Vibrational spectroscopy differentiates between multipotent and pluripotent

Vibrational spectroscopy differentiates between multipotent and pluripotent stem cells †Jacek Klaudiusz Pijanka,a Deepak Kumar,a Tina Dale,a Ibraheem Yousef,b Gary Parkes,c Val e rie Untereiner,d Ying Yang,a Paul Dumas,b David Collins,e Michel Manfait,d Ganesh Dhruvananda Sockalingum,d Nicholas Robert Forsyth a and Josep Sul e -Suso *afReceived 16th July 2010,Accepted 5th October 2010DOI:10.1039/c0an00525hOver the last few years,there has been an increased interest in the study of stem cells in biomedicine for therapeutic use and as a source for healing diseased or injured organs/tissues.More recently,vibrational spectroscopy has been applied to study stem cell differentiation.In this study,we have used both synchrotron based FTIR and Raman microspectroscopies to assess possible differences between human pluripotent (embryonic)and multipotent (adult mesenchymal)stem cells,and how O 2concentration in cell culture could affect the spectral signatures of these cells.Our work shows that infrared spectroscopy of embryonic (pluripotent)and adult mesenchymal (multipotent)stem cells have different spectral signatures based on the amount of lipids in their cytoplasm (confirmed withcytological staining).Furthermore,O 2concentration in cell culture causes changes in both the FTIR and Raman spectra of embryonic stem cells.These results show that embryonic stem cells might be more sensitive to O 2concentration when compared to mesenchymal stem cells.While vibrational spectroscopy could therefore be of potential use in identifying different populations of stem cells further work is required to better understand these differences.IntroductionThere is growing interest and urgency placed upon the biomed-ical application of stem cells.It has now been over a decade since the first descriptions of pluripotent human embryonic stem cells (hESC)and the multipotent characterization of human mesen-chymal stem cells (hMSC)emerged.1,2An entire industry has developed since that time with a single commonality,the thera-peutic use of stem cells in regenerative medicine applications.Clinical trials and in-market therapeutic products based on the replication-limited hMSC have now emerged and become rela-tively commonplace.However,replication-unlimited hESC are yet to be involved in patient-based clinical trials.A difficulty which presents itself in the therapeutic use of hESC lies in their inherent characteristics.The hESC is an immortal,self-renewing cell type with a pluripotent differentiation capacity.In addition to this ability to differentiate into cell type’srepresentative of the three germ layers,undifferentiated hESC can also differentiate in situ to form tumours called teratomas.Proposed therapeutic use of hESC is focused on the partially or terminally-differentiated cell,not on the tumour-capable undif-ferentiated cell.However hESC differentiation relies on chemical or biochemical stimulation and as a result is asynchronous in nature.There is however a paucity of reliable markers of differentiation state which enable clear differentiated/undiffer-entiated ratios to be established.Alternatively hMSC are mul-tipotent stem cells which differentiate predominantly into cell types representative of the mesoderm:bone,cartilage,fat,etc.2Unlike hESC,hMSC are mortal and are not tumourigenic.3An objective,non-disruptive,measure to allow distinction between pluripotent and multipotent stem cells in potential stem cell-based regenerative medicine products could represent an invaluable addition to the field.A further compounding complication in making classifications regarding stem cell types comes from multiple observations that stem cell behavior and biology are modified under physiological oxygen conditions.Under these conditions reductions in cell size,reductions in spontaneous differentiation rate,global transcrip-tional alterations,epigenetic modifications,and translational alterations are all observed.4–7These conditions better reflect the oxygen levels found in the in vivo niche of the cells and may more accurately reflect cell behavior and biology in a post-trans-plantation model.This is also of greater value in making reliable assertions about the presence and potency of stem cells in biopsies from cancer patients.Over the last few years,there has been an increased interest in the study of stem cells using Fourier Transform Infrared (FTIR)8–15and Raman spectroscopy.16–18Most of this work hasaInstitute for Science and Technology in Medicine,Guy Hilton Research Centre,Keele University,Stoke on Trent,UK bSOLEIL Synchrotron,BP48,L’Orme des Merisiers,Gif sur Yvette,France cDepartment of Histopathology,Central Pathology Laboratory,University Hospital of North Staffordshire,Stoke on Trent,UK dM eDIAN,CNRS UMR6237-MEDyC,Universit e de Reims Champagne-Ardenne,UFR de Pharmacie,51096Reims cedex,France eSchool of Computing and Mathematics,Keele University,Keele,UK fCancer Centre,University Hospital of North Staffordshire,Stoke on Trent,Staffordshire,ST46QG,UK†This article is part of a themed issue on Optical Diagnosis.This issue includes work presented at SPEC 2010Shedding Light on Disease:Optical Diagnosis for the New Millennium,which was held in Manchester,UK June 26th–July 1st 2010.PAPER /analyst |AnalystP u b l i s h e d o n 18 O c t o b e r 2010. D o w n l o a d e d b y S u n Y a t -S e n (Z h o n g s h a n ) U n i v e r s i t y o n 04/12/2013 02:52:11.View Article Online / Journal Homepage / Table of Contents for this issuebeen aimed at studying stem cell differentiation,19showing that vibrational spectroscopy has the potential to characterize the biochemical make-up of stem cells in a label-free manner.19In the present work,we decided to assess whether both FTIR microspectroscopy and Raman spectroscopy were able to differentiate between two different types of human stem cells with different intrinsic potencies,i.e.pluripotent (hESC)and multipotent (hMSC).Furthermore,we have investigated how two different concentrations of oxygen (2%and 21%)would influence the FTIR and Raman spectral signatures of both cell types.Synchrotron based FTIR (S-FTIR)microspectroscopy was used due to its intrinsic high brightness,which makes it possible to record high-quality spectra at diffraction-limited spot sizes.Synchrotrons are accelerator facilities that provide extremely high-flux and high-brightness electromagnetic radiation,at energies ranging from the IR through the ultraviolet to the X-ray regions.The use of synchrotron in IR micro-spectroscopy has been previously described in the study of biological samples at both cellular and subcellular levels.20,21ExperimentalCell culturehESC (SHEF1)were cultured using feeder-free Matrigel Ô(BD Biosciences,UK)methodologies as described previously.22Briefly,hESC media,80%Knock-out DMEM (Gibco-Invi-trogen,UK)supplemented with 20%KO-Serum replacement (Gibco-Invitrogen,UK),1%L -glutamine,1%Non-essential amino acids,4ng ml À1human basic fibroblastic growth factor (hbFGF)(Gibco-Invitrogen,UK)and 0.1mM b -mercapto-ethanol (Peprotech,UK)was preconditioned on mouse embry-onic fibroblasts (MEF).MEF-conditioned hESC media was supplemented with a further 4ng ml À1hbFGF and filter sterilized before use.hESC were maintained in both oxygen tensions (2%O 2modified Galaxy R +incubator including modular gas loss preventing design:RS Biotech,and 21%O 2incubator:Thermo Electron Corporation,Heraeus Cytoperm 2).5,6Oxygen levels were controlled throughout the experimentation with a NG4000A Laboratory Nitrogen Generator (Peak Scientific,UK).hMSC were cultured on fibronectin (Sigma Aldrich,UK)coated flasks (10ng ml À1)using standard hMSC media consisting of low glucose DMEM (Lonza-BioWhitakker,UK)supple-mented with 2%fetal bovine serum (FBS)(Gibco-Invtirogen,UK),1%L -glutamine (Lonza,UK)and 1%non-essential amino acids (Lonza,UK).The media was changed twice weekly.Sample preparationCells were detached from culture flasks with trypsin/EDTA,counted and brought to a concentration of 105cells ml À1.Cells were deposited on both low-e MirrIR slides,Kevley Technolo-gies (S-FTIR experiments)and Aluminium coated glass slides (Raman experiments)by cytospinning at 1500rpm for 1min,followed by fixation with 4%paraformaldehyde in 0.9%NaCl.After fixation,slides were briefly washed with 0.9%NaCl and water and air-dried.It has been previously shown that formalin fixation preserves lipid,phosphate,and protein components without significantly influencing the IR spectrum of the cell.23S-FTIR spectroscopyThe S-FTIR spectra of hESC and hMSC were recorded at the SMIS beamline at the SOLEIL Synchrotron facilities (Saint-Aubin,France).A Nicolet Nexus FTIR spectrometer coupled to a Nicolet Continuum XL IR microscope fitted with a 32infinity-corrected Schwarzschild objective and equipped with a liquid nitrogen-cooled 50m m MCT/A detector was used.The spectra were collected at 4cm À1resolution using a double-path single masking aperture size of 15Â15m m 2(covering the full synchrotron IR beam size).The spectra were processed and corrected for Mie scatter using Extended Multiplicative Signal Correction.24Principal component analysis (PCA)of the whole spectral region was performed using The Unscrambler v9.8(CAMO software As,Oslo,Norway).Raman spectroscopyThree Raman spectra were obtained from different areas withinthe nucleus for each individual cell.Measurements were per-formed in the point-mode with a LabRam microspectrometer (Horiba Scientific,France).The excitation source was a diode laser (Toptica Photonics,Germany)generating a 300mW single mode line at 660nm.The microspectrometer was equipped with an Olympus microscope (model BX40)and a long working distance Â100objective,which delivered 3mW at the sample.Scattered light was collected by the same objective and a 950lines/mm grating dispersed it on a CCD detector.The spectral acquisition time was 2Â30s.The confocal aperture was set to 150m m and an optical density filter of 0.3was used to reduce the laser power down to 3mW.Spectra were preprocessed using Matlab 7.2(The Mathworks,Natick,MA).All spectra were smoothed using the Savistsky-Golay function (second order polynomial)and corrected for instrument response.The baseline was corrected using a poly-nomial function (order 2)and vector normalized on the whole spectral range.PCA was carried out on the spectral range from 500to 1800cm À1.Staining for lipidsThe staining of lipids in the study cells was carried out following standard protocol at the Pathology department,University Hospital of North Staffordshire (UHNS).Briefly,samples were stained using Oil Red O solution and counterstained in Mayer’s haematoxylin.Cells expressing higher amounts of lipids stained bright red.Results and discussionThe identification of stem cells together with the characterisation of stem cell differentiation requires a number of molecular biology techniques.15Among them,RT-PCR and microarray technologies;RNA in situ hybridisation and immunocytochem-istry make it possible to monitor the expression of specific genes.While these techniques make it possible to obtain detailed information on single differentiation events,non-invasive and rapid methods to better identify stem cells and better characterize stem cell differentiation would be of great relevance and impact in biomedicine.15P u b l i s h e d o n 18 O c t o b e r 2010. D o w n l o a d e d b y S u n Y a t -S e n (Z h o n g s h a n ) U n i v e r s i t y o n 04/12/2013 02:52:11.Vibrational spectroscopy could be a valuable methodology in the study of stem cells as has been previously reported.8–18On this basis,we used both S-FTIR and Raman micro-spectroscopy to assess whether this technique could differentiate between hESC and hMSC.Synchrotron based FTIR microspectroscopy showed differences between hESC and hMSC in the lipid region (Fig.1a and b).The peaks at 2920cm À1,2850cm À1and 1740cm À1exhibit a higher intensity in hESC when compared to hMSC.Further-more,we saw a shift of the 2920cm À1and 2850cm À1peaks towards lower wavenumbers in the case of hESC when compared to hMSC of around 4cm À1wavenumbers.PCA of hESC and hMSC cultured in 2%or 21%O 2showed a separation between these 2cell lines when cultured in either O 2concentration (data not shown)as it could be expected from the differences seen in their S-FTIR spectra (Fig.1a and b).The peaks at 2850cm À1and 2920cm À1correspond mainly to CH 2stretching modes of methylene chains in membrane lipids.25–27Absorptions in the 1740cm À1spectral region arise from the carbonyl C ]O stretching mode of phospholipids.28,29The presence of this peak in the spectra of cells indicates the higher phospholipid concen-trations in the cellular membrane and cytosol (membrane vesi-cles,endosomes,and secretory vesicles).27In fact,isolation of cell nuclei which is associated with the removal of cytoplasm and cellular membrane causes a marked decrease in all these peak areas mentioned above.21In order to validate these data,stainingof lipids with Oil Red O and Mayer’s haematoxylin solutions was carried out.This staining test confirmed the presence of more lipids in hESC all over their cytoplasm when compared to hMSC (Fig.2a and b).It has been previously described that the amount of lipids present in ESC decreases during differentiation.11Our observation that the more differentiated hMSC has reduced lipid content as compared to hESC would,to some extent,confirm and extend this observation.The question remains as to why lipids should be linked to the potency of the stem cell.A recent observation has detailed that inhibition of the eicosanoid pathway is associated with the maintenance of the pluripotent ground state with murine ESC (mESC).30The eicosanoid pathway promotes the hydrolysis of membrane phospholipids releasing lipid messengers into the cytoplasm.31This is suggestive of lipid messengers playing a key role in the control of differ-entiation and perhaps even as determinants of cell fate.However,further work is required in order to assess the functional role of lipids and mechanisms controlling their reduction during stem cell differentiation.hESC and hMSC were also studied with Raman micro-spectroscopy.In this case,3point spectra were obtained from the nuclear area of each cell.The reason to study just the nuclear area lies in the fact that S-FTIR spectroscopy already showed differences in the lipid content of hESC and hMSC by covering the whole cell with the IR beam.However,it was also important for us to assess whether the nuclear content of the 2study cells could also cause changes in their spectra.To this purpose we concentrated on using Raman spectroscopy to study only the nuclear region.It could be argued that studying thecytoplasmicFig.1Mean of 50S-FTIR spectra from 50individual hMSC cells (bottom spectrum)and hESC cells (top spectrum)cultured in 2%O 2(a)and 21%O 2(b).Spectra are offset forclarity.Fig.2Cytology image of hESC (a)and hMSC (b)stained with Oil Red O and Mayer’s haematoxylin.The presence of lipids is indicated by the darker spots (red spots in colour images in the online version)in hESC.P u b l i s h e d o n 18 O c t o b e r 2010. D o w n l o a d e d b y S u n Y a t -S e n (Z h o n g s h a n ) U n i v e r s i t y o n 04/12/2013 02:52:11.region with Raman spectroscopy(where most lipids are together with those forming the cellular membrane)could also confirm the differences in lipids’concentration.However,we carried out preliminary tests and it was difficult with our instrument to get good quality Raman spectra of the cytoplasmic area.The reason behind this is that cells were prepared as cytopsin,as it was carried out for S-FTIR spectroscopy experiments,and,there-fore,the cytoplasm became very thin giving a signal that was too weak for Raman analysis.On the other hand,using Raman microspectroscopy to assess the cell nucleus reduces the possible distortion in the spectral line shape caused by resonant Mie scattering.21,24,32,33,34Fig.3a and b show the mean spectra of hESC and hMSC cultured in2%and21%O2,respectively.The main difference was an increased intensity of the peak at780 cmÀ1for hESC when compared to hMSC when cultured either in 2%or21%O2(Fig.3a and b).This peak together with the peak at1095cmÀ1corresponds to DNA.35It has been previously described that changes in DNA correlate with stem cell differ-entiation with a decrease in DNA during differentiation.15,35,36,37 Although this could explain the more intense DNA bands in the less differentiated hESC when compared to hMSC,we believe that further work is required to better understand these changes. Nevertheless,our data indicates that DNA content seems increased in the more undifferentiated stem cells.On the other hand,the peak at760cmÀ1which corresponds to tryptophan,35 was more intense in hMSC cells when compared to hESC both in 2%and21%O2culture.The increase of tryptophan has also been observed during aging of eye lenses,reflecting local variations in protein structure and a maturational shift.36While it could be argued that the more immature hESC had a decreased amount of tryptophan based on the760cmÀ1peak this was not confirmed when analysing other tryptophan peaks such as880cmÀ1and 1560cmÀ1.38Further work is required in order to assess whether tryptophan could be a marker for stem cell differentiation.We also carried out PCA in order to confirm any possible differences between hESC and hMSC when cultured in2%O2 and21%O2(Fig.4a and b).PCA of hESc and hMSC when cultured in2%O2showed differences between these two cell types(Fig.4a).While PC1and PC2showed a clear difference between hESC and hMSC cultured in2%O2,this was not the case of PC1and PC2for hESC and hMSC cultured in21%O2 (data not shown).However,higher order PCAs(PC2and PC3) did show a difference between these cells when cultured in21% O2(Fig.4b).Furthermore,the loading plots for these PCAs confirmed that the differences lied mainly in the700cmÀ1and the 800cmÀ1region(Fig.5shows a representative example).This would confirm the differences seen in the peaks at760cmÀ1and 780cmÀ1as described above.Another important parameter we investigated was how different O2concentrations could affect the spectra of these cells. Previous reports have detailed the multiple‘improvements’to the hESC culture system through the use of physiological oxygen although the precise molecular signaling mechanisms controlling these outcomes remain unclear.We,and others,have shown that physiological oxygen reduces spontaneous differentiation, promotes transcriptional homogeneity,decreases the frequency of genomic DNA damage,has epigenetic consequences,and results in the generation of a smaller,less complex cell.4–7 Therefore,we assessed whether S-FTIR and Raman spectros-copies could identify differences in these2cell types based on the O2concentration in the culture media.PCA showed separation between the S-FTIR spectra of hESC cultured in2%and21%O2 Fig.3Mean of50Raman spectra from50individual hMSC(bottomspectrum)and hESC(top spectrum)cultured in2%O2(a)and21%O2(b).Spectra are offset forclarity.Fig.4PCA of hESC(filled circles)and hMSC(open squares)Ramanspectra grown in2%O2(a)and21%O2(b).Publishedon18October21.DownloadedbySunYat-Sen(Zhongshan)Universityon4/12/2132:52:11.(Fig.6a).However,differences were not so clear in the case of hMSC cultured in these two different O 2concentrations (Fig.6b).In the case of Raman microspectroscopy,PCA analysis also showed separation in the Raman spectra of hESC cultured in 2%and 21%O 2(Fig.7a)but no major separation in the Raman spectra of hMSC cultured in 2%and 21%O 2(Fig.7b).We looked at the loading plots and they showed that the possible changes fell around the protein bands in both S-FTIR and Raman microspectroscopies,specially around amide I,indi-cating a higher protein content in the hESC cultured with 21%O 2when compared to hESC cultured in 2%O 2.The data here presented using both S-FTIR and Raman spectroscopy shows clearly that O 2concentration in the culture environment could affect the biochemical profile of hESC.Further work is required to better understand the mechanisms controlling these alterations and how these culture conditions could affect stem cell differentiation.The possibility of using both FTIR and Raman spectroscopies to differentiate between different stem cells and to monitor their growth is tantalizing.To be able to better characterise these cells in a simple way and without further sample manipulation would open up an exciting avenue in the study of stem cells for tissue regeneration.ConclusionsThe work presented here shows that S-FTIR spectroscopy is able to differentiate between hESC and hMSC mainly due to an increased presence of lipids in the cytoplasm of hESC.On the other hand,changes in the DNA as shown with Raman spec-troscopy could also be used to differentiate between these 2cell types.To our knowledge this is the first reported observation that stem cells of adult and embryonic origin differ in their lipid composition.The possibility to differentiate using vibrational spectroscopy between pluripotent (hESC)and multipotent (hMSC)cells has important implications inbiomedicalFig.5Loading plots for PCA of hESC and hMSC grown in 2%O 2.PC1plot corresponds to black line.PC2plot corresponds to greyline.Fig.6PCA of hESC (a)and hMSC (b)S-FTIR spectra grown in 2%O 2(closed circles)and 21%O 2(opensquares).Fig.7PCA of hESC (a)and hMSC (b)Raman spectra grown in 2%O 2.(closed circles)and 21%O 2.(open squares).P u b l i s h e d o n 18 O c t o b e r 2010. D o w n l o a d e d b y S u n Y a t -S e n (Z h o n g s h a n ) U n i v e r s i t y o n 04/12/2013 02:52:11.implications such as regenerative medicine.Furthermore,it seems that hESC are more sensitive to O 2concentration in culture conditions when compared to hMSC.Therefore,vibra-tional spectroscopy could also help in assessing the best culture conditions for these cells.AcknowledgementsWe acknowledge SOLEIL synchrotron for provision of synchrotron radiation facilities at beamline SMIS.This project was supported by EPSRC ‘‘Bridging the Gaps’’grant to support 3ME Initiative,the Cancer Centre,UHNS Charitable Fund,the Franco-British Partnership Programme (Alliance 2010),the M.Gibbons Memorial Fund,and the Take A Breath Productions CIC.References1J.A.Thomson,J.Itskovitz-Eldor,S.S.Shapiro,M.A.Waknitz,J.J.Swiergiel,V.S.Marshall and J.M.Jones,Embryonic Stem Cell Lines Derived from Human Blastocysts,Science ,1998,282,1145–1147.2M. F.Pittenger, A.M.Mackay,S. C.Beck,R.K.Jaiswal,R.Douglas,J. 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