assessing differentiation status of human embryonic stem cells noninvasively using raman...

Assessing Differentiation Status of HumanEmbryonic Stem Cells Noninvasively Using RamanMicrospectroscopy

H. Georg Schulze,† Stanislav O. Konorov,†,‡ Nicolas J. Caron,† James M. Piret,†,§

Michael W. Blades,*,‡ and Robin F. B. Turner*,†,‡,|

Michael Smith Laboratories, The University of British Columbia, 2185 East Mall, Vancouver, BC, Canada, V6T 1Z4,Department of Chemistry, The University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada, V6T 1Z1,Department of Chemical & Biological Engineering, The University of British Columbia, 2360 East Mall, Vancouver, BC,Canada, V6T 1Z3, and Department of Electrical & Computer Engineering, The University of British Columbia, 2332Main Mall, Vancouver, BC, Canada, V6T 1Z4

Raman microspectroscopy is an attractive approach forchemical imaging of biological specimens, including livecells, without the need for chemi-selective stains. Usingamicrospectrometer,near-infraredRamanspectrathrough-out the range 663 cm-1 to 1220 cm-1 were obtainedfrom colonies of CA1 human embryonic stem cells(hESCs) and CA1 cells that had been stimulated todifferentiate for 3 weeks by 10% fetal bovine serumon gelatin. Distributions and intensities of spectralbands attributed to proteins varied significantly be-tween undifferentiated and differentiated cells. Impor-tantly, compared to proteins and lipids, the bandintensities of nucleic acids were dominant in undif-ferentiated cells with a dominance-reversal in dif-ferentiated cells. Thus, we could identify intensityratios of particular protein-related bands (e.g., 757cm-1 tryptophan) to nucleic acid bands (784 cm-1

DNA/RNA composite) that were effective in discrimi-nating between spectra of undifferentiated and dif-ferentiated cells. We observed no discernible negativeeffects due to the laser exposure in terms of morphol-ogy, proliferation, or pluripotency of the stem cells. Weconclude that Raman microscopy and complementarydata processing procedures provide a rapid, noninva-sive approach that can distinguish hESCs from dif-ferentiated cells. This is the first report to identifyspecific Raman markers for the differentiation statusof hESCs.

Embryonic stem cells arise from the inner cell mass of themammalian blastocyst and are self-renewing and pluripotent cells,differentiable into cells from any of the three germ layers. In vitro,

pluripotency can be maintained indefinitely,1-3 spontaneous dif-ferentiation can occur,2-5 directed differentiation and targetedtissue formation can be obtained,2,6-11 and induced pluripotentstem cells can be derived from somatic cells.12 Together, thesecapabilities have spawned tremendous research interest worldwideaimed at directing the differentiation and development of stemcells. Thus, self-renewal and directed differentiation representpowerful new tools for tissue engineering,4,7,9,13 regenerativemedicine,2-4,7,13,14 cell therapies,4,15 pharmaceutical screening,2,4

and related fields.Raman and infrared absorption spectroscopy (IR) are informa-

tion-rich vibrational spectroscopies16-18 with noninvasive potentialable to reveal the chemical composition, structure, and microen-vironment of cellular components.19-27 Infrared spectroscopy is

* Corresponding authors. E-mail: (R.F.B.T.) [email protected], (M.W.B.)[email protected]. Fax: (R.F.B.T.) 604-822-2114, (M.W.B.) 604-822-2847.

† Michael Smith Laboratories, The University of British Columbia.‡ Department of Chemistry, The University of British Columbia.§ Department of Chemical & Biological Engineering, The University of British

Columbia.| Department of Electrical & Computer Engineering, The University of British

Columbia.

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10.1021/ac902697q 2010 American Chemical Society5020 Analytical Chemistry, Vol. 82, No. 12, June 15, 2010Published on Web 05/19/2010

hampered by water absorption and, generally more than Raman,by diffraction-limited spatial resolution.11,24,28,29 Although cellfixation is commonly used to avoid interference by water,11,30

fixatives degrade the quality of IR spectra.31 Raman spectroscopiesare well suited for biological samples in their native state due torelatively weak water interference in the information-rich “finger-print” region. Successful cytological applications include cellsorting and classification,23,32-34 monitoring differentiation andassessing differentiation status,11,19,22,25 imaging, mapping, andidentification of cellular components,24,35-38 following cell cycledynamicsandmitosis27,28,30 aswellasothermetabolicprocesses,39,40

and tissue mapping and characterization.34,36

Krafft et al.11 articulated the principle that each cell ischaracterized by specific molecular constituents yielding sensitivespectroscopic signatures (IR in their case, but this principle isequally applicable to Raman). Cellular spectral signatures mustbe well characterized to avoid misinterpretation due to potentialmodifications by variables such as the cell cycle or the cellularcontext (e.g., medium composition). However, a complete char-acterization of the spectral signatures as they apply to hESCsremains necessary. It also remains to be shown that the laserirradiation required to implement such spectroscopic assaymethods does not impact these signatures, and more importantly,the research or therapeutic value of these cells.

We report here on efforts to characterize the Raman spectralsignatures of hESCs and their spontaneously differentiatedprogeny and to identify specific, robust spectral indicators ofdifferentiation status based on them. In particular, we have focusedon spectral bands that can be linked to specific biochemical

changes within the cells. We also report on the morphological,functional, and proliferative consequences of laser irradiation forhESCs. This is the first report to identify specific Raman markersof differentiation status in hESCs and to show that these markerscan be obtained under conditions that do not compromisepluripotency. Our results complement the only other full reporton the utility of Raman microscopy for use with hESCs.41

MATERIALS AND METHODShESC Culture and Differentiation. The CA1 hESC line was

graciously provided by Dr. Andras Nagy (Mount Sinai HospitalToronto, ON, Canada) and maintained in Matrigel-coated (BDBiosciences, Mississauga, ON, Canada) culture dishes withmTeSR1 medium (STEMCELL Technologies, Vancouver,Canada). Culture dishes (Sarstedt, Montreal, QC, Canada) or12.7 mm diameter silver mirrors with a 100 nm layer of glass(ThorLabs, Inc., Newton, NJ) were prepared by adding Matrigeldiluted 1/30 in DMEM/F12 (Invitrogen, Burlington, ON,Canada) followed by a 1 h incubation at room temperature.CA1 hESCs were maintained with daily medium changes andpassaged every 6 or 7 days using a combination of Dispase(STEMCELL Technologies) and physical sheer to detachhESCs aggregates according to the manufacturer’s protocol.CA1S, a subline of CA1 adapted to enzymatic dissociation42,43

and used for irradiation assessment, was also propagated onMatrigel-coated dishes and maintained undifferentiated usingmTeSR1. Passaging of the CA1S subline differed from CA1passaging as follows: CA1S were enzymatically dissociated tosingle cells using TrypLE Express (Invitrogen) and passagedevery 4 to 5 days. Undifferentiated CA1 cells were dissociatedwith TrypLE Express into small clumps and single cells beforethey were differentiated for 3 weeks on gelatin-coated T75 flasks(Sarstedt) in DMEM/F12 (Invitrogen) containing 10% fetalbovine serum (FBS). The status of both undifferentiated anddifferentiated groups were verified with procedures providedin the Supporting Information. Approval for the use of hESCswas obtained from the Canadian Stem Cell Oversight Committee.

Biochemical Analyses. CA1 cells were plated in Matrigel-coated culture dishes with mTeSR1 medium and harvested 6 daysafter plating for analysis of protein and DNA content. A secondgroup of CA1 cells were also plated in Matrigel-coated culturedishes with mTeSR1 medium. On day 3 after plating, the mediumwas changed to DMEM/F12 with 10% FBS for differentiation. Cellswere passaged on day 6 as described above and harvested onday 18 (i.e., after 15 days in differentiation medium). Forharvesting, cells were rinsed with PBS, dissociated from thesurface with TrypLE, transferred to a microcentrifuge tube, spundown for 2 min at 2500 rpm, resuspended in lysis buffer (470 µLof H2O, 25 µL of CyQUANT 20×, and 5 µL of 10% SDS), andkept at -20 °C until analysis. Using the CyQuant cell prolifera-tion kit following the manufacturer’s protocol for lysed cellsamples (Invitrogen), DNA was determined in triplicate with

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5021Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

a Pharos SX laser scanner (BioRad, Mississauga, ON, Canada)with a fluorescein filter and protein was determined in duplicatewith a BCA protein assay kit (Invitrogen).

Immunochemistry. Immunochemistry was performed aspreviously described.43 Briefly, for assessing laser toxicity, CA1Scells were plated and grown on Matrigel-coated 40 mm diameterglass-bottom Petri dishes (Willco Wells B.V., Amsterdam, TheNetherlands). Cells, from both exposed and nonexposed groups,were fixed with 4% paraformaldehyde, permeabilized with 0.1%Triton in PBS, and then stained with 10 µg/mL monoclonal anti-human/mouse Oct-4 rat IgG (R&D Systems, Minneapolis, MN)and 10 µg/mL Goat F(ab′)2 PE-Cy5 anti-rat IgG (Cedarlane,Hornby, ON, Canada) diluted in 0.1% BSA/PBS with PBS washesbetween each step. Nuclei were stained with 100 ng/mL DAPI(Invitrogen) before the final wash. CHO-K1 cells were used asnegative controls. Cells were examined using an epifluorescenceinverted microscope (Motic, Hong Kong, PRC) and photomicro-graphs obtained using a Nikon D100 digital camera (Mississauga,ON, Canada).

Raman Microscopy on hESCs. Spectra were obtained atroom temperature from colonies of hESCs and differentiatedhESCs with a Raman microscope system (RM 1000, Renishaw,Gloucestershire, U.K.). Before spectral acquisition, the growth/maintenance medium was removed, samples were rinsed, andthen covered with identical amounts of basal medium (DMEM/F12). A Petri dish containing the colony was placed under themicroscope and a 20× objective lens was used to focus the laserbeam to a spot of approximately 6 µm × 50 µm. Raman scattering,generated at 785 nm and 100 mW, was collected for 200 s perspectrum. Spectra (n ) 10) were obtained from both undifferenti-ated and differentiated colonies on the same day. Each spectrumtypically included contributions from at least three cells; therefore,at least 30 cells total per differentiation state were interrogated.Background spectra (n ) 3) were obtained for each sample afterrecording all the spectra from cells. To reduce collection timesand cellular stress during acquisition, spectra were obtained onlyfor the fingerprint region from ca. 660 to 1220 cm-1.

Colonies were subsequently dry-fixed to increase the numberof spectra available for analysis: the medium was decanted andthe remaining moisture evaporated by placing the Petri dishescontaining the colonies in a fume hood for approximately 2 min.The colonies were then allowed to stand a further 2 to 3 days atroom temperature until completely desiccated. Raman spectrawere then obtained from the dried colonies by mapping a 40 µm× 20 µm area, using a 2 µm step, for a total of 231 spectra perculture.

Irradiation Impact on hESCs. To investigate the toxicity oflaser radiation on whole hESC colonies, we used CA1S hESCs.The larger areas of the CA1 colonies could not be irradiatedcompletely and so this would have complicated analyses of theradiation impact. Since the CA1S subline of CA1 is adapted toenzymatic dissociation, smaller undifferentiated colonies can beformed from single cells making it possible to irradiate a wholeisolated colony.

Matrigel was dotted on a custom 50-mm glass-bottom culturedish with a quartz bottom 150 µm thick and CA1S hESCs wereplated at a low density in small clumps of 1-6 cells that had beendissociated with TrypLE. The day after plating, colonies were

circled, photographed, and identified before exposing half thecolonies to laser radiation. The culture dish was placed on a 2Dmanual translation stage for irradiation using an 80 µm diameterexpanded beam from a 2 W continuous wave Ti:Sapphire laser(Mira 900-D, Coherent, Santa Clara, CA) to expose all cells in acolony for 200 s to 785 nm radiation at the same intensities as forthe Raman spectrometry of cells. Visualization was effected on amonitor with sublasing superluminescence from the laser, a 3.5×objective situated above the Petri dish, and a CCD camera. Aftercentering the beam on a colony, the Petri dish was covered withan aluminum plate mask to admit targeted radiation and preventcollateral damage. Cells were returned to the incubator after lasertreatment and maintained for 84 h to allow growth and colonyformation. They were then fixed with 4% paraformaldehyde anda few days later stained for Oct-4 and DAPI.

Data Analysis. Raman spectra were inspected individually forgross inconsistencies; artifacts such as cosmic spikes wereremoved. No spectra were rejected. Spectra were aligned to thephenylalanine ring-stretching peak at 1003 cm-1 and adjustedwhere necessary, smoothed by an automated smoothing filterusing 30 iterations per spectrum,44 and normalized to anunassigned peak in the basal medium at 1040 cm-1. The threebackground spectra for each culture were averaged, smoothed,and normalized as above, and the processed spectrum wassubtracted from each of the 10 processed culture spectra. Thespectrum baseline was then flattened by a 50-point movingaverage, peak stripping, semiautomated procedure.45 Flatteninginvolved 50 iterations per spectrum followed by a second roundof 25 iterations. In spectra from differentiated samples, abackground flattening overcorrection near 800 cm-1 wasavoided by segmentation at this point, flattening each segmentseparately, and recombining the results. After processing,spectra were of high quality and peaks of different cellularcomponents identifiable. A list of relevant peak assignments,assembled from the literature, is given in Table S-1 of theSupporting Information.

Two-sample F-tests were performed to test peak variances ofspectra from hESCs and their progeny for heteroscedasticity.Significance tests of peak intensities were performed with two-tailed t-tests using either equal or unequal variances, the latter asdetermined with the F-tests.

Principal component analysis (PCA) is a multivariate methodthat identifies combinations of spectral variables or principalcomponents (PCs) that are orthogonal and account for the majorsources of variance within a data set. Every spectrum is thenassigned a score for each principal component. We used thesescores for segregation of spectra by differentiation status group.In addition, between-group variances were compared to within-group variances to highlight those variances uniquely related todifferentiation status. Two-dimensional correlation spectroscopy(2DCOS) analyses were also performed and are provided in theSupporting Information.

(44) Schulze, H. G.; Foist, R. B.; Ivanov, A.; Turner, R. F. B. Appl. Spectrosc.2008, 62, 1160–1166.

(45) Schulze, G.; Jirasek, A.; Yu, M. M. L.; Lim, A.; Turner, R. F. B.; Blades,M. W. Appl. Spectrosc. 2005, 59, 545–574.

5022 Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

RESULTS AND DISCUSSIONDifferentiation of hESC Cultures. CA1 hESCs were induced

to differentiate toward neural-like, gut-like, fibroblast-like, andadipocyte-like cells by culturing in feeder-free DMEM/F12 and10% FBS. After 3 weeks under these conditions, less than 7% ofthe remaining cells expressed significant levels of the pluripotencymarker SSEA3. After only 12 days, other markers of pluripotency(Oct-4, Nanog) were already only present at minimal levels. Takentogether, these results (data shown in Figure S-1 of SupportingInformation) justify our use of the term “differentiated” in thefollowing comparisons with undifferentiated CA1 hESCs.

Raman Microscopy. The laser spot size was estimated tocover at least three cells and was used to illuminate undifferenti-ated and differentiated cells at random positions within confluentand dense colonies to collect Raman spectra from similar amountsof material. We further used a peak in the basal medium (1040cm-1, unassigned) to gauge comparable amounts of materialbecause marked differences in this peak would have indicatedrelative differences in scattering from basal medium coveringthe colonies, thus, differences in colony thicknesses. Nomarked differences were observed between groups (datashown in Figure S-1 of Supporting Information). We opted notto use vector normalization because it is inappropriate if theaggregate Raman scattering cross sections differ across groups.Normalization is further addressed in the Supporting Information.Before preprocessing, Raman spectra exhibited high backgrounds,sloping baselines, and signal-to-noise ratios around 40 based onthe phenylalanine band at 1003 cm-1.

After preprocessing, the relative mean intensities of peaksbetween groups were observed to differ as shown in Figure 1.Generally, contributions from nucleic acids (the cytosine, thymine,and uracil ring breathing modes at 782 cm-1 overlapping withthe O-P-O stretch in DNA at 788 cm-1, and the O-P-Ostretch in RNA at 811 cm-1) were more prominent in undif-ferentiated cells and contributions from proteins (the tryp-

tophan ring breathing at 757 cm-1, the protein C-C stretch at937 cm-1, and the phenylalanine ring breathing mode at 1003cm-1) were more prominent in differentiated cells. Figure 1demonstrates that Raman microscopy was capable of capturinginformation related to cell differentiation status and that dif-ferentiation status could be indicated by protein to nucleic acidratios, for example, the 757 to 784 cm-1 bands. Biochemicalanalyses supported this interpretation; they and other poten-tially informative ratios are addressed in separate sectionsbelow.

The individual spectra in each of the groups shown in Figure1 were highly reproducible (see Figure S-3 of Supporting Informa-tion) suggesting that between- and within-group variances con-tained more than merely ‘noise’. Specifically, standard deviationsin part reflected changes in content or concentration, thus, aspectsof cellular dynamics, whereas the means reflected average contentor concentration. Spectral standard deviations are shown in Figure2 where the standard deviations were scaled by a constant to thesame intensities as the 784 cm-1 peak of the respective spectral

Figure 1. The means of 10 spectra each from hESCs (green, lower)and differentiated hESCs (red, upper) with their respective envelopesof standard errors of the mean (SEM, black lines) obtained by Ramanmicrospectroscopy of living cells using 100 mW of 785 nm excitation.Means were estimated to contain Raman scattering from at least 30cells. The spectra were background corrected, baseline flattened, andsmoothed. Non-nucleic acid spectral components dominated thespectra from differentiated cells, while nucleic acid peaks were moreprominent in the spectra from undifferentiated cells. Asterisks indicatesignificant differences between peaks at a confidence level of 95%or higher (t-tests, unequal variances).

Figure 2. Raman spectra means, shown in Figure 1, from theundifferentiated and differentiated groups, respectively, were plottedalong with their standard deviations. The latter were scaled to matchthe DNA bands of the mean spectra at 784 cm-1 to assess whetherthe standard deviations were simple multiples of the mean spectra.For differentiated spectra, the variances for nucleic acid bands (DNAat 784 cm-1 and RNA at 811 cm-1) appeared attenuated relative tothose for proteins (e.g., 1003 cm-1) and lipids (e.g., 718 cm-1).Significantly different variances between peaks of hESCs and theirdifferentiated progeny are indicated on the differentiated peaks by a# (F-tests, p < 0.05); variance at 784 cm-1 reduced, others increased.


means (this provided similar utility to that of the percentagerelative standard deviation).

The standard deviations revealed more prominent variationsin cell composition in undifferentiated hESCs, relative to theintensity of the same peak in the mean spectrum, for the nucleicacid peak at 784 cm-1 and the overlapping nucleic acid andprotein peak at 811 cm-1. With the exception of the phenyla-lanine peaks at 1003 cm-1 and 1031 cm-1, protein-related peakswere less prominent at 757, 853, 937, and 1174 cm-1. Thiscontrasted with differentiated hESCs, where protein-relatedbands at 757, 853, 935, and 1003 cm-1 (even more so in thelatter case than for undifferentiated cells) had greater promi-nence. In addition, relative to hESCs, their progeny hadstatistically significant (p < 0.05) increased variances in protein-related peaks at 757, 827, 874, and 1174 cm-1, while thevariance for the nucleic acid peak at 784 cm-1 was significantlyreduced. Thus, when taken together and comparing undif-ferentiated with differentiated hESCs, both a different patternof cellular composition and a different pattern of variation incellular composition emerged.

Although Raman microscopy was sufficiently sensitive to revealdifferentiation status in the presence of different cell types withdifferent cellular dynamics and cellular compositions, thesedifferent patterns of variation suggested that some indices ofdifferentiation status based on ratios would be more affected thanothers. For example, relatively more variation was evident (Figure2) in the 811 cm-1 peak (RNA, proteins) than the 784 cm-1 peak(DNA, RNA), making the former less desirable for selectionas a differentiation state indicator component due to its greatervariability. Thus, the ratio 757/784 cm-1 was more sensitivethan 827/811 cm-1 as an indicator of differentiation state orstatus. Furthermore, we surmised that the type of differentiationthat occurred may have affected these ratios. Thus, forexample, the 757/784 cm-1 ratio may be a good general hESCdifferentiation state indicator, while the 827/811 cm-1 ratio mayprovide a better indicator of the type of differentiation that isoccurring. These considerations are outside the scope of ourcurrent work, but are of future interest.

Multivariate Analyses. Principal components are rankedorthogonal reductions of the original Raman scattering variables(i.e., the spectra shown in Figure S-3 of the Supporting Informa-tion). The first and second PCs, shown in Figure 3, captured 89%of the variance in the combined data (10 spectra from undifferenti-ated and 10 spectra from differentiated hESCs). PC1 representedvariance (71%) mostly due to proteins and lipids and PC2represented variance (18%) mostly related to nucleic acids. FurtherPCs each described less than 3% of the variance in the dataindividually and collectively less than 11%. This confirmed visualobservations (based on Figure S-3) where, all spectra consideredjointly, the biggest spectrum-to-spectrum differences occurred inprotein (e.g., 757, 853, 937, 1003 cm-1) and lipid (e.g., 718 cm-1)bands followed by nucleic acid bands (e.g., 784, 811 cm-1),the latter especially within the undifferentiated group.

When PCA was applied separately to the spectra from undif-ferentiated and differentiated hESCs to remove the influence ofthe differentiation process itself, the first PCs resembled closelythe standard deviations of the respective groups because the firstPC in each group captured a large portion (∼83%) of the variance

in that group. The remainder of the PCs each captured 6% or lessof the variance. The semblance is shown in Figure S-4 of theSupporting Information where the first PCs were plotted alongwith the Figure 2 standard deviations obtained from the spectraof undifferentiated and differentiated cells, respectively. The PCand standard deviation results were supplementary and differedbecause of minor contributions by instrumental and samplingnoise to the standard deviations, but not to the major PCs.

PCA and standard deviations thus suggested that undifferenti-ated and differentiated hESCs had different cellular compositions(reflected by the variance between groups) and different cellulardynamics (in part reflected by the variances within groups).However, in the differentiated group, cellular dynamics wereconfounded with heterogeneous phenotypes. Nevertheless, thePCs emphasized the relative dominance of nucleic acid-relatedchanges in hESCs and the relative dominance of protein-relatedchanges in their differentiated progeny, supporting the utility ofprotein/nucleic acid ratios as indices of differentiation status.2DCOS results (shown in the Supporting Information) furtherstrengthened this view.

Formulation and Evaluation of Differentiation State In-dicators. Although the foregoing results suggested that protein/nucleic acid ratios had good potential as differentiation stateindicators, other indicators were also examined. Table 1 listspotential indicators based on Raman peaks and their statisticalsignificance levels. The first terms in the ratios are from peaksthat increase and the second terms from peaks that decrease inintensity with differentiation. Most of the first terms are protein-related, while the second terms are nucleic acid-related (assign-ments in Table S-1, also Figure 1).

At present, we favor ratios involving peaks within the same“spectral neighborhood” since preprocessing is likely to distorttheir relative values least. The distribution diagram of two suchintensity ratios (R4 ) 757/784 cm-1 and R7 ) 853/784 cm-1) isgiven in Figure 4. The R4 and R7 ratios changed from 0.45 and0.94, respectively, in hESCs to 1.83 and 3.02, respectively, indifferentiated cells (Table 1). Clear separation between the groups

Figure 3. Principal components are orthogonal reductions of theoriginal Raman scattering variables (i.e., the spectra shown in FigureS-3) that capture most of the variance in the original data; the firstand second principal components shown here indicated that most ofthe variance (71%) in the combined data (10 spectra from undif-ferentiated and 10 spectra from differentiated hESCs) was due toproteins and lipids (PC1) followed by (18%) nucleic acids (PC2).


was evident along both dimensions in Figure 4. Thus, a thresholdof approximately 1.00 allowed discrimination between undifferenti-ated and differentiated cells using R4 only, while a threshold ofapproximately 2.00 permitted the same discrimination potentialusing R7 only. A similarly effective discrimination was observedwhen using the lipid peak R8 ) 718/784 cm-1 (data not shown).It should be noted that these thresholds are not absolute butwill depend on the preprocessing methods employed; it istherefore important to maintain consistency once thresholdsare determined. Future investigations will examine the otherpotential differentiation state indicators shown in Table 1 withmore widely separated peaks as well as ratios not listed in Table1 such as R17 ) 729/718 cm-1 (adenine/phospholipids).

The same general patterns of relative dominance of nucleicacid bands in undifferentiated hESCs and protein bands indifferentiated cells persisted robustly in numerous other samples;we have now repeated the protocol tens of times on differentcolonies with similar results. For example, in a much larger sampleof 462 spectra obtained from the same colonies (i.e., 231 spectraeach), these patterns remained after dry-fixing. Applying adifferentiation state indicator (e.g., R4) to the entire sample aftersuch treatment gave us an indication of how robust it was given

that dry fixing could have distorted the spectra somewhat. Whenthe R4 < 1.00 criterion was used to classify these spectra, 229were hits (classified as undifferentiated cells when the spectrawere obtained from undifferentiated cells), 2 were misses (clas-sified as differentiated cells when the spectra were obtained fromundifferentiated cells), 231 were correct rejections (classified asdifferentiated cells when the spectra were obtained from differenti-ated cells), and 0 were false alarms (classified as undifferentiatedcells when the spectra were obtained from differentiated cells).At face value, this amounted to an accuracy of better than 99%assuming that the groups were uniformly undifferentiated anduniformly differentiated. It is worth remarking here that thesespectra did not derive from 462 cells, but from regularly spacedfields arrayed over two (undifferentiated and differentiated) 20µm × 40 µm areas of dense colonies scanned in 2 µm increments;a monolayer would contain ∼25 or more cells per area.

For comparison with simple ratios, a score plot of the first andsecond PCs for the combined data (i.e., undifferentiated anddifferentiated) is also given in Figure 4. This result demonstratedthat multivariate analyses were not required to discriminate hESCsfrom fully differentiated progeny. However, multivariate analysismay well be needed to identify cells in the early stages of

Table 1. Potential Differentiation State Indicator Ratios Based on Raman Peaks Sorted by Decreasing StatisticalSignificancea

ratio first term (significance level) second term (significance level) ratio (hESCs) ratio (progeny) ratio multiple

R1 827 cm-1 (p < 0.001) 784 cm-1 (p < 0.001) 0.17 1.51 9.09R2 1174 cm-1 (p < 0.001) 784 cm-1 (p < 0.001) 0.27 1.44 5.27R3 1003 cm-1 (p < 0.001) 784 cm-1 (p < 0.001) 2.49 9.71 3.90R4 757 cm-1 (p < 0.001) 784 cm-1 (p < 0.001) 0.45 1.83 4.05R5 874 cm-1 (p < 0.001) 784 cm-1 (p < 0.001) 0.37 1.41 3.80R6 1125 cm-1 (p < 0.001) 784 cm-1 (p < 0.001) 1.10 3.75 3.42R7 853 cm-1 (p < 0.01) 784 cm-1 (p < 0.001) 0.94 3.02 3.20R8 718 cm-1 (p < 0.01) 784 cm-1 (p < 0.001) 1.09 3.42 3.13R9 827 cm-1 (p < 0.001) 811 cm-1 (p < 0.05) 0.48 3.85 8.04R10 1174 cm-1 (p < 0.001) 811 cm-1 (p < 0.05) 0.78 3.66 4.66R11 1003 cm-1 (p < 0.001) 811 cm-1 (p < 0.05) 7.16 24.70 3.45R12 757 cm-1 (p < 0.001) 811 cm-1 (p < 0.05) 1.29 4.64 3.59R13 874 cm-1 (p < 0.001) 811 cm-1 (p < 0.05) 1.07 3.58 3.36R14 1125 cm-1 (p < 0.001) 811 cm-1 (p < 0.05) 3.15 9.54 3.03R15 853 cm-1 (p < 0.01) 811 cm-1 (p < 0.05) 2.71 7.68 2.83R16 718 cm-1 (p < 0.01) 811 cm-1 (p < 0.05) 3.14 8.69 2.77

a First terms are from peaks that increase in intensity with differentiation and second terms are from peaks that decrease in intensity withdifferentiation.

Figure 4. The distribution diagram of two protein/nucleic acid intensity ratios (R4 ) 757/784 cm-1 and R7 ) 853/784 cm-1) showed clearseparation between groups along both dimensions. For comparison, a score plot of the first and second principal components for the combineddata (i.e., undifferentiated and differentiated) is also given. This result demonstrated that multivariate analyses were not required to discriminatebetween undifferentiated and fully differentiated hESCs.


differentiation when discrimination would be more difficultbecause the Raman signatures of differentiation are potentiallyobscured by Raman signatures deriving from the cell cycle or fromstress effects.

Preprocessed spectra (e.g., Figure 1) revealed a number ofsingle variables that contained information about differentiation.For example, the protein-related peak (phenylalanine) was stron-ger in spectra from differentiated cells than in spectra fromundifferentiated cells and the opposite pattern occurred for thenucleic acid-related peak. Indeed, the most robust single variablediscriminators were the composite nucleic acid peak ca. 784 cm-1

(with means ± standard deviations for undifferentiated anddifferentiated groups, respectively, of 0.307 ± 0.076 and 0.144± 0.042) and the sharp phenylalanine peak at 1003 cm-1 (0.797± 0.207 and 1.497 ± 0.318). This raised the possibility that, atleast under some circumstances, the absolute intensity of asingle variable could be used as an effective indicator ofdifferentiation status or possibly the state of differentiation, evenin raw spectra (e.g., Figure S-2C of the Supporting Information).

Biochemical Interpretation. Undifferentiated cells have ahigher average complement of nucleic acids than differentiatedcells; conversely, differentiated cells potentially have a higheraverage complement of proteins than undifferentiated cells. Wefound a statistically significant (p < 0.0146) increase in the ratio(w/w) of proteins to DNA by biochemical analyses of cell lysates.This ratio increased by ∼30% from 44 (99% C.I. 38-52) for CA1cells to 58 (99% C.I. 53-64) for 15-day differentiated progenycompared to CA1 cells, providing independent empirical supportto our Raman analyses.

Undifferentiated hESCs proliferate at a high rate, doubling onaverage every 15 to 16 h,47 form rounded colonies of small tightlypacked cells (Figure S-1A), and have high nucleus to cytoplasmratios.48 Compared to somatic cells, hESCs have a much shorterG1 phase (∼25% of somatic cells), while the other stages are ofsimilar duration.47 There is an added effect from the fact thatduring interphase the nucleus is less dense and Raman scatteringis weaker than in mitosis.28,49 Relatively prominent nucleic acidsignals are therefore anticipated from a generally denser28,49 andproportionately larger nuclear compartment.29 The 2-fold (0.307vs 0.144) higher scattering observed for the 784 cm-1 nucleicacid bands in undifferentiated cells (Figure 2) was compatiblewith these results. Moreover, in murine embryonic stem cells(mESCs), the same Raman marker of nucleic acids (e.g., seeFigure 4 of ref. 25) and IR bands of nucleic acids19 decrease inrelative intensity over several days of differentiation.

Also of interest, protein marker bands in hESCs spectrareflected changes in cellular composition (standard deviations,PCs, 2DCOS slices) that specifically overlapped with those ofcollagen and its major constituents proline and hydroxyproline(see assignments in Table S-1), consistent with downregulationof the Col1a1 gene in mESCs within 18 h of leukemia inhibitingfactor (LIF) removal.8 In contrast, protein composition varies byphenotype in hESC progeny.11,19 Our results were consistent with

these considerations showing that in differentiated cells proteinbands dominated nucleic acids bands but appeared different fromprotein bands in undifferentiated cells (Figure S-7).

Radiation Damage. Because little is known about the adverseeffects on hESC pluripotency induced by prolonged tightly focusedIR and near IR (NIR) wavelengths,41 we conducted a preliminarystudy of its effects on the expression of the hESC POU transcrip-tion factor Oct-4, as well as effects on cell morphology, cellproliferation, and colony formation. Oct-4 is required to sustainhESC self-renewal. We confirm here earlier observations41 on thenoninvasive nature of NIR Raman spectroscopy on hESCs andprovide additional results suggesting that pluripotency is notaffected.

Comparative results from NIR laser-irradiated colonies andnonexposed colonies showed that, on the first day after irradiation,cells had normal morphologies (see Figure S-8, top panel, of theSupporting Information). Cells survived and proliferated to gener-ate colonies of the expected size. Cell expansion of approximately20-fold over 3.5 days corresponded to the expected cell doublingtime of ∼20 h generally observed with this hESC line undermaintenance conditions. Colonies were stained for DNA (FigureS-8, middle panel) and Oct-4 (>90%, Figure S-8, bottom panel) andshowed that no significant differences between treated andnontreated hESCs were evident. Taken together, these werestrong indications that this specific laser dose and exposureprotocol was relatively nonperturbing since even small changeswould have been observed; significant cytotoxic effects would haveyielded decreased cell numbers or differentiation toward Oct-4negative cell progeny.

CONCLUSIONSWe have demonstrated here that the very high information

content of Raman microspectra of hESCs and fully differentiatedheterogeneous progeny renders Raman microspectroscopy suit-able for assessing differentiation status. Attendant irradiationappeared to have innocuous effects on morphology, proliferation,and pluripotency. Therefore, in our opinion, Raman microscopyis a comparatively fast, noninvasive, and rich optical interrogationmethod suitable for use with hESCs.

Specifically, although not fully quantitative, our results allowedus to identify Raman bands that had potential to serve as indicesof differentiation state or that could be used in ratios to generatesuch indices. Ratios of neighborhood peaks were deemed espe-cially valuable since they were subject to similar data processingeffects, thus, minimizing relative distortions. It is a first step towardfully characterizing NIR Raman spectra of hESCs and theirprogeny, thus, extending the knowledge base to aid furtherinvestigations as this important field develops.

ACKNOWLEDGMENTWe thank Chris Sherwood for his assistance with some of the

cell culture and analysis work. Instrumentation and infrastructurewere provided by the UBC Laboratory for Advanced Spectroscopyand Imaging Research (LASIR) and Laboratory for MolecularBiophysics (LMB). Funding was provided by the Natural Sciencesand Engineering Research Council (NSERC), the CanadianInstitutes of Health Research (CIHR), the Michael Smith Founda-tion for Health Research (MSFHR), the UBC Centre for Blood

(46) Fieller, E. C. J. R. Stat. Soc. 1940, 7 (Supplement), 1–64.(47) Becker, K. A.; Ghule, P. N.; Therrien, J. A.; Lian, J. B.; Stein, J. L.; van

Wijnen, A. J.; Stein, G. S. J. Cell. Physiol. 2006, 209, 883–893.(48) Sathananthan, H.; Pera, M.; Trounson, A. Reprod. Biomed. Online 2002,

4, 56–61.(49) Mohlenhoff, B.; Romeo, M.; Diem, M.; Wood, B. R. Biophys. J. 2005, 88,

3635–3640.


Research, the Canada Foundation for Innovation (CFI) and theBritish Columbia Knowledge Development Fund (BCKDF).

SUPPORTING INFORMATION AVAILABLEIn the Supporting Information we provide evidence for the

efficacy of the differentiation protocol, further rationalize, with textand figures, our data preprocessing, provide results from 2DCOSas additional support for the use of protein:nucleic acid ratios asdifferentiation state indices, discuss the optimal Raman excitation

wavelength for use with hESCs to avoid deleterious effects, andshow the evidence that irradiation at 785 nm on hESCs isnoninvasive. This material is available free of charge via theInternet at http://pubs.acs.org.

Received for review November 25, 2009. Accepted April28, 2010.

AC902697Q


assessing differentiation status of human embryonic stem cells noninvasively using raman...

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