标题: Monitoring Differentiation of Human Embryonic Stem Cells Using Real-Time PCR [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:47 标题: Monitoring Differentiation of Human Embryonic Stem Cells Using Real-Time PCR
b TATAA Biocenter, Lundberg Laboratory, G?teborg, Sweden;" B s T+ Y' D
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c Cellular Neurobiology Branch, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland, USA; % h. Y. z+ |- W5 b' z& g . i+ M" Y" s) Ad Laboratory of Neurosciences, National Institute of Aging, Department of Health and Human Services, Baltimore, Maryland, USA;* H6 H1 `6 U4 j4 R
0 t* x. @6 X4 ?5 ce Section of Endocrinology, Lund University, Lund, Sweden) T$ B3 R" c: @4 G
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Key Words. Human embryonic stem cell ? Differentiation ? Real-time polymerase chain reaction ? Gene expression- g0 R9 \: N) J2 E% B4 o+ N5 l
! ]4 D' @/ L) b/ u2 [+ LABSTRACT; y: c7 f0 |4 z1 I6 i; {% J2 f
1 e1 M. E# {: C5 ZPopulations of pluripotent human embryonic stem cells (hESCs) can be derived from the inner cell mass of blastocysts and have the capacity for indefinite, undifferentiated proliferation in vitro . Differentiation of hESCs may occur spontaneously in vitro, especially during suboptimal culture conditions. In addition, hESCs can be coaxed to differentiate in a directed fashion along specific pathways forming a variety of specialized cell types. However, relatively little is currently known about how to control and manipulate hESC differentiation to produce exclusive populations of specific cell types. Besides their importance in basic research, promising future applications of hESCs and their derivatives include cell-replacement therapies . In addition, the hESC technology platform holds tremendous potential in novel approaches for drug discovery and in vitro toxicology . 4 Z7 b: |3 \, e : f2 A1 d) D4 x, J+ rTo maintain hESCs in an undifferentiated state in vitro, the cells are usually cultured on top of a feeder layer obtained either from animal or human sources . In addition, feeder-free conditions for hESC culture have also been reported . Furthermore, efficient propagation of undifferentiated hESCs is also critically dependent on timely passaging of the cells. Normally, this time interval is between 4–7 days, depending on the culture conditions . Despite controlled and standardized culture conditions, hESCs may undergo spontaneous differentiation during in vitro propagation. Differentiating hESCs can be identified based on changes in the morphology of the cells, their downregulation of expression of stem cell–specific markers, and concomitant upregulation of markers for differentiated cell types . Consequently, visual inspection of hESC colonies in concert with immunohistochemical evaluation of the cells is instrumental for quality control of hESC cultures. Furthermore, various labor-intensive and time-consuming tests can be performed in vitro and in vivo to demonstrate the pluripotency of hESCs .8 Z+ v6 b0 e0 R6 l
6 i: |! B' |+ g7 F+ JGene-expression analysis of hESCs is a valuable complement to the approaches indicated above. Global gene-expression profiling has been performed by several independent investigators using a variety of hESC lines in attempts to define a set of universal "stemness" genes . Although some progress in defining genes associated with the pluripotent state has been made, based on the results from these studies it is obvious that there are indeed substantial differences in the gene-expression profiles between individual hESC lines. This is, however, not surprising since all hESC lines are derived from different embryos, each representing a unique genetic background. In addition, the differences in culture conditions used by the various laboratories obscure the interpretation of the data. Thus, the list of genes that can be considered as common molecular markers for undifferentiated hESC is currently relatively short, and among them are the transcription factors Oct-4 and Nanog . On the other hand, derivatives of hESCs can be identified by a number of genes that are expressed exclusively by differentiated cells.: G3 m. o4 `# U5 n1 p
; s* i2 ?5 \- m6 BThe future use of hESCs in drug development and for in vitro toxicity testing will require sensitive and quantitative methods for determination of the differentiation state of the cells. Importantly, these assays should be possible to implement in high-throughput analysis. Quantitative real-time polymerase chain reaction (QPCR) fits these requirements and has emerged as a very attractive large-scale screening technique . & v2 M0 e! G$ E) B B5 t, V9 Y- H. I* y3 d8 {5 K/ ~
Here we describe an approach, based on QPCR, for the quantitative evaluation of differentiating hESCs. By measuring the relative mRNA levels of Oct-4, Nanog, Cripto, and -fetoprotein (AFP) in the same hESC sample and combining these values into an expression index, it is possible to discriminate between undifferentiated hESCs and their early derivatives. We evaluated the method using several independent hESC lines maintained in feeder-dependent and feeder-free conditions and demonstrated that the method is very robust and generally applicable for all cell lines tested. The combination of QPCR and hESC technologies provides novel opportunities for high-throughput analysis of hESCs. 4 K# G8 q% i6 [& U' a) W4 M. l - Y- ~0 G$ w: w4 d: i, Q' N) @MATERIALS AND METHODS 2 g0 Z, u# v9 R7 g6 @6 [' e4 r- q) p% z
Culture and Differentiation of hESCs' c; I0 H m% f$ u7 O0 m. y6 d
- z6 e Y& S% `3 J; OThe hESC lines maintained on MEF and used in this study have been extensively characterized previously, and they express cell-surface antigens and transcriptional markers expected for undifferentiated hESCs as well as exhibiting in vivo and in vitro pluripotency (Cellartis AB, unpublished results). As illustrated in Figures 1A and 1D, 5-day-old hESC colonies displayed the morphology characteristic for undifferentiated hESCs (i.e., large, compact, multicellular colonies of cells with a high nucleus-to-cytoplasm ratio). At this time point, the hESC cultures are normally passaged by mechanical dissociation. However, upon extended in vitro culture, without passaging, the hESCs differentiate spontaneously and generate heterogeneous populations of cells with a variety of morphologies. Figure 1 (1B, 1C, 1E, and 1F) shows differentiating cells at days 14 and 21 after passage. The expression of markers indicative of endo-, ecto-, and mesodermal derivatives has previously been demonstrated in these cells . In addition, we observed that undifferentiated hESC colonies efficiently formed simple and cystic embryoid bodies when placed in suspension cultures , whereas this ability was substantially lower in differentiating cells (data not shown). Taken together, these data indicate that the hESCs remain pluripotent at least up to 5 days after passage, whereas the cultures at days 14 and 21 consist of heterogeneous populations of undifferentiated and differentiating cells. % h$ @6 V1 R4 l9 V3 H0 Y, Q( d, X2 q9 M
Figure 1. Representative illustrations of the morphology of undifferentiated and differentiating human embryonic stem cells (hESCs): (A) SA002 at day 5, (B) SA002 at day 14, (C) SA002 at day 21, (D) AS034 at day 5, (E) AS034 at day 14, and (F) AS034 at day 21. The cells were cultured on mouse embryonic fibroblasts using VitroHES medium supplemented with basic fibroblast growth factor and passaged mechanically every 4–5 days. Extended culture of the hESCs without passaging initiated differentiation, and a mixture of early hESC-derivatives was obtained. Magnification x 100./ [/ ^! K0 r" w1 Y
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Immunohistochemical Analysis of Differentiating hESCs& r0 y& \$ l# a5 H) A! {
7 _/ X8 h( o& @$ C( |The temporal expression of several frequently used hESC markers was evaluated using immunohistochemistry. The hESCs maintained on MEF were fixed at different time points after passage. Subsequently, the cells were stained using antibodies directed against SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Oct-4, Nanog, SSEA-1, and AFP. Representative staining patterns obtained for SSEA-3 and TRA-1-60, SSEA-1, and AFP are shown in Figure 2. Semiquantitative evaluation of the staining intensities was performed as described in Materials and Methods, and the results are presented in Figure 3. The results showed that SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Oct-4, and Nanog were expressed by the vast majority of the cells in the undifferentiated hESC colonies at 4–5 days after passage. During differentiation, the expression of these antigens decreased as expected, although the kinetics of disappearance appeared to be different between the antigens. Whether these results reflect the actual expression levels in the differentiating hESCs or a difference in turnover of the antigens remains to be investigated. Interestingly, none of these antigens disappeared completely in the hESC colonies within the time span for differentiation used in this study. Importantly, markers for differentiated hESCs, such as SSEA-1 and AFP, were not detected in undifferentiated hESCs (5-day-old colonies), but at 9 days after passage these antigens were observed in some regions of differentiating colonies (Figs. 2, 3). Extended in vitro culture resulted in increased expression of SSEA-1 and AFP, and at day 22, the majority of the cells in approximately 50% of the hESC colonies expressed these antigens. Interestingly, based on morphological evaluation and immunohistochemical analysis, undifferentiated hESCs were also identified in certain regions of differentiating colonies even after 24 days of in vitro culture, suggesting that hESCs can undergo several cell divisions in "differentiation-promoting" culture conditions while maintaining their pluripotent phenotype . 0 `$ J3 } x% W# r3 @5 A% G4 ` w6 \; D u I- p" g7 ~
Figure 2. Undifferentiated and spontaneously differentiated human embryonic stem cell colonies maintained on mouse embryonic fibroblasts were analyzed using immunohistochemistry as described in Materials and Methods. The panels show representative 4,6-diamidino-2-phenylindole (DAPI) stainings and the corresponding specific antibody staining for SSEA-3 (SA181), TRA-1-60 (SA181), SSEA-1 (SA181), and AFP (SA121) as indicated. The cells were fixed and analyzed at the time points indicated on the left. Magnification x 100." P% I$ Z: t* Y2 Z" z+ J. T( _+ W
- H' V! D* w: D7 JFigure 3. Semiquantitative evaluation of the immunohistochemical staining of undifferentiated and differentiating human embryonic stem cells (hESCs) was performed as described in Materials and Methods: (A) SA181, (B) SA202, and (C) SA121. For each data point, 12–32 individual hESC colonies were evaluated. Abbreviation: AFP, -fetoprotein. / U6 j0 B& @# n1 s* o6 P9 C m! ?$ f; q) S" B! o* ~
QPCR Analysis of Undifferentiated and Spontaneously Differentiated hESCs 1 `& \% O+ D( | * v) w5 f) N- C% R s! Y) B0 IQPCR systems were designed and optimized for the group of genes indicated in Materials and Methods. Subsequently, we analyzed the mRNA levels of these genes in undifferentiated and differentiating hESCs maintained on MEF. The cells were harvested at days 5, 14, and 21 after passage and total RNA was extracted. After reverse transcription and QPCR analysis, Ct values were obtained for all individual samples (data not shown). Among the genes tested, Cripto, Oct-4, and Nanog were all significantly downregulated upon differentiation of the hESCs. On the other hand, the mRNA level of AFP was substantially increased during differentiation of the hESCs. Due to their consistent and reproducible expression patterns in all five hESC lines tested, we included these four genes as reporter genes in the final QPCR assay. The remaining genes analyzed displayed either inconsistent expression profiles when comparing the different hESC lines or there were little or no changes in their relative mRNA levels during the timeframe used here. a% {; d; H2 K# E# v . |" |% d) ?) r+ D8 Y! cQuantifying the Differentiation State of hESCs ? To obtain a quantitative measure of the relative level of differentiation of the hESCs, the mathematical model (Equation 1) described in Materials and Methods was applied. The input in the equation is the Ct value for each individual reporter gene and the corresponding PCR efficiency, and the output is an index based on the geometric averages between the mRNA levels of down- and upregulated genes. By including several genes, the assay becomes very robust, while maintaining a high precision, and less sensitive to minor fluctuations in the expression levels of the individual genes. Figure 4 shows the calculated expression indexes from the QPCR analyses of hESCs maintained on MEF. For the five cell lines tested, the expression indexes ranged between 150 and 350 for undifferentiated hESCs (day 4–5) and between 0.6 and 10 for differentiating hESCs at day 14. In differentiated progenies from hESCs at day 21, the expression indexes were further decreased and ranged between 0.05 and 2. The quantitatively most striking changes in the expression indexes occurred during the first week of differentiation. The fold changes observed in the expression indexes were between 26 and 415 when comparing cells at day 5 and 14, and the fold changes ranged between 1.2 and 15 when comparing cells at days 14 and 21.- U. R# v8 g4 X1 Q# e- |" x/ E
2 s! w/ k) g# QFigure 4. Quantitative real-time polymerase chain reaction (QPCR) analysis of undifferentiated and differentiating human embryonic stem cells (hESCs) maintained on mouse embryonic fibroblasts: (A) SA001, (B) SA002, (C) AS034, (D) AS034.1, and (E) SA121. The cells were cultured as described in Materials and Methods and harvested at the time points indicated on the x-axis. After RNA extraction, the relative mRNA levels of Oct-4, Nanog, Cripto, and -fetoprotein were determined using QPCR. The expression index was subsequently calculated using the equation indicated in Materials and Methods (Equation 1). The data are presented as the mean plus SD (n = 4). 9 F/ U* k; S- B ( d- w, E" ^- D K5 ^/ ^2 PBy using monoclonal antibodies immobilized on magnetic beads, we separated SSEA-4–positive and SSEA-4–negative fractions of hESCs from a heterogeneous cell population harvested at day 7 after passage. These cells represent a mixture of undifferentiated hESCs and early progenies thereof. Approximately 60% of the cells were captured by the SSEA-4 antibody–coated beads. These results correlate well with the semiquantitative evaluation of the SSEA-4 immunostainings (Fig. 2B). The fractionated cells were subsequently analyzed using QPCR as indicated above. The results are shown in Figure 5 and demonstrate that the expression index of SSEA-4–positive cells is about 15-fold higher than the expression index of SSEA-4–negative cells. 7 a/ T' E! k( I2 _1 p , i7 t- \ u3 ZFigure 5. Quantitative real-time polymerase chain reaction (QPCR) analysis of SSEA-4–positive and SSEA-4–negative human embryonic stem cells. The cells (SA121) were cultured as described in Materials and Methods, harvested at day 7, and fractionated using SSEA-4-antibody–coated magnetic beads. After RNA extraction, the relative mRNA levels of Oct-4, Nanog, Cripto, and -fetoprotein were determined using QPCR. The expression index was subsequently calculated using Equation 1. The data are presented as the mean plus SD (n = 2).. B* i+ L/ y2 m; {6 J) C: F: I
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Finally, we sought to investigate if the QPCR assay was generally applicable also for hESC lines other than the ones maintained on MEF at Cellartis AB. For this purpose, we determined the expression indexes of three independent hESC lines (BG01, BG02, and BG03) that were maintained and differentiated in feeder-free conditions. Importantly, these cell lines were established and cultured in laboratories separate from Cellartis AB, and the samples were analyzed blindly. Despite the substantial differences in the general procedures of culturing and passaging of the hESCs, it was possible to accurately discriminate between the undifferentiated and differentiating cells using the QPCR method (Fig. 6). The expression indexes for the undifferentiated hESCs ranged between 72 and 100 and were significantly higher compared with the corresponding expression indexes for the differentiating hESCs. In addition, based on the expression indexes, it was possible to correctly rank the group of samples from differentiating hESCs (day 5–15), although a few samples deviated slightly from the expected trend (BG01 at day 7, BG02 at day 9, and BG03 at day 7). Interestingly, these apparent outliers were present within a rather small and specific time interval (7–9 days of differentiation). Further studies are necessary to elucidate the possible biological significance of this observation. - K j6 r) ]% X/ Y % n: J! ?6 y, ?' i( K: ~7 eFigure 6. Quantitative real-time polymerase chain reaction (QPCR) analysis of undifferentiated (Undiff.) and differentiating human embryonic stem cells maintained in feeder-free conditions: (A) BG01, (B) BG02, and (C) BG03. The cells were cultured as described in Materials and Methods and harvested at the different time points indicated on the x-axis. After RNA extraction, the relative mRNA levels of Oct-4, Nanog, Cripto, and -fetoprotein were determined using QPCR. The expression index was subsequently calculated using Equation 1. The data are presented as the mean plus SD (n = 2). ' H. I$ Y" o/ v* Q 1 ^* o; t* r2 j0 m; ]DISCUSSION b% u: d/ `' Z7 Y+ J! x8 G
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This work was supported by Cellartis AB, TATAA Biocenter, and the NIH Intramural Program. The work related to hESC lines SA001 and SA002 (listed on the NIH Human Embryonic Stem Cell Registry) was partly supported by NIH grant R24RR019514-01 awarded to Cellartis AB. Dr. Xianmin Zeng is currently at the Buck Institute for Age Research, 8001 Redwood Blvd., Novato, CA 94945, USA.( k2 j% A6 p! [7 }( W2 V# b
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DISCLOSURES 6 C1 t- u" Z- h " Z. o u8 a( V8 @0 X* FThe authors indicate no potential conflicts of interest. 6 F6 H) o1 \" L5 S$ t* a- _! @" E + D' q) V! G* z& R) F% EREFERENCES ; v* U5 z& E! @( F; b- P, T+ b* R
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3 @' u# f& m) g5 [! rRichards M, Tan SP, Tan JH et al. The transcriptome profile of human embryonic stem cells as defined by SAGE. STEM CELLS 2004;22:51–64.(a Cellartis AB, G?teborg,)作者: 张佳 时间: 2015-5-24 21:25