Research Article Angioblast Derived from ES Cells...

Research ArticleAngioblast Derived from ES Cells Construct Blood Vessels andAmeliorate Diabetic Polyneuropathy in Mice

Tatsuhito Himeno12 Hideki Kamiya34 Keiko Naruse5 Zhao Cheng2

Sachiko Ito2 Taiga Shibata1 Masaki Kondo12 Jiro Kato1

Tetsuji Okawa12 Atsushi Fujiya16 Hirohiko Suzuki2 Tetsutaro Kito2

Yoji Hamada6 Yutaka Oiso1 Kenichi Isobe2 and Jiro Nakamura14

1Department of Endocrinology and Diabetes Nagoya University Graduate School of Medicine 65 Tsurumai-choShowa-ku Nagoya 466-8550 Japan2Department of Immunology Nagoya University Graduate School of Medicine 65 Tsurumai-cho Showa-ku Nagoya 466-8550 Japan3Department of Chronic Kidney Disease Initiatives Nagoya University Graduate School of Medicine 65 Tsurumai-choShowa-ku Nagoya 466-8550 Japan4Division of Diabetes Department of Internal Medicine Aichi Medical University School of Medicine 21 KarimataYazako Nagakute Aichi 480-1195 Japan5Department of Internal Medicine School of Dentistry Aichi Gakuin University 1-100 Kusumoto-choChikusa-ku Nagoya 464-8650 Japan6Department of Metabolic Medicine Nagoya University Graduate School of Medicine 65 Tsurumai-choShowa-ku Nagoya 466-8550 Japan

Correspondence should be addressed to Hideki Kamiya hkamiyaaichi-med-uacjp

Received 16 December 2014 Revised 24 March 2015 Accepted 25 March 2015

Academic Editor Raffaele Marfella

Copyright copy 2015 Tatsuhito Himeno et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Background Although numerous reports addressing pathological involvements of diabetic polyneuropathy have been conducted auniversally effective treatment of diabetic polyneuropathy has not yet been established Recently regenerative medicine studiesin diabetic polyneuropathy using somatic stemprogenitor cell have been reported However the effectiveness of these celltransplantations was restricted because of their functional and numerical impairment in diabetic objects Here we investigatedthe efficacy of treatment for diabetic polyneuropathy using angioblast-like cells derived frommouse embryonic stem cellsMethodsand Results Angioblast-like cells were obtained from mouse embryonic stem cells and transplantation of these cells improvedseveral physiological impairments in diabetic polyneuropathy hypoalgesia delayed nerve conduction velocities and reduced bloodflow in sciatic nerve and plantar skin Furthermore pathologically the capillary number to muscle fiber ratios were increased inskeletal muscles of transplanted hindlimbs and intraepidermal nerve fiber densities were ameliorated in transplanted plantar skinTransplanted cells maintained their viabilities and differentiated to endothelial cells and smooth muscle cells around the injectionsites Moreover several transplanted cells constructed chimeric blood vessels with recipient cellsConclusionsThese results suggestthat transplantation of angioblast like cells induced from embryonic stem cells appears to be a novel therapeutic strategy for diabeticpolyneuropathy

1 Introduction

Large prospective clinical studies have shown a strong rela-tionship between hyperglycemia and diabetic microvascular

complications in both type 1 diabetes and type 2 diabetes[1ndash3] Several hypotheses about diabetic complications havebeen formulated increased polyol pathway flux [4] increasedadvanced glycation end-product formation [5ndash10] increased

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2015 Article ID 257230 17 pageshttpdxdoiorg1011552015257230

2 Journal of Diabetes Research

PKC activation [11ndash14] and increased hexosamine pathwayflux [15] The involvement of these chronic hyperglycemia-mediated metabolic aberrances has also been proven indiabetic polyneuropathy (DPN) [16 17]

DPN is the most common peripheral neuropathy anddegeneration of distal axons of peripheral neurons progressesslowly and symmetrically in DPN Along with HbA1c andduration of diabetes baseline cardiovascular disease andsmoking have been indicated as risk factors for DPN [18]Additionally it has been reported that in DPN the thicknessof endoneurial microvessels of sural nerves increased [19 20]while endoneurial nutritive blood flow of sciatic nerves dec-reased [21 22] The vascular dysfunction in DPN is consid-ered to be one of the most important pathologies

Evidence accumulated in the past two decades indicatesthat peripheral blood cells contain a subpopulation with pro-perties similar to embryonic angioblast [23ndash26] These cellscould be differentiated into mature endothelial cells There-fore these cells were termed ldquoendothelial progenitor cellsrdquo(EPCs) It has been attested that EPCs migrated to ischemialesions and played a pivotal role in vascular endothelialfunction and angiogenesis [27 28] Several studies havedemonstrated the interventional effects of EPC transplan-tation on myocardial ischemiainfarction and preventiveeffects on plaque progression in mice [29 30] However thenumber of circulating EPCs decreased in diabetic patientsand EPCs isolated from diabetic mice exhibited decreasedfunctions includingmigration adhesion and tube formation[31ndash35] Recent studies have shown that oxidative stresselevated by hyperglycemia decreased EPC survival throughinhibition of cell proliferation and NO production [36 37]Marrotte et al reported that EPCs isolated from diabeticmice were less effective than EPCs from nondiabetic miceat accelerating wound closure in diabetic mice [38] Theyalso demonstrated that decreased expression of manganesesuperoxide dismutase in EPCs obtained from diabetic micecontributes to impairment of wound healing These func-tional and metabolic obstacles inhibit clinical applications ofEPCs on diabetic vascular complications

In current regenerative medicines cells used for trans-plantations are expected to have two primary abilities one isdifferentiation abilities to specific cells or tissues and the otheris paracrine supportive effects to impaired tissues Certaincell transplantation therapies have already been applied tosome clinical diseases using various stemprogenitor cellsfrom somatic tissues EPC mesenchymal stem cell (MSC)skin-derived precursor cell neural stem cell and neural creststem cell [39 40] In particular MSC has been applied tothe treatment of certain diseases myocardial infarction [41]stroke [42] and graft versus host disease [43] Although theparacrine mechanisms in damaged tissues and the immuno-modulatory properties of MSC have been proven in theseclinical applications the probability of survival of trans-planted MSCs is small and the therapeutic effects on a long-term basis remain unclear

Recent advances of stem cell biology permit the deriva-tion of induced pluripotent stem (iPS) cell lines at an indi-vidual level in mammalians including humans Although EScells are attractive in their pluripotency and availability of

intact and stable undifferentiated status there are difficultiesin clinical applications of human ES cells because of safetyand ethics Although there are still safety problems that haveto be solved the autologous use of human iPS cells mayresolve ethical issues In this circumstance problematic issueswith using ES cells could be circumvented by employing anequivalent substance iPS cells in the future However thereis no standard for iPS cells the number of obtainable iPS celllines is vast and themethods to obtain them are innumerableAdditionally there are heterogeneous properties among theiPS cells Therefore we utilized ES cells in this current study

We have reported that transplantation of EPCs amelio-rated DPN [44] We hypothesized that if angioblast-like cellscould be obtained from iPS or ES cells these cells wouldoperate like EPCs and restore numerous impairments inDPN It has been shown that the vascular endothelial growthfactor receptor-2 (VEGFR2 known also Flk1) was expressedin angioblasts and its function is essential for the differ-entiation of endothelial cells and hematopoietic cells [45]Therefore Flk1 positive (Flk1+) cells derived from ES cellsmay have similar characteristics to angioblasts and similar oradditional functions of EPCs in adults This is the first reportthat demonstrates the therapeutic effects of transplantation ofangioblast-like cells derived from mouse ES cells on DPN

2 Research Design and Methods

21 Cell Culture BRC6 mouse ES cells derived fromC57BL6 female embryo and B6G02 mouse ES cells express-ingGFPderived fromaC57BL6male embryo were obtainedfrom theRikenCell Bank (Ibaragi Japan) ES cells weremain-tained in DMEM (Invitrogen Van Allen Way Carlsbad CA)containing 10 Knockout Serum Replacement (Invitrogen)1 FBS (Sigma-Aldrich St Louis MO) nonessential aminoacids (Invitrogen) 55mmolL 2-mercaptoethanol (GIBCOBurlington ON) 50UmL penicillin (GIBCO) and 50mgmL streptomycin (GIBCO) on feeder layers of mitomycin C-inactivated SNL767 cells (the European Collection of CellCultures Salisbury UK) which were clonally derived froman STO cell line transfected with a G418 resistance cassetteand a leukemia inhibitory factor expression construct [46]The STO cells were established from fibroblasts derived frommale and female embryos

Cell differentiation was induced as previously described[47] In brief a differentiationmedium (120572-MEM (Invitrogen)supplemented with 10 FBS and 5times 10minus5molL 2-mercaptoe-thanol) was used for ES cell differentiation For the inductionES cells were plated at 17 times 103 cellscm2 in the differentiationmedium on gelatin-coated dishes which were coated with01 gelatin (Sigma-Aldrich) solution and cultured for 4 days

Immortalized Schwann cells (IMS32) established by along-term culture of adult mouse DRGs and peripheralnerves [48] were a kind gift from Dr Watabe IMS32s werecultured in DMEM (Sigma-Aldrich) containing 55mM D-glucose penicillin- (100UmL) streptomycin (100mgmL)and 5 FBS (Moregate Biotech Bulimba QLD Australia)PA6 cells a type of mouse MSC were obtained from

Journal of Diabetes Research 3

the Riken Cell Bank The PA6 cells were cultured in 120572-MEMsupplemented with 10 FBS and 5 times 10minus5molL 2-merca-ptoethanol

22 Cell Sorting Differentiated cells were detached usingAccutase (Sigma-Aldrich) after a 4-day culture Dissociatedcells were reacted with Allophycocyanin conjugated anti-mouse Flk1 antibody (eBioscience San Diego CA) followedby sorting with a magnetic cell separation system (MACS)(Miltenyi Biotec Bergisch Gladbach Germany) The purityof the sorted cell population was confirmed in a fluorescence-activated cell sorting (FACS) analysis (BD FACS Canto BDFranklin Lakes NJ) A portion of the separated cells wastransferred to RNAlater Solution (Invitrogen) followed bya freezing preservation for reverse-transcription PCR (RT-PCR)

23 In Vitro Induction of Endothelial Cell and Smooth MuscleCell Sorted Flk1+ cells were plated on gelatin-coated dishesin the differentiation medium For induction to an endothe-lial cell differentiation medium supplemented with 100 ngmL human VEGF

165(RampD Systems Minneapolis MN) was

used The medium was replaced every 2 days After a 4-dayculture cells were fixed with 4 PFA (Wako Pure ChemicalOsaka Japan) for 20 minutes at 4∘C

Then specimens were blocked with 3 goat serum (Vec-tor Laboratories Burlingame CA) with PBS (GIBCO) for30 minutes at room temperature and the following primaryantibodies were applied to the sections at 4∘C overnightrabbit polyclonal anti-120572-SMA antibody (1 200 Santa CruzBiotechnology Inc Santa Cruz CA) or rabbit polyclonalanti-PECAM antibody (1 200 Santa Cruz BiotechnologyInc) After washing the following secondary antibodies wereloaded for 1 hour at room temperature in a dark box AlexaFluor 488-coupled goat anti-rabbit IgG antibody (1 200Invitrogen) or Alexa Fluor 594-coupled goat anti-rabbitantibody (1 300 Invitrogen) Finally nucleus staining wasperformed using DAPI (Merck Tokyo Japan) The stainedsections were observed using a fluorescence microscope(BX51 Olympus Optical Tokyo Japan) and images wereobtained by a CCD camera (DP70 Olympus Optical)

24 Tube Formation Assay For the tube formation assaythe cells separated by MACS were plated on 4-well slides(Iwaki Glass Tokyo Japan) coated thickly with BD MatrigelBasementMembraneMatrix (250 120583L Becton-Dickinson CoFranklin Lakes NJ) at 3 times 104 cellscm2 following culture for48 hours in EBM-2 medium with EGM-2 Bulletkit (LONZABasel Switzerland) After the culture cells were fixedwith 4PFA and stained with Alexa Fluor 594 conjugated isolectinGS-IB4 (Invitrogen) and DAPI (Merck)The tube formationswere assessed with a fluorescence microscope (BX51 Olym-pus Optical) and images were obtained by a CCD camera(DP70 Olympus Optical)

25 Animals and Induction of Diabetes Five-week-old maleC57BL6 mice (Chubu Kagaku Shizai Nagoya Japan) withan initial body weight of 24ndash26 g were allowed to adapt to

the experimental animal facility for 7 days The animals werehoused in an aseptic animal roomunder controlled lightdarkand temperature conditions with food and water available adlibitum Diabetes was induced by intraperitoneal injection(ip) of streptozotocin (STZ) (150mgkg Sigma-Aldrich)Control mice received an equal volume of citric acid buffer(Wako) One week after STZ administration the mice withplasma glucose concentrations of gt16mmolL were definedas diabeticmice Twelve weeks after the induction of diabetes1 times 105 cellslimb of purified Flk1+ cells in 02mL saline wereinjected into the right thigh and soleus muscles of both thenormal and the diabetic mice The left hindlimb muscleswere treated with saline alone Four weeks later the followingparameters were bilaterally measured Before transplantationof Flk1+ cells fasting blood glucose levels and hemoglobinA1c were examined by a FreeStyle Freedom Glucose Meter(Nipro Osaka Japan) and a RAPIDIA Auto HbA1c-L assaykit using latex agglutination (Fujirebio Inc Tokyo Japan)respectively The Nagoya University Institutional AnimalCare and Use Committee approved the protocols of this exp-eriment

26 Measurement of Current Perception Thresholds (CPTs)Using a Neurometer To determine a nociceptive thresholdCPTs were measured in 12- and 16-week diabetic and age-matched normal mice using a CPTLAB Neurometer (Neu-rotron Denver CO) according to the method by Shibataet al [49] with minor modifications The electrodes (SRE-0405-8 Neurotron) for stimulation were attached to plantarsurfaces of the mice Each mouse was kept in a Ballmancage (Natsume Seisakusho Tokyo Japan) suitable for lightrestraint to keep the mice awake Three transcutaneous-sine-wave stimuli with different frequencies (2000 250 and 5Hz)were applied to the plantar surfaces of the mice to determinethe CPT of sensory perceptions (pressure pain and painand temperature resp) The intensity of each stimulationwas automatically increased in gradual increments (001mAfor 5 and 250Hz and 002mA for 2000Hz) The minimumintensity at which each mouse withdrew its paw was definedas the CPT Six consecutive measurements were conducted ateach frequency

27 Nerve Conduction Velocities (NCVs) After intraperi-toneal injection of sodium pentobarbital (5mg100 g Kyorit-suseiyaku Corporation Tokyo Japan) mice were placed on aheated pad in a roommaintained at 25∘C to ensure a constantrectal temperature of 37∘C Motor nerve conduction velocity(MNCV)was determined between the ankle and sciatic notchusing a Neuropak NEM-3102 instrument (Nihon-KodenOsaka Japan) as previously described [42 49 50] Thesensory nerve conduction velocity (SNCV) was measuredbetween the knee and ankle with retrograde stimulation

28 Blood Flows of Sciatic Nerve and Plantar Skin After eval-uation of the MNCV and SNCV blood flows of the sci-atic nerve and the plantar skin were measured by laserDoppler flowmetry (FLO-N1Omegawave Inc Tokyo Japan)To determine sciatic nerve blood flow the thigh skin of


Table 1 Primer sequences

Accession number Gene Forward primer (51015840rarr 31015840) Reverse primer (31015840rarr 51015840)NM 0010252503 Vegfa CAGGCTGCTGTAACGATGAA TTTCTTGCGCTTTCGTTTTTNM 0080062 Fgf2 GTGGATGGCGTCCGCGAGAA ACCGGTTGGCACACACTCCCNM 0088083 Pdgfa GAGATACCCCGGGAGTTGAT TCTTGCAAACTGCAGGAATGNM 0010481391 Bdnf GCCACCGGGGTGGTGTAAGC CATGGGTCCGCACACCTGGGNM 0011126981 Ngf GTGAAGATGCTGTGCCTCAA GCGGCCAGTATAGAAAGCTGNM 0102752 Gdnf CGGACGGGACTCTAAGATGA CGTCATCAAACTGGTCAGGANM 0011640341 Ntf3 CGAACTCGAGTCCACCTTTC AGTCTTCCGGCAAACTCCTTNM 1707862 Cntf GCAATCACCTCTGACCCTTC ACGGTAAGCCTGGAGGTTCTNM 0106122 Kdr GGCGGTGGTGACAGTATCTT GTCACTGACAGAGGCGATGANM 0115872 Tie1 TCAACTGCAGCTCCAAAATG TGACAGCTCTGTCCAAAACGNM 0136902 Tek AAGCATGCCCATCTGGTTAC GTAGGTAGTGGCCACCCAGANM 0010323781 Pecam1 ATGACCCAGCAACATTCACA AAAACGCTTGGGTGTCATTCNM 0098684 Cdh5 ACCGGATGACCAAGTACAGC TTCTGGTTTTCTGGCAGCTTNM 0102283 Flt1 CCAAGGCCTCCATGAAGATA ATACTGTCAGGGGCTGGTTGNM 0093092 T GGGGTATTCCCAATGGGGGTGGC GCCAGGCACTCCGAGGCTAGANM 0115272 Tal1 GTCTCTCAGCGAGAGCCGGGA CGCTCCGTCATCCTGGGGCATANM 0113551 Sfpi1 TCCCATGGTGCCACCCCACA TTGCTGCCTGTCTCCCCGTGNM 0011423351 Lmo2 TGGATGAGGTGCTGCAGATA GGATGCACAGAGACCATCCTNM 0073933 Actb CATCCGTAAAGACCTCTATGCCAAC ATGGAGCCACCGATCCACA

an anesthetized mouse was cut along femur and then anincision through the fascia was carefully performed to exposethe sciatic nerve Five minutes after this procedure the bloodflow was measured by a laser Doppler probe placed 1mmabove the nerve The skin blood flow was determined by themean of the flow at 3 spots During this measurement themouse was placed on a heated pad in a room maintained at25∘C to ensure a constant rectal temperature of 37∘C

29 Tissue Collection Four weeks after the transplantationmice were sacrificed by an overdose of pentobarbital (10mg100 g) Soleus muscles and plantar skin tissue were obtainedfrom normal and diabetic mice Some tissues were snap-frozen in liquid nitrogen followed by preservation at minus80∘Cuntil use and others were transferred to RNAlater Solution(Invitrogen) followed by freezing preservation for RT-PCR

210 Real-Time RT-PCR RNA was extracted from frozensamples of cells and tissues using ISOGEN (Nippon GeneToyama Japan) according to the manufacturerrsquos instructionsand the concentrations were quantified spectrophotometri-cally (NanoDrop ND-100 Nanodrop Tec Wilmington DE)DNA was digested with RNase-free DNase I (Wako PureChemical) which was then inactivated by incubation at 80∘Cfor 10min

Starting from 1 120583g of RNA cDNA was synthesizedusing ReverTra Ace (TOYOBO Osaka Japan) accordingto the manufacturerrsquos descriptions Primers were designedby Primer3 software (httpfrodowimitedu) and testedfor specificity with NCBI-BLAST (httpwwwncbinlmnihgovtoolsprimer-blast) Real-time quantitative RT-PCRwas performed andmonitored usingMx3000PQPCRSystem(STRATAGENE La Jolla California USA) The PCR master

mix was based on SYBR Green PCR Master Mix (AppliedBiosystems Foster City CA) In reaction wells cDNA sam-ples (5 120583L for a total volume of 25 120583L per reaction) wereanalyzed for the gene of interest All data were normalizedto an internal standard 120573-actinThe PCR products were ana-lyzed by agarose gel (Takara Bio Inc Otsu Japan)ethidiumbromide (Sigma-Aldrich) to confirm these predicted lengthsRelative quantity was calculated by the ΔΔCt method Theprimer sequences are shown in Table 1

211 Immunohistochemistry Four weeks after the transplan-tation themice were anesthetized with sodium pentobarbital(5mg100 g) and perfused with 50mL of 4 PFA fixativeAfter perfusion soleusmuscles and plantar skin sampleswereexcised andfixed in 4PFAat 4∘Covernight Specimenswereimmersed in PBS containing 20 sucrose (Wako) embeddedwithin an OCT compound (Sakura Finetechnical TokyoJapan) and cut into 5 120583m sections with a sliding cryostat(CM1800 Leica Microsystems AG Wetzler Germany) After5 minutes of microwave irradiation in citrate buffer (pH60 Wako) the cryostat sections were blocked with 3 goatserum with PBS for 30 minutes at room temperature Priorto being stored at 4∘C overnight the following primaryantibodies were applied to the sections rabbit polyclonalanti-protein-gene-product 95 (PGP 95) antibody (1 500Millipore Billerica MA) rabbit polyclonal anti-120572-SMA anti-body (1 200 Santa Cruz Biotechnology Inc) and anti-rabbitPECAM antibody (1 200 Santa Cruz Biotechnology Inc)After the sections were rinsed with PBS the following sec-ondary antibodies were loaded Alexa Fluor 594-coupled goatanti-rabbit IgG antibody (1 200 Invitrogen) Finally nucleusstaining was performed using DAPI (Merck) The stainedsections were observed using a fluorescence microscope


(BX51OlympusOptical) and imageswere obtained by aCCDcamera (DP70 Olympus Optical)

212 Capillary Number toMuscle Fiber Ratio Capillary num-ber tomuscle fiber ratio was calculated as previously reported[44 49] with minor modifications In brief the sections ofsoleusmuscles fixedwith PFAwere used for immunostainingThe vascular capillaries were stained by Alexa Fluor 594conjugate isolectin GS-IB4 (invitrogen) and counted undera fluorescence microscope (BX51 Olympus Optical) andimages were obtained by a CCD camera (DP70 OlympusOptical) The muscle fibers were concomitantly counted todetermine the capillary number to muscle fiber ratio Fivefields from each section were randomly selected for thecapillary counts

213 Measurement of Intraepidermal Nerve Fiber Densities(IENFDs) Nerve fibers stained with anti-PGP 95 antibodywere counted as previously reported [51] In brief the numberof nerve fibers was counted at the point of intersectionwith the basal membrane regardless of branching within thedermis or epidermis Six captured images from each sectionwere randomly selected for IENFDs IENFDs were derivedand expressed as epidermal nerve fiber numbers per lengthof an epidermal basal membrane (fibersmm)

214 Statistical Analysis All of the group values were exp-ressed as means plusmn SD Statistical analyses were made by one-way ANOVA with the Bonferroni correction for multiplecomparisons All analyses were performed by personnelunaware of the animal identities

3 Results

31 Flk1+ Cells Obtained from ES Cells Expressed Mesodermaland Angioblastic Markers After culturing 4 days on gelatinthe shape of the ES cell colonies changed from sphericalto planar and the population of cells expressing Flk1 wasestimated at 10ndash20 using FACS analysis (Figures 1(a) and1(b)) Purification mediated by Flk1 antibody was validatedand Flk1 mRNA expression in Flk1+ cells was measured atalmost 30 times higher than that in Flk1 negative (Flk1minus) cells(Figure 1(c)) RT-PCRanalyses showedbothmesodermal andangioblast markers that is Brachyury Flk1 Flt1 Tie1 Tie2VE-cad PU1 SCLTal1 and Lmo2 were expressed in Flk1+cells purified employing MACS (Figure 1(d))

32 Flk1+ Angioblastic Cells Expressed Angiogenic and Neu-rotrophic Factors RT-PCR analyses revealed that sortedangioblast-like cells induced from ES (hereafter referred toas ES-AB) expressed angiogenic and neurotrophic factorsVEGF-A PDGF-A FGF2 NGF brain-derived neurotrophicfactor (BDNF) glial cell line-derived neurotrophic factor(GDNF) Neurotrophin-3 (NT-3) and ciliary neurotrophicfactor (CNTF) (Figure 2) Expression of FGF2 NGF andGDNF was significantly increased in ES-ABs compared withFlk1minus cells It is widely known that Schwann cells pro-vide mechanical protection and paracrine effects relying on

the production of neurotrophic and angiogenic factorsTherefore we evaluated the relative expression levels of thesegrowth factors in ES-ABs compared with a mouse Schwanncell line IMS32 Although the expressions of GDNF NGFFGF2 PDGF-A and CNTF evidenced a significant decreasein ES-ABs compared with IMS32 the expressions of VEGF-A and NT-3 in ES-ABs were comparable to those in IMS32(Figure 2) Furthermore the expression of BDNF signifi-cantly increased in ES-ABs compared to IMS32 In additionwe compared the relative expression levels of these growthfactors between ES-ABs and mouse MSCs which have beenwidely applied in ischemic diseases with an expectation oftheir paracrine effects Although expression levels of GDNFand FGF2 in ES-ABs showed a significant decrease comparedwith those in PA6 cells a cell line of mouse MSCs there wasno significant difference in levels of VEGF-A NGF BDNFand CNTF expressions between ES-ABs and MSCs Addi-tionally therewere significant increases ofNT-3 andPDGF-Aexpression levels in ES-Abs compared with PA6 cells

33 ES-ABs Purified with MACS Differentiated to EndothelialCells and Smooth Muscle Cells In Vitro Following purifica-tion with MACS the ES-ABs were cultured on gelatin foradditional 4 days to assess their abilities to differentiate toendothelial cells and smooth muscle cells After the culturecells formed numerous colonies The use of the immunocy-tochemistry method revealed that some colonies expressedan endothelial marker PECAM and the other coloniesexpressed a smooth muscle cell marker 120572-SMA (Figure 3)

34 Structured Tube Formation and ldquoCobblestonerdquo MonolayerWere Constructed from ES-ABs To elucidate whether ES-ABs themselves are enabled to form blood vessels sortedES-AB and Flk1minus cells were separately seeded on MatrigelAfter culturing for 2 days on Matrigel cells were visualizedwith isolectin IB4 Although endothelial cells stained withIB4 were rarely induced from Flk1minus cells significantly heavynumbers of endothelial cells were induced from Flk1+ cellsIn addition a portion of these endothelial cells restructuredinto a vessel-like tube formation and the remaining portiondeployed into a cobblestone-like configuration (Figures 4(a)and 4(b))

35 Body Weights Blood Glucose Levels and HbA1c At 12weeks diabetic mice showed severe hyperglycemia (randomblood glucose levels119875 = 00003 HbA1c119875 lt 00001) and sig-nificantly decreased body weight gain (119875 = 0003) (Table 2)After the transplantation of ES-ABs there was no signifi-cant change between transplanted and nontransplanted mice(Table 2)

36 Some Blood VesselWalls and CapillariesWere Constructedwith Transplanted Cells To detect the distribution of trans-planted cells several mice were injected with GFP-expressingES-ABs To determine the existence of teratomas four weeksafter the transplantation the muscles brains hearts lungsand livers of these mice were collected and sectioned GFPpositive (GFP+) cells were nonexistent except in muscles of


Undifferentiated colony Differentiated colony

(a)

168 603

Presorted Postsorted

0 50K 100K 150K 200K 250KFSC

0

102

103

104

105

Flk1

-APC

0 50K 100K 150K 200K 250KFSC

0

102

103

104

105

Flk1

-APC

(b)

0

05

1

15

Flk1positive Flk1negative

Relat

ive e

xpre

ssio

n

lowastlowast

(c)

VE-cad

Flt-1

Tie-1

Tie-2T

SCLTal1

PU1

Lmo2

PECAM

Flk-1

(d)

Figure 1 Differentiation and purification of angioblast-like cells from mouse ES cells (a) Spherical colonies of ES cells (left) were flatlyoutspread (right) after 4 days of culture on gelatin Bar 30120583m (b) Fluorescence-activated cell sorting analyses showed that a populationof cells expressing Flk1 was estimated at 10ndash20 (left) and came up to 55ndash63 by magnet associated cell separation (right) (c) Reverse-transcription PCR analyses showed that Flk1 mRNA expression in population selected as Flk1 positive cell was almost 30 times higher thanthat in population selected as Flk1 negative cell (119875 lt 0001) (d) Reverse-transcribed PCR analyses showed both mesoderm and angioblastmarkers that is Brachyury (T) Flk1 Flt1 Tie1 Tie2 VE-cad PU1 SCLTal1 and Lmo2 which were expressed in Flk1 positive cells On theother hand PECAM which is considered as a marker of relatively differentiated endothelial cell was not detected in Flk1 positive cells afterthe 4 days of differentiation


0

05

1

15

VEGF-A

Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

0

2

4

6

8

10

12

FGF 2

lowastdaggerdagger

daggerdagger

Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

0

2

4

6

8

PDGF-A

daggerdaggerdaggerdagger

daggerdagger

Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

0

2

4

6

8

NGF

Relat

ive e

xpre

ssio

n

lowastdaggerdagger

daggerdagger

daggerdagger

Flk1+

Flk1minusPA6

IMS32

0

05

1

15

2

BDNF

daggerdagger


Flk1+

Flk1minusPA6

IMS32

Rela

tive e

xpre

ssio

n

0

5

10

15

CNTF

daggerdagger daggerdaggerdaggerdagger

Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

Figure 2 Continued


0

10

20

30

40

50

60

GDNF

Relat

ive e

xpre

ssio

n

lowast

daggerdagger

daggerdagger

daggerdagger

Flk1+

Flk1minusPA6

IMS32

0

05

1

15

2

NT-3

Relat

ive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

Figure 2 Transcript levels of angiogenic and neurotrophic factors in angioblast-like cells induced from ES cells (ES-ABs) Sorted ES-ABsexpressed angiogenic and neurotrophic factors VEGF-A PDGF-A FGF2 NGF brain-derived neurotrophic factor (BDNF) glial cell line-derived neurotrophic factor (GDNF) Neurotrophin-3 (NT-3) and ciliary neurotrophic factor (CNTF) Each expression level of the factorscompared Flk1 positive cells with Flk1 negative cells PA6 cells and IMS32 cells PA6 a cell line of mouse mesenchymal stem cell and IMS32 acell line of immortalized mouse Schwann cell Flk1+ Flk1 positive cells and Flk1minus Flk1 negative cells lowast119875 lt 005 versus Flk1minus cells 119875 lt 0005versus PA6 cells and daggerdagger119875 lt 0005 versus IMS32 cells

120572-SMA

(a)

PECAM

(b)

Figure 3 In vitro differentiation potential of angioblast-like cells induced from ES cells (ES-ABs) After a 4-day culture on gelatin ES-ABsformed many colonies Immunocytochemistry a smooth muscle cell marker 120572-SMA was detected at some colonies (a) and an endothelialmarker PECAM was at the other colonies (b) Red 120572-SMA and green PECAM Bar 100 120583m

the transplanted hindlimbs GFP+ cells resided in the gapsbetween muscle fibers and some GFP+ cells were observed inthe walls of blood vessels interspersedwithGFP negative cells(Figure 5) Some of the GFP+ cells foundwithin the constructof the vessel walls were smooth muscle cells expressing 120572-SMA (Figure 5(a)) while the others were endothelial cellsexpressing PECAM (Figure 5(b))

37 Capillary Number to Muscle Fiber Ratio The vascula-tures were visualized by Alexa594-conjugated isolectin IB4

a marker for endothelial cells (Figure 6(a)) Quantitativeanalyses revealed that the capillary number to muscle fiberratios in the saline-injected diabetic mice were significantlyreduced compared with those in normal mice (119875 = 00004)(Figure 6(b)) Transplantation of ES-ABs significantly aug-mented the ratio in ES-AB transplanted limbs (ES-ABipsi)compared with the ratio in the saline-injected side limbs(ES-ABcontra) in diabetic mice (119875 = 00276) (Figure 6(b))Transplantation of ES-ABs into normal mice showed no sig-nificant differences (119875 = 09609) (Figure 6(b))


Flk1 positive Flk1 negative

(a)


(b)

Figure 4 Tube forming assay of angioblast-like cells induced from ES cells (ES-ABs) on Matrigel To elucidate whether ES-ABs themselvesare enabled to form blood vessels sorted ES-ABs and Flk1 negative cells were respectively seeded on Matrigel Two days later cells werevisualized with isolectin IB4 a stain of endothelial cell (a) Although endothelial cells stained with IB4 were rarely induced from Flk1 negativecells (right) heavy numbers of endothelial cells were induced from ES-ABs and deployed cobblestone-like pattern (left) Bar 100120583m Redisolectin IB4 (b) In ES-AB culture a part of cells stained with IB4 structured vessel like tube formation (left 2 images) On the other handcells forming a line were not stained with IB4 in culture of Flk1 negative cells (right 2 images) Bar 100 120583m Red isolectin IB4 and blue DAPI

Table 2 Body weight blood glucose and HbA1c levels

Normal mice Diabetic mice

Before transplantation After transplantation Before transplantation After transplantationSaline ES-AB Saline ES-AB

HbA1c () 40 plusmn 01 41 plusmn 02 41 plusmn 02 73 plusmn 14 77 plusmn 21 77 plusmn 25

Blood glucose (mmolL) 91 plusmn 16 92 plusmn 16 91 plusmn 16 231 plusmn 28 258 plusmn 80 242 plusmn 90

Body weight (g) 306 plusmn 27 313 plusmn 30 319 plusmn 37 261 plusmn 31 253 plusmn 32 276 plusmn 16

Results are means plusmn SD ES-AB mice transplanted angioblast-like cells induced from embryonic stem cell 119875 lt 0005 versus pretreatment normal mice119875 lt 0005 versus posttreatment normal mice

38 Transplantation of ES-ABs Increased Blood Flow in theSciatic Nerve and Plantar Skin After 12 weeks of diabetesthe blood flow in both the sciatic nerve and plantar skin indiabetic mice decreased significantly compared with that innormal mice (sciatic nerve 119875 = 00431 plantar skin 119875 =00359) and the decrease was ameliorated by transplantationof ES-ABs (sciatic nerve 119875 = 00003 represents DM-S versusDM-ES-ABipsi plantar skin 119875 = 00211 represents DM-Sversus DM-ES-ABipsi) However administration of ES-ABsdid not alter the blood flow in normal mice (sciatic nerve119875 = 09525 represents N-S versus N-ES-ABipsi plantar skin119875 = 02523 represents N-S versus ES-ABipsi) (Figure 7)

39 Reduced Sensory Perception in Diabetic MiceWas Amelio-rated by ES-AB Transplantation After 12 weeks of diabetesCPTs at 5 250 and 2000Hz had significantly increased com-pared with those in normal mice (5Hz 119875 = 0015 250Hz

119875 = 0019 2000Hz 119875 = 0028) representing hypoalgesiain diabetic mice The fourth week after the transplantation ofES-ABs these deficits in sensation had significantly improvedin diabetic mice compared with saline-treated diabetic con-trols (5Hz119875 = 0071 represents DM-S versus DM-ES-ABipsi250Hz 119875 = 00018 represents DM-S versus DM-ES-ABipsi2000Hz119875 lt 00001 represents DM-S versus DM-ES-ABipsi)The transplantation of ES-ABs into normal mice did notinduce significant changes in CPTs (5Hz 119875 = 0934 repre-sents N-S versus N-ES-ABipsi 250Hz 119875 = 0212 representsN-S versus N-ES-ABipsi 2000Hz 119875 = 0260 represents N-Sversus N-ES-ABipsi) (Figure 8)

310 ES-ABs Improved Delayed NCVs in Diabetic MiceMNCVand SNCVof diabeticmice were significantly delayedcompared with those of normal mice (Figure 9) The delayin MNCV and SNCV was significantly restored by ES-AB


VV V

MMM

GFP Merge120572-SMA

(a)

GFP MergePECAM

(b)

Figure 5The transplanted angioblast-like cells induced from ES cells (ES-ABs) constructed blood vessel walls and capillaries Immunohisto-chemical staining revealed that transplanted GFP-expressing ES-ABs were still engrafted 4 weeks after transplantation (a)The engrafted cellsdistributed in vessel wall between hindlimb muscle fibers and coexpressed 120572-SMA Green GFP and red 120572-SMA M muscle and V lumenof a blood vessel Bar 20 120583m (b) Some of GFP positive cells resided in gaps between soleus muscle fibers and coexpressed PECAM GreenGFP and red PECAM White arrows GFP negative capillaries and yellow arrowheads GFP positive capillaries Bar 20 120583m

DM -saline DM-AB

(a)

1

12

14

16

18

2

Nondiabetic Diabetic

Capi

llary

mus

cle fi

ber r

atio

lowast

dagger

S AB+ ABminus S AB+ ABminus

(b)

Figure 6 Capillary number to muscle fiber ratios in soleus muscles (a) The vasculatures were visualized by Alexa594-conjugated isolectinIB4 in soleus muscles More capillaries existed in soleus muscle of transplanted diabetic mice (right) compared to those of nontransplantedcontrol diabetic mice (left) Red isolectin IB4 Bar 50 120583m DM-saline saline-injected limb in diabetic mice and DM-AB limbs transplantedangioblast-like cell derived fromES cells in diabeticmice (b) Quantitative analyses revealed that the capillary number tomuscle fiber ratios inthe saline-injected sides of diabetic mice were significantly reduced compared with those of normal mice (N) Transplantation of angioblast-like cell derived from ES cells (ES-ABs) significantly augmented the ratios in transplanted limbs compared with those in saline-injected sidelimbs in diabetic mice (DM) Transplantation of ES-ABs into normal mice showed no significant differences S saline treated limbs AB+limbs transplanted ES-ABs and ABminus contralateral limbs transplanted ES-ABs Results are means plusmn SD lowast119875 lt 005 versus S in N (N-S) anddagger

119875 lt 005 versus S in DM (DM-S)119873 = 4 in N-S and 119899 = 3 in DM-S (119875 = 00004)119873 = 3 of AB+ in DM and 119899 = 5 of ABminus in DM (119875 = 00276represents DM-S versus AB+ in DM)119873 = 4 of AB+ in N and 119899 = 4 of ABminus in N (119875 = 09609 represents N-S versus AB+ in N)


15

20

25

30

After treatmentN DM

N DMBefore treatment

dagger

Plantar skin blood flow


lowast

(mL

min

100

g)

(a)

10

15

20

25

30

After treatmentN DM


Sciatic nerve blood flow

dagger

lowast


(mL

min

100

g)

(b)

Figure 7 Blood flow of sciatic nerves and plantar skins were ameliorated by transplantation of angioblast-like cell derived from ES cells(ES-ABs) At a time point of 12 weeks of diabetes (before treatment) blood flows of plantar skins (a) and sciatic nerves (b) in diabeticmice significantly decreased compared with those in normal mice and 4 weeks after transplantation (after treatment) the decreases wereameliorated in transplanted limbs of diabetic mice However administration of ES-ABs did not alter blood flow in those of normal miceN normal mice limbs DM diabetic mice limbs S saline-injected limbs AB+ limbs transplanted ES-ABs and ABminus contralateral limbstransplanted ES-ABs Results are means plusmn SD 119875 lt 005 versus pretreatment N lowast119875 lt 005 versus posttreatment S-treated N dagger119875 lt 005 versusposttreatment S-treated DM 119899 = 4ndash6 in each pretreatment group and 119899 = 7ndash10 in each posttreatment group

transplantation (MNCV 119875 = 00289 represents DM-S versusDM-ES-ABipsi SNCV 119875 = 00201 represents DM-S versusDM-ES-ABipsi) (Figure 9) However administration of ES-ABs did not alter NCVs in normal mice (MNCV 119875 = 07604represents N-S versus ES-ABipsi SNCV 119875 = 06016 rep-resents N-S versus N-ES-ABipsi) (Figure 9)

311 Nerve Fibers in the Epidermis Were Preserved by ES-ABsUtilization of fluorescent imaging showed that nerve fibers inthe plantar skin were evidenced in both the epidermis andthe dermis (Figure 10(a)) After 12 weeks of diabetes IENFDsdecreased in diabetic mice (119875 = 00011) (Figure 10(b)) Thisdecreasewas significantly ameliorated by ES-ABs (119875 = 00355represent DM-S versus DM-ES-ABipsi) Administration ofES-ABs did not change IENFDs in normal mice (119875 = 03212represents N-S versus N-ES-ABipsi) (Figure 10(b))

4 Discussion

The present study demonstrated that angioblast-like cellscould be obtained from mouse ES cells and the transplan-tation of the angioblast-like cells ES-AB improved severalphysiological impairments in DPN hypoalgesia delayedNCVs and reduced blood flow in sciatic nerves and plantarskin The immunohistological assessment revealed that thecapillary number to muscle fiber ratios increased in skeletalmuscles of the transplanted hindlimbs and intraepidermalnerve fiber densitieswere also ameliorated in the transplantedplantar skin samples Four weeks after the transplantationtransplanted cells maintained their viabilities and differen-tiated to endothelial cells and smooth muscle cells around

the injected sites Moreover several transplanted cells con-structed chimeric blood vessels with recipient cells

One of the most significant characteristics of the currenttransplantation therapy was the utilization of the heteroge-neous graft cells which were at the early stage of differentia-tion of the pluripotent stem cells We expected some advan-tages of a utilization of cells at the early stage for example amultipotent differentiation ability a high migratory capacityhigh adaptability tomicroenvironments of host tissues a highproliferation ability and a capacity to produce various growthfactors In previous studies Flk1+ cells derived from mousepluripotent stem cells have been shown to differentiate intoa number of cell types including endothelial cells smoothmuscle cells and cardiomyocytes in vitro [47 52] In the cur-rent study it was demonstrated that the adhesive Flk1+ cellsderived frommouse ES cells had amultipotent differentiationability into endothelial cells and smooth muscle cells similarto the previous studies and expressed several angioblastmarkers including Flt1 Tie2 and VE-cad Furthermore tra-nsplanted cells engrafted themselves in gaps between musclefibers around transplanted sites and were incorporated intothe tissue structures of recipient animals These results indi-cate that some of the therapeutic effects obtained in this studywere mediated through the differentiation of ES-ABs

Additionally we demonstrated that ES-ABs secreted awide array of growth factors such as FGF2 VEGF PDGFNGF BDNF GDNF NT-3 and CNTF In previous studiesMSC transplantation has been performed in the field of ische-mic diseases and it has been suggested that its plausibleeffects would be mediated largely through paracrine actionsof locally released arteriogenic cytokines including FGF2 and


10

12

14

16

18

20

22

After treatmentN DM


dagger

CPT with 2000 Hz(m

A)


lowast

(a)

06

07

08

09

10

11

After treatmentN DM


dagger

CPT with 250 Hz

(mA

)


lowast

(b)

07

08

09

10

11

12

13

After treatmentN DM


dagger

CPT with 5 Hz

(mA

)


lowast

(c)

Figure 8 Impaired sensory perceptions in diabetic mice were ameliorated by angioblast-like cells induced from ES cells (ES-ABs)transplantation Stimuli with frequencies of 5Hz (a) 250Hz (b) and 2000Hz (c) evoked excitations of C-fiber A120575-fiber and A120573-fiberrespectively Before cell transplantation CPTs with all kinds of stimuli in diabetic mice had significantly increased compared with thosein normal mice representing hypoalgesia in diabetic mice Four weeks after the transplantation of ES-ABs these deficits in sensation weresignificantly improved in diabetic mice compared with saline-treated diabetic controls The transplantation of ES-ABs into normal mice didnot induce significant changes in CPTs N normal mice limbs DM diabetic mice limbs S saline-injected limbs AB+ limbs transplantedES-ABs and ABminus contralateral limbs transplanted ES-ABs Results are means plusmn SD 119875 lt 005 versus pretreatment N lowast119875 lt 005 versusposttreatment S-treated N and dagger119875 lt 005 versus posttreatment S-treated DM 119899 = 8ndash10 in each nondiabetic group and 119899 = 7ndash9 in eachdiabetic group

VEGF [53ndash55] Furthermore FGF2 andVEGFhave been rep-orted to have neurosupportive effects [56 57] In our studyFGF2 VEGF and PDGF were expressed in ES-ABs andin particular PDGF expression of ES-ABs was significantlyhigher than that of MSCs In addition to these conventionalangiogenic factors neurotrophic factors such asNGF BDNFGDNF NT-3 and CNTF were expressed in ES-ABs In theperipheral nervous system these neurotrophic factors are

predominantly secreted by Schwann cells Although NGFand GDNF expressions in ES-ABs were significantly lowerthan those in Schwann cells NT-3 and BDNF expressionsin ES-ABs were favorably compared with those in Schwanncells These observations suggest that the interventionaleffects of ES-AB transplantation on DPN demonstrated inthis study would be partially achieved by the paracrineactions of growth factors and these factors would work as


30

35

40

45

50M

NCV

(ms

)

After treatmentN DM

S AB+ ABminus S AB+ ABminusN DMBefore treatment

dagger

lowast

(a)

25

30

35

40

45

$

After treatmentN DM


dagger

SNCV

(ms

)

lowast


(b)

Figure 9 Transplantation of angioblast-like cells induced from ES cells (ES-ABs) improved delayed nerve conduction velocities (NCVs)in diabetic mice Motor nerve conduction velocity (MNCV) (a) and sensory nerve conduction velocity (SNCV) (b) of diabetic mice weresignificantly delayed compared with those of normal mice after 12 weeks duration of diabetes The delays in MNCV and SNCV weresignificantly restored by ES-ABs transplantation However administrations of ES-ABs did not alter NCVs in normal mice N normal micelimbs DM diabetic mice limbs S saline-injected limbs AB+ limbs transplanted ES-ABs and ABminus contralateral limbs transplanted ES-ABsResults are means plusmn SD 119875 lt 005 versus pretreatment N lowast119875 lt 005 versus posttreatment S-treated N and dagger119875 lt 005 versus posttreatmentS-treated DM 119899 = 6 in each pretreatment group and 119899 = 7ndash10 in each posttreatment group

both angiogenic and neurotrophic factors However furtherstudies to elucidate which growth factor is relevant to theparacrine action should be considered

Our study revealed that the capillary number to musclefiber ratios in hindlimb skeletal muscles were significantlylower in diabetic mice than in normal mice which is consis-tent with human studies [58] and the ES-AB transplantationmight restore the decrease through induction and construc-tion of neovascularization The level of therapeutic vasculo-genesis prompted by the ES-ABs transplantation was similarto that prompted by EPC transplantation as we previouslyreported [44] and the differentiation and incorporation ofES-ABs to vessels were consistent with this report Howeverit remains unknown why ES-AB transplantation did not haveany effect on normal mice It can be speculated that diabeticconditions such as hyperglycemia and impaired local micro-circulationmight cause or require the transplanted ES-ABs tomore strongly produce growth factors and promote vasculo-genesis Further studies are in progress in our laboratory

The impairment of nerve blood flow (NBF) is one ofthe major pathogenic factors in the development of DPNAlthough there are some disagreements as to whetherreduced NBF can account for diabetic neuropathy [59 60]this notion is clinically and experimentally supported bymany other studies which reported that the amelioration ofNBF by various treatments improved impaired nerve func-tions [61ndash63] The beneficial effects of ES-AB transplantationon NCVs and NBFs in this study could be comparableto those of the therapeutic modalities reported previouslyReduced capillary number to muscle fiber ratios and bloodflows of plantar skin in diabetic mice were ameliorated bytransplantation of ES-ABs Therefore these ameliorations of

NCVs and NBFs may be due in part to vasculogenesis ren-dered byES-ABs and asmentioned above paracrine effects ofangiogenetic and neurotrophic factors produced by ES-ABsHowever well-conceived studies aiming to clarify molecularmechanisms of ES-Abs on each cell type in vascular andnervous systems should be performed in the future Further-more it should be also considered to determine the durationof the beneficial effects using a lengthened intervention byrepetitive transplantations

In the present study we evaluated sensory nerve func-tions using the Neurometer CPTLAB The Neurometer is adevice that measures selectively the CPTs in three classes ofafferent fibers by applying transcutaneous sine-wave electricstimuli to the skin at three frequencies of 2000 250 and5Hz via surface electrodes at a current intensity in therange of 001ndash99mA The pulses at 2000 250 and 5Hzmainly stimulate large myelinated (A120573-) small myelinated(A120575-) and small unmyelinated (C-) fibers respectively TheNeurometer is now widely used clinically to evaluate theeffects of analgesic drugs and peripheral nerve function invarious painful neuropathies including DPN [50 64ndash66]Twelve weeks after the induction of diabetes diabetic miceshowed hypoalgesia in CPTs at all stimuli with differentialfrequencies and transplantation of ES-ABs improved theseabnormalities The aforementioned results were consistentwith the results regarding the impaired IENFDs restored bythe transplantation of ES-ABs in diabetic mice

In conclusion we have demonstrated the beneficial effectsof transplantation of angioblast-like cells derived from EScells on DPN This ES-AB is relatively easy to obtain and canbe repeatedly expanded to sufficient numbers for cell therapyAlthough further studies designed to reveal other useful


DM-ES-AB

DM-saline

(a)

25

30

35

40

45

50

IEN

F (fi

bers

mm

)

After treatmentN DM


dagger


lowastlowast

(b)

Figure 10 Intraepidermal nerve fibers (IENFs) were preserved by angioblast-like cells induced from ES cells (ES-ABs) (a) IENFs in plantarskins were visualized with PGP95 antibody (red) After 15 weeks of diabetes IENF densities were decreased in diabetic mice (upper) and theimpairmentwas restored by ES-ABs transplantation (lower) DM-saline plantar skin of saline-injected limbs in diabeticmice andDM-ES-ABplantar skin of limbs transplanted ES-ABs in diabetic mice White arrowheads IENFs (b) Quantification of IENF densities demonstratedthat IENFs were significantly decreased in diabetic mice compared with those in normal mice at 12 weeks of diabetes and this decreasewas significantly ameliorated by transplantation of ES-ABs However administration of ES-ABs did not change IENF densities in normalmice N normal mice limbs DM diabetic mice limbs S saline-injected limbs AB+ limbs transplanted ES-ABs and ABminus contralaterallimbs transplanted ES-Abs Results are means plusmn SD 119875 lt 0005 versus pretreatment N lowastlowast119875 lt 0005 versus posttreatment S-treated N anddagger

119875 lt 005 versus posttreatment S-treated DM 119899 = 3-4 in each group

aspects of ES-AB transplantation on DPNmight be requiredtransplantation of angioblast-like cells induced from pluripo-tent cells appears promising as a novel therapeutic strategy forDPN

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Acknowledgments

This research was supported in part by Grant-in-Aid for Sci-entific Research (21592506) from the Ministry of EducationCulture Sports Science and Technology (MEXT) and in partby the ldquoStrategic Research AGU-Platform Formation (2008ndash2012)rdquo Project for PrivateUniversitiesmatching fund subsidyfrom MEXT of Japan The authors thank Michiko YamadaKeiko Shimamoto Naoko Furukawa Chikako Nagase andMayumi Katagiri for technical assistance and they thankMinoru Tanaka in the Division for Medical Research Engi-neering Nagoya University Graduate School ofMedicine formaintenance of the FACS instrument

References

[1] F Ismail-Beigi T Craven M A Banerji et al ldquoEffect of inten-sive treatment of hyperglycaemia onmicrovascular outcomes intype 2 diabetes an analysis of the ACCORD randomised trialrdquoThe Lancet vol 376 no 9739 pp 419ndash430 2010

[2] TheDiabetes Control and Complications Trial Research GroupldquoThe effect of intensive treatment of diabetes on the develop-ment and progression of long-term complications in insulin-dependent diabetes mellitusrdquo The New England Journal ofMedicine vol 329 no 14 pp 977ndash986 1993

[3] R Turner ldquoIntensive blood-glucose control with sulphony-lureas or insulin compared with conventional treatment andrisk of complications in patients with type 2 diabetes (UKPDS33)rdquoThe Lancet vol 352 no 9131 pp 837ndash853 1998

[4] D K Wilson K M Bohren K H Gabbay and F A QuiocholdquoAn unlikely sugar substrate site in the 165 A structure ofthe human aldose reductase holoenzyme implicated in diabeticcomplicationsrdquo Science vol 257 no 5066 pp 81ndash84 1992

[5] F G Soriano L Virag P Jagtap et al ldquoDiabetic endothelial dys-function the role of poly(ADP-ribose) polymerase activationrdquoNature Medicine vol 7 no 1 pp 108ndash113 2001

[6] I Giardino D Edelstein and M Brownlee ldquoNonenzymaticglycosylation in vitro and in bovine endothelial cells altersbasic fibroblast growth factor activity a model for intracellularglycosylation in diabetesrdquo The Journal of Clinical Investigationvol 94 no 1 pp 110ndash117 1994

[7] K Horie T Miyata K Maeda et al ldquoImmunohistochemicalcolocalization of glycoxidation products and lipid peroxidationproducts in diabetic renal glomerular lesions Implication forglycoxidative stress in the pathogenesis of diabetic nephropa-thyrdquo The Journal of Clinical Investigation vol 100 no 12 pp2995ndash3004 1997

[8] M Lu M Kuroki S Amano et al ldquoAdvanced glycation endproducts increase retinal vascular endothelial growth factorexpressionrdquoThe Journal of Clinical Investigation vol 101 no 6pp 1219ndash1224 1998

[9] A M Schmidt O Hori J X Chen et al ldquoAdvanced glycationendproducts interacting with their endothelial receptor induceexpression of vascular cell adhesion molecule-1 (VCAM-1)in cultured human endothelial cells and in mice a potentialmechanism for the accelerated vasculopathy of diabetesrdquo TheJournal of Clinical Investigation vol 96 no 3 pp 1395ndash14031995

[10] M Shinohara P J Thornalley I Giardino et al ldquoOverexpres-sion of glyoxalase-I in bovine endothelial cells inhibits intra-cellular advanced glycation endproduct formation and prevents

hyperglycemia-induced increases in macromolecular endocy-tosisrdquo The Journal of Clinical Investigation vol 101 no 5 pp1142ndash1147 1998

[11] P A Craven R K Studer and F RDeRubertis ldquoImpaired nitricoxide-dependent cyclic guanosine monophosphate generationin glomeruli from diabetic rats Evidence for protein kinase C-mediated suppression of the cholinergic responserdquo Journal ofClinical Investigation vol 93 no 1 pp 311ndash320 1994

[12] H Ishii M R Jirousek D Koya et al ldquoAmelioration of vasculardysfunctions in diabetic rats by an oral PKC 120573 inhibitorrdquoScience vol 272 no 5262 pp 728ndash731 1996

[13] D Koya and G L King ldquoProtein kinase C activation and thedevelopment of diabetic complicationsrdquo Diabetes vol 47 no 6pp 859ndash866 1998

[14] P Xia R M Kramer and G L King ldquoIdentification of themechanism for the inhibition of Na+K+-adenosine triphos-phatase by hyperglycemia involving activation of protein kinaseC and cytosolic phospholipase A2rdquo The Journal of ClinicalInvestigation vol 96 no 2 pp 733ndash740 1995

[15] V Kolm-Litty U Sauer A Nerlich R Lehmann and E DSchleicher ldquoHigh glucose-induced transforming growth factorbeta1 production is mediated by the hexosamine pathway inporcine glomerular mesangial cellsrdquo The Journal of ClinicalInvestigation vol 101 no 1 pp 160ndash169 1998

[16] K A Kles and A I Vinik ldquoPathophysiology and treatment ofdiabetic peripheral neuropathy the case for diabetic neurovas-cular function as an essential componentrdquo Current DiabetesReviews vol 2 no 2 pp 131ndash145 2006

[17] DW Zochodne ldquoDiabetes mellitus and the peripheral nervoussystem manifestations and mechanismsrdquo Muscle and Nervevol 36 no 2 pp 144ndash166 2007

[18] S Tesfaye N Chaturvedi S E M Eaton et al ldquoVascular riskfactors and diabetic neuropathyrdquo The New England Journal ofMedicine vol 352 no 4 pp 341ndash350 2005

[19] C Giannini and P J Dyck ldquoUltrastructural morphometricabnormalities of sural nerve endoneurial microvessels in dia-betes mellitusrdquo Annals of Neurology vol 36 no 3 pp 408ndash4151994

[20] H Yasuda and P J Dyck ldquoAbnormalities of endoneurialmicrovessels and sural nerve pathology in diabetic neuropathyrdquoNeurology vol 37 no 1 pp 20ndash28 1987

[21] N E Cameron M A Cotter V Archibald K C Dinesand E K Maxfield ldquoAnti-oxidant and pro-oxidant effects onnerve conduction velocity endoneurial blood flow and oxygentension in non-diabetic and streptozotocin-diabetic ratsrdquo Dia-betologia vol 37 no 5 pp 449ndash459 1994

[22] L J Coppey J S Gellett E P Davidson J A Dunlap DD Lund and M A Yorek ldquoEffect of antioxidant treatmentof streptozotocin-induced diabetic rats on endoneurial bloodflowmotor nerve conduction velocity and vascular reactivity ofepineurial arterioles of the sciatic nerverdquo Diabetes vol 50 no8 pp 1927ndash1937 2001

[23] T Asahara T Murohara A Sullivan et al ldquoIsolation of putativeprogenitor endothelial cells for angiogenesisrdquo Science vol 275no 5302 pp 964ndash967 1997

[24] Y Lin D J Weisdorf A Solovey and R P Hebbel ldquoOriginsof circulating endothelial cells and endothelial outgrowth frombloodrdquo The Journal of Clinical Investigation vol 105 no 1 pp71ndash77 2000

[25] M Peichev A J Naiyer D Pereira et al ldquoExpression ofVEGFR-2 and AC133 by circulating human CD34+ cells identifies


a population of functional endothelial precursorsrdquo Blood vol95 no 3 pp 952ndash958 2000

[26] M C Yoder L E Mead D Prater et al ldquoRedefining endothe-lial progenitor cells via clonal analysis and hematopoieticstemprogenitor cell principalsrdquo Blood vol 109 no 5 pp 1801ndash1809 2007

[27] M Massa V Rosti M Ferrario et al ldquoIncreased circulatinghematopoietic and endothelial progenitor cells in the earlyphase of acute myocardial infarctionrdquo Blood vol 105 no 1 pp199ndash206 2005

[28] S Shintani T Murohara H Ikeda et al ldquoMobilization ofendothelial progenitor cells in patients with acute myocardialinfarctionrdquo Circulation vol 103 no 23 pp 2776ndash2779 2001

[29] J George A Afek A Abashidze et al ldquoTransfer of endothelialprogenitor and bone marrow cells influences atheroscleroticplaque size and composition in apolipoprotein E knockoutmicerdquoArteriosclerosisThrombosis andVascular Biology vol 25no 12 pp 2636ndash2641 2005

[30] Y-S Yoon A Wecker L Heyd et al ldquoClonally expanded novelmultipotent stem cells from human bone marrow regeneratemyocardiumaftermyocardial infarctionrdquoThe Journal of ClinicalInvestigation vol 115 no 2 pp 326ndash338 2005

[31] M L Balestrieri M Rienzo F Felice et al ldquoHigh glucosedownregulates endothelial progenitor cell number via SIRT1rdquoBiochimica et Biophysica Acta vol 1784 no 6 pp 936ndash9452008

[32] G P Fadini MMiorinM Facco et al ldquoCirculating endothelialprogenitor cells are reduced in peripheral vascular complica-tions of type 2 diabetesmellitusrdquo Journal of the AmericanCollegeof Cardiology vol 45 no 9 pp 1449ndash1457 2005

[33] N Krankel V Adams A Linke et al ldquoHyperglycemia reducessurvival and impairs function of circulating blood-derivedprogenitor cellsrdquo Arteriosclerosis Thrombosis and VascularBiology vol 25 no 4 pp 698ndash703 2005

[34] C J M Loomans E J P de Koning F J T Staal et alldquoEndothelial progenitor cell dysfunction a novel concept inthe pathogenesis of vascular complications of type 1 diabetesrdquoDiabetes vol 53 no 1 pp 195ndash199 2004

[35] O M Tepper R D Galiano J M Capla et al ldquoHumanendothelial progenitor cells from type II diabetics exhibitimpaired proliferation adhesion and incorporation into vascu-lar structuresrdquoCirculation vol 106 no 22 pp 2781ndash2786 2002

[36] O Awad C Jiao N Ma M Dunnwald and G C SchattemanldquoObese diabeticmouse environment differentially affects primi-tive andmonocytic endothelial cell progenitorsrdquo StemCells vol23 no 4 pp 575ndash583 2005

[37] K A Gallagher Z-J Liu M Xiao et al ldquoDiabetic impairmentsin NO-mediated endothelial progenitor cell mobilization andhoming are reversed by hyperoxia and SDF-1120572rdquo Journal ofClinical Investigation vol 117 no 5 pp 1249ndash1259 2007

[38] E J Marrotte D-D Chen J S Hakim and A F ChenldquoManganese superoxide dismutase expression in endothelialprogenitor cells accelerateswoundhealing in diabeticmicerdquoTheJournal of Clinical Investigation vol 120 no 12 pp 4207ndash42192010

[39] P Koch Z Kokaia O Lindvall and O Brustle ldquoEmerging con-cepts in neural stem cell research autologous repair and cell-based disease modellingrdquo The Lancet Neurology vol 8 no 9pp 819ndash829 2009

[40] D G Phinney and D J Prockop ldquoConcise review mesenchy-mal stemmultipotent stromal cells the state of transdifferen-tiation and modes of tissue repairmdashcurrent viewsrdquo Stem Cellsvol 25 no 11 pp 2896ndash2902 2007

[41] C Stamm B Westphal H-D Kleine et al ldquoAutologous bone-marrow stem-cell transplantation for myocardial regenerationrdquoThe Lancet vol 361 no 9351 pp 45ndash46 2003

[42] M Chopp and Y Li ldquoTreatment of neural injury with marrowstromal cellsrdquo The Lancet Neurology vol 1 no 2 pp 92ndash1002002

[43] K le Blanc I Rasmusson B Sundberg et al ldquoTreatment ofsevere acute graft-versus-host disease with third party hap-loidentical mesenchymal stem cellsrdquo The Lancet vol 363 no9419 pp 1439ndash1441 2004

[44] K Naruse Y Hamada E Nakashima et al ldquoTherapeuticneovascularization using cord blood-derived endothelial pro-genitor cells for diabetic neuropathyrdquo Diabetes vol 54 no 6pp 1823ndash1828 2005

[45] J E Ferguson III R W Kelley and C Patterson ldquoMechanismsof endothelial differentiation in embryonic vasculogenesisrdquoArteriosclerosis Thrombosis and Vascular Biology vol 25 no11 pp 2246ndash2254 2005

[46] R L Williams D J Hilton S Pease et al ldquoMyeloid leukaemiainhibitory factor maintains the developmental potential ofembryonic stem cellsrdquo Nature vol 336 no 6200 pp 684ndash6871988

[47] G Narazaki H Uosaki M Teranishi et al ldquoDirected andsystematic differentiation of cardiovascular cells from mouseinduced pluripotent stem cellsrdquo Circulation vol 118 no 5 pp498ndash506 2008

[48] KWatabe T Fukuda J Tanaka H Honda K Toyohara andOSakai ldquoSpontaneously immortalized adultmouse Schwann cellssecrete autocrine and paracrine growth-promoting activitiesrdquoJournal of Neuroscience Research vol 41 no 2 pp 279ndash2901995

[49] T Shibata KNaruseH Kamiya et al ldquoTransplantation of bonemarrow-derived mesenchymal stem cells improves diabeticpolyneuropathy in ratsrdquo Diabetes vol 57 no 11 pp 3099ndash31072008

[50] J J Katims E H Naviasky L K Y Ng M Rendell and M LBleecker ldquoNew screening device for assessment of peripheralneuropathyrdquo Journal of Occupational Medicine vol 28 no 12pp 1219ndash1221 1986

[51] K K Beiswenger N A Calcutt and A P Mizisin ldquoEpidermalnerve fiber quantification in the assessment of diabetic neu-ropathyrdquo Acta Histochemica vol 110 no 5 pp 351ndash362 2008

[52] H Ishitobi AWakamatsu F Liu et al ldquoMolecular basis for FlK1expression in hemato-cardiovascular progenitors in themouserdquoDevelopment vol 138 no 24 pp 5357ndash5368 2011

[53] T Iwase N Nagaya T Fujii et al ldquoComparison of angiogenicpotency between mesenchymal stem cells and mononuclearcells in a rat model of hindlimb ischemiardquo CardiovascularResearch vol 66 no 3 pp 543ndash551 2005

[54] T Kinnaird E S Burnett M Shou et al ldquoLocal delivery ofmarrow-derived stromal cells augments collateral perfusionthrough paracrinemechanismsrdquoCirculation vol 109 no 12 pp1543ndash1549 2004

[55] I Masaki Y Yonemitsu A Yamashita et al ldquoAngiogenic genetherapy for experimental critical limb ischemia accelerationof limb loss by overexpression of vascular endothelial growthfactor 165 but not of fibroblast growth factor-2rdquo CirculationResearch vol 90 no 9 pp 966ndash973 2002


[56] M Nakae H Kamiya K Naruse et al ldquoEffects of basicfibroblast growth factor on experimental diabetic neuropathyin ratsrdquo Diabetes vol 55 no 5 pp 1470ndash1477 2006

[57] P Schratzberger D H Walter K Rittig et al ldquoReversal ofexperimental diabetic neuropathy by VEGF gene transferrdquoTheJournal of Clinical Investigation vol 107 no 9 pp 1083ndash10922001

[58] P Marin B Andersson M Krotkiewski and P BjorntorpldquoMuscle fiber composition and capillary density in women andmen with NIDDMrdquo Diabetes Care vol 17 no 5 pp 382ndash3861994

[59] J M Kennedy and DW Zochodne ldquoInfluence of experimentaldiabetes on the microcirculation of injured peripheral nervefunctional and morphological aspectsrdquo Diabetes vol 51 no 7pp 2233ndash2240 2002

[60] D W Zochodne ldquoNerve and ganglion blood flow in diabetesan appraisalrdquo International Review of Neurobiology vol 50 pp161ndash202 2002

[61] N E Cameron M A Cotter and S Robertson ldquoAngiotensinconverting enzyme inhibition prevents development of mus-cle and nerve dysfunction and stimulates angiogenesis instreptozotocin-diabetic ratsrdquoDiabetologia vol 35 no 1 pp 12ndash18 1992

[62] I G Obrosova C Van Huysen L Fathallah X Cao M JStevens andDAGreene ldquoEvaluation of1205721-adrenoceptor anta-gonist on diabetes-induced changes in peripheral nerve func-tion metabolism and antioxidative defenserdquo The FASEB Jour-nal vol 14 no 11 pp 1548ndash1558 2000

[63] T Okawa H Kamiya T Himeno et al ldquoTransplantationof neural crest-like cells derived from induced pluripotentstem cells improves diabetic polyneuropathy in micerdquo CellTransplantation vol 22 no 10 pp 1767ndash1783 2013

[64] E A Masson A Veves D Fernando and A J M BoultonldquoCurrent perception thresholds a new quick and reproduciblemethod for the assessment of peripheral neuropathy in diabetesmellitusrdquo Diabetologia vol 32 no 10 pp 724ndash728 1989

[65] R Matsutomo K Takebayashi and Y Aso ldquoAssessment ofperipheral neuropathy using measurement of the current per-ception threshold with the neurometer in patients with type 2diabetesmellitusrdquo Journal of InternationalMedical Research vol33 no 4 pp 442ndash453 2005

[66] A Veves M J Young C Manes and A J M BoultonldquoDifferences in peripheral and autonomic nerve function mea-surements in painful and painless neuropathy a clinical studyrdquoDiabetes Care vol 17 no 10 pp 1200ndash1202 1994

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


PKC activation [11ndash14] and increased hexosamine pathwayflux [15] The involvement of these chronic hyperglycemia-mediated metabolic aberrances has also been proven indiabetic polyneuropathy (DPN) [16 17]

DPN is the most common peripheral neuropathy anddegeneration of distal axons of peripheral neurons progressesslowly and symmetrically in DPN Along with HbA1c andduration of diabetes baseline cardiovascular disease andsmoking have been indicated as risk factors for DPN [18]Additionally it has been reported that in DPN the thicknessof endoneurial microvessels of sural nerves increased [19 20]while endoneurial nutritive blood flow of sciatic nerves dec-reased [21 22] The vascular dysfunction in DPN is consid-ered to be one of the most important pathologies

Evidence accumulated in the past two decades indicatesthat peripheral blood cells contain a subpopulation with pro-perties similar to embryonic angioblast [23ndash26] These cellscould be differentiated into mature endothelial cells There-fore these cells were termed ldquoendothelial progenitor cellsrdquo(EPCs) It has been attested that EPCs migrated to ischemialesions and played a pivotal role in vascular endothelialfunction and angiogenesis [27 28] Several studies havedemonstrated the interventional effects of EPC transplan-tation on myocardial ischemiainfarction and preventiveeffects on plaque progression in mice [29 30] However thenumber of circulating EPCs decreased in diabetic patientsand EPCs isolated from diabetic mice exhibited decreasedfunctions includingmigration adhesion and tube formation[31ndash35] Recent studies have shown that oxidative stresselevated by hyperglycemia decreased EPC survival throughinhibition of cell proliferation and NO production [36 37]Marrotte et al reported that EPCs isolated from diabeticmice were less effective than EPCs from nondiabetic miceat accelerating wound closure in diabetic mice [38] Theyalso demonstrated that decreased expression of manganesesuperoxide dismutase in EPCs obtained from diabetic micecontributes to impairment of wound healing These func-tional and metabolic obstacles inhibit clinical applications ofEPCs on diabetic vascular complications

In current regenerative medicines cells used for trans-plantations are expected to have two primary abilities one isdifferentiation abilities to specific cells or tissues and the otheris paracrine supportive effects to impaired tissues Certaincell transplantation therapies have already been applied tosome clinical diseases using various stemprogenitor cellsfrom somatic tissues EPC mesenchymal stem cell (MSC)skin-derived precursor cell neural stem cell and neural creststem cell [39 40] In particular MSC has been applied tothe treatment of certain diseases myocardial infarction [41]stroke [42] and graft versus host disease [43] Although theparacrine mechanisms in damaged tissues and the immuno-modulatory properties of MSC have been proven in theseclinical applications the probability of survival of trans-planted MSCs is small and the therapeutic effects on a long-term basis remain unclear

Recent advances of stem cell biology permit the deriva-tion of induced pluripotent stem (iPS) cell lines at an indi-vidual level in mammalians including humans Although EScells are attractive in their pluripotency and availability of

intact and stable undifferentiated status there are difficultiesin clinical applications of human ES cells because of safetyand ethics Although there are still safety problems that haveto be solved the autologous use of human iPS cells mayresolve ethical issues In this circumstance problematic issueswith using ES cells could be circumvented by employing anequivalent substance iPS cells in the future However thereis no standard for iPS cells the number of obtainable iPS celllines is vast and themethods to obtain them are innumerableAdditionally there are heterogeneous properties among theiPS cells Therefore we utilized ES cells in this current study

We have reported that transplantation of EPCs amelio-rated DPN [44] We hypothesized that if angioblast-like cellscould be obtained from iPS or ES cells these cells wouldoperate like EPCs and restore numerous impairments inDPN It has been shown that the vascular endothelial growthfactor receptor-2 (VEGFR2 known also Flk1) was expressedin angioblasts and its function is essential for the differ-entiation of endothelial cells and hematopoietic cells [45]Therefore Flk1 positive (Flk1+) cells derived from ES cellsmay have similar characteristics to angioblasts and similar oradditional functions of EPCs in adults This is the first reportthat demonstrates the therapeutic effects of transplantation ofangioblast-like cells derived from mouse ES cells on DPN

2 Research Design and Methods

21 Cell Culture BRC6 mouse ES cells derived fromC57BL6 female embryo and B6G02 mouse ES cells express-ingGFPderived fromaC57BL6male embryo were obtainedfrom theRikenCell Bank (Ibaragi Japan) ES cells weremain-tained in DMEM (Invitrogen Van Allen Way Carlsbad CA)containing 10 Knockout Serum Replacement (Invitrogen)1 FBS (Sigma-Aldrich St Louis MO) nonessential aminoacids (Invitrogen) 55mmolL 2-mercaptoethanol (GIBCOBurlington ON) 50UmL penicillin (GIBCO) and 50mgmL streptomycin (GIBCO) on feeder layers of mitomycin C-inactivated SNL767 cells (the European Collection of CellCultures Salisbury UK) which were clonally derived froman STO cell line transfected with a G418 resistance cassetteand a leukemia inhibitory factor expression construct [46]The STO cells were established from fibroblasts derived frommale and female embryos

Cell differentiation was induced as previously described[47] In brief a differentiationmedium (120572-MEM (Invitrogen)supplemented with 10 FBS and 5times 10minus5molL 2-mercaptoe-thanol) was used for ES cell differentiation For the inductionES cells were plated at 17 times 103 cellscm2 in the differentiationmedium on gelatin-coated dishes which were coated with01 gelatin (Sigma-Aldrich) solution and cultured for 4 days

Immortalized Schwann cells (IMS32) established by along-term culture of adult mouse DRGs and peripheralnerves [48] were a kind gift from Dr Watabe IMS32s werecultured in DMEM (Sigma-Aldrich) containing 55mM D-glucose penicillin- (100UmL) streptomycin (100mgmL)and 5 FBS (Moregate Biotech Bulimba QLD Australia)PA6 cells a type of mouse MSC were obtained from




























3 Results










(a)

168 603


0 50K 100K 150K 200K 250KFSC

0

102

103

104

105

Flk1

-APC

0 50K 100K 150K 200K 250KFSC

0

102

103

104

105

Flk1

-APC

(b)

0

05

1

15


Relat

ive e

xpre

ssio

n

lowastlowast

(c)

VE-cad

Flt-1

Tie-1

Tie-2T

SCLTal1

PU1

Lmo2

PECAM

Flk-1

(d)



0

05

1

15

VEGF-A

Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

0

2

4

6

8

10

12

FGF 2

lowastdaggerdagger

daggerdagger

Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

0

2

4

6

8

PDGF-A


daggerdagger

Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

0

2

4

6

8

NGF

Relat

ive e

xpre

ssio

n

lowastdaggerdagger

daggerdagger

daggerdagger

Flk1+

Flk1minusPA6

IMS32

0

05

1

15

2

BDNF

daggerdagger


Flk1+

Flk1minusPA6

IMS32

Rela

tive e

xpre

ssio

n

0

5

10

15

CNTF


Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

Figure 2 Continued


0

10

20

30

40

50

60

GDNF

Relat

ive e

xpre

ssio

n

lowast

daggerdagger

daggerdagger

daggerdagger

Flk1+

Flk1minusPA6

IMS32

0

05

1

15

2

NT-3

Relat

ive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32


120572-SMA

(a)

PECAM

(b)







(a)


(b)














VV V

MMM

GFP Merge120572-SMA

(a)

GFP MergePECAM

(b)


DM -saline DM-AB

(a)

1

12

14

16

18

2


Capi

llary

mus

cle fi

ber r

atio

lowast

dagger


(b)




15

20

25

30

After treatmentN DM


dagger



lowast

(mL

min

100

g)

(a)

10

15

20

25

30

After treatmentN DM



dagger

lowast


(mL

min

100

g)

(b)




4 Discussion






10

12

14

16

18

20

22

After treatmentN DM


dagger

CPT with 2000 Hz(m

A)


lowast

(a)

06

07

08

09

10

11

After treatmentN DM


dagger

CPT with 250 Hz

(mA

)


lowast

(b)

07

08

09

10

11

12

13

After treatmentN DM


dagger

CPT with 5 Hz

(mA

)


lowast

(c)





30

35

40

45

50M

NCV

(ms

)

After treatmentN DM


dagger

lowast

(a)

25

30

35

40

45

$

After treatmentN DM


dagger

SNCV

(ms

)

lowast


(b)









DM-ES-AB

DM-saline

(a)

25

30

35

40

45

50

IEN

F (fi

bers

mm

)

After treatmentN DM


dagger


lowastlowast

(b)







Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































3 Results










(a)

168 603


0 50K 100K 150K 200K 250KFSC

0

102

103

104

105

Flk1

-APC

0 50K 100K 150K 200K 250KFSC

0

102

103

104

105

Flk1

-APC

(b)

0

05

1

15


Relat

ive e

xpre

ssio

n

lowastlowast

(c)

VE-cad

Flt-1

Tie-1

Tie-2T

SCLTal1

PU1

Lmo2

PECAM

Flk-1

(d)



0

05

1

15

VEGF-A

Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

0

2

4

6

8

10

12

FGF 2

lowastdaggerdagger

daggerdagger

Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

0

2

4

6

8

PDGF-A


daggerdagger

Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

0

2

4

6

8

NGF

Relat

ive e

xpre

ssio

n

lowastdaggerdagger

daggerdagger

daggerdagger

Flk1+

Flk1minusPA6

IMS32

0

05

1

15

2

BDNF

daggerdagger


Flk1+

Flk1minusPA6

IMS32

Rela

tive e

xpre

ssio

n

0

5

10

15

CNTF


Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

Figure 2 Continued


0

10

20

30

40

50

60

GDNF

Relat

ive e

xpre

ssio

n

lowast

daggerdagger

daggerdagger

daggerdagger

Flk1+

Flk1minusPA6

IMS32

0

05

1

15

2

NT-3

Relat

ive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32


120572-SMA

(a)

PECAM

(b)







(a)


(b)














VV V

MMM

GFP Merge120572-SMA

(a)

GFP MergePECAM

(b)


DM -saline DM-AB

(a)

1

12

14

16

18

2


Capi

llary

mus

cle fi

ber r

atio

lowast

dagger


(b)




15

20

25

30

After treatmentN DM


dagger



lowast

(mL

min

100

g)

(a)

10

15

20

25

30

After treatmentN DM



dagger

lowast


(mL

min

100

g)

(b)




4 Discussion






10

12

14

16

18

20

22

After treatmentN DM


dagger

CPT with 2000 Hz(m

A)


lowast

(a)

06

07

08

09

10

11

After treatmentN DM


dagger

CPT with 250 Hz

(mA

)


lowast

(b)

07

08

09

10

11

12

13

After treatmentN DM


dagger

CPT with 5 Hz

(mA

)


lowast

(c)





30

35

40

45

50M

NCV

(ms

)

After treatmentN DM


dagger

lowast

(a)

25

30

35

40

45

$

After treatmentN DM


dagger

SNCV

(ms

)

lowast


(b)









DM-ES-AB

DM-saline

(a)

25

30

35

40

45

50

IEN

F (fi

bers

mm

)

After treatmentN DM


dagger


lowastlowast

(b)







Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

168 603


0 50K 100K 150K 200K 250KFSC

0

102

103

104

105

Flk1

-APC

0 50K 100K 150K 200K 250KFSC

0

102

103

104

105

Flk1

-APC

(b)

0

05

1

15


Relat

ive e

xpre

ssio

n

lowastlowast

(c)

VE-cad

Flt-1

Tie-1

Tie-2T

SCLTal1

PU1

Lmo2

PECAM

Flk-1

(d)



0

05

1

15

VEGF-A

Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

0

2

4

6

8

10

12

FGF 2

lowastdaggerdagger

daggerdagger

Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

0

2

4

6

8

PDGF-A


daggerdagger

Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

0

2

4

6

8

NGF

Relat

ive e

xpre

ssio

n

lowastdaggerdagger

daggerdagger

daggerdagger

Flk1+

Flk1minusPA6

IMS32

0

05

1

15

2

BDNF

daggerdagger


Flk1+

Flk1minusPA6

IMS32

Rela

tive e

xpre

ssio

n

0

5

10

15

CNTF


Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

Figure 2 Continued


0

10

20

30

40

50

60

GDNF

Relat

ive e

xpre

ssio

n

lowast

daggerdagger

daggerdagger

daggerdagger

Flk1+

Flk1minusPA6

IMS32

0

05

1

15

2

NT-3

Relat

ive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32


120572-SMA

(a)

PECAM

(b)







(a)


(b)














VV V

MMM

GFP Merge120572-SMA

(a)

GFP MergePECAM

(b)


DM -saline DM-AB

(a)

1

12

14

16

18

2


Capi

llary

mus

cle fi

ber r

atio

lowast

dagger


(b)




15

20

25

30

After treatmentN DM


dagger



lowast

(mL

min

100

g)

(a)

10

15

20

25

30

After treatmentN DM



dagger

lowast


(mL

min

100

g)

(b)




4 Discussion






10

12

14

16

18

20

22

After treatmentN DM


dagger

CPT with 2000 Hz(m

A)


lowast

(a)

06

07

08

09

10

11

After treatmentN DM


dagger

CPT with 250 Hz

(mA

)


lowast

(b)

07

08

09

10

11

12

13

After treatmentN DM


dagger

CPT with 5 Hz

(mA

)


lowast

(c)





30

35

40

45

50M

NCV

(ms

)

After treatmentN DM


dagger

lowast

(a)

25

30

35

40

45

$

After treatmentN DM


dagger

SNCV

(ms

)

lowast


(b)









DM-ES-AB

DM-saline

(a)

25

30

35

40

45

50

IEN

F (fi

bers

mm

)

After treatmentN DM


dagger


lowastlowast

(b)







Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

05

1

15

VEGF-A

Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

0

2

4

6

8

10

12

FGF 2

lowastdaggerdagger

daggerdagger

Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

0

2

4

6

8

PDGF-A


daggerdagger

Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

0

2

4

6

8

NGF

Relat

ive e

xpre

ssio

n

lowastdaggerdagger

daggerdagger

daggerdagger

Flk1+

Flk1minusPA6

IMS32

0

05

1

15

2

BDNF

daggerdagger


Flk1+

Flk1minusPA6

IMS32

Rela

tive e

xpre

ssio

n

0

5

10

15

CNTF


Rela

tive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32

Figure 2 Continued


0

10

20

30

40

50

60

GDNF

Relat

ive e

xpre

ssio

n

lowast

daggerdagger

daggerdagger

daggerdagger

Flk1+

Flk1minusPA6

IMS32

0

05

1

15

2

NT-3

Relat

ive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32


120572-SMA

(a)

PECAM

(b)







(a)


(b)














VV V

MMM

GFP Merge120572-SMA

(a)

GFP MergePECAM

(b)


DM -saline DM-AB

(a)

1

12

14

16

18

2


Capi

llary

mus

cle fi

ber r

atio

lowast

dagger


(b)




15

20

25

30

After treatmentN DM


dagger



lowast

(mL

min

100

g)

(a)

10

15

20

25

30

After treatmentN DM



dagger

lowast


(mL

min

100

g)

(b)




4 Discussion






10

12

14

16

18

20

22

After treatmentN DM


dagger

CPT with 2000 Hz(m

A)


lowast

(a)

06

07

08

09

10

11

After treatmentN DM


dagger

CPT with 250 Hz

(mA

)


lowast

(b)

07

08

09

10

11

12

13

After treatmentN DM


dagger

CPT with 5 Hz

(mA

)


lowast

(c)





30

35

40

45

50M

NCV

(ms

)

After treatmentN DM


dagger

lowast

(a)

25

30

35

40

45

$

After treatmentN DM


dagger

SNCV

(ms

)

lowast


(b)









DM-ES-AB

DM-saline

(a)

25

30

35

40

45

50

IEN

F (fi

bers

mm

)

After treatmentN DM


dagger


lowastlowast

(b)







Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

50

60

GDNF

Relat

ive e

xpre

ssio

n

lowast

daggerdagger

daggerdagger

daggerdagger

Flk1+

Flk1minusPA6

IMS32

0

05

1

15

2

NT-3

Relat

ive e

xpre

ssio

n

Flk1+

Flk1minusPA6

IMS32


120572-SMA

(a)

PECAM

(b)







(a)


(b)














VV V

MMM

GFP Merge120572-SMA

(a)

GFP MergePECAM

(b)


DM -saline DM-AB

(a)

1

12

14

16

18

2


Capi

llary

mus

cle fi

ber r

atio

lowast

dagger


(b)




15

20

25

30

After treatmentN DM


dagger



lowast

(mL

min

100

g)

(a)

10

15

20

25

30

After treatmentN DM



dagger

lowast


(mL

min

100

g)

(b)




4 Discussion






10

12

14

16

18

20

22

After treatmentN DM


dagger

CPT with 2000 Hz(m

A)


lowast

(a)

06

07

08

09

10

11

After treatmentN DM


dagger

CPT with 250 Hz

(mA

)


lowast

(b)

07

08

09

10

11

12

13

After treatmentN DM


dagger

CPT with 5 Hz

(mA

)


lowast

(c)





30

35

40

45

50M

NCV

(ms

)

After treatmentN DM


dagger

lowast

(a)

25

30

35

40

45

$

After treatmentN DM


dagger

SNCV

(ms

)

lowast


(b)









DM-ES-AB

DM-saline

(a)

25

30

35

40

45

50

IEN

F (fi

bers

mm

)

After treatmentN DM


dagger


lowastlowast

(b)







Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)


(b)














VV V

MMM

GFP Merge120572-SMA

(a)

GFP MergePECAM

(b)


DM -saline DM-AB

(a)

1

12

14

16

18

2


Capi

llary

mus

cle fi

ber r

atio

lowast

dagger


(b)




15

20

25

30

After treatmentN DM


dagger



lowast

(mL

min

100

g)

(a)

10

15

20

25

30

After treatmentN DM



dagger

lowast


(mL

min

100

g)

(b)




4 Discussion






10

12

14

16

18

20

22

After treatmentN DM


dagger

CPT with 2000 Hz(m

A)


lowast

(a)

06

07

08

09

10

11

After treatmentN DM


dagger

CPT with 250 Hz

(mA

)


lowast

(b)

07

08

09

10

11

12

13

After treatmentN DM


dagger

CPT with 5 Hz

(mA

)


lowast

(c)





30

35

40

45

50M

NCV

(ms

)

After treatmentN DM


dagger

lowast

(a)

25

30

35

40

45

$

After treatmentN DM


dagger

SNCV

(ms

)

lowast


(b)









DM-ES-AB

DM-saline

(a)

25

30

35

40

45

50

IEN

F (fi

bers

mm

)

After treatmentN DM


dagger


lowastlowast

(b)







Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















VV V

MMM

GFP Merge120572-SMA

(a)

GFP MergePECAM

(b)


DM -saline DM-AB

(a)

1

12

14

16

18

2


Capi

llary

mus

cle fi

ber r

atio

lowast

dagger


(b)




15

20

25

30

After treatmentN DM


dagger



lowast

(mL

min

100

g)

(a)

10

15

20

25

30

After treatmentN DM



dagger

lowast


(mL

min

100

g)

(b)




4 Discussion






10

12

14

16

18

20

22

After treatmentN DM


dagger

CPT with 2000 Hz(m

A)


lowast

(a)

06

07

08

09

10

11

After treatmentN DM


dagger

CPT with 250 Hz

(mA

)


lowast

(b)

07

08

09

10

11

12

13

After treatmentN DM


dagger

CPT with 5 Hz

(mA

)


lowast

(c)





30

35

40

45

50M

NCV

(ms

)

After treatmentN DM


dagger

lowast

(a)

25

30

35

40

45

$

After treatmentN DM


dagger

SNCV

(ms

)

lowast


(b)









DM-ES-AB

DM-saline

(a)

25

30

35

40

45

50

IEN

F (fi

bers

mm

)

After treatmentN DM


dagger


lowastlowast

(b)







Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















15

20

25

30

After treatmentN DM


dagger



lowast

(mL

min

100

g)

(a)

10

15

20

25

30

After treatmentN DM



dagger

lowast


(mL

min

100

g)

(b)




4 Discussion






10

12

14

16

18

20

22

After treatmentN DM


dagger

CPT with 2000 Hz(m

A)


lowast

(a)

06

07

08

09

10

11

After treatmentN DM


dagger

CPT with 250 Hz

(mA

)


lowast

(b)

07

08

09

10

11

12

13

After treatmentN DM


dagger

CPT with 5 Hz

(mA

)


lowast

(c)





30

35

40

45

50M

NCV

(ms

)

After treatmentN DM


dagger

lowast

(a)

25

30

35

40

45

$

After treatmentN DM


dagger

SNCV

(ms

)

lowast


(b)









DM-ES-AB

DM-saline

(a)

25

30

35

40

45

50

IEN

F (fi

bers

mm

)

After treatmentN DM


dagger


lowastlowast

(b)







Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















10

12

14

16

18

20

22

After treatmentN DM


dagger

CPT with 2000 Hz(m

A)


lowast

(a)

06

07

08

09

10

11

After treatmentN DM


dagger

CPT with 250 Hz

(mA

)


lowast

(b)

07

08

09

10

11

12

13

After treatmentN DM


dagger

CPT with 5 Hz

(mA

)


lowast

(c)





30

35

40

45

50M

NCV

(ms

)

After treatmentN DM


dagger

lowast

(a)

25

30

35

40

45

$

After treatmentN DM


dagger

SNCV

(ms

)

lowast


(b)









DM-ES-AB

DM-saline

(a)

25

30

35

40

45

50

IEN

F (fi

bers

mm

)

After treatmentN DM


dagger


lowastlowast

(b)







Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















30

35

40

45

50M

NCV

(ms

)

After treatmentN DM


dagger

lowast

(a)

25

30

35

40

45

$

After treatmentN DM


dagger

SNCV

(ms

)

lowast


(b)









DM-ES-AB

DM-saline

(a)

25

30

35

40

45

50

IEN

F (fi

bers

mm

)

After treatmentN DM


dagger


lowastlowast

(b)







Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DM-ES-AB

DM-saline

(a)

25

30

35

40

45

50

IEN

F (fi

bers

mm

)

After treatmentN DM


dagger


lowastlowast

(b)







Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgments


References












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Angioblast Derived from ES Cells...

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Transcript of Research Article Angioblast Derived from ES Cells...