i ed neu ons.
We demons a ed he impo ance o he laminin α5-chain in in i o
cul u es o hPSC-de i ed neu ons. O he comme cially a ailable sub-
s a es, LN521 con aining he α5-chain and he LN511-E8 agmen
con aining he C- e minal egion o he α5-chain (Fig. 5A), we e s udied
nex . These specific laminin subs a es ha e been used o in i o cul-
u es o hPSCs (Miyazaki e al., 2012b;Rodin e al., 2014a), bu a com-
pa ison o hPSC-de i ed neu ons has no been pe o med.
He e, hPSC-de i ed neu ons we e cul u ed on LN511-E8, LN521,
LN511 and mouse laminin. Cell a achmen , mo phology, and ne wo k
o ma ion we e simila on all subs a es, as de ec ed by phase con as
mic oscopy (Fig. 5B). Rep esen a i e images o immunos ained cells
on di e en subs a es a e p esen ed in Fig. 5C. The o al cell coun s
and numbe o MAP-2- and β- ubulinIII-posi i e cells we e quan ified
as desc ibed abo e. The highes o al cell coun was de ec ed on
LN511 (mdn 1287 cells/image), bu he app oxima ely same o al cell
coun was obse ed on LN521 (mdn 1264 cells/image) (Fig. 5D). The
o al cell coun s on LN511-E8 and mouse laminin we e lowe , bu no
significan ly di e en (mdn 998 and 930 cells/image, espec i ely),
compa ed o cul u es on LN511 and LN521. A simila end was de ec ed
when he neu on coun s we e compa ed in di e en cul u es. Neu on
coun s on LN511 (mdn 896 neu ons/image) was significan ly highe
compa ed o cul u es on LN511-E8 (603 neu ons/image, p b0.005)
and mouse laminin (585 neu ons/image, p b0.01) (Fig. 5E). Howe e ,
he p opo ion o neu ons was simila in all cul u es, anging om
76% (mouse laminin) o 71% (LN511).
3.6. Simila de elopmen o neu onal ne wo k unc ionali y is de ec ed on
all laminin α5 subs a es
Neu onal ne wo k unc ionali y was measu ed as desc ibed abo e,
and spon aneous ac i i y in cul u es on LN511-E8 and LN521 was com-
pa ed o cul u es on LN511 and mouse laminin. The beha io s o cells
g own on hese subs a es on MEA was simila o he esul s desc ibed
abo e, de ec ed on polys y ene (Fig. 6A).
Neu onal ne wo ks gene a ed on LN511-E8, LN521, and LN511 p e-
sen ed simila ends in he de elopmen o ac i i y (Fig. 6B–C), wi h
highe spike- and bu s -de ec ing elec ode le els compa ed o cul u es
on o he subs a es (Fig. 4A–B). The highes pe cen ages o ac i e spike-
and bu s -de ec ing elec odes we e obse ed in cul u es on LN521,
wi h he excep ion o he bu s -de ec ing elec ode le els a one week.
Fig. 3. Rela i e changes in gene exp ession in hPSC-de i ed neu ons on di e en laminin
iso o ms. The exp ession p ofiles o genes encoding o human ECM and adhesion
molecules we e examined in hPSC-de i ed neu ons cul u ed on LN211, LN332, LN411,
LN511, mouse laminin and human laminin o one week. A) The mean old changes
ela i e o he suspension sample a e p esen ed as a hea map in he log
2
scale. B) The
mean old changes in he exp ession, ela i e o suspension sample, o genes wi h ei he
s a is ically significan di e ences o a minimum o a h ee- old di e ence in ela i e
exp ession be ween cells cul u ed on LN411 and LN511 a e p esen ed as floa ing ba s
(min-max) on he le y-axis. S uden 's - es (*p ≤0.05; **p ≤0.01; ***p ≤0.005).
Fu he mo e, he mean old changes in cells cul u ed on LN511 ela i e o cells cul u ed
on LN411 a e p esen ed as do s on he igh y-axis. The da a om h ee biological
expe imen s a e p esen ed.
122 A. Hyysalo e al. / S em Cell Resea ch 24 (2017) 118–127

Pe cen ages o spike- and bu s -de ec ing elec odes inc eased om
10% o 44% and om 0% o 20%, espec i ely, o e ime. The lowes pe -
cen ages o ac i e elec odes we e de ec ed on mouse laminin, wi h
he excep ion o he bu s -de ec ing elec ode le el a one week.
Pe cen ages o spike- and bu s -de ec ing elec odes inc eased om
4% o 30% and om 1% o 8%, espec i ely, o e ime. A he wo and
h ee week ime poin s, he spike- (bo h p b0.05) and bu s -de ec ing
(p b0.05, p b0.01 a wo and h ee weeks, espec i ely) elec ode le els
in cul u es on LN521 we e significan ly highe han in cul u es on mouse
laminin. No significan di e ences in he ac i e elec ode le els we e de-
ec ed in cul u es on LN521 and LN511-E8 compa ed o cul u es on LN511.
The highes spon aneous spiking equency in ac i e elec odes was
also obse ed in cul u es on LN521 a e e y ime poin , anging om
0.09 Hz o 0.22 Hz (Fig. 6D). The spon aneous spiking equency in-
c eased o e ime in cul u es on e e y subs a e. No significan di e -
ences we e de ec ed a one o wo weeks, bu a h ee weeks, cells
cul u ed on LN521 p esen ed significan ly highe spiking equencies
han cells on LN511 (0.14 Hz, p b0.05) o mouse laminin (0.12 Hz, p b
0.01) (Fig. 6D). No significan di e ences we e obse ed in he o al
bu s coun s in bu s -de ec ing elec odes be ween cul u es a one o
wo weeks, bu a he h ee week ime poin , cells on mouse laminin
showed significan ly lowe bu s coun s (0.9 bu s s/min) han cells on
LN511-E8 (2.4 bu s s/min, p = 0.01) o LN521 (2 bu s s/min, p =
0.03) (Fig. 6E).
Taken oge he , he esul s indica e ha on all laminin α5 subs a es
neu ons de eloped isually indis inguishable ne wo ks. The dis ibu-
ion o ac i e neu ons in he cul u es, as well as spike and bu s equen-
cies, we e ela i ely simila in neu ons cul u ed on LN511-E8 and LN521
compa ed o cul u es on LN511. These esul s we e confi med wi h ad-
di ional hESC line Regea 11/013 and hIPSC line 04511WTs (Supplemen-
al Fig. 5). Al hough he o e all ac i i y o di e en cell lines was sligh ly
a ying, simila pa e n o neu onal ne wo k ac i i y on laminin α5sub-
s a es was de ec ed wi h all s udied cell lines.
4. Discussion
In his s udy, we fi s compa ed cell a achmen , iabili y and neu o-
nal ne wo k o ma ion on LN211, LN332, LN411, and LN511. The hPSC-
de i ed neu ons exhibi ed he mos e ficien a achmen and neu onal
ne wo ks o ma ion on LN511. The highes cell iabili y, li e cell
coun , and li e cell co e age, indica ing e ficien sp eading o he iable
cells in he cul u es, we e obse ed in cul u es on LN511. The lowes cell
a achmen and li e cell coun , wi h limi ed ne wo k o ma ion, we e
de ec ed on LN411. Cells on LN211 and LN332 showed mode a e cell a -
achmen , li e cell coun , and ne wo k o ma ion. Cell iabili y pe cen -
age was significan ly highe on LN511 compa ed o all o he cul u es.
Howe e , he e ec o cul u e subs a e on cell iabili y pe cen age is
challenging o de ec as he de ached dead cells a e mos ly emo ed
om he cul u es du ing media changes. Thus, i was conside ed ha
he ac ual amoun o li e cells in he cul u es be e eflec ed he di e -
ences be ween he cul u e subs a es. The o al cell coun s analyzed
om immunocy ochemical images co esponded o he neu on coun s
on all laminin subs a es, indica ing ha he emaining neu al p ecu so
cells in he popula ion did no influence he p e e ence o hPSC-de i ed
Fig. 4. De elopmen o spon aneous neu onal unc ionali y in cul u es on di e en laminin
iso o ms. The hPSC-de i ed neu ons we e cul u ed on MEAs coa ed wi h LN211, LN332,
LN411, LN511, mouse laminin and human laminin o h ee weeks and
elec ophysiological ac i i y was measu ed once a week. A) Pe cen ages o spike-
de ec ing elec odes and B) bu s -de ec ing elec odes in he cul u es on di e en
laminin iso o ms a e one, wo, and h ee weeks. The esul s a e p esen ed as medians
wi h in e qua ile anges om h ee biological expe imen s. C) De elopmen o he
spon aneous spiking equency in ac i e elec odes and D) o al bu s coun s in he
bu s -de ec ing elec odes. The da a om h ee biological expe imen s p esen ed as
Tukey boxplo s. Mann-Whi ney U es (*p ≤0.05; **p ≤0.01; ***p ≤0.005). All iso o ms
and human laminin we e fi s compa ed pai wise wi h mouse laminin. Fu he mo e,
LN211, LN332 and LN411 we e hen compa ed pai wise wi h LN511. Compa isons we e
pe o med a e e y ime poin .
123A. Hyysalo e al. / S em Cell Resea ch 24 (2017) 118–127
neu ons o he laminin iso o ms. Neu onal di e en ia ion, which was
quan ified as he neu on pe cen age (neu on coun / o al cell coun ),
was e ficien and simila o ou p e iously published esul s
(Lappalainen e al., 2010). Bo h he o al cell and neu on coun s we e
clea ly highes on LN511 and he lowes on LN411. Di e ence be ween
LN511 and LN411 occu s in he α-chain, and p e ious in i o s udies in-
dica e he impo ance o he laminin α5-chain in CNS de elopmen
(Mine e al., 1998) and he basemen memb ane o he hippocampus
(Indyk e al., 2003). In i o s udies ha e also shown he supe io i y o
LN511 o e LN211 and LN411 as subs a es o oden p ima y neu on
cul u es (Fusaoka-Nishioka e al., 2011;Plan man e al., 2008). Ou e-
sul s a e consis en wi h hese findings, indica ing ha he laminin
α5-chain has an impo an unc ion in human CNS neu ons. Despi e
he di e ences be ween oden and human cells, as well as p ima y
and s em cell-de i ed neu ons, he same laminin chain o iso o m e fi-
cien ly suppo s neu onal g ow h in i o.
O he comme cially a ailable subs a es con aining he α5-chain a e
he LN521 iso o m and LN511-E8 agmen . Laminin iso o ms −511
and −521 di e in he s uc u es o hei βchains, bu he unc ional
di e ences in i o a e no known (Rodin e al., 2014b). The LN511-E8
agmen is a unca ed p o ein composed o he C- e minal egions o
he α5, β1andγ1 chains, including he in eg in-binding si e, bu lacking
some biological ac i i ies o he in ac laminins. I is, howe e , easie
and mo e e ficien o p oduce he agmen han ull-leng h laminin
(Miyazaki e al., 2012b). LN521 and LN511-E8 ha e been used o di e -
en ia e hPSC-de i ed neu ons (Miyazaki e al., 2012b;Rodin e al.,
2014a) bu a de ailed compa ison be ween he e ec s o he LN511,
LN521, and LN511-E8 subs a es on hPSC-de i ed neu ons has no
been pe o med. We s udied his aspec and did no obse e any di e -
ences be ween neu onal cul u es on LN511 and LN521 in e ms o cell
a achmen , su i al, di e en ia ion, o ne wo k o ma ion. Mino de-
c eases in he o al cell and neu on coun s we e de ec ed in cul u es
on LN511-E8 compa ed o cul u es on LN511 and LN521. Collec i ely,
hPSC-de i ed neu ons beha e simila ly on all laminin subs a es ha
con ain he α5-chain.
In his s udy, gene exp ession p ofiles we e analyzed in cells cul-
u ed on LN211, LN332, LN411 and LN511. In gene al, se e al genes e-
la ed o human ECM and adhesion molecules we e up egula ed in cells
cul u ed on all subs a es compa ed o he suspension sample, sugges -
ing o adhesion, neu onal mig a ion and ma u a ion p ocesses ollow-
ing cell a achmen o coa ed su aces. The exp ession p ofiles in
neu ons cul u ed on LN411 and human laminin we e he mos s iking-
ly di e gen om he o he g oups, possibly as a consequence o he de-
ec ed weak a achmen and iabili y o neu ons on hese coa ing
subs a es. A mo e de ailed compa ison o cul u es on LN511 and
LN411, he mos and he leas suppo i e cul u e subs a es, espec i e-
ly, was pe o med o gain insigh s in o he possible mechanisms unde -
lying he de ec ed laminin iso o m p e e ence. Mul iple genes encoding
ECM p o eins (se e al collagen α1chains,LAMB3,FN1,andECM1)we e
ei he up egula ed in he neu onal cul u es on all coa ing subs a es
compa ed o he suspension sample o conside ably up egula ed in
he LN511 cul u es compa ed o he cul u es on LN411. This esul
could indica e ac i e ECM p oduc ion by he human neu ons unde
ou cul u e condi ions, as neu onal ECM p oduc ion has p e iously
been documen ed in di e en expe imen al designs using oden p i-
ma y neu ons in i o (Fudge and Mea ow, 2013;Lande e al., 1998).
In eg ins a e impo an ecep o s o laminins ha ha e specifici ies
o ce ain laminin iso o ms. LN511 has been iden ified as he mos p e-
e ed ligand o se e al laminin-binding in eg ins, whe eas LN411 only
has modes a fini y o wo in eg in ecep o s (Nishiuchi e al., 2006).
In eg in subuni s αV,β4,andα1we e up egula ed in cul u es on
LN511 compa ed o cul u es on LN411, o which αVand β4 o m
in eg in ecep o s wi h epo ed specifici y o LN511 (Nishiuchi e al.,
2006;Sasaki and Timpl, 2001). In eg in subuni α1exp ession has
been obse ed in he de eloping cen al and pe iphe al ne ous sys-
ems and cul u ed oden DRG neu ons in i o (Duband e al., 1992;
Fig. 5. Cell mo phology and immunocy ochemical cha ac e iza ion o hPSC-de i ed
neu ons on LN511-E8 and LN521. P o ein exp ession o he neu onal ma ke s MAP-2
and β- ubulin
III
we e de e mined in hPSC-de i ed neu ons cul u ed on LN511-E8,
LN521, LN511, and mouse laminin o one week. A) Schema ic ep esen a ion o he
in ac LN511/LN521 and he LN511-E8 s uc u es. B) Rep esen a i e images o neu onal
ne wo ks o med on di e en laminin subs a es. Scale ba : 100 μm. C) Rep esen a i e
images o MAP-2/β- ubulin
III
/GFAP-s aining o hPSC-de i ed neu ons on di e en
laminin subs a es. Scale ba : 100 μm. D) To al cell coun s and E) neu on coun s
quan ified in cul u es on LN511-E8, LN521, LN511, and mouse laminin a e one week.
The da a om h ee biological expe imen s p esen ed as Tukey boxplo s. Mann-
Whi ney U es (*p ≤0.05; **p ≤0.01; ***p ≤0.005). LN511, LN521, and LN511-E8 we e
fi s compa ed o mouse laminin, and hen LN521 and LN511-E8 we e compa ed o
LN511.
124 A. Hyysalo e al. / S em Cell Resea ch 24 (2017) 118–127
Tomaselli e al., 1993). Howe e , α1 in eg ins ha e no been p e iously
iden ified as ecep o s o LN511 o LN411. In eg ins can also bind o he
ECM p o eins such as TGFBI and ICAM1. ICAM1 and TGFBI we e signifi-
can ly up egula ed in cul u es on LN511 compa ed o cul u es on
LN411 in all cell lines included in his s udy. TGFBI con ains RGD mo i
ha in e ac s wi h α β3 in eg in and modula es se e al in eg in-medi-
a ed cellula unc ions in humans (Son e al., 2013). ICAM1 in e ac s
wi h a ie y o ecep o s, including in eg in subuni s, αL, αM, and β2
(E ienne-Manne ille e al., 1999). Thus, some in eg in subuni s can be
up egula ed in esponse o he exp ession o ECM p o eins p oduced
by he cul u ed cells. P o einases a e in ol ed in b eaking down he
ECM (We b, 1997), and he de ec ed di e ences in he exp ession le els
o ADAMTS13 and MMP10 be ween he LN511 and LN411 cul u es could
indica e a ia ions in ECM emodeling in cul u es on di e en laminin
subs a es. ECM p o eins also bind g ow h ac o s, he eby egula ing
hei bioa ailabili y o he cells (B izzi e al., 2012). Toge he , ou esul s
sugges ha molecules in ol ed in de e mining he laminin iso o m
p e e ence in hPSC-de i ed neu ons could be ela ed o cell adhesion
and ECM emodeling. Especially ICAM1 and TGFBI could be po en ial a -
ge s o u he s udies in es iga ing he mechanisms suppo ing
g ow h and de elopmen o hPSC-de i ed neu ons on LN511 in i o.
Despi e he c ucial impo ance o spon aneous unc ionali y and he
unc ional de elopmen o neu onal ne wo ks in i o, he e ec s o spe-
cific laminin iso o ms on neu onal ne wo k ac i i y ha e no p e iously
been s udied. When neu onal unc ionali y was compa ed in cells on
LN211, LN332, LN411, and LN511, he highes spon aneous ne wo k ac-
i i y, in e ms o spike- and bu s -de ec ing elec ode pe cen ages, was
de ec ed in cul u es on LN511. P e ious s udies wi h oden p ima y
Fig. 6. De elopmen o spon aneous neu onal unc ionali y in cul u es on LN511-E8 and LN521. The hPSC-de i ed neu ons we e cul u ed on MEAs coa ed wi h LN511-E8, LN521, LN511,
and mouse laminin o h ee weeks and elec ophysiological ac i i y was measu ed once a week. A) Rep esen a i e phase con as images o he neu onal ne wo ks o med on MEAs
coa ed wi h di e en laminin subs a es a he one week ime poin . Scale ba : 100 μm. B) Pe cen ages o spike-de ec ing elec odes and C) bu s -de ec ing elec odes a e one, wo,
and h ee weeks. The esul s a e p esen ed as medians wi h in e qua ile anges om six pa allel cul u es. D) De elopmen o he spon aneous spiking equency in ac i e elec odes
and E) o al bu s coun in bu s -de ec ing elec odes. The da a om six pa allel cul u es p esen ed as Tukey boxplo s. Mann-Whi ney U es (*p ≤0.05; **p ≤0.01; ***p ≤0.005).
LN511, LN521, and LN511-E8 we e fi s compa ed o mouse laminin, and hen LN521 and LN511-E8 we e compa ed o LN511. Compa isons we e pe o med a e e y ime poin .
125A. Hyysalo e al. / S em Cell Resea ch 24 (2017) 118–127
neu ons ha e shown ha he neu onal ne wo k densi y a ec s he
unc ional cha ac e is ics o he ne wo k (Bi fie al., 2013;Wagenaa
e al., 2006). A highe cell ne wo k densi y esul s in as e inc ease in
he ac i e elec ode coun s and bu s ing a es. He e, we showed ha
in addi ion o sp eading e enly on he cul u e a ea and o ming neu o-
nal ne wo ks wi h highes densi y on LN511, he cul u es showed he
wides dis ibu ion o elec ophysiologically ac i e neu ons. The com-
pa ison o he laminin α5 subs a es LN511-E8, LN521, and LN511 e-
ealed ha he e we e no subs an ial di e ences in he de elopmen
o unc ional neu onal ne wo ks, and hese subs a es we e supe io
o he o he laminin iso o ms. Clea di e ences be ween he spon ane-
ous spiking o bu s ing equencies we e no de ec ed in he cul u es on
he di e en laminin subs a es. This esul sugges s ha laminin sub-
s a es ha e an impo an ole in he ini ial adhe ence and basemen
memb ane-like suppo o de eloping neu onal ne wo ks, bu hey
do no a ec specific cha ac e is ics o he neu onal ne wo k ac i i y.
Howe e , only he mos gene al pa ame e s o elec ophysiological ac-
i i y a e co e ed he e; hus, his aspec should be add essed in mo e
ex ensi e elec ophysiological s udies.
Defined laminin subs a es p o ide a mo e con olled cell cul u e
en i onmen o hPSC-de i ed neu ons, which can be u ilized o in
i o disease modeling, oxicological s udies, and d ug disco e y. Re-
combinan iso o ms a e a ele an op ion o p e iously used, unde-
fined, and he e ogeneous human o mouse laminin. In addi ion,
ecombinan human laminin iso o ms a e also a p omising app oach
o he p oduc ion o hPSC-de i ed cells o egene a i e medicine
(Rodin e al., 2014a). Thus, hPSC cul u e and di ec ed di e en ia ion
p o ocols u ilizing same ecombinan laminin iso o ms o agmen (s)
as cell cul u e subs a es would be beneficial. Bo h LN511 and LN521
can suppo he sel - enewal o hPSCs (Rodin e al., 2010;Rodin e al.,
2014b), and pa ien -specific iPSCs can be de i ed, expanded, and u -
he di e en ia ed in o dopamine gic neu ons on LN521 (Lu e al.,
2014). Ou unpublished obse a ions a e simila in e ms o e ficien
hPSC cul u ing on LN521, ollowed by neu onal di e en ia ion in sus-
pension cul u e (unpublished da a). E ficien hPSC cul u ing (Miyazaki
e al., 2012b;Rodin e al., 2014a) and u he di e en ia ion in o dopa-
mine gic neu ons ha e also been epo ed on LN511-E8 (Doi e al.,
2014;Nakagawa e al., 2014). Howe e , conflic ing esul s ha e been e-
po ed because LN511-E8 has shown o be an ei he mo e (Miyazaki e
al., 2012b)o less(Rodin e al., 2014a) suppo i e cul u e subs a e o
hPSCs compa ed o he ull-leng h LN511 iso o m. Ou esul s show
ha neu onal di e en ia ion and ma u a ion in o unc ional neu ons
can be pe o med on LN511, LN521, and LN511-E8, bu none o hese
laminin α5 subs a es is supe io .
5. Conclusions
In conclusion, we showed ha acco ding o he mo phological, bio-
chemical, and unc ional analyses, laminin α5 subs a es p o ide e fi-
cien suppo and a defined in i o en i onmen o he gene a ion o
hPSC-de i ed neu onal ne wo ks, ega dless o he used hPSC line.
Ou esul s p o ide no el insigh s in o e ec s o di e en laminin iso-
o ms on hPSC-de i ed neu ons in i o and can be u he u ilized o
enhanced p oduc ion o unc ional hPSC-de i ed neu ons o esea ch
and clinical applica ions.
Supplemen a y da a o his a icle can be ound online a h p://dx.
doi.o g/10.1016/j.sc .2017.09.002.
Acknowledgemen s
We hank s a is ician Heini Huh ala, M.Sc. and Lau a Ylä-Ou inen,
Ph.D. o hei assis ance wi h s a is ical analyses and MEA. Tanja
Paa ilainen, M.Sc. and Teemu Ihalainen, Ph.D. a e acknowledged o
hei assis ance in image quan ifica ion and M.Sc. s uden s Elina
Haukka aa a and Emilia Hen inen o hei assis ance wi h immunocy-
ochemical analyses. This wo k was financially suppo ed by Doc o al
P og amme in Biomedicine and Bio echnology (BioMediTech, Uni e si-
y o Tampe e), he Finnish Funding Agency o Inno a ion (Tekes), and
Finnish Cul u al Founda ion.
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