The Endothelial Cell Line bEnd.3 MaintainsHuman Pluripotent Stem Cells

Katherine Joubin,* Amelia Richardson,* Natalia Novoa, Edmund Tu, and Mark J. Tomishima

Endothelial cells line blood vessels and coordinate many aspects of vascular biology. More recent work hasshown that endothelial cells provide a key niche in vivo for neural stem cells. In vitro, endothelial cells secrete afactor that expands neural stem cells while inhibiting their differentiation. Here, we show that a transformedmouse endothelial cell line (bEnd.3) maintains human pluripotent stem cells in an undifferentiated state. bEnd.3cells have a practical advantage over mouse embryonic fibroblasts for pluripotent stem cell maintenance sincethey can be expanded in vitro and engineered to express genes of interest. We demonstrate this capabilityby producing fluorescent and drug-resistant feeder cells. Further, we show that bEnd.3 secretes an activity thatmaintains human embryonic stem cells without direct contact.

Introduction

Endothelial cells are specialized cells lining bloodvessels that orchestrate many biological functions. The

endothelium coordinates many aspects of vascular biology asmight be predicted from their close apposition to circulatingblood. More recently, it was shown that endothelial cellsprovide a niche for neural precursors in vivo [1–4]. Culturedendothelial cells release a soluble activity that promotesneural precursor expansion, inhibit their differentiation andmaintain neuronogenic potential after differentiation in vitro[5,6]. The identity of the soluble factor(s) responsible for thisactivity has not yet been identified.

Previous work on neural progenitors and endothelial cellswas performed using mouse cells. We wondered whetherendothelial factors also expanded human embryonic stemcell (hESC)–derived neural progenitors. However, ESCsmust first be directed to neural cells before we could testtheir responsiveness to the endothelial factor. Previous workshowed that many cells contained a so-called stromal de-rived induction activity (SDIA) and therefore could causeESCs to adopt a neural cell fate [7,8]. While bone marrowstromal cells cause neural induction, neural progenitorsrapidly differentiate into neurons; stromal cells do not pre-vent their further differentiation to neurons and glia.

Here, we tested the hypothesis that endothelial cells havean stromal derived induction activity (SDIA) activity. If true,then coculture of hESCs with endothelial cells might providean ideal system to convert ESCs to neural progenitorswithout continued neuronal differentiation since endothelialcells secrete factors to inhibit neuronal differentiation and

enhance neural progenitor self-renewal. This could increasethe synchrony and purity of ESC-derived neural cultures, atleast in principle.

In contrast to our original hypothesis, we found that theendothelial cell line bEnd.3 robustly maintained human plu-ripotent cells in the undifferentiated state for at least 20passages, the longest time tested. Coculture with bEnd.3maintained the hESC lines H1 and H9, and human inducedpluripotent stem cells (hiPSCs). Direct coculture was not nec-essary to maintain PSCs, suggesting that bEnd.3 secretes anactivity maintaining pluripotency. One important practicalapplication of using these cells is that bEnd.3 could be passagedat least 10 times without losing the ability to maintain plurip-otent cells; in contrast, primary mouse embryonic fibroblasts(PMEFs) are typically used at passage 3 since they lose theability to maintain pluripotent cells over passage. This allowsthe genetic engineering of feeder lines in vitro instead of theproduction of a transgenic mouse. Such versatility makes thesecells of practical interest for those engineering hPSCs andprovides some biological insight into endothelial cells.

Materials and Methods

PSC culture

H9 (WA09) and H1 (WA01) hESCs and hiPSCs fromMRC-5 fibroblasts (i202) were routinely maintained onPMEFs plated at a density of 11,500–13,500 cells/cm2, de-pending on the lot used (GlobalStem, Inc.). PSCs were feddaily with hPSC media [composed of Dulbecco’s modifiedEagle medium (DMEM)/F12 (11330-032; Gibco), 20%

Developmental Biology Program, SKI Stem Cell Research Facility, Center for Stem Cell Biology, Sloan-Kettering Institute, New York, NewYork.

*These two authors contributed equally to this work.

STEM CELLS AND DEVELOPMENT

Volume 21, Number 12, 2012

� Mary Ann Liebert, Inc.

DOI: 10.1089/scd.2011.0501

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knockout serum replacement (10828-028; Gibco), 3.5 mMglutamine (25030-081; Life Technologies), 0.1 mM MEM NEAA(11140-050; Gibco), 55 mM 2-mercaptoethanol (21985-023; LifeTechnologies), and 6 ng/mL fibroblast growth factor 2(FGF2) (R&D Systems)]. A working protocol for hPSC cul-ture is available at http://stemcells.mskcc.org.

bEnd.3 culture

bEnd.3 cells (ATCC No. CRL-2299) were maintained inDMEM + 10% fetal bovine serum (FBS). Cells were split withtrypsin before 80% confluence at a ratio between 1:6 and1:12, usually every 3–4 days. To prepare cells for use asfeeders, cells were exposed to 103 Gy of radiation beforefreezing as aliquots. bEnd.3 cells were plated at a density of13,000 cells/cm2 before coculture with PSCs.

Engineering bEnd.3 and H9 hESCs

To genetically engineer bEnd.3 and H9 hESCs, 4–5 millioncells were nucleofected with 10mg of the appropriate DNAconstruct using program B-16 and solution V (Amaxa Nu-cleofector; Lonza). Cells were allowed to recover for 2 daysbefore starting selection. Selection was continued until thecontrol nucleofection without DNA lacked viable cells. Formost experiments, the drug-resistant population was thenpassaged together as an engineered line. The mCherry-expressing bEnd.3 cells were sorted 3 times to enrich formCherry-positive cells.

Conditioned media production

MEFs or bEnd.3 cells were plated at 50,000 cells/cm2

in DMEM with 10% FBS overnight. The next day, thesemedia were removed and the cells were washed once withphosphate-buffered saline (PBS). Then hPSC media wereplaced on the cells overnight for conditioning. The media-only control was added to a tissue culture dish for overnightincubation. The next day, the media were removed fromeach condition and FGF2 was added to 10 ng/mL before use.Confluent monolayers were used for up to 3 weeks.

Neural differentiation

H9 colonies were dissociated into smaller clusters withdispase (5 mg/mL; Stem Cell Technologies) and washed oncein hESC media before a 1:30 split and cocultured with MS5 [9],MEFs, and bEnd.3 mouse brain endothelial cells (ATCC No.2299) in serum-replacement medium [8] supplemented withrecombinant Noggin (250 ng/mL); 1mM SB431542 was chan-ged completely on days 3, 6, and 9 to suppress SMAD activityand increase neural induction. Cultures were fixed for im-munofluorescence or harvested for gene expression analysison day 10. Quantitative polymerase chain reaction (qPCR)results are from 6 to 9 technical replicates of 3 independentbiological experiments for each sample.

Directed differentiation

H9 hESCs were fed on days 1 and 2 after passage withRPMI with 10 ng/mL bone morphogenetic protien 4(BMP4) (R&D 314-BP), 1 mM SB431542, glutamine, and 0.5%HyClone FBS. Cells were assayed on day 3 for mesoderm

and trophectoderm differentiation. For endoderm, H9hESCs were fed on days 1 and 2 with Roswell ParkMemorial Institute Medium (RPMI) with 100 ng/mL Acti-vin (R&D 338-AC), glutamine, and 0.5% HyClone FBS. Cellswere assayed on day 3 for endoderm differentiation. Neuralinduction was performed using the dual SMAD inhibitionprotocol [10]. Briefly, cells were dissociated into a single-celled suspension with Accutase before plating on Matrigelin the presence of conditioned media (either MEF orbEnd.3) supplemented with 10 ng/mL FGF2 and 10 mM Y-27632. For qPCR analysis, the Chambers et al. protocol wasfollowed verbatim until harvesting on day 15. qPCR wasperformed as described previously. For immunofluores-cence, confluent monolayers on neural precursors werepassaged using mechanical dissociation (STEMPRO EZPassage; Invitrogen) on day 10 onto Matrigel-coated dishesin N2 media supplemented with 20 ng/mL brain derivedneurotropic factor (BDNF), 200 mM ascorbic acid, 50 ng/mLShh (C25II), and 100 ng/mL FGF8. qPCR results are from 6to 9 technical replicates of 3 independent biological exper-iments for each sample.

Embryoid bodies and teratomas

Embryoid bodies were made by dispase treating hESCsand replating large fragments in hPSC media without FGF2in a low adherence plate. Clusters were fed every 3 days.Teratomas were made by dispase treating cells before cre-ating a near single-cell suspension through pipetting. Cellswere washed and resuspended in DMEM + 10% FBS. Foreach implant, 3 million cells were resuspended in a volumeof 200 mL DMEM + 10% FBS with 20 mM Y-27632 and 30%Matrigel.

Quantitative PCR

Messenger RNA levels were determined by isolatingmRNA with the Qiagen RNeasy kit before cDNA productionwith the Qiagen Quantitect RT kit. Taqman probes wereobtained from Applied Biosystems: pax6 (Hs01088108_m1),cdx2 (Hs00230919_m1), brachyury (Hs00610080_m1), sox17(Hs00751752_s1), and the internal standard hprt (4326321E).qPCR was performed on an Eppendorf Realplex2 with thefollowing cycling conditions: 50�C for 2 min, 95�C for 10 min,and 40 cycles of 95�C for 15 s and 60�C for 1 min. All qPCRresults are from 6 to 9 technical replicates of 3 independentbiological experiments for each sample.

Immunostaining

Immunostaining was performed by fixing cells in 4%paraformaldehyde for 15 min. Primary antibodies used in thisstudy are pax6 (Covance PRB-278P; 1:500), brachyury (R&DAF2085 at 1:40), cdx2 (BioGenex CDX2-88 ready to use), sox17(R&D AF1924 at 1:200), Oct3/4 (Santa Cruz Biotechnologies,sc-5279; 1:100), tra1-60 (Chemicon MAB4360; 1:100), and Na-nog (BD Pharmingen 560482; 1:200); secondary antibodiesconjugated to Alexa488, 568, or 647 (1:1,000) were used tovisualize primary antibody binding, and Hoechst 33342(Molecular Probes H-3570; 1:1,000) was added to stain nuclei.For surface markers, the staining was performed in PBS con-taining 1% PBS. Triton X-100 (0.1%) was added to the stainingbuffer for intracellular antigens.

THE ENDOTHELIAL CELL LINE BEND.3 MAINTAINS HPSCS 2313

FACS analysis

Antibodies against SSEA-3 (conjugated to Alexa 647; BDBiosciences, No. 561145) and SSEA-4 (conjugated to Alexa647; BD Biosciences, No. 560796) were used to quantitatepluripotency markers. In brief, cells were dissociated withAccutase for 40 min, washed with PBS, and then countedbefore a second centrifugation. One million cells were re-suspended in PBS with 0.1% bovine serum albumin (BSA) ina volume of 100mL containing the recommended amount ofantibody (5mL for SSEA-3, 20 mL for SSEA-4 per test). Cellswere incubated in the dark on ice for 1 h before 3 PBS wa-shes. Washed cells were resuspended a final time in PBS with0.1% BSA before analysis on a FACSAria (BD Biosciences).All FACS analysis data were derived from 3 independentexperiments.

hESC marker expression on bEnd.3 feeder layers

H9 GFP.2 hESCs were passaged onto MEFs and bEnd.3 cellsthat had been plated at equal densities (*12,500 cells/cm2)and FACS analysis was used to quantify stem cell marker ex-pression on the GFP + population. As above, FACS analysisdata were derived from 3 independent experiments. Primersused for endpoint PCR are listed in Supplementary Table 1(Supplementary Data are available online at www.liebertonline.com/scd).

Results

Neural induction

We initially tested the hypothesis that coculture of hESCswith endothelial cells would convert hESCs to neural cells;that is, endothelial cells have a ‘‘stromal-cell derived induc-ing activity’’ [7]. To test this hypothesis, we cocultured H9hESCs with PMEFs as a negative control, MS-5 bone marrowstromal cells as a positive control, and bEnd.3 endothelialcells as the experimental condition (Fig. 1). Each feeder linewas irradiated to prevent overgrowth during coculture.hESC cultures were split with dispase before seeding on

feeder monolayers at a relatively low density as previouslydescribed ([9]; see Materials and Methods section). Twelvedays after differentiation, hESCs in the MS-5 condition hadturned predominantly into circular arrangements of neuralprogenitors called neural rosettes (Fig. 1A) [9,11,12]. Colonymorphology looked relatively flat and epithelial in the other2 conditions. Pax6 immunostaining revealed robust neuralinduction in the MS-5–positive control plate (Fig. 1A). Incontrast, both the endothelial cells and the MEFs had onlysmall patches of Pax6 (Fig. 1A) and few neural rosettes.qPCR (Fig. 1B) provided further evidence for a lack of neuralinduction in endothelial conditions. Pax6 expression was lowin the bEnd.3 and MEF coculture relative to the MS-5 co-culture (n = 3 independent experiments). Taken together,these data suggested that endothelial cells are inefficient inneuralizing hESCs.

hESC maintenance

We explored the use of endothelial cell coculture for hESCmaintenance because the hESCs continued to appear mor-phologically undifferentiated after coculture with bEnd.3cells during the neural induction experiment described pre-viously. In preliminary experiments, hESCs (H1- and GFP-expressing H9) and an iPSC line (i202) were maintained onMEFs as a control (Fig. 2A–C) and in parallel on bEnd.3 cells(Fig. 2A¢–C¢). The colony morphology for all 3 lines wasstable over passage and retained sharp borders as would beexpected if bEnd.3 cells maintained PSCs in the undifferen-tiated state. To examine this more closely, we maintained H9hESCs for over 20 passages on bEnd.3 feeder cells. Indirectimmunofluorescence analysis for Oct4, Nanog, and Tra1-60showed similar staining between the 2 different conditions(Fig. 2D, E and D¢, E¢). Karyotype analysis was normal forbEnd.3- and MEF-maintained H9 hESCs (SupplementaryFig. S1).

H9 hESCs maintained on endothelial cells retained plur-ipotency since they could be directed to derivatives of all 3germ layers and extraembryonic cell types (Fig. 3A). hESCswere cultured in RPMI with BMP4, low serum, and the TGFbeta/Activin inhibitor SB431542 to create mesoderm

FIG. 1. Human embryonic stem cells (hESCs) are not efficiently converted to neural cells when cocultured with bEnd.3feeder cells. hESCs were cocultured with MS-5 (as a positive control), mouse embryonic fibroblasts (MEFs) (as a negativecontrol), or bEnd.3 cells to determine the ability of each feeder layer to cause neural induction. (A) Indirect immunofluo-rescence using Pax6 antibodies to reveal neural induction (red staining). DAPI counterstain (blue) to demonstrate the presenceof Pax6-negative cells. Scale bar = 100 mM. (B) Quantitative polymerase chain reaction (qPCR) was used to determine the foldchange of Pax6 mRNA relative to hESCs.

2314 JOUBIN ET AL.

(Brachyury + ) and trophectoderm (Cdx2 + ). H9 hESCs thatare cultured in Activin and low serum created endoderm(Sox17 + ), while the neural induction used dual SMAD in-hibition (Pax6 + ) ([10]; see Materials and Methods section fordetails). qPCR showed similar levels of each germ layermarker after directed differentiation from both MEF- andbEnd.3-maintained hESCs (top of Fig. 3; n = 3 independentexperiments). Similarly, indirect immunofluorescence withantibodies against Pax6, Brachyury, Sox17, and Cdx2showed comparable levels of staining with each marker afterdifferentiation (Fig. 3B).

To confirm these results, we also made embryoid bodiesand teratomas with H9 hESCs that had been maintained onbEnd.3 for 20 passages. Embryoid bodies derived from MEF-or bEnd.3-maintained cells had similar growth patterns andmorphologies over the 21-day differentiation (Fig. 4A).Genes characteristic of the 3 germ layers were present in EBscultured on both feeders (Fig. 4B), and bEnd.3-maintained

H9 hESCs also formed teratomas in vivo (Fig. 4C). Takentogether with the in vitro data described previously, theseresults confirmed the pluripotent nature of the bEnd.3-maintained hESCs.

bEnd.3 cell expansion

One factor that dramatically increases the cost and com-plexity associated with using PMEFs is that they cannotbe passaged without losing the ability to maintain ESCs:most labs use MEFs at passage 3. The bEnd.3 mouse brainendothelial cell line was transformed with the polyomamiddle-sized T antigen [13]. Therefore, to see whetherbEnd.3 retained the ability to maintain ESCs after expansion,we performed experiments with passage 0, 5, and 10 bEnd.3cells. A portion of each passage was irradiated beforefreezing down while the rest was used for further expansion.We found that passage-10 bEnd.3 cells were as effective as

FIG. 2. Human pluripotentstem cells (hPSCs) maintainedon MEFs (A–F) or bEnd.3feeder cells (A¢–F¢). (A, A’)GFP-expressing H9 hESCs, (B,B¢) H1 hESCs, and i202 hu-man induced PSCs (hiPSCs)(C, C¢) maintained on bothfeeder types. Immuno-fluorescence of PSC markersNanog (D, D¢), Oct4 (E, E¢),and Tra1-60 (F, F¢) in H9hESCs. Scale bar = 400mM in(A–C) and (A¢-C¢) and81.25mM in (D–F) and (D¢–F¢).

THE ENDOTHELIAL CELL LINE BEND.3 MAINTAINS HPSCS 2315

earlier passage cells (Fig. 5). The morphology of hESC colo-nies maintained the same tightly packed phenotype in eachcondition (Fig. 4A). Immunofluorescence analysis showed noobvious differences in the expression levels of Oct4 and Tra1-60 in each condition (Fig. 5A). To check our subjective ob-servations, we cocultured GFP-expressing H9 cells on pas-sage 0, 5, and 10 cells before performing FACS analysis toquantitate the surface staining of SSEA-3 and - 4. Figure 5Bshows the percentage of SSEA-3 and - 4 on the GFP + frac-tion of cells to eliminate feeders from the analysis. No dif-ferences were observed in the levels of stem cell surfacemarkers between early and late-passage bEnd.3-maintainedhESCs (n = 3 independent experiments). This data suggestedthat bEnd.3 cells could be expanded extensively in vitrobefore using them to maintain hPSCs.

Engineered feeders

As more complicated genetic schemes are employed inhPSC research, feeders containing fluorescent markers ormultiple drug resistance types are desired. The production ofdrug-resistant MEFs is expensive and time consuming sincenew transgenic mice must first be made. Since bEnd.3 is atransformed cell line, we attempted to create transgenic feedercells in vitro. We initially engineered bEnd.3 cells to express

mCherry to visualize and physically separate feeders fromhPSCs using flow cytometry. In a proof-of-concept experi-ment, we cocultured GFP-expressing H9 hESCs on mCherry-expressing bEnd.3 cells (Fig. 6A). Flow cytometry couldseparate the feeders from the hESCs (Fig. 6B, 6C, 6A¢–C¢).

Another important application is the creation of drug-resistant feeders to engineer hPSCs. To this end, we nucleo-fected a plasmid containing a Puromycin resistance cassetteinto bEnd.3 cells and selected Puro-resistant lines (bEnd.3-Puro). Such Puromycin-resistant feeders were capable ofmaintaining hESCs. As a proof of concept, we co-nucleofectedan mCherry-expressing plasmid and a Puromycin-resistanceplasmid into H9 hESCs before selection on bEnd.3-Puro cells.H9 hESCs expressing mCherry were readily obtained andexpanded on bEnd.3-Puro cells (Fig. 6D–F). bEnd.3 cells arealso Neomycin resistant, a remnant from the original trans-formation [13]. Such feeders will be useful for genetic target-ing or transgenesis in hPSCs and can be engineered to expressany drug-resistant cassette desired.

bEnd.3 secretes an activity that maintains hESCs

Endothelial cells, including bEnd.3, secrete an activity thatexpands mouse neural progenitors while inhibiting theirdifferentiation in vitro [5]. We therefore tested whether the

FIG. 3. H9 hESCs maintained on bEnd.3 cells can be directed to all 3 different germ layers and trophoblasts. For differ-entiation details, see Materials and Methods section. (Top) After differentiation, the gene expression levels of markerscharacteristic of different germ layers were compared by qPCR: Pax6 for neural cells, Sox17 for endoderm, Brachyury formesoderm, and Cdx2 for trophoblasts. (Bottom) Indirect immunofluorescence with the same markers at the protein level toverify the qPCR results. Scale bar = 100 mM.

2316 JOUBIN ET AL.

bEnd.3 activity that maintains undifferentiated hESCs is alsosecreted. H9 hESCs were passaged onto Matrigel with un-conditioned media or media conditioned by MEFs or bEnd.3cells. After expansion in conditioned media for 7 days, themorphology of hESCs was noted before FACS analysis.A fraction of the cells were further expanded in the sameconditions to assess further feeder-free expansion in thepresence or absence of conditioned media. Three sequentialrounds of feeder-free passage of H1 and i202 were examinedfor morphology and SSEA-4 expression through FACSanalysis (Fig. 7). Both pluripotent lines expanded in hPSCmedia without conditioning drastically changed morphology(Fig. 7, top), lost SSEA-4 expression (Fig. 7, bottom), andnearly stopped dividing. MEF- and bEnd.3-conditionedmedia expanded cells with the correct morphology (Fig. 7,top) and maintained rapid cell division and SSEA-4 expres-

sion (Fig. 7, bottom). Taken together, our data show thatbEnd.3-conditioned media are competent to expand hPSCs,suggesting that bEnd.3 also secretes a factor(s) that maintainspluripotency.

Discussion

We serendipitously discovered that hPSCs coculturedwith the transformed brain endothelial cell line bEnd.3 aremaintained in their undifferentiated state as well as PMEFs.This discovery is important for a number of reasons: (1)variability—each batch of MEFs can be different and there-fore must be quality controlled. The use of one suboptimalpreparation can differentiate all hPSCs in a lab, costing timeand money. (2) Cost—it is expensive to purchase or makePMEFs. (3) Reduction of animal use—it is preferable to

FIG. 4. H9 hESCs maintained onbEnd.3 cells can make embryoid bodiesand teratomas. (A) Phase-contrast imageof day 9 embryoid bodies derived frombEnd.3- or MEF-maintained H9 hESCs.(B) Semi-qPCR showing gene expressionin day 21 embryoid bodies derived fromH9 hESCs maintained for 20 passageson bEnd.3 or MEF as a control. (C) Ter-atomas derived from bEnd.3-maintainedH9 hESCs. Derivatives of all 3 germ layerswere present. Left: neural crest, neuroe-pithelium, and brain tissue. Middle:endoderm and mesoderm. Right: cartilageand epithelium.

THE ENDOTHELIAL CELL LINE BEND.3 MAINTAINS HPSCS 2317

FIG. 5. Passage of bEnd.3 cells does not hinder their ability to maintain hESCs. bEnd.3 cells were passaged 5 or 10 timesbefore irradiation and used as feeders in hESC culture. (A) Colony morphology, Oct-4 and Tra1-60 staining of GFP + H9hESCs on passage 0, 5, and 10 bEnd.3 feeder cells. (B) Quantitation of stem cell surface markers. Cultures grown on passage 0,5, and 10 bEnd.3 feeder cells were dissociated into single cells before surface staining with stem cell surface marker anti-bodies. Histograms represent the amount of each antibody on the GFP + cells to eliminate feeder cell contamination.

FIG. 6. Genetically engi-neering bEnd.3 feeders.Fluorescent image (A) andFACS analysis (A¢) of GFP +hESCs growing on mCher-ry + bEnd.3 feeder cells. Mixedcultures could be separatedinto red (B, B¢) and greenpopulations (C, C¢) after flowcytometry. Scale bar = 300mM.Puromycin-resistant feederswere used to make mCherry-expressing H9 hESCs (D–F).Transgenic H9s were made bynucleofecting an mCherry andpuromycin-resistance expres-sing plasmid at the sametime. Nucleofected cells wererecovered on Puromycin-resistant bEnd.3 feeder cellsbefore Puromycin selection.Scale bar = 100mM.

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reduce the number of animals used in research. (4) Trans-genic feeders—it is easier to engineer bEnd.3 feeder cellssince they can be expanded without losing their ability tosupport PSCs.

While some labs have moved to feeder-free culture, manylabs (including ours) feel that feeder-based hPSC culture ismore robust than feeder-free culture (eg, see Ref. [14]). This islikely the reason that many influential stem cell labs continueto use mouse feeder layers (for some examples, see Refs.[15–19]). Current feeder-free systems are also expensive forlabs performing extensive cell culture. One reason that labsswitch to feeder-free systems is convenience. To make feed-ers, animals must be bred or ordered and sacrificed on aparticular day. Then, after a dissection, the primary culturemust be briefly expanded since it is thought that MEFs losethe ability to maintain stem cells as they are passaged. Ex-panded MEFs undergo mitotic inactivation and aliquottingbefore quality control to verify that each lot of feeders canadequately maintain hESCs. Because of the effort required tomake and quality control MEFs, many laboratories chooseto purchase MEFs. Commercially purchased MEFs can stilllead to variability and is a major cost associated with hPSCculture. The use of bEnd.3 cells reduces the inconvenienceassociated with feeder production; further, any interestedlaboratory can order them from the nonprofit repositoryATCC (www.atcc.org).

The use of primary, early passage MEFs also restricts thegenetic engineering of feeders since selection in vitro requirescell division beyond what is considered acceptable for hPSCmaintenance. As such, engineered feeders are usually ac-complished by the production of a transgenic mouse colony,but the time and cost associated with this can be prohibitive.

We demonstrate here that bEnd.3 can be engineered to befluorescent so that feeders can be visualized, quantitated, orphysically separated by flow cytometry. Alternatively, drug-resistant feeders were made and used here to select trans-genic hPSCs. Future work could be aimed at engineeringbEnd.3 feeders to express FGF2 or other proteins that helpsupport or expand hPSCs. Such a system has already beendescribed for transformed human fibroblasts [20]. Engineeredfeeders secreting a constant source of growth factors could bemore affordable and effective than is possible with feeder-freeculture systems.

Another reason cited for switching to human- or feeder-free systems is that hPSCs will be used for clinical applications.While clinical applications will likely become increasinglyimportant for PSCs, most studies focus on human develop-ment or disease modeling where mouse cell coculture is lessof a concern. This is likely the reason that many hPSC labscontinue to use mouse feeders as noted previously [13–18].We suggest that bEnd.3 cells are a suitable replacement forMEFs in basic science applications of hPSCs: clinical appli-cations will require human- or feeder-free systems. Ideally, arobust, simple, defined, animal- and feeder-free cultureparadigm that is cost effective would be best for all appli-cations; in our opinion, such a system has yet to emerge.

For clinical applications, others groups have pursuedmany different types of human feeders including trans-formed lines [20], hESC-derived fibroblast-like cells [21–24],and some from human fetuses, foreskin, or adult cells [25–31].We have not tried these other feeder-based systems so wecannot comment on their efficacy. Most of these studies arequalitative and do not include MEF-maintained cultures as areference for comparison. In our opinion, this complicates

FIG. 7. bEnd.3-conditionedmedia maintain PSCs with-out feeders. (Top) Morphol-ogy of H9 hESCs expandedon Matrigel-coated disheswith no conditioning, MEF-or bEnd.3-conditioned hESCmedia. (Bottom) SSEA-4expression with H1 hESCs(black) and i202 hiPSCs (white)over 3 passages withoutfeeders in media withoutconditioning (square) or con-ditioned by MEF ( circle) orbEnd.3 conditioning (invertedtriangle).

THE ENDOTHELIAL CELL LINE BEND.3 MAINTAINS HPSCS 2319

their evaluation since many groups use different qualitativecriteria as being optimal. Furthermore, many of the primarycells cannot be passaged extensively limiting their utility.One of the few quantitative comparisons found that MEF-maintained hESCs were more undifferentiated than humanforeskin–maintained hESCs, as judged by SSEA-3 expression[32]. Further work is needed to rigorously compare differentfeeder systems.

Practicalities aside, the observation that bEnd.3 endothe-lial cells maintain hPSCs raises interesting biological ques-tions. One possibility is that the same factor(s) secreted frombEnd.3 that maintains fetal neural progenitors is also re-sponsible for maintaining PSCs. Alternatively, endothelialcells might secrete different activities that act on different cellpopulations. Future work aimed at identifying these secretedfactor(s) will be interesting from a biological perspective andcould have a major impact on in vitro stem cell culture.

Acknowledgments

This work was funded by a generous grant from the StarrFoundation and from NYSTEM (C024175-01). The authorswould like to thank Margaret Leversha and Kalyani Cha-dalavada for karyotyping (Molecular Cytogenetics Core Labat MSKCC), and Elisa De Stanchina and Xiaodong Huang(Antitumor Assessment Core Facility at MSKCC) and JerroldWard (Global VetPathology, Montgomery Village, MD/HistoServ, Inc.) for help with teratoma formation, cutting,staining, and pathology.

Author Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Mark J. Tomishima

Developmental Biology ProgramSKI Stem Cell Research Facility

Center for Stem Cell BiologySloan-Kettering Institute

1275 York AvenueNew York, NY 10065

E-mail: tomishim@mskcc.org

Received for publication September 2, 2011Accepted after revision January 4, 2012

Prepublished on Liebert Instant Online January 6, 2012

THE ENDOTHELIAL CELL LINE BEND.3 MAINTAINS HPSCS 2321