EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS
Matrix Remodeling Maintains Embryonic Stem Cell Self-Renewal by
LARALYNNE M. PRZYBYLA,aTHOROLD W. THEUNISSEN,bRUDOLF JAENISCH,a,bJOEL VOLDMANc
aDepartment of Biology andcDepartment of Electrical Engineering and Computer Science, Massachusetts Institute
of Technology, Cambridge, Massachusetts, USA;bWhitehead Institute for Biomedical Research, 9 Cambridge
Center, Cambridge, Massachusetts, USA
Key Words. Embryonic stem cell•Extracellular matrix•Matrix metalloproteinase•Self-renewal•Stat3
While a variety of natural and synthetic matrices have
been used to influence embryonic stem cell (ESC) self-
renewal or differentiation, and ESCs also deposit a rich
matrix of their own, the mechanisms behind how extracel-
lular matrix affects cell fate are largely unexplored. The
ESC matrix is continuously remodeled by matrix metallo-
proteinases (MMPs), a process that we find is enhanced by
the presence of mouse embryonic fibroblast feeders in a
paracrine manner. Matrix remodeling by MMPs aids in
the self-renewal of ESCs, as inhibition of MMPs inhibits
the ability of ESCs to self-renew. We also find that addi-
tion of the interstitial collagenase MMP1 is sufficient to
maintain long-term leukemia inhibitory factor (LIF)-
independent mouse ESC (mESC) self-renewal in a dose-
dependent manner. This remarkable ability is due to the
presence of endogenously produced self-renewal-inducing
signals, including the LIF-family ligand ciliary neurotro-
phic factor, that are normally trapped within the ECM
and become exposed upon MMP-induced matrix remodel-
ing to signal through JAK and Stat3. These results
uncover a new role for feeder cells in maintaining self-
renewal and show that mESCs normally produce sufficient
levels of autocrine-acting pro-self-renewal ligands. STEM
Disclosure of potential conflicts of interest is found at the end of this article.
Interactions between stem cells and their extracellular micro-
environment influence tissue generation, maintenance, and
repair , but comparatively little is known about the contri-
bution of endogenous extracellular components to stem cells
cultured in vitro. We previously found that downregulation of
soluble secreted signaling caused mouse embryonic stem cells
(mESCs) to exit their self-renewing state, and that extracellu-
lar matrix (ECM) remodeling was necessary to retain mESC
self-renewal in the absence of soluble autocrine cues . To
further probe the effects of matrix remodeling on maintaining
mESC self-renewal, here we assessed the mechanism and
specificity involved in this phenomenon.
Once an adherent cell is attached to a substrate, it begins
to lay down an ECM composed of several types of proteins
and connective fibers. During cell culture, the matrix is also
continuously remodeled, the most prominent class of endoge-
nously secreted remodeling enzymes being matrix metallopro-
teinases (MMPs) . One function of the matrix is to act as
a reservoir of growth factors and cytokines , and remodel-
ing can allow for the release of these trapped signaling pro-
teins . Matrix-bound proteins, including FGF4, have been
shown to have a profound effect on self-renewal , but the
role of remodeling proteins on mESC self-renewal is
While several methods have recently been described for
maintaining the self-renewal of mESCs [7–10], routine culture
generally involves a feeder layer composed of mitotically
inactivated mouse embryonic fibroblasts (MEFs) coupled with
exogenous addition of the cytokine leukemia inhibitory factor
(LIF) . These conditions have also been used to repro-
gram somatic cells to pluripotency after ectopic expression of
defined factors [12–14]. MEFs are known to contribute to
mESC self-renewal by the secretion of LIF [15, 16], and here
we investigated whether the feeder layer has an additional
role in remodeling the ECM. We then expanded on this to
study the effect of matrix remodeling in nonfeeder-based ESC
cultures, finding that addition of a single collagenase is suffi-
cient to maintain ESC self-renewal and went on to explore
the mechanism behind this remarkable effect.
MATERIALS AND METHODS
Mouse ESCs (CCE, V6.5 , ABJ1 [Oct4-GFP] , Sox2-GFP
, 7?TCF-eGFP , H2A-GFP, and H2B-GFP  lines)
Author contributions: L.M.P.: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript
writing; T.W.T.: collection of data, data analysis and interpretation, and manuscript editing; R.J.: experimental design and manuscript
editing; J.V.: conception and design, assembly of data, data interpretation, and manuscript writing.
Correspondence: Joel Voldman, Ph.D., Department of Electrical Engineering and Computer Science, Massachusetts Institute of
Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA. Telephone: 617-253-2094; Fax: 617-258-5845. e-mail:
firstname.lastname@example.org Received September 11, 2012; accepted for publication January 19, 2013; first published online in STEM CELLS
EXPRESS February 13, 2013. V
STEM CELLS 2013;31:1097–1106 www.StemCells.com
C AlphaMed Press 1066-5099/2013/$30.00/0 doi: 10.1002/stem.1360
were routinely cultured in medium consisting of Dulbecco’s modi-
fied Eagle’s medium (DMEM) supplemented with 15% defined fe-
tal bovine serum (Hyclone, Logan, UT, http://www.hyclone.com),
4 mM L-glutamine, 1 mM nonessential amino acids, 1? penicil-
lin-streptomycin, 100lM b-mercaptoethanol (Sigma, St. Louis,
MO, http://www.sigmaaldrich.com), and 10 ng/ml LIF (ESGRO,
Chemicon, Temecula, CA, http://www.chemicon.com). All cell
culture reagents were from Invitrogen (Carlsbad, CA, http://
www.invitrogen.com) unless otherwise noted. Cells were grown at
37?C in a humidified incubator with 7.5% CO2. For serum-free
culture, N2B27 medium with 10 ng/ml LIF and 10 ng/ml bone
morphogenetic protein 4 (BMP4) (R&D Systems, Minneapolis,
MN, http://www.rndsystems.com) was used  and wells were pre-
coated with gelatin. 129 epiblast stem cells (EpiSCs) derived from
the epiblast of day E5.5 mouse embryos  were cultured in me-
dium consisting of DMEM/F12 supplemented with 15% defined fetal
bovine serum (Hyclone), 5% knockout serum replacement (Invitro-
gen), 2 mM L-glutamine, 1 mM nonessential amino acids, 1? peni-
cillin-streptomycin, 100lM b-mercaptoethanol (Sigma), and 3.5 ng/
ml basic fibroblast growth factor (bFGF). For feeder-free experi-
ments, EpiSCs were cultured in N2B27 medium supplemented with
20 ng/ml Activin A (R&D Systems) and 12 ng/ml bFgf (R&D Sys-
tems)  and wells were precoated with human plasma fibronectin
(Millipore, Billerica, MA, http://www.millipore.com). MEFs were
cultured in DMEM supplemented with 3% fetal bovine serum
(Hyclone), 4 mM L-glutamine, and 1? penicillin-streptomycin.
Transwell assays were performed in six-well plates using 3.0lm
polyester membrane inserts (Corning, Acton, MA, http://www.cor-
Culture Media Additives
The following concentrations of culture media additives were
used for all experiments: MMP1, 50 ng/ml (Peprotech, Rocky
Hill, NJ, http://www.peprotech.com); Ro 32-3555, 50lM (Tocris,
a subsidiary of R&D Systems, http://www.tocris.com); collage-
nase, 20lg/ml (Sigma #C9722); MMP2, 50 ng/ml (Peprotech);
MMP3, 50 ng/ml (Peprotech); retinoic acid (RA), 1lM (Sigma);
PD0325901, 1lM (Stemgent, Cambridge, MA, https://www.stem
gent.com); Bay 11-7085, 50lM (Tocris); Dkk, 100 ng/ml (Pepro-
tech); IWP2, 2lM (Sigma); Wnt3a, 100 ng/ml (Peprotech); JAK
inhibitor I, 1lM (Calbiochem, San Diego, CA, http://www.emd-
biosciences.com); SB431542, 1lM (Sigma); LIF blocking anti-
body, 500 ng/ml (R&D Systems); cardiotrophin-1 (CT-1), 10 ng/
ml (Peprotech); ciliary neurotrophic factor (CNTF), 10 ng/ml
(Peprotech); oncostatin M (OSM), 10 ng/ml (Peprotech); CT-1
blocking antibody, 7.5lg/ml (R&D Systems); CNTF blocking
antibody, 7.5lg/ml (20lg/ml for 4-day experiments) (R&D Sys-
tems); OSM blocking antibody, 7.5lg/ml (R&D Systems). For
short hairpin RNA (shRNA) knockdowns, candidate shRNA hair-
pins were cloned into a packaging vector for transfection into
Phoenix cells and subsequent infection into mESCs. shRNA-con-
taining cells were green fluorescent protein (GFP) sorted and im-
mediately plated for knockdown verification or experimental use.
Stat3 shRNA with a starting nucleotide of 1,346 had the follow-
shRNA with a starting nucleotide of 1,413 had the following
TCCGAGACCCTCTGATGCCTACTGCCTCGGA-30. gp130 sh-
RNA with a starting nucleotide of 909 had the following
Quantitative Reverse Transcription Polymerase
Cells were harvested using TrypLE Express trypsin replacement
(Invitrogen), and total RNA was isolated using the RNeasy Mini
Kit (Qiagen, Hilden, Germany, http://www1.qiagen.com), accord-
ing to manufacturer’s instructions. RNA was converted to cDNA
using the ProtoScript cDNA synthesis kit with Oligo(dT) primer
(New England Biolabs, Ipswich, MA, https://www.neb.com), and
quantitative polymerase chain reaction (PCR) reactions were set
up using the iQ SYBR green supermix (Bio-Rad, Hercules, CA,
http://www.bio-rad.com), according to manufacturer’s instruc-
tions. Reactions were run on a Bio-Rad C1000 thermal cycler
using a CFX96 real-time system. Primers are listed in Supporting
Information Table S1.
Reporter cell lines were harvested and incubated with propidium
iodide to identify dead cells before performing flow cytometry.
Direct intracellular immunostaining was performed with an Alexa
Fluor 647-linked anti-mouse Nanog antibody (eBioscience, San
Diego, CA, http://us.ebioscience.com). Internal fluorescent inten-
sity for all samples was measured on a FACSCaliber flow cytom-
eter (BD Biosciences, San Diego, CA, http://www.bdbiosciences.-
com). Cells sorted for further analysis were sorted on a MoFlo
mRNA libraries were prepared from total RNA isolated with Trizol
(Invitrogen) and purified with Dynabeads mRNA purification kit
(Invitrogen). RNA was fragmented using Ambion RNA Fragmenta-
tion Reagents (Invitrogen), first strand cDNA was prepared using
SuperScript III Reverse Transcriptase (Invitrogen), and second strand
cDNA was prepared using Second Strand Buffer (Invitrogen) and
DNA Polymerase I (New England Biolabs). Whole transcriptome
mRNA sequencing of barcoded samples was performed on an Illu-
mina GAIIx, and data were processed according to the Illumina pipe-
line—Firecrest as the image analysis module, Bustard as the base call-
ing module, and Bowtie for sequence alignment. Reads were mapped
to the mouse reference genome and reads per kilobase per million
(RPKM) values were generated. Heat map was generated using the
freely downloadable software Java Treeview and Cluster 3.0.
For embryoid body formation, ESCs were harvested from culture
and replated at 4?105cells in a 60-mm ultra low attachment
culture dish (Corning). Cells were grown in ESC medium with
no LIF, and medium was replenished every 2 days. For blastocyst
injections, V6.5 ESCs were grown off feeders in serum-contain-
ing media in the presence of LIF or MMP1 for five passages,
then were injected into host blastocysts (C57/B6?DBA F2).
Cells grown under identical conditions in the absence of LIF or
MMP1 were no longer viable by five passages.
Enzyme-Linked Immunosorbent Assay
Enzyme-linked immunosorbent assay (ELISA) was performed on
cells grown in the indicated conditions for 48 hours. Results were
normalized by total protein content of the samples. Conditioned
media was spun down using an Amicon 3 kD cutoff filter spin col-
umn, and ECM samples were collected from cells grown for 5
days in the indicated conditions by adding Liberase DL (Roche,
Basel, Switzerland, http://www.roche-applied-science.com) at 0.13
Wu ¨nsch units/ml for 30 minutes at 37?C and collecting the
digested supernatant. Phospho-extracellular signal-regulated ki-
nase (ERK) ELISA was purchased from R&D Systems, and assay
was performed according to manufacturer’s instructions. Phospho-
Stat3 ELISA was purchased from RayBioTech (Norcross, GA,
http://www.raybiotech.com), and assay was performed according
to manufacturer’s instructions. CNTF ELISA was purchased from
online.com), and assay was performed according to manufac-
turer’s instructions. For MMP activity assay, the Amplite Univer-
sal Fluorimetric MMP Activity Assay Kit was purchased from
AAT Bioquest (Sunnyvale, CA, http://aatbio.com), and assay was
performed according to manufacturer’s instructions.
ESC Matrix Remodeling Maintains Self-Renewal
26 Prowse ABJ, McQuade LR, Bryant KJ et al. Identification of potential
pluripotency determinants for human embryonic stem cells following
proteomic analysis of human and mouse fibroblast conditioned media.
J Proteome Res 2007;6:3796–3807.
27 Berge D ten, Kurek D, Blauwkamp T et al. Embryonic stem cells
require Wnt proteins to prevent differentiation to epiblast stem cells.
Nat Cell Biol 2011;13:1070–1075.
28 Brons IGM, Smithers LE, Trotter MWB et al. Derivation of pluripo-
tent epiblast stem cells from mammalian embryos. Nature 2007;448:
29 Schryver B, Hinck L, Papkoff J. Properties of Wnt-1 protein that ena-
ble cell surface association. Oncogene 1996;13:333–342.
30 Fuerer C, Habib SJ, Nusse R. A study on the interactions between
heparan sulfate proteoglycans and Wnt proteins. Dev Dyn 2010;239:
31 Berendsen AD, Fisher LW, Kilts TM et al. Modulation of canonical
Wnt signaling by the extracellular matrix component biglycan. Proc
Natl Acad Sci USA 2011;108:17022–17027.
32 Hirai H, Karian P, Kikyo N. Regulation of embryonic stem cell self-
renewal and pluripotency by leukaemia inhibitory factor. Biochem J
33 Chen X, Xu H, Yuan P et al. Integration of external signaling path-
ways with the core transcriptional network in embryonic stem cells.
34 Heinrich PC, Behrmann I, Haan S et al. Principles of interleukin (IL)-
6-type cytokine signalling and its regulation. Biochem J 2003;374:1.
35 Bourillot P, Aksoy I, Schreiber V et al. Novel STAT3 target genes
exert distinct roles in the inhibition of mesoderm and endoderm differ-
entiation in cooperation with Nanog. Stem Cells 2009;27:1760–1771.
36 Davey RE, Onishi K, Mahdavi A et al. LIF-mediated control of em-
bryonic stem cell self-renewal emerges due to an autoregulatory loop.
FASEB J 2007;21:2020–2032.
37 Zandstra PW, Le HV, Daley GQ et al. Leukemia inhibitory factor
(LIF) concentration modulates embryonic stem cell self-renewal and
differentiation independently of proliferation. Biotechnol Bioeng 2000;
38 Yoshida K, Chambers I, Nichols J et al. Maintenance of the pluripo-
tential phenotype of embryonic stem cells through direct activation of
gp130 signalling pathways. Mech Dev 1994;45:163–171.
39 Conover JC, Ip NY, Poueymirou WT et al. Ciliary neurotrophic factor
maintains the pluripotentiality of embryonic stem cells. Development
40 Pennica D, Shaw KJ, Swanson TA et al. Cardiotrophin-1. Biological
activities and binding to the leukemia inhibitory factor receptor/gp130
signaling complex. J Biol Chem 1995;270:10915–10922.
41 Keung AJ, Juan-Pardo EM de, Schaffer DV et al. Rho GTPases medi-
ate the mechanosensitive lineage commitment of neural stem cells.
Stem Cells 2011;29:1886–1897.
42 Wells RG, Discher DE. Matrix elasticity, cytoskeletal tension, and
TGF-b: The insoluble and soluble meet. Sci Signal 2008;1:pe13.
43 Vu TH, Werb Z. Matrix metalloproteinases: Effectors of development
and normal physiology. Genes Dev 2000;14:2123–2133.
44 Alexander CM, Hansell EJ, Behrendtsen O et al. Expression and func-
tion of matrix metalloproteinases and their inhibitors at the maternal-
embryonic boundary during mouse embryo implantation. Development
45 Heissig B, Hattori K, Dias S et al. Recruitment of stem and progenitor
cells from the bone marrow niche requires MMP-9 mediated release
of Kit-ligand. Cell 2002;109:625–637.
46 Guo Y, Barbara G-E, Hal EB. Murine embryonic stem cells secrete
cytokines/growth modulators that enhance cell survival/anti-apoptosis
and stimulate colony formation of murine hematopoietic progenitor
cells. Stem Cells 2006;24:850–856.
47 Ghosh S, Basu M, Roy SS. ETS-1 protein regulates vascular endothe-
lial growth factor-induced matrix metalloproteinase-9 and matrix met-
alloproteinase-13 expression in human ovarian carcinoma cell line
SKOV-3. J Biol Chem 2012;287:15001–15015.
48 Kim E-S, Kim M-S, Moon A. TGF-beta-induced upregulation of
MMP-2 and MMP-9 depends on p38 MAPK, but not ERK signaling
in MCF10A human breast epithelial cells. Int J Oncol 2004;25:
49 Guo W, Pylayeva Y, Pepe A et al. Beta 4 integrin amplifies ErbB2 sig-
naling to promote mammary tumorigenesis. Cell 2006;126:489–502.
50 Hawkins K, Mohamet L, Ritson S et al. E-cadherin and, in its absence,
N-cadherin promotes Nanog expression in mouse embryonic stem cells
via STAT3 phosphorylation. Stem Cells 2012;30:1842–1851.
ESC Matrix Remodeling Maintains Self-Renewal