Flow Cytometric Cell Cycle Analysis of Cultured Porcine Fetal Fibroblast Cells1

10131014BOQUEST ETAL.FIG.1.A histogram of DNA (red fluorescence)and protein content (green fluorescence)and a scatterplot of protein versus DNA obtained using flow cytometry of cycling cultured porcine fetal fibroblast cells.Single-parameter histogram of DNA allows for the discrimination of cell populations existing in G0ϩG1(2C DNA content),S (between 2C and 4C),and G2ϩM (4C)phases of the cell cycle,while the scatterplot of DNA versus protein also allows for the distinction between G0and G1cell populations.Cell Fixation and StainingFetal cells were analyzed for protein and DNA content by ethanol fixation and staining with propidium iodide and fluorescein isothiocyanate using a modified method de-scribed by Crissman and Steinkamp [16].Trypsinized cells were resuspended in DMEM with 10%FBS and dispensed into 15-ml tubes so that each tube contained 0.5million cells.Cells were pelleted by centrifugation at 500ϫg for 10min and thoroughly resuspended in 1ml of cold ‘‘saline GM’’(6.1mM glucose,137mM NaCl,5.4mM KCl,1.5mM Na 2HPO 4·7H 2O,0.9mM KH 2PO 4,0.5mM EDTA;all reagents were purchased from Sigma),after which 3ml of ethanol (4ЊC)was slowly added to each tube while vortex-ing.After ethanol fixation (at least 12h at 4ЊC),cells were pelleted,then washed once with PBS containing 5mM EDTA.Pelleted cells were stained by adding 1ml PBS containing 30␮g/ml propidium iodide (Molecular Probes,Eugene,OR),0.005␮g/ml fluorescein isothiocyanate (Mo-lecular Probes),and 0.3mg/ml RNase A (Sigma).Staining was achieved at room temperature for at least 1h.Stained cells were filtered through a 30-␮m nylon mesh (Spectrum,Los Angeles,CA)just prior to flow cytometry.Flow Cytometry and Confocal MicroscopyStained cells,suspended in the staining solution,were analyzed using a Becton Dickinson (Rutherford,NJ)flow cytometer as described by Crissman and Steinkamp [16].Fluorescence data were collected using 488-nm excitation and filter combination consisting of a 600-nm long-pass dichroic filter,with 515-and 590-nm long-pass filters.Cells were gated on forward light scatter versus side light scatter such that only cells with a normal DNA and protein content were assayed (see Fig.4).Fluorescence data were obtained from 10000viable cells per sample.Histogram plots of red fluorescence (DNA)and green fluorescence (protein)were created using the Cell Quest program (Becton Dickinson,San Jose,CA).Percentages of cells existing within the var-ious phases of the cell cycle were calculated using Cell Quest by gating on G0,G1,S,and G2ϩM cell populations visualized using the scatterplot of green fluorescence versus red fluorescence (see Fig.1).The effect of cell size on the distribution of cells in the various stages of the cell cycle was also determined.This was achieved using forward light scatter to separately gate on small-,medium-,and large-sized cells (7–15␮m,16–23␮m,24–32␮m,respectively;see Fig.4)and subsequent recalculation of G0,G1,S,and G2ϩM percentages.Confocal microscopy of stained cells was performed us-ing a Bio-Rad MRC-600(Richmond,CA)equipped with a krypton-argon ion laser and mounted on a Optiphoto II Ni-kon (Garden City,NY)microscope.Images were obtained at ϫ400magnification by repeated laser scanning (eight times during 8sec)to improve the signal:noise ratio.Cell TreatmentsCell cycle comparisons were made between cycling cells,serum-starved cells,and cells cultured to confluency.Flasks were seeded with fetal cells (passages 5–10)at a concentration of 105cells/ml.After 2days of culture,cells were allocated to one of the following treatments before being fixed in ethanol as described above:1)immediate fixation (cycling cells);2)replacement of growth medium with DMEM ϩ0.5%FBS and culture for either 5or 10additional days (serum-starved cells);3)changing ofgrowth medium every 3days for an additional 10days of culture (culture to confluency).Fetal cells were also exposed to cell cycle inhibitors.After culture for 1day,medium was removed from flasks and replaced with growth medium containing colchicine (Sigma;0␮M,0.5␮M,1.0␮M,1.5␮M),mimosine (Sig-1015CELL CYCLE ANALYSIS OF SERUM-STARVEDCELLS FIG.2.Typical histograms of DNA (red fluorescence)and protein content (green fluorescence)and scatterplots of protein versus DNA,obtained using flow cytometry of porcine fetal fibroblast cells cultured under a variety of conditions.ma;0mM,0.4mM,0.8mM,1.2mM),or DMSO (Sigma;0,0.5%,1.0%,2.5%).Cells were cultured with colchicine and mimosine for 24h,and DMSO-treated cells were cul-tured for 48h before fixation.The above-mentioned ex-periments were replicated three times.Cells from each rep-licate were analyzed by flow cytometry on separate occa-sions.On each occasion,two samples of each treatment were analyzed (n ϭ6).Statistical AnalysisStatistical analysis was performed using the General Lin-ear Models procedure in the Statistical Analysis System (Cary,NC).Differences between treatments were deter-mined using the Student’s t -test and were judged to be sig-nificant when p Ͻ0.05.RESULTSFigure 1represents the distribution of cycling fetal cells existing in the various phases of the cell cycle as deter-mined by flow cytometry.Single-parameter histograms of DNA (red fluorescence)provided data for percentages of cells existing in G0ϩG1(2C DNA content),S (between 2C and 4C),and G2ϩM (4C).The distinction between G0and G1cell populations was made possible by computer-gen-erated scatterplots showing the 2-dimensional distributionof dots whose horizontal and vertical displacement from the origin represents the relative red (DNA)and green (protein)fluorescence intensity,respectively,of individual cells.Rectangular gates set on dots depicting cells enabled the calculation of percentages of cells existing in G0,G1,S,and G2ϩM.The G0/G1gate border was set on cells having protein levels lower than the vast majority of S-phase cells and estimated to not include G1transient cells,which have levels of RNA (or protein)between G0and G1[12,15].Once this gate was set,it was not altered for the entire course of these experiments.As made evident by the prominent G0ϩG1DNA peaks (Figs.1and 2),the majority of fetal cells at a given point in time were in G1or pared to values in cycling cells,however,higher percentages of cells in G0ϩG1were apparent when cells were serum starved or cultured to con-fluency (Table 1).Conversely,cycling cultures contained greater percentages of cells in G2ϩM than other treatments.In comparison to growth to confluency and cycling treat-ments,serum starvation resulted in lower percentages of cells existing in S phase.Scatterplots of protein versus DNA and single-parameter histograms of protein both clearly show a marked decline in the levels of cellular protein of fetal cells as a conse-quence of serum deprivation (Fig.2).As made apparent by the wide-spreading green fluorescence histograms,the char-1016BOQUEST ETAL.FIG.3.Confocal micrographs of porcine fetal fibroblast cells from cy-cling cultures (A )and cultures serum starved for 5days (B ).Insets in A and B show high magnification of some of the cells.Green images show cellular protein,whereas red images show DNA.Yellow images are the overlay of green and red (DNA and protein)images.Bar ϭ50␮m.FIG.4.Scatterplot of side light scatter (particle density)versus forward light scatter (particle size)of cycling porcine fetal fibroblast cells allowing for gating of only the viable cell population excluding cellular debris and cell doublets.The effect of cell size on the distribution of cells in the various phases of the cell cycle was obtained by resetting gates to only include small-,medium-,and large-sized viable cells.TABLE 2.Percentages (ϮSD)of porcine fetal fibroblast cells in the var-ious phases of the cell cycle when forward light scatter gating was set to only include small,medium or large size cells.*Cell cycle phaseCell sizeSmallMedium Large Cells from cycling cultures G0G1G0ϩG1S G2ϩM 7.1Ϯ3.1a 79.7Ϯ3.8a 86.8Ϯ1.4a 6.6Ϯ1.1a 6.6Ϯ0.8a0.3Ϯ0.5b 69.1Ϯ3.6b 69.4Ϯ3.8b 8.2Ϯ1.0b 22.4Ϯ3.5b0.0Ϯ0.0b 28.7Ϯ8.7c 28.7Ϯ8.7c 10.3Ϯ2.3b 61.1Ϯ9.3c Cells from cultures serum starved for 5days G0G1G0ϩG1S G2ϩM 72.2Ϯ12.0a 23.1Ϯ11.8a 95.2Ϯ0.3a 2.1Ϯ0.4a 2.7Ϯ0.5a 28.5Ϯ13.1b54.9Ϯ11.1b 83.5Ϯ2.2b3.9Ϯ1.1a12.8Ϯ2.7b 0.8Ϯ0.9c 45.6Ϯ4.4b 46.5Ϯ4.9c 8.5Ϯ3.1b 45.1Ϯ7.0c Cells from cultures serum starved for 10daysG0G1G0ϩG1S G2ϩM 64.2Ϯ20.3a 30.1Ϯ19.8a 94.3Ϯ0.81a 2.0Ϯ0.4a 3.7Ϯ0.7a 19.5Ϯ15.4b 57.7Ϯ15.0b77.1Ϯ1.8b 4.6Ϯ1.6b18.3Ϯ1.0b 0.5Ϯ0.6c 39.5Ϯ2.3a 40.0Ϯ2.2c 7.5Ϯ2.5c 52.5Ϯ4.0c Cells from cultures grown to confluency G0G1G0ϩG1S G2ϩM 15.8Ϯ14.1a 77.6Ϯ13.8a 93.4Ϯ1.0a 3.8Ϯ1.3a 2.7Ϯ0.5a 1.9Ϯ2.0b 82.2Ϯ3.1a 84.1Ϯ2.8b 6.6Ϯ2.7a 9.3Ϯ1.0b0Ϯ0b 59.7Ϯ7.1b 59.7Ϯ7.1c 11.8Ϯ5.5b 28.4Ϯ3.2c*Percentages with different superscripts within rows differ significantly (p Ͻ0.05).TABLE 1.Percentages (ϮSD)of porcine fetal fibroblast cells in the var-ious phases of the cell cycle after a variety of culture treatments.*Cell cycle phase Culture treatmentCycling 5Days serum starvation 10Days serum starvation Culture to confluency G0G1G0ϩG1SG2ϩM2.8Ϯ1.2a 71.3Ϯ3.5a 74.1Ϯ3.0a 7.8Ϯ1.0a 18.2Ϯ2.7a48.3Ϯ9.7b 39.2Ϯ9.4b 87.5Ϯ0.7b 3.3Ϯ0.9b 9.3Ϯ1.4b48.5Ϯ17.6b 38.9Ϯ17.1b 87.4Ϯ1.6b 3.0Ϯ0.7b 9.7Ϯ1.7b6.0Ϯ5.3a 79.1Ϯ6.6a 85.1Ϯ2.8b 6.1Ϯ2.5a 8.8Ϯ1.6b*Percentages with different superscripts within rows differ significantly (p Ͻ0.05).acteristic feature of cells from cycling cultures and cultures grown to confluency was cell heterogeneity with respect to protein content.As a result,percentages of cells in G0did not differ between cycling and confluent treatments,even though confluent cultures contained higher G1percentages (Table 1).By comparison,histograms of green fluorescence of starved cells displayed a shift toward the axis indicating that the majority of these cells had low protein content.Confocal micrographs of stained cells clearly illustrate the lower protein content of starved cells compared with cy-cling cells (Fig.3).Accordingly,serum starvation markedly increased percentages of cells in G0.Approximately 45%more starved cells were in G0as compared to their cycling counterparts.However,prolonging starvation for an addi-tional 5days did not increase percentages of cells shiftinginto G0.Scatterplots of serum-starved cultures also showed that low protein content was evident not only for cells with 2C DNA content but also for cells with 2ϽC Ͻ4and 4C DNA content.Based on forward light scatter,a measurement of particle (cell)size,percentages of cells in the various stages of the1017CELL CYCLE ANALYSIS OF SERUM-STARVED CELLSTABLE 3.Percentages (ϮSD)of porcine fetal fibroblast cells gated using forward light scatter to only include small,medium or large size cells under a variety of culture conditions.*Cell size Culture treatmentCycling 5Days serum starvation 10Days serum starvation Culture to confluency Small Medium Large41.7Ϯ5.2a 51.4Ϯ5.5a 6.9Ϯ4.1ab50.9Ϯ4.3b 44.5Ϯ3.6b 4.6Ϯ0.9b66.1Ϯ5.5c 30.7Ϯ4.7c 3.2Ϯ1.0b41.3Ϯ8.5a 48.8Ϯ3.7ab 10.0Ϯ5.1a*Percentages with different superscripts within rows differ significantly (p Ͻ0.05).TABLE 5.Percentages (ϮSD)of porcine fetal fibroblast cells existing within the various phases of the cell cycle after treatment with colchicine for 24h.*Cell cycle phase Colchicine (␮M)00.5 1.0 1.5G0G1G0ϩG1SG2ϩM4.4Ϯ2.6a 69.4Ϯ0.9a 73.8Ϯ2.4a 9.1Ϯ1.617.1Ϯ3.9a2.4Ϯ1.1a 52.3Ϯ6.0b 54.7Ϯ5.7b 8.0Ϯ2.237.4Ϯ4.6b3.5Ϯ2.1a 48.7Ϯ1.8b 52.1Ϯ3.4b 8.7Ϯ2.839.1Ϯ5.4b1.4Ϯ0.7b 49.8Ϯ4.2b 51.2Ϯ4.1b 8.1Ϯ2.440.9Ϯ2.0b*Percentages with different superscripts within rows differ significantly (p Ͻ0.05).TABLE 6.Percentages (ϮSD)of porcine fetal fibroblast cells existing within the various phases of the cell cycle after treatment with mimosine for 24h.*Cell cycle phase Mimosine (mM)00.40.8 1.2G0G1G0ϩG1SG2ϩM5.7Ϯ5.369.4Ϯ4.9ab 75.2Ϯ1.2a 7.5Ϯ0.7a 17.4Ϯ1.0ab3.5Ϯ2.764.4Ϯ1.2b 67.9Ϯ2.1b 13.9Ϯ4.3b 18.3Ϯ3.0b2.5Ϯ2.273.1Ϯ5.4ac 75.5Ϯ4.1a 10.2Ϯ0.9a 14.2Ϯ4.1ac2.2Ϯ2.375.8Ϯ4.1c 78.0Ϯ4.2a 10.4Ϯ3.2a 11.7Ϯ3.9c*Percentages with different superscripts within rows differ significantly (p Ͻ0.05).TABLE 4.Percentages (ϮSD)of porcine fetal fibroblast cells existing within the various phases of the cell cycle after treatment with DMSO for 48h.*Cell cycle phase DMSO (%)00.5 1.0 2.5G0G1G0ϩG1SG2ϩM4.7Ϯ1.6a 79.7Ϯ1.9a 84.2Ϯ0.9a 4.0Ϯ0.6a 11.8Ϯ0.9a7.9Ϯ3.2a 78.6Ϯ1.8a 86.5Ϯ2.3b 3.6Ϯ1.0a 9.9Ϯ1.6b24.8Ϯ20.0b 63.9Ϯ19.6b 88.7Ϯ1.3c 2.4Ϯ0.5b 9.0Ϯ1.0b29.3Ϯ9.6b 56.8Ϯ10.5b 86.0Ϯ1.7b 2.0Ϯ0.4b 12.1Ϯ1.4a*Percentages with different superscripts within rows differ significantly (p Ͻ0.05).cell cycle were calculated when gates were set to include only small-,medium-,or large-sized cells (Fig.4).Micro-scopic measurement of a population of trypsinized cycling cells showed that cell diameters ranged between 7␮m and 32␮m with the average being 18␮m.For every culture treatment,it is clearly evident that as the cell size decreased from large to small,percentages of cells existing in G0ϩG1and G0alone increased significantly,whereas percentages of S-and G2ϩM-phase cells decreased (Table 2).In cul-tures that were serum starved for 5days,72.2%of small cells existed in G0,whereas in cycling cultures,79.7%of small cells were in parisons of percentages of cells gated as small,medium,or large sized between culture treatments showed that starved cultures contained greater percentages of small-sized cells than cycling and confluent cultures (Table 3).Serum starvation for 10days resulted in significantly higher percentages of smaller cells than 5days starvation.Cell cycle inhibitors DMSO,mimosine,and colchicine were added to cultures in an attempt to synchronize fetal cells in G0,G1,and G2ϩM phases of the cell cycle,re-spectively.Treatment of cultures with DMSO for 48h in-creased percentages of G0cells in a dose-dependent fashion (Table 4).Optimal G0arrest of 24.8%of the cell population occurred when DMSO was added at a concentration of 1%.Cultures also responded to colchicine treatment:G2ϩM percentages were higher than in their untreated (0␮M col-chicine)counterparts when colchicine was added at a con-centration of 0.5␮M,although concentrations above 0.5␮M did not further increase percentages of cells in G2ϩM (Table 5).Fetal cells were found to be relatively unrespon-sive to mimosine treatment (Table 6).Significantly greater percentages of G1cells occurred in comparison to those in control cultures (0mM)only when mimosine was added at a high concentration of 1.2mM,with the difference being only about 5%.DISCUSSIONThe development of reconstructed embryos following nuclear transfer appears to be dependent upon a variety of factors.The most important factor identified thus far is cell cycle synchrony of donor nuclei with recipient enucleated oocytes (reviewed in [1–3]).Donor nuclei must be in G1or G0,when transferred to fresh oocytes with high levels of maturation-promoting factor,in order to condense nor-mally and maintain correct ploidy of subsequent embryos at the end of the first cell cycle.In an attempt to improve donor nuclei treatment prior to nuclear transfer,we used flow cytometry to study the cell cycle characteristics of porcine fetal cells cultured under a variety of cell cycle-arresting conditions.The prominent G0ϩG1DNA peaks suggest that,like other nontransformed fibroblasts [18],porcine fetal fibro-blasts have an inherently long G1phase.Alterations to cul-ture conditions,however,changed the dynamics of their cell cycle.Serum-starved cultures and cultures grown to con-fluency contained higher and lower percentages of G0ϩG1and G2ϩM cells,respectively,compared to cycling cul-tures.One can therefore predict that the use of nuclei from serum-starved or confluent cultures will result in higher percentages of reconstructed embryos containing the cor-rect complement of DNA.It is known that,for certain cell types,serum starvation is not a viable method for synchronizing cells into G0,ei-ther because the cells arrest in G1instead or because they undergo apoptosis [13,19].Since quiescent cells contain characteristically low levels of protein [10,13],we mea-sured cellular protein levels in concert with DNA content and found that porcine fetal fibroblasts do enter G0in re-sponse to serum starvation.Measurement of green fluores-cence clearly indicated a marked decline in levels of cel-lular protein when cells were deprived of serum.We found that starved cultures contained approximately 45%more cells in G0than their cycling counterparts and that 5days1018BOQUEST ET AL.of starvation was sufficient for obtaining the highest per-centages.The results also demonstrate,however,that only a subpopulation of cells were able to enter G0in response to serum starvation.The majority of other cells appeared to be induced into quiescence while in S and G2ϩM phas-es,since scatterplots of serum-starved cells displayed many with2ϽCϽ4and4C DNA content also containing low levels of protein.Quiescent cultures of other cell types have also been found to contain noncycling populations of S and G2cells[14].Culture to confluency is another strategy used to shift cells into G0in response to overcrowding[20,21].In con-trast to observations in other nontransformedfibroblasts [20],we did not see greater G0percentages in cultures grown to confluency;instead percentages of G1cells in-creased.This result highlights the importance of usingflow cytometry to assess the outcome of a particular cell cycle-arresting strategy for each cell line.By gating on small-,medium-,and large-sized cells,we found that percentages of cells existing in the various phas-es of the cell cycle differed due to cell size.As the cell size decreased from large to small,percentages of cells in G0ϩG1and G0alone increased,whereas S and G2ϩM percentages decreased.This result was expected because cellular volume increases from G1through G2ϩM phases of the cell cycle[13,22].Accordingly,starved cultures contained higher percentages of small-sized cells as com-pared with cycling cultures.A60%reduction in the cell size of proliferating3T3cells has been reported to occur after8h of serum starvation due to the rapid decline in protein synthesis[10].This may help to explain why star-vation for an additional5days further increased percent-ages of small cells.Since the vast majority of small cells contained a2C complement of DNA,it is envisaged that the use of small donor cells will lead to higher embryonic development rates after nuclear transfer.Indeed,nuclear transfer work in our laboratory has shown that rates of chromosome con-densation and pronuclear formation doubled in reconstruct-ed pig embryos with use of small fetalfibroblasts as com-pared to largefibroblasts(unpublished results).Moreover, since72.2%of small starved cells were in G0whereas 79.7%of small cycling cells were in G1,future experiments comparing the use of small cycling and small starved cells may shed light on the importance of G0in normal devel-opment of reconstructed embryos.We also tested the response of fetalfibroblasts to cell cycle inhibitors DMSO,mimosine,and colchicine,which have been shown to arrest cultured cells in G0,G1,and G2ϩM,respectively[15,20,23].Addition of1%DMSO to cycling cultures for48h resulted in24.8%of cells en-tering G0and therefore offers an alternative strategy to se-rum starvation for obtaining G0cells.Fibroblasts respond-ed to colchicine treatment(0.5␮M),since percentage of G2ϩM cells increased to37.4%.However,higher concen-trations of colchicine did not further increase G2ϩM per-centages.It has been reported that mimosine added to cul-tures of a variety of cell lines at0.2mM reversibly arrests cells late in G1[24]by reducing the activity of the protein synthesis initiation factor eIF-5A[20].In contrast,our re-sults show that mimosine treatment had little effect in in-creasing G1percentages of fetalfibroblasts even when add-ed at a high concentration of1.2mM.Once again,this result highlights the importance of verifying the respon-siveness of each cell line to a particular cell cycle-arresting treatment.In this study,we usedflow cytometry to examine the cell cycle characteristics of porcine fetalfibroblast cells. 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