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A NONINVASIVE METHOD FOR COUNTING HUMAN PLURIPOTENT STEM CELL NUMBERS BY LIVE CELL IMAGING Mika Suga 1, Hiroaki Kii 2, Takayuki Uozumi 2, Yasujiro Kiyota 2 and Miho K Furue 1
1 Dept. of Disease Bioresources Research, Laboratory of Stem Cell Cultures, National Inst. of Biomedical Innovation, Osaka, Japan 2 Instruments company, NIKON CORPORATION, Yokohama, Japan
Background Celldensity isacritical factorforcontrollingbothgrowthanddifferentiationof
humanpluripotentstem(hPS)cellsincludingembryonicstem(ES)cellsandinduced
pluripotentstem (iPS)cells.Despite the factof theevolutionofhPScellculture
techniques,countingcellnumbersisstillproblematic.Therefore,wehavedeveloped
anoninvasivecellcountingmethodforhPScellsthroughanalyzinglivecellimages.
Outline AJapaneseadulthumanskin fibroblast (JCRB0534:TIG-114) -derived iPS-
TIG114-4f1human iPScells (JCRB1437,JCRBCellBank,NIBIO,Osaka,Japan),
whichwasestablishedbyYamanaka’sgroup (CiRA,KyotoUniversity)1)2) and
H9humanEScells (WA09,WISCBank,WiCellResearch Institute,Madison,
WI,USA)3)4)wereseededon inactivatedmouseembryonic fibroblasts (MEF)as
describedpreviously 4), and thenstainedwithacell-permeantSYTO24green
fluorescentnucleicacidstain(Invitrogen).Phasecontrastandfluorescentimagesof
thecellsobtainedintermittentlyinacellincubatorobservationsystem,BioStation CT (NikonCorporation)wereanalyzedbyautomated imageanalysissoftware
CL-Quant(NikonCorporation).AreaofES/iPScellcolonycoveragewasmeasuredfromphasecontrast images.Numberofstainednucleiwascounted fromgreen
fluorescent images. Immediatelyafter imaging, theconventionalcellcountingby
hemocytometerwasperformedtocomparewiththenumbersoffluorescentnuclei.
Materials and Methods Cell culture H9and iPS-TIG114-4f1weremaintainedon irradiatedmouseembryo
fibroblastfeedercells(MEF,MilliporeCo.) inanKSR-basedmediumsupplemented
with5ng/mlhumanrecombinantFGF-2(KatayamaKagakuKogyoLTD.).H9were
passagedwith1mg/mlDispase(Roche)andaplasticscraper (SumitomoBakelite
Co.LTD.).iPS-TIG114-4f1weremechanicallypassagedwithEZPassage(Invitrogen).
Thecellsweresplitataratioof1:5–1:8every6days.HumanEScellswereused
followingtheGuidelinesforutilizationofhumanembryonicstemcellsoftheMinistry
ofEducation,Culture,Sports,ScienceandTechnologyofJapan.Thisresearchusing
hES/iPScellswasapprovedby the institutionalethical reviewboardatNational
InstituteofBiomedicalInnovation.
Live cell imaging Thecellsseededona6-wellplatewereculturedintheBioStation
CTat37°C/5%CO2,andmonitoredby time lapse live imaging.Phasecontrast
imageswerecapturedevery12hoursautomaticallyatamagnificationof4x.
Todetectnucleiof livingcells, thecellswerestainedwith1µMSYTO24green
fluorescentdye (Invitrogen).Fluorescent imagesof thestainednucleiandphase
contrastimageswereobtainedbyBioStationCT,andanalyzedbyCL-Quantwiththe
custommaderecipe.Thisrecipeenablestodetectindividualcoloniesandmeasure
theareacoveredbythesecoloniesfromthephasecontrastimageandmeasurethe
numberofnucleifromthefluorescentimage.
Conventional cell counting Thecellsweredissociatedusing0.025%trypsinand
0.01%EDTA inPhosphateBufferedSaline (Gibco) andcountedbydisposal
hemocytometer,OneCellCounter(BioMedicalScience,LTD.).
Results Thefluorescentnucleuscountingwithimagesandtheconventionalcellcountingbyhemocytometershowedsimilarresults.Inthecaseoftotalcellnumbersabove
1x104,cellnumbersbynucleuscountingweresimilarandreproduciblewiththosebyconventionalcellcounting.(seeFigure1.)
Therewasasignificantcorrelationbetweenthecolonycoverageareaandthenucleus/cellcounting.Thecorrelationcurveswerespecificforeachcellline.(seeFigure2.)
Figure 1. Daily cell growth of hPS cells was quantified by imaging.
(A)Thegreenfluorescentandphasecontrast imagesof livinghPScellsstained
withSYTO24. (Left)Merged imageofSYTO24fluorescenceandphasecontrast.
(Right)DetectionofhPScoloniesfromphasecontrastimage.NotethatMEFswere
separatedfromhPScolonies.(B)Eachnucleus(right,markedasgreenpoints)was
detectedandsegmentedfromtheSYTO24fluorescentimage(left)usingCL-Quant.
Allobjectswithinthewellweremeasuredtocountcellnumbers. (C)Comparison
ofcellcountingbetweenconventionalmethod (hemocytometer)and the image
analysis(nucleistaining).(D)Dailyincreaseofcolonycoverageareawasquantified
byimageanalysis.
Figure 2. Colony coverage area was analyzed from phase contrast images.
(A)Colonycoverageareawasanalyzedfromphasecontrastimages.Yellowregion
isthedetectedhPScolonies.MEFsweresuccessfullyremovedbyourhPScolony
extractingprocedure. (B)Correlationbetweencellcountbynucleistainingand
colonycoveragearea.Centerarea(7680x7820pixel;240.23mm2)ofeachwellwas
analyzed.NotethatthecorrelationcurvesaredifferentbetweeniPS-TIG114-4f1and
H9.
Conclusions Hereweshowedasignificantcorrelationbetweenthecellnumberscountedbynucleifluorescentstainingandthecolonycoverageareameasuredfromphasecontrastimaging.Theseresultsshow
thatnumbersofhumanPScellscanbeestimatedfromthetotalcolonycoverageareathroughphasecontrastimaging.Thuswehavedevelopedanewnoninvasivecellcountingmethod.Furthermore,
obtainingtime-lapsephasecontrastimagesenablesustomonitorcolonymorphologicalchangesandtocalculategrowthrateduringhumanPScellculture.Foraccurateestimationofcellnumbers,itis
neededtodeterminethecorrelationcurveofthecellnumbersandthecolonycoverageareaforeachhumanPScelllines.ThiscorrelationratiomightcharacterizeasthepropertyofeachhPScelllines.
Ournoninvasivetechniquecouldbeusefulforqualitycontrolorhigh-throughputscreeninganalysiswithoutwastingordamagingthecells.
References1)Takahashi,et al.(2007)Cell30;131(5):861-72.2)ISCI(2011)Nat Biotechnol. 27;29(12):1132-44.3)Thomson,et al.(1998)Science282:1145-1147.4)Amit,et al.(2000) Developmental Biology227:271-278.
Acknowledgement ThisresearchissupportedbyNEDO(NewEnergy
andIndustrialTechnologyDevelopmentOrganization)
ontheproject“FundamentalTechnologyforPromoting
theIndustrialApplicationofHumanStemCells”.
1000000
100000
10000
10000 20 40 60 80 100 120 140
y = 20.754x2 + 794.39xR2 = 0.9748
y = 14.293x2 + 438.19xR2 = 0.9777 iPS-TIG114-4f1
H9(per
240
.23
mm
2 )C
ell c
ount
Colony coverage area (mm2)
iPS-TIG114-4f1
H9
(A)(A)
(B)
(B)
(D)
(C)
1×107
1×106
1×105
1×104
1 2 3 4 5 6 7 8 9
5×108
5×107
5×106
5×105
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Day
Day1 2 3 4 5 6 7 8 9
hemocytometer
nuclei staining
cell
coun
t p
er w
ell
colo
ny c
over
age
area
(pix
el p
er w
ell)
iPS-TIG114-4f1 H9
iPS-TIG114-4f1 H9