EFFECTS OF DEFINED AND UNDEFINED EXTRACELLULAR MATRICES ON HUMAN IPSC MORPHOLOGY,EXPANSION AND DIFFERENTIATION
Xi Lu, Sol Degese, Susan Tousey, David Galitz, Marnelle Anderson, Jeff Cooper and Kevin Flynn

I N T RO D U C T I O NVN

VNBMEFN

48h72h-96hFigure 1. Colony Morphology is Influenced by Adherence Substrate. Brightfield images of small andlarge (long axis > 400 µm) iPSC colonies cultured on VN, FN and BME at 48 h and 72-96 h. Moresymmetrical, “rounder” and seemingly denser packed colonies with VN compared to other surfaces.
AB
C
20VN

VN

BME

FN

105VN

FN

BME

SOX17/DAPI

Figure 2. Increased F-Actin Localization to Colony Perimeter with VN. A. F-actin “fence” at edge ofcolony edge formed in small (top) and large (bottom row) colonies on B. all substrates. C.
Quantification of % F-actin fence area of iPSC colonies.
B

SRC

Integrin αV

FAK

0.025

0.025

0.1

0.02

0.02

0.015

0.015

0.05

0.01

0.01

0.005

0.005

000.15

0VN

FN

VNBME

FN

BME

VN

FN

pSRC/SRCChemiluminescence
(AU)

0.03

Paxillin/F-actin/DAPI

BME

0.02

pFAK/FAK

0.4

0.1

0.3

0.08
0.06

0.2
0.01

0.02

0VN

FN

BME

120
100806040200VN

FNBME

VN

FN

BME

120
10080
6040
200Figure 5. Functional Pluripotency Maintained Across ECM Substrates. iPSC grown on VN, FN, or BME
expressed positive markers of definitive endoderm (SOX17, top row) or ectoderm (Otx2, bottom row)
lineages after differentiation. Quantification of showed no significant differences in percentage ofdifferentiated cells between substrates.

0.04

0.1

0

OCT3/4-APC

OTX2/DAPIf

β1 /F-actin/DAPI

Integrin β5

SSEA1-PerCP%OTX2+ Cells

Chemiluminescence(AU)

A

SSEA4-FITC

Figure 4. Pluripotent Stemness Marker Expression is Maintained and Similar Across ECM
Substrates. Stemness markers of iPSC on VN, FN, and BME characterized by flow cytometry. Cells
expressed positively for Oct-3/4, Sox2, and SSEA-4 and negatively for SSEA-1. No significant
differences were observed between cells cultured on any of the substrates.
VNFNBME150BME

SOX2-PE%SOX17+ Cells

F-actin/Oct3

METHODSiPSC Maintenance: hiPSC (IBJ6) were maintained in Bio-Techne’s animal free iPSC media and passaged at
70% confluence using Versene™. Glass coverslips or 6 well plates were coated with Recombinant Human
Vitronectin (R&D Systems, Catalog # 2308-V0N, 10 µg/mL), Fibronectin (R&D Systems, Catalog # 1918-FN,
10 µg/mL), or Cultrex® Reduced Growth Factor BME, Type R1 (R&D Systems Catalog # 3433-010-R1, 1:40
dilution). Cells were passaged > 3 times prior to analysis via flow cyotometry, immunocytochemistry, ordifferentiation.
Flow Cytometry: iPSCs were dissociated using Accutase™ and stained for stemness markers such as Oct3/4 and SSEA-1 using H/M Pluripotent Stem Cell Multi-Color Flow Cytometry Kit (R&D Systems, Catalog #FMC001). Cells were then processed with BD LSRFortessa and data analyzed in FlowJo.
Cell Differentiation: iPSC were differentiated to ectoderm or endoderm lineages using StemXVivo ®
Ectoderm (R&D Systems, Catalog# SC031B) or Endoderm Differentiation Kit (R&D Systems, Catalog #SC019B). SOX17+ (endoderm) and Otx2+ (ectoderm) cells were quantified using Operetta.
Immunocytochemistry: Cells were fixed with 4% PFA and permeabilized with 0.3% Triton-X-100. Primary
antibodies include Oct-3/4 (R&D Systems, Catalog # AF1759), Paxillin (R&D Systems, Catalog # AF4259),
β1 (R&D Systems, Catalog # MAB17781), and F-actin. Differentiated cells were stained with primary
antibodies against SOX17 (R&D Systems, Catalog # AF1924) or Otx2 (R&D Systems, Catalog # AF1979)
Cells were then washed with PBS and stained with appropriate NorthernLights™ secondary antibodies(R&D Systems, Catalog # NL001, NL007) and DAPI.
Simple Western: Colony lysates were harvested using cold RIPA buffer and then analyzed using Protein
Simple’s Wes/Jess. Antibodies include FAK (R&D Systems, Catalog# AF4467), pFAK (R&D Systems,Catalog# MAB4528), SRC (R&D Systems, Catalog# AF3389), pSRC (R&D Systems, Catalog# AF2685),
Integrin αv (R&D Systems, Catalog# AF1219), or Integrin β5 (R&D Systems, Catalog# AF3824) andappropriate HRP secondary antibodies (R&D Systems, Catalog# HAF005, HAF016, 042-205, 043-522,042-206)

FNBME

Human induced pluripotent stem cells (iPSC) have great potential as a cell source for regenerative
medicine, drug discovery and disease modeling. The quality and consistency of iPSC culture is critical forbasic research and clinical therapy development. To improve reproducibility and safety, there have beensignificant effort in developing fully controlled iPSC systems such as using defined xeno-free media andfeeder free substrates such as basement membrane extracts (BME). IPSC are anchorage dependent
cells whose survival, pluripotency, and differentiation potential are critically dependent upon extracellularmatrix (ECM). BME is a soluble form of the basement membrane that contains undefined ratios of ECM
proteins. Proteins such as human recombinant vitronectin (VN) and fibronectin (FN) pose an attractive
alternative to BME and have been shown to support iPSC culture. IPSC attach to the ECM via integrin
receptors which are linked to focal adhesions and the cytoskeleton. Depending on the nature of thesubstrate, different integrin receptors may be recruited to promote iPSC adhesion and activate various
survival, mitotic or differentiation pathways. It is critically important to understand the influences thatdiverse substrates may have on iPSC colony morphology or differentiation potential. Identifying andcharacterizing a chemically-defined surface for culturing iPSCs will be paramount for establishingconsistency in stem cell cultures. In this work, we report differences in human iPSC colony morphology
when cultured on different ECM substrates. Subsequently, iPSC colonies on VN, FN, or BME werecharacterized by assessing cellular identities, cytoskeletal structure, integrin/FA signaling, anddifferentiation potential.

R E S U LT S

F-Actin (% Area)

Poster # TSC3450VNFNBMEVN

FN

BME

Figure 3. IPSC Colonies on Different Substrate Show Similar FAK/SRC Profiles. A. Simple Westernanalysis of focal adhesion and integrin proteins. B. Representative images of focal adhesion(paxillin) formation (top) and β1 integrins (bot) for colonies on all substrates.

C O N C LU S I O N
Despite differences in morphology and the F-actin fence on the edges of iPSC colonies, defined(VN,FN) and undefined ECM (BME) all supported growth and propagation of iPSC colonies formultiple passages without adversely affecting their differentiation potential or stemness markers.

CONTACT: Xi.lu@bio-techne.com

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