human embryonic stem cell-derived neural crest

8/13/2019 Human Embryonic Stem Cell-Derived Neural Crest

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Human embryonic stem cell-derived neural crest

cells capable of expressing markers of osteochondral

or meningealchoroid plexus differentiation

Human pluripotent stem (hPS) cells such ashuman embryonic stem (hES) and induced pluri-potent stem cells possess the capacity to cascadethrough all somatic cell lineages during differenti-ation [1]. hPS cells therefore offer new possibilitiesfor modeling human development in vitro, as wellas a means of manufacturing rare and valuable celltypes useful in regenerative medicine. However,clinical-grade cellular formulations will likelyrequire standards of purity and identity exceedingthose observed in most published differentiationprotocols. The majority of published manufactur-ing methods intended to generate clinical-gradecells entail the steps of: scale up of the undiffer-entiated hPS cells; differentiation under a denedprotocol to generate an enriched population of the

desired cell type; and a purication process (com-monly utilizing afnity methods). However, thethousand-fold complexity of cell types emergingfrom hES cell cultures [2], the inherent variabilityintroduced in each of the multiple differentiationfate decisions made by the cells in the process, andthe current inability to identify the majority ofcell types contaminating the formulations makesthe manufacture of hPS cell-derived therapeuticschallenging.

An alternative to this heterogeneous differentia-tion method is the expansion of clonal embryonic

progenitor (EP) cell types. We previously reported

that >140 diverse human EP (hEP) cell types arecapable of extensive clonal expansion when uti-lizing diverse culture conditions [3]. The cells aredesignated embryonic progenitors to reect thefact that they typically display patterns of geneexpression corresponding to early development,can be extensively passaged in vitro,then have thepotential to respond to various cues to terminallydifferentiate into adult cell types. Like hPS cells,hEP cells may not represent naturally occurringstem cells, but instead, like hPS cells, may rep-resent progenitors that display a developmentalstasis while showing proliferative capacity incertain culture conditions. Unlike adult-derivedstem cells such as bone marrow mesenchymalstem cells (MSCs), which currently lack rigor-

ous clonal data demonstrating differentiated fatesbeyond hypertrophic chondrocytes, adipocytes,broblasts, and perhaps reticular and osteoblastcells, clonal hEPs could be a strategy for obtain-ing scalable progenitors with greater diversity andsite specicity.

We previously reported that screening of100 diverse hEP lines for chondrogenic potentialin the presence of TGFb3 in micromass (MM)culture identied seven lines capable of induc-tion of COL2A1 and associated osteochondralmarkers [4]. Here we describe two novel neural

crest clones that did not induce COL2A1 in

Aims:The transcriptome and fate potential of three diverse human embryonic stem cell-derived clonalembryonic progenitor cell lines with markers of cephalic neural crest are compared when differentiatedin the presence of combinations of TGFb3, BMP4, SCF and HyStem-C matrices. Materials & methods:Thecell lines E69 and T42 were compared with MEL2, using gene expression microarrays, immunocytochemistryand ELISA. Results:In the undifferentiated progenitor state, each line displayed unique markers of cranialneural crest including TFAP2Aand CD24; however, none expressed distal HOXgenes including HOXA2orHOXB2, or the mesenchymal stem cell marker CD74. The lines also showed diverse responses whendifferentiated in the presence of exogenous BMP4, BMP4 and TGFb3, SCF, and SCF and TGFb3. The clonesE69 and T42 showed a profound capacity for expression of endochondral ossification markers when

differentiated in the presence of BMP4 and TGFb3, choroid plexus markers in the presence of BMP4 alone,and leptomeningeal markers when differentiated in SCF without TGFb3. Conclusion:The clones E69 andT42 may represent a scalable source of primitive cranial neural crest cells useful in the study of cranialembryology, and potentially cell-based therapy.

KEYWORDS: adipocyte nAPOD nbone ncartilage nchoroid plexus nclonal embryonicprogenitor cells ncraniofacial nCYP26B1 nembryonic stem cells nmeninges nneuralcrest nPTGDS nTTR

Hal Sternberg1,

Jianjie Jiang1,

Pamela Sim1,

Jennifer Kidd1,

Jefrey Janus1,

Ariel Rinon2,

Ron Edgar2,

Alina Shitrit2

,David Larocca3,

Karen B Chapman4,

Francois Binee5

& Michael D West*1

1BioTime, Inc., 1301 Harbor Bay,

Parkway, Alameda, CA 94502, USA2LifeMap Sciences, Tel Aviv, Israel3Mandala Biosciences, La Jolla,

CA, USA4OncoCyte Corporaon, Alameda,

CA, USA5OrthoCyte Corporaon, Alameda,

CA, USA

*Author for correspondence:

Tel.: +1 510 521 3390 ext. 303Fax:+1 510 521 3389

[email protected]

53ISSN 1746-075110.2217/RME.13.86 2014 Future Medicine Ltd Regen. Med.(2014) 9(1), 5366

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RESEARCHARTICLE Sternberg, Jiang, Sim et al.

the presence of TGFb3 in the previous study,but when differentiated in an expanded com-bination of differentiation factors, show mark-ers for osteochondral, choroid plexus, as well asleptomeningeal differentiation.

Materials & methodsn Cell lines & growth factors

The hEP cell clones E69 (cat. no. ES-101, Bio-Time, CA, USA), T42 (cat. no. ES-210, Bio-Time) and MEL2 (cat. no. ES-268, BioTime)were derived as previously described [3]. T42 andMEL2 were derived from the hES cell line H9(NIH registered as WA09). The line E69 wasderived from the hES cell line ACT03 (MA03).The hEP cell lines were routinely cultured incorresponding ESpan medium as recommendedby the manufacturer (BioTime). The cells when

maintained in the undifferentiated state werecultured at 37C in a humidied atmosphereof 10% CO

2and 5% O

2on gelatinized culture

vessels. The relatively high concentration of CO2

provided physiological pH in relatively low con-centrations of O

2. The hEP cell lines were serially

passaged as previously described, while conu-ence was carefully prevented for more than 2 daysto prevent differentiation while in the progenitorstate. When assaying gene expression of the undif-ferentiated progenitors, cells were synchronized inquiescence by growing to conuence, then wereswitched to medium containing only 10% of the

normal serum concentration and held for 5 daysto induce quiescence. TGFb3 was obtained fromLonza (NJ, USA, cat. no. PT-4124) or R&D Sys-tems (MN, USA, cat. no. 243-B3-010). BMP4was obtained from HumanZyme (IL, USA).SCF was obtained from R&D Systems (cat. no.255-SC-010).

n MM-induced differentiationCells were cultured in the undifferentiated state,and upon conuence detached with 0.25% w/vtrypsin/EDTA (Invitrogen, CA, USA), which

was diluted 1:3 with phosphate-buffered saline(PBS; Ca, Mg free). The trypsin was deactivatedupon the addition of growth medium, cells werecounted, spun at 150 g for 5 min, supernatantremoved and cells resuspended at a cell densityof 2.0 107cells/ml in their respective growthmedium. Twenty-five or more MM aliquots(200,000 cells/10 l aliquot) were seeded ontoCorning tissue culture-treated polystyrene platesor dishes (Corning, MA, USA). The seeded MMswere placed in a humidied incubator at 37Cwith 5% O

2and 10% CO

2for 2 h for attachment.

The growth medium for each respective cell line

was added. The following morning, the mediumwas aspirated, cells were rinsed with PBS (Caand Mg free), which was replaced with Factor-Containing Differentiation medium as per themanufacturers instructions (differentiation kitES-K210; BioTime). Factor-containing differen-

tiation medium consisted of BioTime factor-freemedium plus TGFb3 or other TGFbfamily mem-bers as described herein. Briey, sterile lyophilizedTGFb3 was reconstituted with the addition ofsterile 4 mM HCl containing 1 mg/ml bovineserum albumin to prepare a 2000 stock solutionwith a concentration of 20 g/ml. The stock solu-tion was stored after aliquoting at -80C. Factor-containing differentiation medium (10 g/mlTGFb3) was prepared just before use by the addi-tion of 1.0 l of stock TGFb3 for each 2 ml offactor-free differentiation. Cells were maintained

in a humidied incubator at 37C with 5% O2and 10% CO2in factor-containing differentiation

medium, which was replaced with freshly pre-pared medium every 23 days. At the designatedtime points, RNA was extracted using QiagenRNeasy Kits (cat. no. 74104, Qiagen, CA, USA)according to the manufacturers instructions.RNA yield was maximized using QIAshredders(cat. no. 79654, Qiagen) to homogenize samplesfollowing the lysis of the MMs with RLT bufferbefore RNA extraction.

n Differentiation in HyStem-4D bead

arraysHyStem-C (BioTime) was prepared according tothe manufacturers instructions. The Hystem-Ckit consists of three reagents that need to be recon-stituted in degassed deionized water. Briey, theHyStem component (thiol-modied hyaluronan,10 mg) was dissolved in 1 ml to prepare a 1% w/vsolution, the Gelin-S component (thiol-modiedgelatin, 10 mg) was also dissolved in 1 ml waterto prepare a 1% w/v solution, and a polyethyleneglycol diacrylate (PEGDA, 10 mg) crosslinkerwas dissolved in 0.5 ml to prepare a 2% w/v

solution. Then, 1 ml HyStem was mixed with1 ml Gelin-S just before use. Pelleted cells wereresuspended in the HyStem:Gelin-S (1:1 v/v)mix, followed by the addition of the crosslinkerPEGDA, to yield a nal cell suspension concen-tration of 2.0 107cells/ml. The cell suspensionwas aliquoted at 25 l/bead into six-well plates(Corning 3516) after partial gelation (typicallyve beads per well). Differentiation medium wasadded to each well following complete gelation(2040 min). Plates were placed in a humidiedincubator at 37C with ambient O

2and 10%

CO2, and the cells were fed fresh differentiation

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Clonal human embryonic stem cell-derived neural crest RESEARCHARTICLE

medium three-times weekly. The hydrogel con-structs are either xed for immunohistochemicalanalysis, or lysed using RLT (Qiagen), for totalRNA to analyze transcript expression (usingquantitative real-time PCR (qRT-PCR) and/orwhole genome microarray), at the desired time

points.

n ELISAThe concentration of transthyretin protein in con-ditioned medium was determined by ELISA. E69,T42 and MEL2 cells were cultured for 14 days inMM culture in the presence of 10 ng/ml BMP4(50 MMs at 200,000 cells each in 10 cm dishes,with 20 ml serum-free differentiation medium).After 3 days, conditioned medium was removed,and concentrated using Amicon spin concentra-tors (cat. no. UFC901024, Millipore Corp., CA,

USA). Concentrates were assayed in duplicateat three dilutions (100, 200, and 400 forboth T42 and E69; and 50, 100 and 200 forMEL2) using an Abcam transthyretin ELISAkits (cat. no. ab108895, Abcam, MA, USA). Theconcentration (ng/ml) of transthyretin in theconditioned medium was calculated upon takinginto account the fold concentration in Amiconconcentrators.

n Immunofluorescent detectionof KRT17EP cells grown under quiescence-inducing culture

conditions were washed three times with PBS andxed in 4% paraformaldehyde for 20 min at roomtemperature (RT). Fixed cells were washed threetimes in PBS, permeabilized and blocked by incu-bation in a blocking buffer (10% normal donkeyserum and 0.1% Triton X-100 in PBS) overnightat 4C. The cells were incubated for 1 h at RTwith primary rabbit anti-KRT17 monoclonalantibody EP1623 (ab109725, Abcam) at a dilu-tion of 1:100 in 0.5 blocking buffer. Next theywere washed three times with PBS, and incubatedfor 1 h at RT with Alexa Fluor 568 donkey anti-

rabbit IgG antibody (Invitrogen, A10042) at a1:1000 dilution in PBS. Control cells were stainedunder identical conditions except that total rabbitIgG (0111-01, Southern Biotech, AL, USA) wasused as the primary antibody (30 g/ml). Cellswere counterstained with DAPI at 0.1 mg/ml for15 min at RT.

n Gene expression analysisTotal RNA was extracted directly from cells usingQiagen RNeasy Mini Kits according to the manu-facturers instructions. RNA concentrations were

obtained using a Beckman DU 530 (Beckman

Coulter, CA, USA) or NanoDrop (DE, USA)spectrophotometer, and RNA integrity was deter-mined by denaturing agarose gel electrophoresisor by an Agilent 2100 bioanalyzer (Agilent, CA,USA). Whole-genome expression analysis wasperformed using Illumina HumanHT-12 v4 Bea-

dArrays (Illumina, CA, USA), and RNA expres-sion levels for certain genes were veried by qRT-PCR. For the Illumina BeadArrays, total RNAwas linearly amplied and biotin-labeled usingIllumina TotalPrep kits (Life Technologies, CA,USA). The cRNA quality was controlled usingan Agilent 2100 Bioanalyzer, and was hybridizedto Illumina BeadChips, processed, and read by aBeadStation array reader according to the manu-facturers instructions. Values under 130 relativeuorescence units were considered as nonspecicbackground signal.

n Data analysis for clusteringRaw microarray data of the cell clones was nor-malized with the R beadarray library [5]. Oneprobe was selected for each gene by using thedefault parameters of collapse Rows functionfrom the weighted correlation network analysis(WGCNA) library [6]. For each cell culture sam-ple, the differential expression values were calcu-lated against a wide selection of EP cell clones.Genes that were upregulated by more than two-fold in at least one specic cell line in comparisonwith the progenitor reference were used in cluster-

ing. The dendrogram was created using hierarchi-cal cluster analysis using average agglomerationmethod.

n qRT-PCR analysisSamples for assessment were prepared in standardOptical 96-well reaction plates (Applied Biosys-tems, CA, USA, PN 4306737). They consistedof 30 ng of RNA equivalent of cDNA, 0.8 Mper gene-specic custom oligonucleotide primerset (Invitrogen), and ultrapure distilled water(cat. no. 10977015, Invitrogen), which was

diluted 1:1 with 12.5 l of Power SYBR GreenPCR Master Mix (cat. no. 4367659, AppliedBiosystems) incorporating AmpliTaq Gold DNApolymerase for a total reaction volume of 25 l.An Applied Biosystems 7500 Real-Time PCRSystem employing SDS2.0.5 software was usedto run qRT-PCR. Amplication conditions wereset at 50C for 2 min (stage 1), 95C for 10 min(stage 2), 40 cycles of 95C for 15 s then 60C for1 min (stage 3), with a dissociation stage (stage 4)at 95C for 15 s, 60C for 1 min, and 95C for 15 s.Cycle threshold (Ct) values for the amplicons of

genes COL2A1,COL10A1,ACANand CTRAC1

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were normalized to the average Ct value of threehousekeeping genes (GAPDH,RPS10and GUSB)and the normalized gene expression was thencalculated relative to that of early passage knee-normal human articular chondrocytes (NHACs)(Lonza). Gene expression for the gene TTRacross

samples was analyzed from the average Ct valueof the amplication product, which was nor-malized using the average Ct value of GAPDH.Primers used included:ACAN (NM_013227.2)f. TGAGTCCTCAAGCCTCCTGT, r. CCTC-TGTCTCCTTGCAGGTC (185 bp); COL2A1(NM_001844.4) f. TGGCCTGAGACAGC-ATGA, r. AGTGTTGGGAGCCAGATTG(373 bp); COL10A1 (NM_000493.3) f. GGG-CCTCAATGGACCCACCG, r. CTGGGC-CTTTGGCCTGCCTT (150 bp); CRTAC1(NM_018058.4) f. ATCCGTAGAGAGCAC-

GGAGA, r. GGACTCTCCATGGGACAAGA(144 bp); GAPDH (NM_002046.3) f. GGC-CTCCAAGGAGTAAGACC, r. AGGGG-TCTACATGGCAACTG (147 bp); GUSB(NM_000181.2) f. AAACGATTGCAGGGT-TTCAC, r. CTCTCGTCGGTGACTGTTCA(171 bp); TTR (NM_000371.3) f. TGCAG-AGGTGGTATTCACAGC, r. GGTGGAA-TAGGAGTAGGGGC (85 bp); and KRT17(NM_000422.2) f. TCCTGCAGGCTGGG-ATCT, r. GGTGGCTGTGAGGATCTTGT(333 bp).

ResultsWe previously reported that when 100 diversehES-derived clonal hEP cell lines were differ-entiated in MM conditions in the presence ofTGFb3, dexamethasone, and insulintrans-ferrinselenium medium for 14 days, seven ofthe lines showed evidence of chondrogenic dif-ferentiation as measured by the upregulation ofchondrocyte-specic markers such as the tran-script for COL2A1. These seven clonal progeni-tors designated 4D20.8, 7PEND24, 7SMOO32,E15, MEL2, SK11, and SM30 showed diverse

site-specific homeobox gene expression anddiverse differentiation responses to a panel ofcombinations of TGFbfamily members [7].

Since the screening of randomly cloned popu-lations of hES-derived progenitors in diverse dif-ferentiation conditions appeared to reveal noveland valuable cell fates for the cells, we undertooka large-scale screening of the cells in the presenceof diverse growth factors. We differentiated thecells in HyStem-4D bead arrays, a condition thatin our hands leads to improved uniformity andreproducibility of the differentiation [8]. In addi-

tion, since clinical-grade HyStem-C is available

and associated clinical trials are underway testingthe matrix as a device for the therapeutic deliveryof anchorage-dependent cells, novel discoveriesmade with these cell/matrix combinations in vitromay be expected to have a greater probability of asimilar performance when the same cell/matrix

formulation is used in vivo.Since BMP4 alone orin combination with TGFb3 is known to be animportant differentiation factor in neural crestdifferentiation [9], we initially examined theeffects of these factors on two EP cell clones desig-nated E69 and T42 that expressed cranial neuralcrest markers and that were previously reportedto be negative for COL2A1expression when dif-ferentiated in MM conditions in the presence ofTGFb3 alone [4].

n Comparison of gene expression in

the undifferentiated cell clonesdesignated E69, T42 & MEL2As shown in FIGURE1, all three cell clones dis-played a mesenchymal morphology with subtlecell line-specic differences in shape and growth(F IGURE 1A1C). The previously reported cloneMEL2 was chosen as a control as it shared withE69 and T42 cells a lack of HOXgene expressionand like E69 and T42 cells expressed markers ofcephalic neural crest. The clones E69 and T42along with MEL2 were expanded in vitroandthen rendered quiescent at conuence for 5 daysin the presence of relatively low levels of serum to

synchronize the cells and thereby minimize cellcycle artifacts in the gene expression analysis. Iso-lated RNA for the three cell types were then ana-lyzed by gene expression microarray analysis usinghuman HT-12 v4 microarrays and the LifeMapDiscovery(LMD), a database [101]that corre-lates the unique molecular markers with thoseappearing during normal developmentin vivo[2].

As shown in FIGURE 2 & SUPPLEMENTARY TABL E 1(see online at www.futuremedicine.com/doi/suppl/10.2217/RME.13.86), sheet 12, thethree clones shared the cranial neural crest

markers TFAP2A [10]and CD24 [11], and lackeddistal HOXgene expression including HOXA2and HOXB2. The clone E69 (passages rangingfrom 14 to 22) expressed KRT17, TRIM4andZIC2while T42 (passages ranging from 17 to20) expressed TRIML2, EPDR1, ZIC2 andlower but detectable levels of KRT17.ZIC2 isnaturally expressed during neural crest, cranio-facial and axial skeleton development [12]. Asshown in LMD, ZIC2 is expressed in somiticdermomyotome, sclerotome, maxillary, man-dibular and limb bud mesenchyme [102,103]. The

relative levels of KRT17 transcript in E69 and



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T42 were conrmed by qRT-PCR and proteinby immunocytochemistry (FIGURE3).

MEL2 cells (passages 1819) expressedHAND2 (expressed in branchial arch mesen-chyme and, as shown in LMD, expressed in cra-nial and cardiac neural crest cells) [104106]. MEL2

also expressed the retinoid metabolizing enzymeALDH1A2, which has been shown in the LMDto be expressed selectively in the maxillary andmandibular processes [107,108]. Additional mark-ers of undifferentiated MEL2 cells were CRABP1and DLX5.

n Comparative gene expression in EPclones E69, T42 & MEL2 whendifferentiated in the presence of TGFb3& BMP4We next compared the clones E69 (passage 14),T42 (passage 17) and MEL2 (passage 23) when

differentiated for 14 days in HyStem-4D beadarrays. HyStem-C is a biocompatible hydrogelcomprised of thiolated hyaluronan and gela-tin. The crosslinker utilized was PEGDA. Thecells were mixed with reconstituted HyStem-Ccomponents, then 25-l beads were incubatedin differentiation medium supplemented withdiverse growth factors, a protocol referred to asHyStem-4D bead arrays [8]. The differentiationmedium was supplemented with 10 ng/ml BMP4and 10 ng/ml TGFb3, RNA was isolated after14 days and analyzed by Illumina gene expres-

sion microarrays. Mean relative uorescence unitvalues were calculated (SUPPLEMENTARYTABLE1, sheet 3)and compared with the mean values of the cells inthe original progenitor state. As shown in FIGURE4& SUPPLEMENTARYTABLE1, sheet 4, differentiation inHyStem-C for 14 days in the presence of a combi-nation of BMP4 and TGFb3 resulted in a markedinduction of osteochondral gene expression. Theclones E69 and T42 upregulated COL2A1expres-sion (38,675- and 87,955-fold higher expressioncompared with cultured NHACs, respectively)as determined by qRT-PCR (SUPPLEMENTARYTABLE2,

sheet 1), while MEL2 induced COL2A1 at a

much lower level (154-fold over cultured NHACs(SUPPLEMENTARYFIGURE1 & SUPPLEMENTARYTABLE2,sheet 1).In addition, replicate microarray analysis of E69and T42 showed an induction of ACAN andCOL10A1(also conrmed by qRT-PCR), andthe osteochondral markers EPYC,COMP,and

SPP1, as determined solely by microarray analysis(FIGURE4).The previously characterized EP clones

4D20.8, 7PEND24, 7SMOO32, E15, SK11,and SM30 showed variable expression of chon-drogenic versus osteogenic markers depending onthe combinations of BMP used, but rarely lev-els of osteogenic markers comparable to MSCs.The line MEL2 was previously reported to showrelatively high levels of osteogenic markers butlow levels of COL2A1. However, E69 and T42cells differentiated in HyStem-C with BMP4and TGFb3 showed relatively strong expres-

sion of the osteogenic markers SCINandALPL(SUPPLEMENTARYTABLE1), simultaneously with relativelyhigh expression of COL2A1. The expression oftissue-nonspecic ALPL is frequently associatedwith mineralization [13]. Additional markers ofosteogenesis expressed by the differentiated E69,T42 and MEL2 cells, as determined by micro-array analysis, included BMP2with only MEL2cells expressing the calvarial marker GPC3 [14].

n Comparison of differentiated fates ofthe clonal EP cell lines E69, T42 & MEL2

in HyStem-C with BMP4The clones E69 (passage 14), T42 (passage 17)and MEL2 (passage 23) were then incubated for14 days in HyStem-4D bead arrays in the presenceof 10 ng/ml of BMP4, insulintransferrinsele-nium and dexamethasone but in the absence ofTGFb3. As shown in FIGURE5 & SUPPLEMENTARYTABLE1,sheets 56, the clones E69 and T42 upregulatedthe relatively rarely expressed transcript TTRaswell as KITupon differentiation, while upregu-lation of the expression of these genes was notdetectable in the clone MEL2. The TTRgene

encodes transthyretin, an abundant protein in the

Figure 1. Phase contrast images of the human embryonic progenitor cell lines E69, T42 andMEL2. (A)E69 (passage 13);(B)T42 (passage 14); and(C)MEL2 (passage 14) are shown in anundifferentiated state in log growth conditions. Scale bars: 100 m.



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Figure 2. Microarray-based analysis of unique gene expression markers in the clonal human embryonic progenitor cell linesE69, T42 and MEL2.Expression of the genes KRT17,TRIML2,DLX5,TFAP2A,EPDR1,ALDH1A2,TRIM4,ZIC2and HAND2are shown asRFUs in the clones E69 (passages ranging from 14 to 22, n = 5), T42 (passages ranging 1720, n = 7) and MEL2 (passages ranging from18 to 19, n = 4) cultured in the undifferentiated state and for 5 days in quiescence-inducing conditions. Data are displayed as meanvalues of two or more biological replicates. Error bars represent standard deviation. RFU values


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cerebrospinal uid (CSF), transporting thyroidhormones, retinol, and potentially b-amyloid oli-gomers [1517]. The expression of TTRin the E69(passage 14) and T42 (passage 17) clones in thepresence of HyStem-C and BMP4 was conrmedby qRT-PCR, with T42 expressing the highest lev-

els of the transcript (FIGURE6 & SUPPLEMENTARYTABLE2,sheet 1) and no transcript detectable in MEL2(passage 23). To determine whether transthyretinwas also expressed on a protein level, 3-day serum-free conditioned medium was collected fromparallel cultures of E69 (passage 16), T42 (pas-sage 17) and MEL2 (passage 19) in MM condi-tions with BMP4, and analyzed by ELISA for thepresence of soluble secreted protein. As shown inFIGURE6& SUPPLEMENTARYTABLE2, sheet 2, the T42 lineexpressed the highest levels of secreted protein,reaching approximately 8.9 ng/ml at 3 days, E69

reached approximately 0.7 ng/ml, while no tran-sthyretin was detectable in the media of culturedMEL2 cells.

TTRis reported to be expressed relatively rarelyin vivoin tissues such as the retina, liver, and cho-roid plexus of the brain. We therefore examinedRNA from >130 diverse cultured somatic celltypes and observed TTRexpression in cells fromonly these tissues (SUPPLEMENTARYTABLE 1, sheet 8).Specically, cultured hES-derived retinal pigmentepithelial (RPE) cells expressed high levels ofTTR, while we did not observe TTRexpression inundifferentiated hES cells or human iris pigment

epithelial cells. We also observed TTRexpression

in primary cultures of human choroid plexusepithelium and human choroid plexus-derivedstromal cells, but did not observe expression incultured human choroid plexus vascular endothe-lial cells. Lastly, we observed TTRexpression inprimary hepatocytes, but not hepatic sinusoidal

endothelial or stellate cells (SUPPLEMENTARYTABLE 1,sheet 8). Since neither E69 or T42 cells, differen-tiated in BMP4 in either HyStem or MM condi-tions, expressed the numerous common markersof RPE cells such as TYRP1or TYR, or markers ofhepatocytes such as FOXA2or FGG, we concludethat the gene expression pattern of differentiatedE69 and T42 cells corresponds more closely tochoroid plexus-related cells than to either RPEcells or hepatocytes.

n Effects of HyStem-C matrix & SCF on

E69 & T42 differentiationHyStem-C is composed of crosslinked gelatin andhyaluronic acid, each component capable of bind-ing or otherwise modifying the activity of manybasic growth factors. Therefore, it is reasonable toquestion how the differentiation of E69 and T42cells may be inuenced by HyStem-C versus MMconditions. We therefore examined the differen-tiation of the clones E69 and T42 in MM condi-tions wherein 10-l aliquots at 2.0 107cells/mlgrowth medium were allowed to settle and attach,with subsequent incubation in the same dif-ferentiation medium as used previously in the

HyStem-4D bead arrays.

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Figure 3. KRT17expression in the human embryonic progenitor cell clones E69, T42 and MEL2 cultured in theundifferentiated state. (A)Mean KRT17expression relative to GAPDHmeasured by quantitative real-time PCR in the undifferentiated,5-day quiescent cell clones E69, T42 and MEL2. Error bars represent standard deviation. (BD)Immunofluorescent detection of KRT17 inE69, T42 and MEL2 cells cultured in an undifferentiated state using rabbit anti-KRT17 antibody and Alexa Fluor 568-conjugated goatanti-rabbit antibody. (EG)E69, T42 and MEL2 cells cultured in an undifferentiated state and stained with control preimmune rabbit IgGpolyclonal antiserum and Alexa Fluor 568-conjugated goat anti-rabbit antibody. Cell nuclei were stained using DAPI.



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As shown in FIGURE 7 & SUPPLEMENTARY TABL E 1,sheet 6, the clones E69 and T42, when differ-entiated in HyStem-C in the presence of BMP4alone, showed relatively high levels of the adipo-cyte markers FABP4and CD36, and relativelylower levels of TTRcompared with the cells dif-ferentiated under MM conditions. In additionto expressing higher levels of TTR, cells differ-

entiated in MM conditions with BMP4 showed

increased induction of KIT(the receptor for c-kitligand, also known as SCF), a known marker ofchoroid plexus cells, in particular, the choroidplexus epithelium.

To examine whether activation of KITaffectedE69 and T42 cell differentiation, we differenti-ated E69 and T42 cells for 14 days in 10 ng/mlSCF alone or SCF and BMP4, with each condi-

tion performed in both MM and HyStem-4D

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Figure 4. Microarray-based gene expression analysis of E69, T42 and MEL2 differentiated in HyStem-4D bead arrays inBMP4 and TGFb3.The mean relative expression of COL2A1,COL10A1,ACAN,EPYC,COMPand SPP1are shown in control conditionsand 14 days of differentiation of E69 (passage 14, n = 2), T42 (passage 17, n = 2) and MEL2 (passages ranging from 23 to 26, n = 2) in

10 ng/ml BMP4 and 10 ng/ml TGFb3. Data are displayed as mean values of two or more biological replicates. Error bars representstandard deviation. RFU values


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bead arrays. As shown in FIGURE7& SUPPLEMENTARYTABLE1, sheet 7, culture of the cells in SCF aloneresulted in a marked induction of the lepto-meningeal markers PTGDS and ISLR in bothE69 and T42 in both MM and HyStem-4Dbead arrays. PTGDS encodes a glutathione-

independent PGD synthase converting PGH2to PGD

2in the brain where it is also known as

b-trace, the second most abundant protein of theCSF after albumin. In humans, it appears to beexpressed at its highest levels in the arachnoidbarrier cells, followed by arachnoid trabecular andpia cells, but is not expressed in the dura mater[18,19]. T42 appeared to differ from E69 cells inthat it induced a higher amount of SLC6A1andAPODwhen differentiated in the presence of SCFwithout BMP4.

The combination of BMP4 and SCF led to an

outcome similar to BMP4 and TGFb3, namelyTTR-expressing cells (FIGURE7 & SUPPLEMENTARYTABLE1,sheet 7). As before, HyStem-C in these condi-tions led to a higher expression of the adipocytemarkers FABP4and CD36.

It has been reported that cultured mamma-lian neural crest cells express low-afnity NGFR[4]. However, we did not detect NGFR in E69,T42 or MEL2 in any differentiation conditionby microarray analysis.

DiscussionWe previously reported that combinatorial clon-

ing of hES-derived progenitor cell lines is capableof generating a diversity of >140 distinguishablecell types by non-negative matrix factorization.When 100 of these were screened in MM condi-tions in the presence of TGFb3, only seven linesupregulated COL2A1expression. In this study, weexpanded this screening to include two candidateTFAP2A+,CD24+cranial neural crest cells desig-nated E69 and T42 that previously failed to showosteochondral differentiation in TGFb3 and MMconditions. The clones E69 and T42 were differ-entiated in parallel with the line MEL2, which

also expressed similar cranial neural crest markersto E69 and T42, but unlike them did undergoosteochondral differentiation in TGFb3 and MMconditions. Neither E69, T42 or MEL2 expressedmarkers of bone marrow MSCs such as CD74, amarker reported to distinguish MSCs from otherbroblastic cell types (SUPPLEMENTARYTABLE1)[20].

An examination of gene expression during dif-ferentiation of the three clones in HyStem-4Dbead arrays cultured in the presence of BMP4and TGFb3 showed that both E69 and T42robustly upregulated markers of endochondral

ossication (COL2A1andALPL), while MEL2

exhibited markers of ossication with low expres-sion of COL2A1. When all three cell clones were

E69HyStemB

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Figure 5. Microarray-based gene expression analysis of E69, T42 and MEL2differentiated in HyStem-4D bead arrays and MM in BMP4 and TGFb3.The relative expressions of COL2A1,TTRand KITare shown in: control conditions;14 days of differentiation of E69 (passage 14, n = 2), T42 (passage 17, n = 2) andMEL2 (passages ranging from 23 to 26, n = 2) in 10 ng/ml BMP4 and 10 ng/mlTGFb3 in HyStem-4D bead arrays; 14 days of differentiation of E69 (passage 14,n = 2), T42 (passage 17, n = 2) and MEL2 (passages ranging from 23 to 26, n = 2)in 10 ng/ml BMP4 in HyStem-4D bead arrays; and 14 days of differentiation of E69(passages ranging from 15 to 22, n = 5), T42 (passages ranging from 17 to 20,n = 5) and MEL2 (passage 19, n = 2) in 10 ng/ml BMP4 in MM conditions. Data aredisplayed as mean values of two or more biological replicates. Error bars representstandard deviation. RFU values


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differentiated in HyStem-C with BMP4 alone,no detectable COL2A1 expression ocurredin E69 and T42, but instead, the cells mark-edly upregulated TTR, a marker of the choroid

plexus, retinal pigment epithelium, and hepato-cytes. Since neither E69 or T42 expressed othermarkers of hepatocytes or RPE such as FGGorBEST1,respectively, we conclude that E69 andT42 likely correlate more closely with neural crestcells capable of participating in the formation ofthe choroid plexus as opposed to RPE or hepato-cytes. These results are also consistent with thereport that hES cells differentiated en masse inthe presence of BMP4 leads to cells with markersof choroid plexus epithelial cells [21].

The choroid plexus is normally thought to be

formed from contributions from two germ lay-ers; epithelial cells derived directly from the neu-roepithelium; and stromal cells from the cephalicneural crest [22]. The expression of KRT17in theundifferentiated E69 and T42 clones suggeststhat they may be capable of generating choroidplexus epithelial cells despite the mesenchymalmorphology of the cells cultured in vitro [23].However, the expression of the cranial neuralcrest marker TFAP2Amay suggest that the cellscorrespond more closely with the stromal com-ponents of the choroid plexus. Further study will

be required to accurately determine what cells of

the choroid plexus in vivo the TTR+differentiatedprogeny of E69 and T42 correspond to, as wellas the normality and functionality of the cells.

Since BMP4 induced the expression of KITin

both E69 and T42, we examined the effects ofSCF in combination with BMP4 on the differen-tiation of the cells. Interestingly, TGFb3 appearsto have a dominant effect over BMP4 in determin-ing an osteochondral fate to E69 and T42, whileBMP4 appears to have a dominant effect overSCF in determining a choroid plexus fate. Onlyin the presence of SCF alone in either HyStem-Cor MM conditions, did we observe the markedupregulation of leptomeningeal markers such asISLR,PTGDS,SLC6A1andAPOD. While ISLRand PTGDSare relatively constitutively expressed

in developing murine leptomeninges, the genesSLC6A1andAPODare expressed at higher levelsin the mesenchyme of the choroid plexus, menixprimitiva, or mesenchyme associated with themesencephalic exure [24]. Interestingly, the cloneE69 expressed relatively lower levels of SLC6A1andAPOD, perhaps indicative of T42 represent-ing cells of a differing anatomical location in thedeveloping human. In addition, both E69 andT42 expressed relatively high levels of CYP26B1in conditions paralleling TTRexpression, whiledifferentiation in SCF alone led to a loss of TTR

signal, as well as a loss of CYP26B1 expression.

Concentration(ng/ml)

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Figure 6. Mean relative levels of expression ofTTRin cells as determined by quantitativereal-time PCR and ELISA.(A)RNA from the human embryonic progenitor cell clones E69, T42 andMEL2 differentiated for 14 days in HyStem-4D bead arrays in the presence of 10 ng/ml BMP4 wasanalyzed by quantitative real-time PCR for the TTRtranscript. (B)Three-day serum-free conditionedmedium from 14-day HyStem-4D bead arrays was analyzed by ELISA to quantitate the concentrationof transthyretin. Error bars represent standard deviation.



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The localized expression of CYP26B1and TTRin the developing mouse embryo may aid in theidentication of the clones E69 and T42. Theretinoid-metabolizing protein CYP26B1 playsan important role in metabolizing and putativelyinactivating the morphogen retinoid acid. In the

region of the midbrain/hindbrain exure, itsexpression begins at approximately E8.0 in themouse [25]corresponding to approximately humanembryonic day 1719. Therefore, this may suggestthat the clones E69 and T42 are in a developmen-tal stasis corresponding to approximately humanembryonic day 1719, although additional molec-ular markers will need to be identied to clarifythe embryonic stage of development of these cellswhen propagated in the undifferentiated state.

The differentiation of the meninges and cho-roid plexus from mesodermal and ectodermal

progenitors leads to the separation of the CNSfrom the rest of the body, as well as likely play-ing a role in directing neuronal differentiationand axon guidance. Mesencephalic and metence-phalic meninges are believed to be derived fromparaxial mesoderm [22]. Telencephalic meninges

Ctrl

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Figure 7. Heat map of osteochondral, meningeal, adipose, and choroid plexus markers in E69, T42 and MEL2 cells culturedin diverse differentiation conditions.RFU values of select markers of osteochondral, meningeal, adipose and choroid plexusdifferentiation obtained by microarray analysis are displayed. E69, T42 and MEL2 cells were differentiated for 14 days in eitherHyStem-4D bead arrays or MM conditions in the presence of added growth factors shown. Color key shows associated ranges of RFUvalues.Ctrl: Control; MM: Micromass; RFU: Relative fluorescence unit.

from mesencephalic neural crest. The twin ori-gins of meninges is similar to the twin origins ofthe craniad axial skeleton where the spinal verte-brae and occipital bones are derived from paraxialmesoderm, and the remaining bones and cartilageof the cranium are of neural crest origin (LMD).

Assuming the neural crest origin of E69 and T42,it is logical to conclude that the osteochondralfate of the cells when differentiated in the pres-ence of BMP4 and TGFb3 therefore representsneurocranial endochondral ossication.

The primitive markers expressed by these cells,including their differentiation into cells withmarkers of developing meninges, may provide amodel of both normal meningeal developmentas well as meningiomas, the latter being thoughtto derive from primitive leptomeningeal precur-sors [26]. In particular, while the role of Nf2gene

inactivation has been rmly established in theetiology of meningiomas, the precise timing ofthe effects of the inactivation with cell differentia-tion remains to be more fully dened. The use ofthese cell types in in vitroNf2 inactivation andoncogenesis may therefore facilitate these studies.



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We hypothesize that the EP cell clones E69 andT42 may represent primitive embryonic neuralcrest mesenchyme with the potential to differ-entiate into leptomeninges of the upper medulla,adjacent bone of the neurocranium, or choroid

plexus of the fourth ventricle. Further denitionof the molecular markers distinguishing thesecell types will aid in identifying pluripotent stemcell-derived cells, such as clonal EPs, as diverseprogenitors of cells of the developing head andface. These cells and the associated molecularmarkers are now included in LMD [101], a data-base designed to navigate the complexities of cellphenotypes in normal human development.

As shown in FIGURE8, E69 and T42 share similarfates when cultured in BMP4 in the presence orabsence of TGFb3 and analyzed by a clustering

algorithm of the entire probe set of the Illumina

microarray. They clustered in a similar mannerwhen cultured in SCF in the absence of BMP4,but in the latter case, with leptomeningealmarkers.

ConclusionTo our knowledge, this is the rst report ofclonal EPs with markers of neural crest capableof expressing markers of both osteochondral andmeningealchoroid plexus differentiation. Theclonality and scalability of these lines may facili-tate process development for the manufactureof reproducible cultures for research in embryo-logy and drug discovery [27], including researchin the molecular origins of meningiomas fromprimitive neural crest progenitors, orthopedic andneurological applications in regenerative medicine

such as Alzheimers disease [16,17], as well as for the

Choroid plexusEndochondralossificationIntramembranousossification Meninges

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Figure 8. Dendrogram of global gene expression of E69, T42 and MEL2.Global RNA transcripts from the clones E69, T42 andMEL2 in the differentiated states cluster as discrete fates correlating with markers of osteochondral, choroid plexus and leptomeningealcells. The y-axis label represents the distance measure between two clusters where samples that are highly correlated will have acorrelation value close to 1 (distance value close to zero).MM: Micromass.



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in vitromanufacture of CSF components such astransthyretin and b-trace. In addition, the cellsmay provide a novel means of delivering diversetherapeutic proteins into the CSF for therapeuticeffect in a number of metabolic or degenerativediseases of the CNS.

Future perspectiveThere is an increasing recognition of the intrin-sic challenges in manufacturing puried andidentied clinical-grade therapeutics from hPScells. While hPS cells offer, in principle, a scal-able source for the manufacture of many rare andvaluable cell types, and there exist many reportsof associated differentiation protocols, methodsof obtaining a reproducible product with the req-uisite purity required for clinical use are rarelyreported. In addition, in many cases it would be

useful to generate puried populations of differ-entiated cells with site-specic homeobox geneexpression, since these genes are often implicatedin the responsiveness of progenitors to growthfactors and sometimes not-so-subtle differencesin function. One potential solution to this bot-tleneck is the clonal expansion of EPs down-stream of hPS cells. Such scalable populations ofcells would be predicted to have lineage-specic

multipotency, as opposed to pluripotency, andtherefore a simpler path to the differentiatedproduct. The establishment of clonally puriedprogenitors of cranial meningeal, neurocranial,and choroid plexus cell types could allow repro-ducible experiments on a uniform population of

cells, thereby facilitating research in bloodbrainbarrier physiology, embryology, and potentially intherapeutic applications.

Financial & competing interests disclosure

This study was funded by BioTime, Inc. and OrthoCyte

Corporation. The authors have no other relevant affiliations

or financial involvement with any organization or entity

with a financial interest in or financial conflict with the

subject matter or materials discussed in the manuscript apart

from those disclosed.

No writing assistance was utilized in the production of

this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate insti-

tutional review board approval or have followed the princi-

ples outlined in the Declaration of Helsinki for all human or

animal experimental investigations. In addition, for investi-

gations involving human subjects, informed consent has been

obtained from the participants involved.

Executive summary

Comparison of gene expression in the undifferentiated cell clones designated E69, T42 & MEL2

The human embryonic stem cell-derived human embryonic progenitor cell clones E69, T42 and MEL2 show a mesenchymalmorphology when cultured in vitroand share the embryonic neural crest markers TFAP2A andCD24.

The clone MEL2 uniquely expressed the differential gene expression markers DLX5,ALDH1A2and HAND2.

The clones E69 and T42 expressedZIC2and KRT17unlike MEL2, while E69 and T42 differed in that E69 alone was DYNLT3+and

TRIM4+, while T42 alone was TRIML2+and EPDR1+.

Differential KRT17 expression on a protein level in the clone E69 was readily confirmed by immunocytochemistry.

The mesenchymal stem cell marker CD74was not expressed in either E69, T42 or MEL2.

Comparative gene expression when differentiated in HyStem-C hydrogels in the presence of BMP4 & TGFb3

When E69, T42 and MEL2 were differentiated in HyStem-4D bead arrays in differentiation media supplemented with TGFb3 and BMP4,

all three lines markedly upregulated the expression of one or more of the osteochondral markers COL2A1,COL9A2,COL10A1,ACAN

or COMP.

Unlike MEL2, which was previously identified as a candidate progenitor capable of intramembranous ossification, the clones E69 and

T42 displayed markers of robust endochondral ossification with E69 showing an average of >30,000-fold and T42 showing

>80,000-fold higher transcript for COL2A1compared with cultured normal human articular chondrocytes.

Like MEL2, the clones E69 and T42 showed marked upregulation of osteogenesis markers such asALPL,IBSPand COL10A1when

differentiated in the presence of TGFb3 and BMP4.

Comparison of differentiated fates of the clonal embryonic progenitor cell lines E69, T42 & MEL2 in HyStem-C with BMP4

When cultured in HyStem-4D bead arrays in the presence of BMP4 without added TGFb3, E69 and T42 cells but not MEL2 cells

markedly upregulated the expression of the choroid plexus markers TTRand KIT.

Transthyretin was detectable in 72-h conditioned medium of BMP4-differentiated E69 and T42, but not MEL2.

Effects of HyStem-C matrix & SCF on E69 & T42 differentiation

The culture of E69 and T42 cells in the presence of differentiation media supplemented with SCF but not TGFb3 led to an upregulation

of leptomeningeal markers PTGDSand ISLR, with T42 markedly upregulating SLC6A1.

The culture of E69 and T42 in HyStem-C, as apposed to micromass conditions, also induced the adipocyte markers FABP4and CD36in

numerous conditions tested.



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RESEARCHARTICLE Sternberg, Jiang, Sim et al.RESEARCHARTICLE

References

1 Thomson JA, Itskovitz-Eldor J, Shapiro SS

et al.Embryonic stem cell lines derived from

human blastocysts.Science 282(5391),

11451147 (1998).

2 Edgar R, Mazor Y, Rinon Aet al.LifeMap

Discovery: the embryonic development, stemcells, and regenerative medicine research

portal.PLoS ONE 8(7), e66629 (2013).

3 West MD, Sargent RG, Long Jet al.The

ACTCellerate initiat ive: large-sca le

combinatorial cloning of novel human

embryonic stem cell derivatives.Regen. Med.

3(3), 287308 (2008).

4 Sternberg H, Murai JT, Erickson IEet al.

A human embryonic stem cell-derived clonal

progenitor cell line with chondrogenic

potential and markers of craniofacial

mesenchyme.Regen. Med. 7(4), 481501

(2012).

5 Dunning MJ, Smith ML, Ritchie ME, Tavare

S. beadarray: R cl asses and methods for

Illumina bead-based data.Bioinformatics

23(16), 21832184 (2007).

6 Miller JA, Cai C, Langfelder Pet al.

Strategies for aggregating gene expression

data: the collapseRows R function.BMC

Bioinformatics 12, 322 (2011).

7 Sternberg H, Kidd J, Murai JTet al.Seven

diverse human embryonic stem cell-derived

chondrogenic clonal embryonic progenitor

cell lines display site-specic cell fates.Regen.

Med.8(2), 125144 (2013).

8 Sternberg H, Janus J, West M. Deningcell-matrix combination products in the era

of pluripotency.Biomatter 3(1), e24496

(2013).

9 Christiansen JH, Coles EG, Wilkinson DG.

Molecular control of neural crest formation,

migration and differentiation.Curr. Opin.

Cell Biol. 12(6), 719724 (2000).

10 Ishii M, Han J, Yen HY, Sucov HM, Chai Y,

Maxson RE Jr. Combined deciencies of

Msx1 and Msx2 cause impaired patterning

and survival of the cranial neural crest.

Development 132(22), 49374950 (2005).

11 Pruszak J, Ludwig W, Blak A, A lavian K,Isacson O. CD15, CD24, and CD29 dene a

surface biomarker code for neural lineage

differentiation of stem cells.Stem Cells

27(12), 29282940 (2009).

12 Elms P, Siggers P, Napper D, Greeneld A,

Arkell R . Zic2 is required for neural crest

formation and hindbrain patterning during

mouse development.Dev. Biol. 264(2),

391406 (2003).

13 Hellingman CA, Koevoet W, van Osch

GJ. Can one generate stable hyaline cartilage

from adult mesenchymal stem cells? A

developmental approach.J. Tissue Eng. Regen.

Med. 6(10), e1e11 (2012).

14 Dwivedi PP, Grose RH, Filmus Jet al.

Regulation of bone morphogenetic protein

signalling and cranial osteogenesis by Gpc1and Gpc3.Bone 55(2), 367376 (2013).

15 Yang DT, Joshi G, Cho PY, Johnson JA,

Murphy RM. Transthyretin as both a sensor

and a scavenger of beta-amyloid oligomers .

Biochemistry 52(17), 28492861 (2013).

16 Buxbaum JN, Ye Z, Reixach Net al.

Transthyretin protects Alzheimers mice from

the behavioral and biochemical effects of

Abeta toxicit y.Proc. Natl Acad. Sci. USA

105(7), 26812686 (2008).

17 Han SH, Jung ES, Sohn JHet al.Human

serum transthyretin levels correlate inversely

with Alzheimers disease.J. Alzheimers Dis .25(1), 7784 (2011).

18 Urade Y, Kitahama K, Ohishi H, Kaneko T,

Mizuno N, Hayaishi O. Dominant expression

of mRNA for prostaglandin D synthase in

leptomeninges, choroid plexus, and

oligodendrocytes of the adult rat brain.Proc.

Natl Acad. Sci. USA 90(19), 90709074

(1993).

19 Yamashima T, Sakuda K, Tohma Yet al.

Prostaglandin D synthase (beta-trace) in

human arachnoid and meningioma cells: roles

as a cell marker or in cerebrospinal uid

absorption, tumorigenesis, and calcication

process.J. Neurosci . 17(7), 23762382(1997).

20 Ishii M, Koike C, Igarashi Aet al.Molecular

markers distinguish bone marrow

mesenchymal stem cells from broblasts.

Biochem. Biophys. Res. Commun. 332(1),

297303 (2005).

21 Watanabe M, Kang Y J, Davies LMet al.

BMP4 sufciency to induce choroid plexus

epithelial fate from embryonic stem

cell-derived neuroepithelial progenitors.

J. Neurosci . 32(45), 1593415945 (2012).

22 Catala M. Embryonic and fetal development

of structures associated with the cerebro-spinal uid in man and other species. Part I:

the ventricular system, meninges and choroid

plexuses.Arch. Anat . Cytol. Pathol. 46(3),

153169 (1998).

23 McGowan KM, Coulombe PA. Onset of

keratin 17 expression coincides with the

denition of major epithelial lineages during

skin development.J. Cell Bio l. 143(2),

469486 (1998).

24 Sanchez D, Ganfornina MD, Martinez S.

Expression pattern of the lipocalin

apolipoprotein D during mouse

embryogenesis.Mech. Dev. 110(12),

225229 (2002).

25 MacLean G, Abu-Abed S, Dolle P, Tahayato

A, Chambon P, Petkovich M. Cloning of a

novel retinoic-acid metabolizing cytochromeP450, Cyp26B1, and comparative expression

analysis with Cyp26A1 during early murine

development.Mech. Dev. 107(12), 195201

(2001).

26 Kalamarides M, Stemmer-Rachamimov AO,

Niwa-Kawakita Met al.Identication of a

progenitor cell of origin capable of generating

diverse meningioma histological subtypes.

Oncogene 30(20), 23332344 (2011).

27 Dragunow M. Meningeal and choroid plexus

cells novel drug targets for CNS disorders.

Brain Res. 1501, 3255 (2013).

n Websites

101 LifeMap Discovery.

http://discovery.lifemapsc.com

102 LifeMap Discovery. Cervical

Dermomyotome Cells.

http://discovery.lifemapsc.com/in-vivo-

development/somite/cervical-

dermomyotome/cervical-dermomyotome-cells

103 LifeMap Discovery. Prechondrocytic

Mesenchymal Cells.


development/somite/lumbar-spinous-process-

of-vertebrae/prechondrocytic-mesenchymal-

cells

104 LifeMap Discovery. Prechordal

Mesenchyme.


development/head-mesenchyme/prechordal-

mesenchyme

105 LifeMap Discovery. Cranial Neural

Crest Cells.


development/head-mesenchyme/branchial-

arch-1/cranial-neural-crest-cells

106 LifeMap Discovery. Cardiac Neural

Crest Cells.


development/neural-crest/cardiac-neural-

crest/cardiac-neural-crest-cells

107 LifeMap Discovery. Max illary Process.


development/bone/maxillary-process

108 LifeMap Discovery. Mandibular Process.


development/bone/mandibular-process

Regen Med (2014) 9(1)66 f t i

human embryonic stem cell-derived neural crest

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