Stem Cells 2005;23:477-482
www.StemCells.com
© 2005 AlphaMed Press
Magnetic Cell Sorting Purification of Differentiated Embryonic Stem Cells Stably Expressing Truncated Human CD4 as Surface Marker
Robert David,
Michael Groebner,
Wolfgang-Michael Franz
University Clinic, Med I, Munich, Germany
Key Words. ES cells • Magnetic cell sorting • CD4 • Cell transplantation
Correspondence: Wolfgang-Michael Franz, M.D., Ph.D, Klinikum Großhadern, Marchioninistraße 15, 81377 München, Germany. Telephone: 49-89-7095-6095; Fax: 49-89-7095-6094; e-mail: wolfgang.franz{at}med.uni-muenchen.de
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ABSTRACT
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Embryonic stem (ES) cells offer great potential in regenerative medicine and tissue engineering. Clinical applications are still hampered by the lack of protocols for gentle, high-yield isolation of specific cell types for transplantation expressing no immunogenic markers. We describe labeling of stably transfected ES cells expressing a human CD4 molecule lacking its intracellular domain (
CD4) under control of the phosphoglycerate kinase promoter for magnetic cell sorting (MACS). To track the labeled ES cells, we fused CD4 to an intracellular enhanced green fluorescent protein domain (CD4EGFP). We showed functionality of the membrane-bound fluorescent fusion protein and its suitability for MACS leading to purities greater than 97%. Likewise, expression of CD4 yielded up to 98.5% positive cells independently of their differentiation state. Purities were not limited by the initial percentage of CD4+ cells, ranging from 0.6%–16%. The viability of MACS-selected cells was demonstrated by reaggregation and de novo formation of embryoid bodies developing all three germ layers. Thus, expression of CD4 in differentiated ES cells may enable rapid, high-yield purification of a desired cell type for tissue engineering and transplantation studies.
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INTRODUCTION
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Embryonic stem (ES) cells offer a huge potential in the field of regenerative medicine as well as tissue engineering because of their capacity to produce every cell and tissue type in vitro. In the future, the treatment of human diseases may be revolutionized by the ability to generate any cell, tissue, or even organ in the laboratory. Yet several obstacles must be overcome to bring ES-derived cell or tissue types to a clinical application. One major hindrance is that no approach has yet yielded a highly pure population of a single functional transplantable cell type. This, however, is an essential prerequisite to avoid the risk of teratoma and tumor formation and further impairment of tissue function, which may result from the implantation of undifferentiated ES cells or of undesired differentiated cell types [1, 2]. Therefore, an efficient means to purify the desired population is required [3, 4].
Methods such as fluorescence-activated cell sorting (FACS) or magnetic cell sorting (MACS) allow such purification but are dependent on the expression of a specific surface marker that can be recognized by a fluorescent or magnetic microbead-tagged antibody. To be fully effective, an endogenous marker needs to be absolutely cell-type specific. In many cases, however, such as cardiomyocytes, an appropriate endogenous marker is not known, and sorting methods have to rely on the introduction of a marker gene under the control of a lineage-specific promoter. In such an effort, we previously labeled and FACS purified ventricular cardiomyocytes expressing enhanced green fluorescent protein (EGFP) [5]. However, cytometry is slow and typically capable of analyzing no more than 3,000 cells per second for sorting purities of greater than 95% with yields of 50%–70%. In cases of myocardial infarction with a 10% necrosis, approximately 40 g of viable myocardium may be necessary. With an average weight of 80 ng per single cardiomyocyte, more than 108 cells are required for transplantation, resulting in a theoretical purification period of more than 500 hours [6]. Therefore, FACS does not seem to provide the capabilities to identify a rare population of cells or to separate large numbers of cells because of the excessive amount of analysis and sorting time.
Alternative approaches relying on the introduction of a drug-resistance gene rather than a fluorescent protein for antibiotic selection [7–9] are critical because of the long incubation period with the hazard of resistance and possible harmful effects of the antibiotic on terminally differentiated cells themselves. Additionally, FACS based on fluorescent markers as well as antibiotic selection rely on the expression of nonhuman proteins that may cause additional immunological problems or even be toxic in patients.
To overcome the above described obstacles, we have established a protocol for the labeling and isolation of stably transfected ES cells based on MACS, which is currently regarded to be the gold standard for mild and time-sparing cell purification. Using MACS, up to 1011 cells can be analyzed in approximately 1 hour, making it possible to separate large cell numbers and to identify even rare populations of cells. Our method relies on the expression of an intracellular truncated human CD4 surface antigen, thereby making an immunogenic potential unlikely. It may become an important tool for high-yield selection of specific cell types, providing the basis for future cell transplantation therapy.
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MATERIALS AND METHODS
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Plasmid Construction
The CD4EGFP cDNA containing a PolyA signal 3' to the stop codon was generated via overlapping polymerase chain reaction (PCR). The corresponding CD4 and EGFP fragments had been derived via proofreading PCR from pMACS 4.I (Miltenyi Bio-tech, Bergisch Gladbach, Germany) and pEGFP (Clontech, Palo Alto, CA). Thereafter, a fragment containing the cytomegalovirus (CMV) promoter, the SP6 promoter, and the 5' globin leader sequence was generated via PCR from pCS2+ [10]. Subsequently, a second overlapping PCR was performed combining the pCS2+-derived fragment with the CD4EGFP cDNA. This PCR fragment was inserted into pCR-XL-Topo (Invitrogen, Carlsbad, CA) via TOPO cloning, leading to pCMV-CD4EGFP. After sequencing, this vector was used for in vitro translation and injections into Xenopus embryos.
The vector pPGK-CD4EGFP was generated via PCR using pCMV-CD4EGFP as template, thereby introducing BamH1 and Not1 sites in the primers. The resulting fragment was used to replace the EGFP coding region in pEGFP-1 (Clontech). The phosphoglycerate kinase (PGK) promoter was then inserted via Xho1 and BamH1. To generate pPGK-CD4, the CD4EGFP cDNA was excised from pPGK-CD4EGFP and replaced with a CD4 cDNA fragment likewise containing BamH1 and Not1 sites. pPGK-CD4EGFP and pPGK-CD4 were used for electroporations of ES cells after verifying their sequences.
In Vitro Translation
In vitro transcription and translation incorporating 35S-methionine was performed using a TNT kit (Promega, Madison, WI) according to the manufacturer’s instructions followed by standard SDS-PAGE and autoradiography.
Xenopus Injections
Injections of Xenopus embryos at two-cell stage were performed as described [11] using 50 pg of non linearized pCMV-CD4EGFP DNA. The embryos were subsequently grown until early tadpole stages for analysis using epifluorescent microscopy.
ES Cell Culture
Electroporation and isolation of stable clones using the murine ES cell line GSES were performed according to standard protocols with minor modifications [5]. Thereby, 5 µg XhoI linearized vector was used for electroporation (240V/500 µF) of 5 x 106 GSES cells. Transgenic ES cells were grown in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated ES-qualified fetal calf serum, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 1 x nonessential amino acids, 0.4 mg/ml Geneticin (G418) (all reagents from GIBCO BRL, Eggenstein, Germany), and 0.1 mM ß-mercaptoethanol (Sigma, Deisenhofen, Germany). They were kept undifferentiated and feeder free by addition of 1,000 U/ml purified recombinant mouse leukemia inhibitory factor (ESGRO, Life Technologies, Inc., Grand Island, NY). Cells were maintained at 37°C in a humidified atmosphere of 5% CO2/95% air. Monolayers were passaged by trypsinization at confluence of 70%–80%. For MACS, undifferentiated cells were dissociated using phosphate-buffered saline (PBS) containing 5 mM EDTA as described below. In vitro differentiation was initiated as follows: GSES cells were harvested with 0.25% trypsin-EDTA, and dissociated cells were transferred to bacteriological dishes at a density of 2 x 105 ES cells/ml in Iscove’s modified Eagle’s medium (Sigma) supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 1 x non-essential amino acids (all reagents from Life Technologies, Inc.), and 450 µM 
-monothioglycerol (Sigma). After 2 days, embryoid bodies (EBs) were transferred to new medium. At day 6, EBs with a similar size were plated onto gelatin-coated tissue culture dishes. The growth medium for the attached differentiation cultures was changed every day. For MACS, the cells were dissociated as described below.
Flow Cytometry and Magnetic Cell Sorting
For FACS and MACS, the cells were dissociated in PBS containing 5 mM EDTA for 15 minutes at 37°C after washing the cells twice in PBS without calcium. Subsequently, the cells were spinned down at 2,500 rpm for 3 minutes in an Eppendorf centrifuge and resuspended in 100 µl ice-cold PBS containing 2 mM EDTA and 0.5% bovine serum albumin (BSA). FACS analyses were performed with a FACS calibur using the evaluation program CellQuest after incubating in phycoerythrin (PE)-conjugated CD4 antibodies (Pharmingen, San Diego). For MACS, the labeling was performed on 3 to 5 x 106 GSES cells by incubating in PE-conjugated CD4 antibodies and a second incubation in magnetic -PE microbeads according to the supplier (Miltenyi Biotech). For magnetic separation, we used mini-MACS columns (Miltenyi Biotech), applying 3 to 5 x 106-labeled cells on one column. For MACS, the buffer (PBS containing 2 mM EDTA and 0.5% BSA) was degassed in an ultrasonic bath and cooled on ice. The positive fraction was applied to a second column before subjecting the cells to FACS analysis.
Reverse Transcription–Polymerase Chain Reaction
Semiquantitiative reverse transcription (RT)–PCR was performed according to standard protocols using RNA isolated from 200 to 250 EBs by the Rneasy-Kit (Qiagen, Hilden, Germany). The amplified fragments corresponded to bp 64-189 of the murine H4, to bp 66-386 of the murine HNF-4, to bp 580–810 of the murine Brachyury, to bp 7–270 of the murine Involucrin, and to bp 315–625 of the murine Neurogenin cDNAs.
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RESULTS
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To establish MACS for genetically engineered ES cells, we generated a CD4EGFP fusion construct simultaneously offering the advantages of in vivo detectability via green fluorescence. This construct consists of CD4 and EGFP (CD4EGFP), the latter thereby replacing the natural intracellular domain of the native CD4 molecule (Fig. 1A
). The plasmid contains an SP-6 promoter for in vitro translation and a CMV promoter for expression in eucaryotic cells. Expression of CD4EGFP in vitro yielded a protein of the correct size of 74 kD (Fig. 2A), and injection into Xenopus embryos clearly led to the expected membrane-bound localization of the fluorescent fusion protein (Figs. 2B, 2C).
Next we planned to transfect murine GSES cells with CD4EGFP. Because viral promoters such as CMV often show extremely high yet unstable activity in ES cells, we generated the plasmid pPGK-CD4EGFP (Fig. 1B
). After electroporation and selection, four stable G418-resistant clones were obtained. As expected, these clones displayed membrane-bound EGFP fluorescence, confirming the functionality of the fusion construct (Figs. 2D, 2E). In the various clones, 50.2%–63.1% of the cells were detectable in FACS via antibodies directed against the CD4 part of the fusion protein. Thereby, all of the EGFP-positive cells were contained within this fraction. However, a small proportion of the positively stained cells displayed no or weak EGFP fluorescence, demonstrating that detection of the fusion construct via the CD4 antibody is more sensitive than its EGFP fluorescence.
We now tested the feasibility of MACS using CD4EGFP-positive cells differentiated for 3 days. To reduce their percentage to a more realistic number with respect to future applications using specific promoters, we diluted the labeled cells to 5.9% with native differentiated GSES before MACS purification. This initial population of differentiated cells displaying the CD4 antigen by FACS was enriched to 96.2% (Fig. 3A). Again, all of the EGFP-positive cells were present within this fraction, and some positively stained cells showed a weak or even no fluorescence. Likewise, use of undifferentiated ES cells yielded results comparable to those described above with a purity of 97.3%, whereby the initial population consisted of 13.7% positive cells (data not shown).
Our next goal was to verify the data using the nonimmunogenic CD4 molecule without an intracellular EGFP part as well as to extend our studies to lower percentages of CD4-positive cells. GSES cells were transfected with pPGK-CD4 (Fig. 1C), and after selection, four stable G418-resistant clones were obtained expressing CD4 on their cell surface as determined via FACS analysis. In the various clones, 37.7%–53.8% were CD4 positive.
Differentiated cells of clone 6, in which CD4 had been detected on 38.5% of the cells, were then subjected to MACS. We hereby investigated the feasibility of MACS using the stably CD4-expressing ES cells at early and late time points of differentiation. Again we compared different percentages of CD4-positive cells before the purification. Figure 3B shows that even 0.6% of positive cells within the whole population of ES cells after 3 days of differentiation (early time point) led to a 98.5% pure population of CD4-expressing cells. When the population before MACS contained 7.3% positive cells in a parallel experiment, a purity of 96.8% was achieved (data not shown). Equivalent results leading to a purity of greater than 96% were achieved at a later time point of differentiation (12 days), whereby the initial positive population was 10.7% (Fig. 3C). Likewise, when we used undifferentiated CD4-positive cells at initial percentages between 3.8% and 15.8%, purities between 97.6% and 98.4% of cells were achieved (data not shown).
To verify the viability of the MACS-selected cells, we recultivated those after the purification procedure, which was performed at day 3 of differentiation. Figure 4A shows single dissociated CD4EGFP-positive cells after the elution of the MACS column. These cells were able to reaggregate and form normal fluorescent EBs (Figs. 4B, 4C). Likewise, CD4-expressing cells formed normal EBs (data not shown). To verify the normal development of these reaggregated EBs, we subjected them to RT-PCR experiments. Figure 4D shows that CD4EGFP as well as CD4-expressing cells at day 4 after the MACS purification did express markers for all three germ layers compared with cells that were not MACS purified.
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DISCUSSION
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As an approach to the current lack of a straightforward purification protocol of transplantable ES cell–derived cell types, we here show labeling and isolation of stably transfected ES cells based on the mild and time-sparing method of MACS. This was achieved after transfection of a nonimmunogenic human CD4 surface molecule lacking its intracellular domain (CD4). CD4 has been successfully applied for MACS selection of various cell types [12–14], but so far the system has merely been used as a transfection marker via viral promoters. Our ultimate goal is to use it as a selection tool for distinct subtypes of ES-derived cells via specific promoters. Because CD4 is only expressed on cells of the immune system, the problem of a contamination with cells endogenously expressing CD4 does not appear in cultures of native ES cells.
To establish MACS for genetically engineered ES cells, we generated a CD4EGFP fusion construct simultaneously offering the advantages of in vivo detectability via green fluorescence. Our work shows that even more than 98% of positive viable cells can be obtained using CD4EGFP independently of their differentiation state and the initial percentage of CD4EGFP-positive cells. This makes the technique equal to the best FACS results with respect to purity, combined with a much higher cellular compatibility and the independence of a cost-intensive cell sorter. CD4EGFP should significantly simplify the characterization of promoter constructs in ES cells, because an active promoter driving its expression will be easily detectable under the epifluorescent microscope, thereby enabling an easy determination of the optimal time point for MACS. Likewise, the purification could be visually tracked simply under epifluorescence microscopy, and after transplantation into animals, donor cells may be detectable without the effort of antibody staining. It must be noted that a tiny proportion of the positively stained cells do not show detectable EGFP fluorescence. However, this slightly weaker sensitivity of the EGFP part of the fusion molecule compared with its detectabilitiy via antibody staining will not abrogate its general advantages described above, because most of the cells are positive for both detection methods.
We next transferred our technique to the human-derived CD4 alone to guarantee the nonimmunogenic properties of this human-derived antigen for future applications in patients. Likewise, this yielded up to 98.5% of positive, viable cells independently of their differentiation state even when very low frequencies of positive cells (0.6%) were present in the initial population, making it likely that the technique could be brought into a clinical application in the near future.
An important obstacle impeding a clinical use of ES cells at present is that no efficient means to purify the desired population is available [1–4]. This is further hampered by the fact that in many cases, an appropriate endogenous surface marker is unknown, leading to attempts of introducing a marker gene under the control of a lineage-specific promoter. In such an attempt, we had previously EGFP-labeled ventricular-like cardiomyocytes using the ventricular-specific 2.1-kb myosin light chain-2v promoter [5], achieving a 97% pure population of fluorescent cells after FACS sorting. However, the FACS procedure may affect the electrophysiological characteristics of these highly specified cells, and it is also of great importance to bear in mind the limitations of FACS with respect to sorting capacity over time. High-speed sorting, which works at rates up to 35,000 cells per second, may be regarded as an alternative. However, using this technique, up to 50% of the cells are lost compared with standard sorting techniques. High-speed sorting, which allows running up to 120 million cells per hour through the sorter to yield a 5- to 10-fold enrichment, can be useful for preselection of a certain cell population. The enriched population may then be either sorted again using standard techniques or used as is if a 50% purity is acceptable for the application. That way, 10-hour sorts may be turned into 1-hour sorts, which would still lead to an unrealistically long sorting time for rare cell populations. Additionally, the increased stress on the cells due to the increased velocity makes fragile cells and cells that do not grow well to poor candidates for high-speed sorting.
Using MACS, up to 1011 cells can be analyzed in approximately 1 hour, making it possible to separate large cell numbers and to identify even very rare populations of cells, which appears unlikely for FACS. Other approaches are based on the introduction of a drug-resistance gene rather than a surface marker to allow for preferential antibiotic selection of subpopulations [7–9]. Disadvantages of this technique comprise the long selection period, bearing the risk of the emergence of resistance as well as possible harmful effects of the antibiotic on terminally differentiated cells themselves. Again, like EGFP fluorescence, an antibiotic resistance requires the expression of nonhuman proteins in the transplanted cells, which may cause additional immunological problems in future clinical applications. Our MACS-based approach relying on the expression of a nonimmunogenic surface marker of human origin may become an important step to overcome these obstacles.
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CONCLUSIONS
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The great medical potentials of ES cells are impeded so far by the lack of an efficient nonimmunogenic way to isolate the desired cell types meant for transplantation. We have labeled ES cells stably expressing human CD4 truncated for its intracellular domain (CD4) for MACS. To track the labeled cells, CD4 was fused to an intracellular EGFP rest (CD4EGFP). We prove functionality of CD4EGFP and its suitability for MACS yielding purities greater than 97% when expressed via the PGK promoter. Likewise, expression of CD4 led to greater than 98% positive viable cells, which was not affected by their differentiation state. Additionally, purities were not influenced by the initial content of CD4-expressing cells, ranging from 0.6%–16%. After the MACS procedure, the cells were able to reaggregate and form normal EB-expressing markers of all three embryonic germ layers, proving their viability. The application of our technique in differentiated ES-derived cell types may allow the rapid purification of a desired cell type with high yields, thereby providing an important basis for tissue engineering and cell transplantation studies.
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ACKNOWLEDGMENTS
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We are very grateful to Christiane Gross for expert technical assistance as well as to Professor Ralph Rupp for providing the Xenopus facilities. This project is funded by the Deutsche Forschungsgemeinschaft (FR705/11-1) and the Fritz-Bender-Stiftung. Robert David and Michael Groebner contributed equally to this work.
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Received August 3, 2004;
accepted for publication January 19, 2005.
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Abstract
Introduction
