- Email: [email protected]
A novel method for purification of inner cell mass and trophectoderm cells from blastocysts using magnetic activated cell sorting
A novel method for purification of inner cell mass and trophectoderm cells from blastocysts using magnetic activated cell sorting Manabu Ozawa, Ph.D., and Peter J. Hansen, Ph.D. Department of Animal Sciences and D.H. Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, Florida
Objective: To develop a simple method to purify blastomeres of inner cell mass (ICM) and trophectoderm (TE) lineage using magnetic activated cell sorting. Design: Prospective laboratory study. Setting: Embryology research laboratory. Patient(s): None. Intervention(s): Trophectoderm cells of zona-free blastocysts were labeled with concanavalin A conjugated to FITC, and every nucleus in the blastocyst was labeled with Hoechst 33342. The labeled blastocyst was disaggregated to single cells by trypsin treatment followed by pipetting using a finely drawn, flame-polished micropipet. Disaggregated blastomeres were incubated with anti-FITC antibody conjugated to magnetic microbeads and subjected to magnetic cell sorting to separate cells into FITC-positive and -negative fractions. Main Outcome Measure(s): Purity and gene expression. Result(s): In the FITC-positive fraction, an average of 91.2% of cells was dual-labeled with FITC and Hoechst, whereas only 7.8% of FITC negative fractions were labeled with FITC. Expression of CDX2, a trophectoderm marker, was significantly higher in the FITC-positive fraction, whereas expression of NANOG, an inner cell mass marker, was significantly higher in the FITC-negative fraction. Conclusion(s): Highly purified trophectoderm cells or inner cell mass cells can be collected using magnetic activated cell sorting. This method can be useful for understanding differentiation and function of cell lineages in the blastocyst. (Fertil Steril 2011;95:799–802. 2011 by American Society for Reproductive Medicine.) Key Words: Blastocyst, inner cell mass, trophectoderm, lineage, purification
The first distinct lineage differentiation in the mammalian embryo occurs at the blastocyst stage, when blastomeres are segregated into inner cell mass (ICM) or trophectoderm (TE) (1, 2). Transcription factors such as NANOG and POU5F1 are expressed in ICM cells and act to maintain pluripotency. Conversely, trophectoderm expresses transcription factor genes, such as CDX2 and EOMES, to direct differentiation (3). Obtaining purified TE and ICM is beneficial for the study of early development and differentiation, because it allows study of lineagespecific gene expression and cell function. Moreover, purified TE and ICM populations could have application for tissue engineering and regenerative medicine. Stem cell lines from ICM and TE have been established in vitro, such as embryonic stem cells from ICM (4) and trophoblast stem cells from TE (5, 6). There are limitations to current methods for the separation of TE and ICM from blastocysts, which have involved immunosurgery (7), mechanical dissection using a micromanipulator (8), and manual Received June 22, 2010; revised and accepted October 7, 2010; published online November 5, 2010. M.O. has nothing to disclose. P.J.H. has nothing to disclose. Supported by Agriculture and Food Research Initiative Competitive Grant no. 2009-65203-05732 from the U.S.D.A. National Institute of Food and Agriculture, the University of Florida Research Foundation Research Opportunity Fund and Japanese Society for Promotion of Science Fellowship 21-717 (to M.O.). Reprint requests: Peter J. Hansen, Ph.D., P.O. Box 110910, Department of Animal Sciences, University of Florida, Gainesville, FL 32601 (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.10.006
selection following trypsinization (9, 10). Among the limitations are cell damage (immunosurgery), requirements for a high degree of technical proficiency (micromanipulator), and low throughput (manual selection). In this study, we describe a simple and effective method to sort cells of the blastocyst using magnetic activated cell sorting (MACS), following disaggregation of the blastocyst into single cells using trypsin. Our results show that the population of sorted cells was highly enriched, based on fluorescent staining patterns and expression of lineage-specific marker genes.
MATERIALS AND METHODS Bovine blastocysts were produced in vitro as described previously (11). The day of IVF was defined as day 0, and blastocysts were collected on day 7. Embryos were cultured using SOF-BE1 medium, which is based on a modified synthetic oviduct fluid (12) that has been additionally changed to contain 1 mM alanyl-glutamine, 0.5 mM sodium citrate, 2.77 mM myoinositol, 5.3 mM sodium lactate, and 4 mg/mL essentially fatty-acid free bovine serum albumin (BSA). Disaggregation of blastocysts into single blastomeres was modified from a previously described method using concanavalin A (ConA) (9). ConA binds high-mannose oligosaccharides expressed on the cell membrane, but does not penetrate junctional complexes between trophoblast cells, so that it does not enter into the inner layers of the blastocyst. This characteristic of ConA interaction with the blastocyst is the basis for the separation technique used
Fertility and Sterility Vol. 95, No. 2, February 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
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in this study. The zona pellucida was removed from the blastocyst by a short (approximately 1 minute) exposure to acidic Tyrode’s solution (Millipore, Billerica, MA). All subsequent incubation steps were performed in 100-mL microdrops covered in oil and at room temperature. Blastocysts were washed three times in MACS buffer (Dulbecco’s PBS [Invitrogen, Carlsbad, CA] containing 0.5% [w/v] BSA and 2 mM ethylenediaminetetraacetic acid [EDTA], pH 7.2), and incubated with ConA conjugated to FITC (Sigma-Aldrich, St. Louis, MO; ConA-FITC, 1 mg/mL in MACS buffer) for 10 min in the dark to label the outer layer of the blastocyst. The blastocysts were then washed three times in MACS buffer and incubated with 1 mg/mL Hoechst 33342 (Sigma-Aldrich) in MACS buffer for 3 minutes to label the nuclei of all blastomeres. Blastocysts were then washed three times using MACS buffer, transferred to PBS containing 1 mM EDTA, and incubated for 5 minutes. Subsequently, blastocysts were transferred to 0.05% (w/v) trypsin0.53 mM EDTA solution (Invitrogen) and incubated 10 min at 38.5 C. A group of 25–30 trypsin-treated blastocysts was transferred to a 100-ml drop of MACS buffer and disaggregated into single blastomeres by repeated pipetting using a finely drawn, flame-polished mouth micropipette. The drop containing disaggregated blastomeres was transferred into a 1.5-mL tube and mixed with 500 mL of PBS containing 1 mM EDTA and 10% (v/v) fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) to inactivate trypsin. Cells were then washed twice in MACS buffer by centrifugation (500 g for
5 minutes). Subsequently, large clusters of cells were eliminated by passing the suspension over a cell strainer (BD Biosciences, San Jose, CA), and the single blastomeres passing through the strainer collected. Blastomeres were centrifuged (500 g for 5 minutes) and resuspended in 110 mL of MACS buffer. Ten microliters of the sample was used for cell counting, and the remainder was used for MACS. A suspension of single blastomeres from 25–30 blastocysts was incubated with 10-mL magnetic microbeads conjugated to mouse anti-FITC IgG1 (Miltenyi Biotec, Auburn, CA) for 15 minutes on ice. Blastomeres were washed three times with MACS buffer by centrifugation (500 g for 5 minutes), resuspended in 500 ml MACS buffer, and loaded into a MACS separation column (Miltenyi Biotec) attached to a magnetic board (Spherotech, Lake Forest, IL). The column was washed three times with MACS buffer (500 mL each) to obtain the FITC-negative fraction in the eluate. The column was then detached from the magnetic board and washed three times using MACS buffer (500-ml each) to recover the FITC-positive fraction eluate. A 200-mL sample of each fraction of cells was observed under a Zeiss Axioplan microscope (Zeiss, G€ ottingen, Germany) with green and blue filter sets. The cells remaining after MACS were subjected to mRNA extraction using the PicoPure RNA Isolation Kit (Molecular Devices, Sunnyvale, CA) followed by DNase (New England Biolabs, Ipswich, MA) treatment and reverse transcription (High Capacity cDNA
FIGURE 1 Differential labeling of ICM and TE. (A–C) Labeling of a representative blastocyst with ConA-FITC (A, outer cells) and Hoescht 33342 (B, all nuclei). The merged image is shown in (C). The area circled by a dotted line represents an ICM. (D) Individual blastomeres of the blastocyst after disaggregation by trypsinization followed by repeated pipetting using a finely drawn, flame-polished mouth micropipette. Note that one cell, dual-labeled with ConA-FITC and Hoescht 33342, is a trophoblast cell. The other cells, labeled with Hoescht 333442 only, are ICM cells.
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TABLE 1 RNA recovery and cell purity after MACS.
Intact blastocyst Disaggregated blastocyst before MACS MACS-positive fraction MACS-negative fraction
Replicates
Percent of cells that were dual labeled
Percent of cells that were labeled with Hoechst only
Extracted RNA per embryo (ng)
4 4
— 62.6 2.0a
— 37.4 2.0a
14.3 0.2 —
4 4
91.2 2.0b 7.8 2.0c
8.8 2.0b 92.2 2.0c
7.1 0.5 4.4 0.6
Note: Values are expressed as the least-squares mean SEM of data from four replicates. a,b,c Values within columns with different superscripts are different (P<0.05 or less). Ozawa. Purification of TE and ICM. Fertil Steril 2011.
Reverse Transcription Kit, Applied Biosystems, Foster City, CA). The NANOG and CDX2 mRNA were quantified by a 7300 Fast Real-Time PCR System (Applied Biosystems) using SYBR Green PCR Master Mix reagent (Applied Biosystems). PCR conditions were set to the following parameters: 2 minutes at 50 C and 10 min at 95 C followed by 50 cycles each of 15 seconds at 95 C and 1 min at 60 C. To obtain the fold difference, data were analyzed using the delta-delta cycle threshold method described previously (13). GAPDH was used as a reference gene. The primers were as follows: NANOG (GenBank accession No. DQ069776): 50 -GACACCCTCGACACG GACAC-30 , 50 -CTTGACCGGGACCGTCTCTT-30 , CDX2 (GenBank accession No. XM_871005): 50 -GCCACCATGTACGTGAGCTAC30 , 50 -ACATGGTATCCGCCGTAGTC-30 , and GAPDH (GenBank accession No. XM_001252511.2): 50 -ACCAGAAGACTGTGGA TGG-30 , 50 -CAACAGACACGTTGGGAGTG-30 .
recovered from cells isolated by MACS; this represents 80% of the RNA present in intact blastocysts and suggests a high rate of recovery of blastomeres during the purification process.
FIGURE 2 Expression of CDX2 (Top) and NANOG (Bottom) mRNA in the ConA-FITC positive fraction (ie., trophoblast; TE), ConA-FITC negative fraction (i.e., inner cell mass; ICM) and in intact blastocysts. Each gene was quantified by real-time RT-PCR, and the amounts were standardized by the amount of GAPDH in the same sample. Data are least-squares means SEM from four replicates. Values without a common letter (a–b) in each figure differ significantly (P<0.05).
Statistical Analysis Twenty-five to 30 blastocysts were used as a group for one replicate, and four replicates were performed for each experiment. All percentage data were analyzed after arcsine transformation. Data were analyzed by least-squares analysis of variance using the Proc GLM Procedure of the Statistical Analysis System version 9.2 (SAS Institute Inc., Cary, NC) followed by Duncan’s multiple range test. Data are expressed as least-squares means SEM.
RESULTS Purities of the Blastomeres and Recovery Efficiency before and after MACS A representative blastocyst labeled with ConA-FITC and Hoechst 33342 is shown Figure 1. Note that outer cells are the only ones labeled with ConA-FITC (Fig. 1A). The area circled by a dotted line in Figure 1C represents an inner cell mass. After disaggregation of the blastocyst, two types of single blastomeres are observed—cells that were positive for FITC and Hoescht 33342 (TE cells) and cells that were negative for FITC but positive for Hoechst 33342 (ICM cells; Fig. 1D). Purity and amount of RNA obtained from cells subjected to MACS are summarized in Table 1. Before MACS, approximately two thirds of the disaggregated blastomeres labeled with Hoescht 33342 were also labeled with ConA-FITC, whereas one third were ConA-FITC negative. After MACS, the percentage of dual-labeled cells in the ConA-FITC–positive fraction was 91.2%, whereas the incidence of dual-labeled cells in the ConA-FITC–negative fraction was only 7.8 3.0%. A total of 11.5 ng RNA per blastocyst was Fertility and Sterility
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Expression of Lineage Marker Genes in Separated Cells after MACS Results are shown in Figure 2. NANOG mRNA expression was lower in the ConA-FITC–positive fraction than in the ConA-FITC–negative fraction. Conversely, mRNA for CDX2 was lower in the ConAFITC–positive fraction than in the ConA-FITC–negative fraction. For both genes, control blastocysts had intermediate amounts of expression.
DISCUSSION In this study, a simple method to separate ICM from TE has been established using a common cell sorting method, MACS. This method does not require expensive equipment and can be learned readily. ConA-FITC was used to label the outermost cells in the blastocyst (i.e., the trophectoderm). ConA binds high-mannose oligosaccharides expressed on the cell membrane, but does not penetrate into cytoplasm or, as shown in this study, into the inner layers of cells in the blastocyst. This characteristic of ConA interactions with the blastocyst is the basis for the separation technique used. Using labeling with ConA-FITC and Hoescht 33342, a high degree of enrichment of the two cell populations could be established. In addition, NANOG, which is considered a marker of ICM (14), was more highly expressed in the ConA-FITC–negative cell
population, whereas CDX2, which is highly expressed by TE cells but is either not expressed or only expressed to a slight degree by ICM cells in mouse (15) and bovine (16), was more highly expressed in the ConA-FITC–positive cell population. The fact that there was a not complete absence of NANOG mRNA in the ConA-FITC–positive cell population, or of CDX2 mRNA in the ConA-FITC–negative cell population, is to be expected because cell purity was approximately 91%–92%. In addition, some bovine TE cells limited amounts of NANOG protein (17). Similarly, weak CDX2 mRNA expression is observed in the bovine ICM (18). Furthermore, human ICM cells isolated by immunosurgery also express CDX2 mRNA, although at lower amounts than for the TE (7). The simple method reported in this study can be used for studying differentiation of the mammalian embryo and control of pluripotency, as well as preparing embryonic cells of specific lineages for cell therapy. Separation of blastomeres into ICM and TE means that it is not only possible to study the effects of treatment on the ratio of ICM to TE in the blastocyst—as has been possible for some time (19)—but that functional and molecular characteristics of ICM and TE can be readily determined. Acknowledgments: The authors thank Dr. Alan D. Ealy, William Rembert, Central Beef Packing Co. (Center Hill, FL), and Scott A. Randell of Southeastern Semen Services (Wellborn, FL) for assistance.
REFERENCES 1. Willadson SM. Nuclear transplantation in sheep embryos. Nature 1986;320:63–5. 2. Rossant J, Tam PPL. Blastocyst lineage formation, early embryonic asymmetries and axis patterning in the mouse. Development 2009;136:701–13. 3. Ealy AD, Yang QE. Control of interferon-tau expression during early pregnancy in ruminants. Am J Reprod Immunol 2009;61:95–106. 4. Murry CE, Keller G. Differentiation of embryonic stem cells to clinical relevant populations: lessons from embryonic development. Cell 2008;132:661–80. 5. Rielland M, Brochard V, Lacroix MC, Renard JP, Jouneau A. Early alteration of the self-renewal/ differentiation threshold in trophoblast stem cells derived from mouse embryos after nuclear transfer. Dev Biol 2009;334:325–34. 6. Douglas GC, VandeVoort CA, Kumar P, Chang TC, Golos TG. Trophoblast stem cells: models for investigating trophectoderm differentiation and placental development. Endocr Rev 2009;30:228–40. 7. Adjaye J, Huntriss J, Herwig R, BenKahla A, Brink TC, Wierling C, et al. Primary differentiation in the human blastocyst: Comparative molecular portraits of inner cell mass and trophectoderm cells. Stem Cells 2005;23:1514–25.
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8. Dahl JA, Reiner AH, Klungland A, Wakayama T, Collas P. Histone H3 lysine 27 methylation asymmetry on developmentally-regulated promoters distinguish the first two lineages in mouse preimplantation embryos. PLoS ONE 2010;5: e9150. 9. Collins J, Fleming T. Specific mRNA detection in single lineage-marked blastomeres from preimplantation embryos. Trends Genet 1995;11:5–7. 10. Kurimoto K, Yabuta Y, Ohinata Y, Saitou M. Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis. Nat Protoc 2007;2:739–52. 11. Loureiro B, Bonilla L, Block J, Fear JM, Bonilla AQS, Hansen PJ. Colony-stimulating factor 2 (CSF-2) improves development and posttransfer survival of bovine embryos produced in vitro. Endocrinology 2009;150:5046–54. 12. Fischer-Brown A, Monson R, Parrish J, Rutledge J. Cell allocation in bovine embryos cultured in two media under two oxygen concentrations. Zygote 2002;10:341–8. 13. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2DDCT method. Methods 2001;21: 402–8.
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14. Zernicka-Goetz M, Morris SA, Bruce AW. Making a firm decision: multifaceted regulation of cell fate in the early mouse embryo. Nat Rev Genet 2009;10: 467–77. 15. Chen L, Yabuuchi A, Eminli S, Takeuchi A, Lu CW, Hochedlinger K, et al. Cross-regulation of the Nanog and Cdx2 promoters. Cell Res 2009;19: 1052–61. 16. Kuijk EW, Puy LD, Van Tol HTA, Oei CHY, Haagsman HP, Colenbrander B, et al. Differences in early lineage segregation between mammals. Dev Dyn 2008;237:918–27. 17. Pant D, Keefer CL. Expression of pluripotencyrelated genes during bovine inner cell mass explant culture. Cloning Stem Cells 2009;11:1–11. 18. Degrelle SA, Campion E, Cabau C, Piumi F, Reinaud P, Richard C, et al. Molecular evidence for a critical period in mural trophoblast development in bovine blastocysts. Dev Biol 2005; 288:448–60. 19. Thouas GA, Korfiatis NA, French AJ, Jones GM, Trounson AO. Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts. Reprod Biomed Online 2001;3:25–9.
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Objective: To develop a simple method to purify blastomeres of inner cell mass (ICM) and trophectoderm (TE) lineage using magnetic activated cell sorting. Design: Prospective laboratory study. Setting: Embryology research laboratory. Patient(s): None. Intervention(s): Trophectoderm cells of zona-free blastocysts were labeled with concanavalin A conjugated to FITC, and every nucleus in the blastocyst was labeled with Hoechst 33342. The labeled blastocyst was disaggregated to single cells by trypsin treatment followed by pipetting using a finely drawn, flame-polished micropipet. Disaggregated blastomeres were incubated with anti-FITC antibody conjugated to magnetic microbeads and subjected to magnetic cell sorting to separate cells into FITC-positive and -negative fractions. Main Outcome Measure(s): Purity and gene expression. Result(s): In the FITC-positive fraction, an average of 91.2% of cells was dual-labeled with FITC and Hoechst, whereas only 7.8% of FITC negative fractions were labeled with FITC. Expression of CDX2, a trophectoderm marker, was significantly higher in the FITC-positive fraction, whereas expression of NANOG, an inner cell mass marker, was significantly higher in the FITC-negative fraction. Conclusion(s): Highly purified trophectoderm cells or inner cell mass cells can be collected using magnetic activated cell sorting. This method can be useful for understanding differentiation and function of cell lineages in the blastocyst. (Fertil Steril 2011;95:799–802. 2011 by American Society for Reproductive Medicine.) Key Words: Blastocyst, inner cell mass, trophectoderm, lineage, purification
The first distinct lineage differentiation in the mammalian embryo occurs at the blastocyst stage, when blastomeres are segregated into inner cell mass (ICM) or trophectoderm (TE) (1, 2). Transcription factors such as NANOG and POU5F1 are expressed in ICM cells and act to maintain pluripotency. Conversely, trophectoderm expresses transcription factor genes, such as CDX2 and EOMES, to direct differentiation (3). Obtaining purified TE and ICM is beneficial for the study of early development and differentiation, because it allows study of lineagespecific gene expression and cell function. Moreover, purified TE and ICM populations could have application for tissue engineering and regenerative medicine. Stem cell lines from ICM and TE have been established in vitro, such as embryonic stem cells from ICM (4) and trophoblast stem cells from TE (5, 6). There are limitations to current methods for the separation of TE and ICM from blastocysts, which have involved immunosurgery (7), mechanical dissection using a micromanipulator (8), and manual Received June 22, 2010; revised and accepted October 7, 2010; published online November 5, 2010. M.O. has nothing to disclose. P.J.H. has nothing to disclose. Supported by Agriculture and Food Research Initiative Competitive Grant no. 2009-65203-05732 from the U.S.D.A. National Institute of Food and Agriculture, the University of Florida Research Foundation Research Opportunity Fund and Japanese Society for Promotion of Science Fellowship 21-717 (to M.O.). Reprint requests: Peter J. Hansen, Ph.D., P.O. Box 110910, Department of Animal Sciences, University of Florida, Gainesville, FL 32601 (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.10.006
selection following trypsinization (9, 10). Among the limitations are cell damage (immunosurgery), requirements for a high degree of technical proficiency (micromanipulator), and low throughput (manual selection). In this study, we describe a simple and effective method to sort cells of the blastocyst using magnetic activated cell sorting (MACS), following disaggregation of the blastocyst into single cells using trypsin. Our results show that the population of sorted cells was highly enriched, based on fluorescent staining patterns and expression of lineage-specific marker genes.
MATERIALS AND METHODS Bovine blastocysts were produced in vitro as described previously (11). The day of IVF was defined as day 0, and blastocysts were collected on day 7. Embryos were cultured using SOF-BE1 medium, which is based on a modified synthetic oviduct fluid (12) that has been additionally changed to contain 1 mM alanyl-glutamine, 0.5 mM sodium citrate, 2.77 mM myoinositol, 5.3 mM sodium lactate, and 4 mg/mL essentially fatty-acid free bovine serum albumin (BSA). Disaggregation of blastocysts into single blastomeres was modified from a previously described method using concanavalin A (ConA) (9). ConA binds high-mannose oligosaccharides expressed on the cell membrane, but does not penetrate junctional complexes between trophoblast cells, so that it does not enter into the inner layers of the blastocyst. This characteristic of ConA interaction with the blastocyst is the basis for the separation technique used
Fertility and Sterility Vol. 95, No. 2, February 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
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in this study. The zona pellucida was removed from the blastocyst by a short (approximately 1 minute) exposure to acidic Tyrode’s solution (Millipore, Billerica, MA). All subsequent incubation steps were performed in 100-mL microdrops covered in oil and at room temperature. Blastocysts were washed three times in MACS buffer (Dulbecco’s PBS [Invitrogen, Carlsbad, CA] containing 0.5% [w/v] BSA and 2 mM ethylenediaminetetraacetic acid [EDTA], pH 7.2), and incubated with ConA conjugated to FITC (Sigma-Aldrich, St. Louis, MO; ConA-FITC, 1 mg/mL in MACS buffer) for 10 min in the dark to label the outer layer of the blastocyst. The blastocysts were then washed three times in MACS buffer and incubated with 1 mg/mL Hoechst 33342 (Sigma-Aldrich) in MACS buffer for 3 minutes to label the nuclei of all blastomeres. Blastocysts were then washed three times using MACS buffer, transferred to PBS containing 1 mM EDTA, and incubated for 5 minutes. Subsequently, blastocysts were transferred to 0.05% (w/v) trypsin0.53 mM EDTA solution (Invitrogen) and incubated 10 min at 38.5 C. A group of 25–30 trypsin-treated blastocysts was transferred to a 100-ml drop of MACS buffer and disaggregated into single blastomeres by repeated pipetting using a finely drawn, flame-polished mouth micropipette. The drop containing disaggregated blastomeres was transferred into a 1.5-mL tube and mixed with 500 mL of PBS containing 1 mM EDTA and 10% (v/v) fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) to inactivate trypsin. Cells were then washed twice in MACS buffer by centrifugation (500 g for
5 minutes). Subsequently, large clusters of cells were eliminated by passing the suspension over a cell strainer (BD Biosciences, San Jose, CA), and the single blastomeres passing through the strainer collected. Blastomeres were centrifuged (500 g for 5 minutes) and resuspended in 110 mL of MACS buffer. Ten microliters of the sample was used for cell counting, and the remainder was used for MACS. A suspension of single blastomeres from 25–30 blastocysts was incubated with 10-mL magnetic microbeads conjugated to mouse anti-FITC IgG1 (Miltenyi Biotec, Auburn, CA) for 15 minutes on ice. Blastomeres were washed three times with MACS buffer by centrifugation (500 g for 5 minutes), resuspended in 500 ml MACS buffer, and loaded into a MACS separation column (Miltenyi Biotec) attached to a magnetic board (Spherotech, Lake Forest, IL). The column was washed three times with MACS buffer (500 mL each) to obtain the FITC-negative fraction in the eluate. The column was then detached from the magnetic board and washed three times using MACS buffer (500-ml each) to recover the FITC-positive fraction eluate. A 200-mL sample of each fraction of cells was observed under a Zeiss Axioplan microscope (Zeiss, G€ ottingen, Germany) with green and blue filter sets. The cells remaining after MACS were subjected to mRNA extraction using the PicoPure RNA Isolation Kit (Molecular Devices, Sunnyvale, CA) followed by DNase (New England Biolabs, Ipswich, MA) treatment and reverse transcription (High Capacity cDNA
FIGURE 1 Differential labeling of ICM and TE. (A–C) Labeling of a representative blastocyst with ConA-FITC (A, outer cells) and Hoescht 33342 (B, all nuclei). The merged image is shown in (C). The area circled by a dotted line represents an ICM. (D) Individual blastomeres of the blastocyst after disaggregation by trypsinization followed by repeated pipetting using a finely drawn, flame-polished mouth micropipette. Note that one cell, dual-labeled with ConA-FITC and Hoescht 33342, is a trophoblast cell. The other cells, labeled with Hoescht 333442 only, are ICM cells.
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TABLE 1 RNA recovery and cell purity after MACS.
Intact blastocyst Disaggregated blastocyst before MACS MACS-positive fraction MACS-negative fraction
Replicates
Percent of cells that were dual labeled
Percent of cells that were labeled with Hoechst only
Extracted RNA per embryo (ng)
4 4
— 62.6 2.0a
— 37.4 2.0a
14.3 0.2 —
4 4
91.2 2.0b 7.8 2.0c
8.8 2.0b 92.2 2.0c
7.1 0.5 4.4 0.6
Note: Values are expressed as the least-squares mean SEM of data from four replicates. a,b,c Values within columns with different superscripts are different (P<0.05 or less). Ozawa. Purification of TE and ICM. Fertil Steril 2011.
Reverse Transcription Kit, Applied Biosystems, Foster City, CA). The NANOG and CDX2 mRNA were quantified by a 7300 Fast Real-Time PCR System (Applied Biosystems) using SYBR Green PCR Master Mix reagent (Applied Biosystems). PCR conditions were set to the following parameters: 2 minutes at 50 C and 10 min at 95 C followed by 50 cycles each of 15 seconds at 95 C and 1 min at 60 C. To obtain the fold difference, data were analyzed using the delta-delta cycle threshold method described previously (13). GAPDH was used as a reference gene. The primers were as follows: NANOG (GenBank accession No. DQ069776): 50 -GACACCCTCGACACG GACAC-30 , 50 -CTTGACCGGGACCGTCTCTT-30 , CDX2 (GenBank accession No. XM_871005): 50 -GCCACCATGTACGTGAGCTAC30 , 50 -ACATGGTATCCGCCGTAGTC-30 , and GAPDH (GenBank accession No. XM_001252511.2): 50 -ACCAGAAGACTGTGGA TGG-30 , 50 -CAACAGACACGTTGGGAGTG-30 .
recovered from cells isolated by MACS; this represents 80% of the RNA present in intact blastocysts and suggests a high rate of recovery of blastomeres during the purification process.
FIGURE 2 Expression of CDX2 (Top) and NANOG (Bottom) mRNA in the ConA-FITC positive fraction (ie., trophoblast; TE), ConA-FITC negative fraction (i.e., inner cell mass; ICM) and in intact blastocysts. Each gene was quantified by real-time RT-PCR, and the amounts were standardized by the amount of GAPDH in the same sample. Data are least-squares means SEM from four replicates. Values without a common letter (a–b) in each figure differ significantly (P<0.05).
Statistical Analysis Twenty-five to 30 blastocysts were used as a group for one replicate, and four replicates were performed for each experiment. All percentage data were analyzed after arcsine transformation. Data were analyzed by least-squares analysis of variance using the Proc GLM Procedure of the Statistical Analysis System version 9.2 (SAS Institute Inc., Cary, NC) followed by Duncan’s multiple range test. Data are expressed as least-squares means SEM.
RESULTS Purities of the Blastomeres and Recovery Efficiency before and after MACS A representative blastocyst labeled with ConA-FITC and Hoechst 33342 is shown Figure 1. Note that outer cells are the only ones labeled with ConA-FITC (Fig. 1A). The area circled by a dotted line in Figure 1C represents an inner cell mass. After disaggregation of the blastocyst, two types of single blastomeres are observed—cells that were positive for FITC and Hoescht 33342 (TE cells) and cells that were negative for FITC but positive for Hoechst 33342 (ICM cells; Fig. 1D). Purity and amount of RNA obtained from cells subjected to MACS are summarized in Table 1. Before MACS, approximately two thirds of the disaggregated blastomeres labeled with Hoescht 33342 were also labeled with ConA-FITC, whereas one third were ConA-FITC negative. After MACS, the percentage of dual-labeled cells in the ConA-FITC–positive fraction was 91.2%, whereas the incidence of dual-labeled cells in the ConA-FITC–negative fraction was only 7.8 3.0%. A total of 11.5 ng RNA per blastocyst was Fertility and Sterility
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Expression of Lineage Marker Genes in Separated Cells after MACS Results are shown in Figure 2. NANOG mRNA expression was lower in the ConA-FITC–positive fraction than in the ConA-FITC–negative fraction. Conversely, mRNA for CDX2 was lower in the ConAFITC–positive fraction than in the ConA-FITC–negative fraction. For both genes, control blastocysts had intermediate amounts of expression.
DISCUSSION In this study, a simple method to separate ICM from TE has been established using a common cell sorting method, MACS. This method does not require expensive equipment and can be learned readily. ConA-FITC was used to label the outermost cells in the blastocyst (i.e., the trophectoderm). ConA binds high-mannose oligosaccharides expressed on the cell membrane, but does not penetrate into cytoplasm or, as shown in this study, into the inner layers of cells in the blastocyst. This characteristic of ConA interactions with the blastocyst is the basis for the separation technique used. Using labeling with ConA-FITC and Hoescht 33342, a high degree of enrichment of the two cell populations could be established. In addition, NANOG, which is considered a marker of ICM (14), was more highly expressed in the ConA-FITC–negative cell
population, whereas CDX2, which is highly expressed by TE cells but is either not expressed or only expressed to a slight degree by ICM cells in mouse (15) and bovine (16), was more highly expressed in the ConA-FITC–positive cell population. The fact that there was a not complete absence of NANOG mRNA in the ConA-FITC–positive cell population, or of CDX2 mRNA in the ConA-FITC–negative cell population, is to be expected because cell purity was approximately 91%–92%. In addition, some bovine TE cells limited amounts of NANOG protein (17). Similarly, weak CDX2 mRNA expression is observed in the bovine ICM (18). Furthermore, human ICM cells isolated by immunosurgery also express CDX2 mRNA, although at lower amounts than for the TE (7). The simple method reported in this study can be used for studying differentiation of the mammalian embryo and control of pluripotency, as well as preparing embryonic cells of specific lineages for cell therapy. Separation of blastomeres into ICM and TE means that it is not only possible to study the effects of treatment on the ratio of ICM to TE in the blastocyst—as has been possible for some time (19)—but that functional and molecular characteristics of ICM and TE can be readily determined. Acknowledgments: The authors thank Dr. Alan D. Ealy, William Rembert, Central Beef Packing Co. (Center Hill, FL), and Scott A. Randell of Southeastern Semen Services (Wellborn, FL) for assistance.
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Vol. 95, No. 2, February 2011