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A Novel Method for Purification of Inner Cell Mass and Trophectoderm Cells

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A Novel Method for Purification of Inner Cell Mass and Trophectoderm Cells 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 SterilÒ 2011;95:799–802. Ó2011 by American Society for Reproductive Medicine.) Key Words: Blastocyst, inner cell mass, trophectoderm, lineage, purification The first distinct lineage differentiation in the mammalian embryo selection following trypsinization (9, 10). Among the limitations occurs at the blastocyst stage, when blastomeres are segregated into are cell damage (immunosurgery), requirements for a high degree inner cell mass (ICM) or trophectoderm (TE) (1, 2). Transcription of technical proficiency (micromanipulator), and low throughput factors such as NANOG and POU5F1 are expressed in ICM cells (manual selection). In this study, we describe a simple and and act to maintain pluripotency. Conversely, trophectoderm effective method to sort cells of the blastocyst using magnetic expresses transcription factor genes, such as CDX2 and EOMES,to activated cell sorting (MACS), following disaggregation of the direct differentiation (3). blastocyst into single cells using trypsin. Our results show that the Obtaining purified TE and ICM is beneficial for the study of early population of sorted cells was highly enriched, based on development and differentiation, because it allows study of lineage- fluorescent staining patterns and expression of lineage-specific specific gene expression and cell function. Moreover, purified TE marker genes. 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 MATERIALS AND METHODS (4) and trophoblast stem cells from TE (5, 6). Bovine blastocysts were produced in vitro as described previously There are limitations to current methods for the separation of TE (11). The day of IVF was defined as day 0, and blastocysts were and ICM from blastocysts, which have involved immunosurgery (7), collected on day 7. Embryos were cultured using SOF-BE1 medium, mechanical dissection using a micromanipulator (8), and manual which is based on a modified synthetic oviduct fluid (12) that has been additionally changed to contain 1 mM alanyl-glutamine, Received June 22, 2010; revised and accepted October 7, 2010; 0.5 mM sodium citrate, 2.77 mM myoinositol, 5.3 mM sodium published online November 5, 2010. lactate, and 4 mg/mL essentially fatty-acid free bovine serum M.O. has nothing to disclose. P.J.H. has nothing to disclose. albumin (BSA). Supported by Agriculture and Food Research Initiative Competitive Grant Disaggregation of blastocysts into single blastomeres was modi- no. 2009-65203-05732 from the U.S.D.A. National Institute of Food and fied from a previously described method using concanavalin A Agriculture, the University of Florida Research Foundation Research (ConA) (9). ConA binds high-mannose oligosaccharides expressed Opportunity Fund and Japanese Society for Promotion of Science on the cell membrane, but does not penetrate junctional complexes Fellowship 21-717 (to M.O.). Reprint requests: Peter J. Hansen, Ph.D., P.O. Box 110910, Department of between trophoblast cells, so that it does not enter into the inner Animal Sciences, University of Florida, Gainesville, FL 32601 (E-mail: layers of the blastocyst. This characteristic of ConA interaction pjhansen@ufl.edu). with the blastocyst is the basis for the separation technique used 0015-0282/$36.00 Fertility and Sterilityâ Vol. 95, No. 2, February 2011 799 doi:10.1016/j.fertnstert.2010.10.006 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc. in this study. The zona pellucida was removed from the blastocyst by 5 minutes). Subsequently, large clusters of cells were eliminated a short (approximately 1 minute) exposure to acidic Tyrode’s by passing the suspension over a cell strainer (BD Biosciences, solution (Millipore, Billerica, MA). All subsequent incubation steps San Jose, CA), and the single blastomeres passing through the were performed in 100-mL microdrops covered in oil and at room strainer collected. Blastomeres were centrifuged (500 Â g for 5 min- temperature. Blastocysts were washed three times in MACS utes) and resuspended in 110 mL of MACS buffer. Ten microliters of buffer (Dulbecco’s PBS [Invitrogen, Carlsbad, CA] containing the sample was used for cell counting, and the remainder was used 0.5% [w/v] BSA and 2 mM ethylenediaminetetraacetic acid for MACS. [EDTA], pH 7.2), and incubated with ConA conjugated to FITC A suspension of single blastomeres from 25–30 blastocysts was (Sigma-Aldrich, St. Louis, MO; ConA-FITC, 1 mg/mL in MACS incubated with 10-mL magnetic microbeads conjugated to mouse buffer) for 10 min in the dark to label the outer layer of the blasto- anti-FITC IgG1 (Miltenyi Biotec, Auburn, CA) for 15 minutes on cyst. The blastocysts were then washed three times in MACS buffer ice. Blastomeres were washed three times with MACS buffer by and incubated with 1 mg/mL Hoechst 33342 (Sigma-Aldrich) in centrifugation (500 Â g for 5 minutes), resuspended in 500 ml MACS buffer for 3 minutes to label the nuclei of all blastomeres. MACS buffer, and loaded into a MACS separation column (Miltenyi Blastocysts were then washed three times using MACS buffer, trans- Biotec) attached to a magnetic board (Spherotech, Lake Forest, IL). ferred to PBS containing 1 mM EDTA, and incubated for 5 minutes. The column was washed three times with MACS buffer (500 mL Subsequently, blastocysts were transferred to 0.05% (w/v) trypsin- each) to obtain the FITC-negative fraction in the eluate. The column 0.53 mM EDTA solution (Invitrogen) and incubated 10 min at 38.5C. was then detached from the magnetic board and washed three times A group of 25–30 trypsin-treated blastocysts was transferred to using MACS buffer (500-ml each) to recover the FITC-positive a 100-ml drop of MACS buffer and disaggregated into single blasto- fraction eluate. A 200-mL sample of each fraction of cells was meres by repeated pipetting using a finely drawn, flame-polished observed under a Zeiss Axioplan microscope (Zeiss, Gottingen,€ mouth micropipette. The drop containing disaggregated blastomeres Germany) with green and blue filter sets. was transferred into a 1.5-mL tube and mixed with 500 mL of PBS The cells remaining after MACS were subjected to mRNA containing 1 mM EDTA and 10% (v/v) fetal bovine serum (Atlanta extraction using the PicoPure RNA Isolation Kit (Molecular Devices, Biologicals, Lawrenceville, GA) to inactivate trypsin. Cells were Sunnyvale, CA) followed by DNase (New England Biolabs, Ipswich, then washed twice in MACS buffer by centrifugation (500 Â g for 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. Ozawa. Purification of TE and ICM. Fertil Steril 2011. 800 Ozawa and Hansen Purification of TE and ICM Vol. 95, No. 2, February 2011 TABLE 1 RNA recovery and cell purity after MACS. Percent of cells that Percent of cells that were Extracted RNA Replicates were dual labeled labeled with Hoechst only per embryo (ng) Intact blastocyst 4 — — 14.3 Æ 0.2 Disaggregated blastocyst 4 62.6 Æ 2.0a 37.4 Æ 2.0a — before MACS MACS-positive fraction 4 91.2 Æ 2.0b 8.8 Æ 2.0b 7.1 Æ 0.5 MACS-negative fraction 4 7.8 Æ 2.0c 92.2 Æ 2.0c 4.4 Æ 0.6 Note: Values are expressed as the least-squares mean Æ 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). recovered from cells isolated by MACS; this represents 80% of The NANOG and CDX2 mRNA were quantified by a 7300 Fast the RNA present in intact blastocysts and suggests a high rate of Real-Time PCR System (Applied Biosystems) using SYBR Green recovery of blastomeres during the purification process.
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