Selection of Embryonic Stem Cell-Derived Enhanced Green Fluorescent Protein-Posi - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:55 |只看该作者 |倒序浏览 |打印

作者：Eva Hedlunda,b,c, Jan Pruszaka,c, Andrew Ferreea,c, Angel Viuelaa,c, Sunghoi Honga,c, Ole Isacsona,c, Kwang-Soo Kima,b作者单位：aUdall Parkinson 3 I9 G1 R8 N& ?0 f! P, U6 I: m
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      【摘要】
   Transplantation of mouse embryonic stem (mES) cells can restore function in Parkinson disease models, but can generate teratomas. Purification of dopamine neurons derived from embryonic stem cells by fluorescence-activated cell sorting (FACS) could provide a functional cell population for transplantation while eliminating the risk of teratoma formation. Here we used the tyrosine hydroxylase (TH) promoter to drive enhanced green fluorescent protein (eGFP) expression in mES cells. First, we evaluated 2.5-kilobase (kb) and 9-kb TH promoter fragments and showed that clones generated using the 9-kb fragment produced significantly more eGFP /TH  neurons. We selected the 9-kb TH clone with the highest eGFP/TH overlap for further differentiation, FACS, and transplantation experiments. Grafts contained large numbers of eGFP  dopamine neurons of an appropriate phenotype. However, there were also numerous eGFP  cells that did not express TH and did not have a neuronal morphology. In addition, we found cells in the grafts representing all three germ layers. Based on these findings, we examined the expression of stem cell markers in our eGFP  population. We found that a majority of eGFP  cells were stage-specific embryonic antigen-positive (SSEA-1 ) and that the genetically engineered clones contained more SSEA-1  cells after differentiation than the original D3 mES cells. By negative selection of SSEA-1, we could isolate a neuronal eGFP  population of high purity. These results illustrate the complexity of using genetic selection to purify mES cell-derived dopamine neurons and provide a comprehensive analysis of cell selection strategies based on tyrosine hydroxylase expression.# `  O: n. ~( ?  R& n7 s% `
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Disclosure of potential conflicts of interest is found at the end of this article. 5 }2 l3 H& `5 n0 _4 c0 @
      【关键词】 Genetic engineering Fluorescence-activated cell sorting Parkinson disease Stage-specific embryonic antigen CD-& ~7 m! }/ [# I' x( t8 x$ }, Q4 C
   INTRODUCTION7 l( Z0 B2 P# R' ?
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Embryonic stem (ES) cells, transplanted either in a naïve or a predifferentiated state, can alleviate symptoms in animal models of Parkinson disease  tyrosine hydroxylase (TH) promoters have indicated that primary dopamine neurons can be purified from embryos by FACS and survive replating into culture and transplantation.
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Transgenic mouse experiments using the rat TH promoter have indicated that 5' sequences 4.5 kilobases (kb) . In our strategy to purify dopamine neurons from mES cells, we compared the ability of a 2.5-kb fragment and a 9-kb fragment of the rat TH promoter to drive eGFP expression in mES cells after in vitro differentiation. Since the integration site could affect the promoter function, we generated multiple mES clones and analyzed their differentiation in vitro by immunofluorescent staining and FACS. One clone was finally selected based on its high overlap in eGFP and TH expression and further characterized in vitro as well as in vivo after FACS and transplantation into naïve mice and 6-hydroxydopamine-lesioned rats.

MATERIALS AND METHODS6 F5 }0 k) g% J2 [, r7 z
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Construction of 2.5-kb TH and 9-kb TH Promoter Plasmids

The 2.5- or 9.0-kb promoter region of the rat TH gene  (National Center for Biotechnology Information accession no. AF014956>) were inserted into the multiple cloning site of the pEGFP-1 promoterless vector (GenBank accession no. U55761; Clontech, Palo Alto, CA, http://www.clontech.com), upstream of the eGFP gene. The 2.5-kb TH promoter fragment was retrieved by digestion with SfiI, and the 9-kb TH promoter fragment was retrieved by digestion with HindIII. Insert orientation was verified by double diagnostic digestion and sequence analysis of boundaries.; d  j' K- H# k, k: q$ _& k7 X
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mES Cell Propagation
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The mouse blastocyst-derived embryonic stem cell line D3 (ATCC, Manassas, VA, http://www.atcc.org) was propagated on mitomycin C-treated (10 µg/ml medium; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) primary murine embryonic fibroblasts (PMEFs) (no. 00321; Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supplemented with 2 mM L-glutamine (Invitrogen), 1 mM ¦Â-mercaptoethanol, 1x nonessential amino acids (Invitrogen), 1x nucleosides (Specialty Media; Chemicon, Temecula, CA, http://www.chemicon.com), 15% fetal bovine serum (FBS) (Sigma-Aldrich), 100 U/ml penicillin, 100 µg/ml streptomycin (Invitrogen), and 2,000 U/ml human recombinant leukemia inhibitory factor (R&D Systems Inc., Minneapolis, http://www.rndsystems.com). D3 cells were passaged four times before transfection and were subsequently purified from PMEFs.( k6 \0 ~+ ?$ _! ~7 `- Z$ w

Stable Transfection, Clonal Expansion, and Differentiation of mES Cells

D3 mES cells were transfected with 2.5-kb TH-pEGFP-1, 9-kb TH-pEGFP-1, or pEGFP-1 alone using Lipofectamine plus (Invitrogen). Stably transfected cells were selected in ES medium containing 500 µg/ml neomycin (Clontech). We screened a large number of independent drug-resistant colonies (108 of the smaller-sized colonies of each construct) and aimed to isolate those exhibiting a faithful co-expression pattern of TH and eGFP after in vitro differentiation on PA6 (Riken, Tsukuba, Japan, http//:www.riken.jp)  (supplemental online data), to compare which protocol would generate appropriate eGFP  neurons for transplantation. Prior to in vitro differentiation, mES cell were purified from STO feeders. Cells used for immunofluorescent staining were fixed in 4% paraformaldehyde for 30 minutes and rinsed with phosphate-buffered saline (PBS). Cells to be used for FACS and transplantation were harvested at days 8¨C11 of differentiation using 0.05% trypsin/EDTA. For both PA6- and MS5-based protocols, the full differentiation time is 14 days. Cells to be further analyzed in vitro after FACS were plated onto primary rat astrocytes (Cambrex, Walkersville, MD, http://www.cambrex.com) and supplemented with 10 ng/ml glial-derived neurotrophic factor (GDNF) and 20 ng/ml brain-derived neurotrophic factor (BDNF).4 a0 K1 k# T  H# O
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FACS

Cells were differentiated for 8¨C11 days, harvested using 0.05% Trypsin/EDTA (Invitrogen), gently dissociated into a single-cell suspension, and resuspended in phenol-free Hanks' balanced salt solution (Invitrogen) containing 20 mM glucose (Sigma-Aldrich), penicillin-streptomycin, and 2% FBS. Samples were filtered, analyzed, and sorted immediately or were subjected to surface marker staining: mouse anti-stage-specific embryonic antigen-positive (anti-SSEA-1) antibody (0.4 µg/ml; Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/dshbwww), incubated for 50 minutes at 4¡ãC, washed, and then incubated with the corresponding secondary antibody followed by washing steps. Cells were analyzed and sorted using a FACSAria cell sorter and FACSDiva software (BD Biosciences, San Diego, http://www.bdbiosciences.com). The population of interest was identified by forward and side scatter gating. Using a 488-nm laser for excitation, GFP positivity was determined according to fluorescence intensity in the GFP channel (490-nm long-pass  filters) against autofluorescence in the yellow fluorescent protein (527 LP, 550/30 BP) or red (595 LP, 610/20 BP) channels. D3 mES cells or EV-1 cells, at the same stage of differentiation, were used as the GFP-negative controls. eGFP positivity was confirmed by reanalysis by FACS, and viability was determined by trypan blue exclusion. SSEA-1 positivity was determined according to fluorescence in the red channel compared with negative controls lacking the primary antibody and/or secondary antibodies. Further flow cytometric analysis was performed using FlowJo software (Tree Star, Ashland, OR, http://www.treestar.com).- O! U, s; G  `' J: l' U8 d" V
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Animal Procedures

All animal procedures were performed in accordance with National Institutes of Health guidelines and were approved by the Animal Institutional Care and Use Committee at McLean Hospital, Harvard Medical School., V' E, i' C2 h  \2 ?6 ^& l

Transplantation of eGFP  Cells to Naïve Mice and 6-Hydroxydopamine-Lesioned Rats

Nine-kb TH-eGFP mES cells, FACS purified for eGFP , were resuspended at 100,000 cells per microliter. Female naïve C57/Bl6 mice (n = 15) were grafted with 1¨C2 µl of cell suspension, and Sprague-Dawley rats with unilateral 6-hydroxydopamine lesions (Charles River Laboratories, Wilmington, MA, http://www.criver.com) were grafted with 3 µl of cell suspension (n = 18) or medium alone (n = 7). Transplantations and immunosuppression were performed as previously described  (supplemental online data). Mice were sacrificed 4 weeks and rats 10 weeks after transplantation, unless needed earlier. Animals were anesthetized by an i.p. overdose of pentobarbital (150 mg/kg) and perfused intracardially with heparinized saline (0.1% heparin) followed by 4% paraformaldehyde. Brains were removed, postfixed for 6 hours in paraformaldehyde, equilibrated in 20% sucrose, and sectioned on a freezing microtome in 40-µm serially collected coronal slices.) b6 R; Q; Q7 F! D5 v! m4 F6 k

Histological and Stereological Procedures

For immunofluorescent staining, cells/sections were incubated with blocking buffer (PBS, 10% normal donkey serum, 0.1% Triton X-100) for 1 hour and subsequently with primary antibodies in blocking buffer overnight. Information on primary antibodies used is given in supplemental online data. The coverslips/sections were subsequently incubated in fluorescent-labeled secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) in blocking buffer for 1 hour, rinsed in PBS, counterstained with Hoechst 33342 (4 µg/ml), and mounted (Gel/Mount; Biomeda Corp., Foster City, CA, http://www.biomeda.com). For light microscopy, a biotinylated secondary antibody (1:300; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) was used, followed by incubation in streptavidin-biotin complex (Vector Laboratories) and 3,3'-diaminobenzidine (Vector Laboratories). Confocal analysis was performed using a Zeiss LSM510/Meta Station (Thornwood, NY, http://www.zeiss.com). Stereology was performed using Stereo Investigator software (MicroBrightField, Williston, VT, http://www.microbrightfield.com) and a Zeiss Axioplan I fluorescent microscope. Graft volumes and TH neuron numbers were calculated using the Cavalieri estimator and Optical fractionator probes. The eGFP/TuJ1 and eGFP/TH overlap in cells after in vitro differentiation (3 coverslips per condition) and the percentage of eGFP  or eGFP¨C TH  cells in the grafts (n = 6) was determined by random sampling using StereoInvestigator. eGFP and TH overlap in purified eGFP /SSEA-1¨C neurons was assessed by randomized quantification of TH expression (647 nm) in cells identified as eGFP  (488 nm) using confocal microscopy (three coverslips were counted).' h$ I! c* t. V4 e$ i# d6 y

Statistical Analysis2 P9 i! T! T9 q* U6 }) X3 v
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The number of eGFP  events in TH-GFP clones versus the original D3 mES clone, as well as the rotations for mES cell-grafted and vehicle-grafted animals, was analyzed using analysis of variance (ANOVA). The extent of overlap between eGFP and TuJ1 or TH after in vitro differentiation of 9-kb TH-eGFP cells was determined by unpaired t test. The percentage of TH  cells in the rat grafts that were eGFP  or eGFP¨C was analyzed by paired t test. InStat3 software (GraphPad Software, Sa Diego, http://www.graphpad.com) was used for all statistical analyses.
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RESULTS

Evaluation of TH-eGFP Promoter Constructs in mES Cells In Vitro
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We transfected naïve D3 mES cells with a 2.5-kb (2.5-kb TH-eGFP) or a 9-kb (9-kb TH-eGFP) rat TH promoter construct driving eGFP expression. G418-resistant clones were screened for overlap between eGFP and TH expression after 14 days of differentiation using the PA6-based protocol ; p
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Figure 1. Evaluation of TH-eGFP promoter constructs in mouse embryonic stem (mES) cells during in vitro differentiation. (A, B): Overlap in eGFP and TH expression in a (A) 2.5-kilobase (kb) TH-GFP clone and in a (B) 9-kb TH-eGFP clone after 14 days of differentiation on PA6. Cultures of 9-kb TH-eGFP clones contained many more eGFP /TH  cells compared with the 2.5-kb TH-GFP cultures (yellow co-expression). (C): FACS analysis for eGFP  events within an FSC/SSC gate of naïve D3 mES cells autofluorescence), one 2.5-kb TH-eGFP clone (number 91) and three 9-kb TH-eGFP clones (numbers 15, 41, and 93) after 9 days of differentiation on PA6. All 9-kb TH clones showed an expected higher proportion of eGFP  events compared with the naïve D3 mES cell line (*, p 1 {8 V% q$ V, w% _

Comparison of MS5- and PA6-Based In Vitro Differentiation Protocols8 I! ?( U6 ?% L3 ?" p$ y, K7 w

To evaluate which differentiation protocol would better generate appropriate eGFP  neurons for transplantation, the 9-kb TH-eGFP clone 15 (9-kb TH-eGFP) was differentiated using the PA6-based  (Fig. 2E). Both coculture systems generated few dopamine ¦Â-hydroxylase-positive cells (Fig. 2F), and no eGFP  cells had a norepinephric phenotype (Fig. 2F). Based on these findings, we used the MS5-based protocol in all subsequent experiments.
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Figure 2. Comparison of MS5- and PA6-based in vitro differentiation protocols. (A¨CH): The 9-kilobase (kb) TH-eGFP mouse embryonic stem cells were analyzed in vitro after 9 days of differentiation using either the PA6 or MS5-based protocols. (A), enlarged in (B): eGFP and TuJ1 overlap was high using either protocol, although it was significantly higher using the MS5-based protocol (PA6, 94.9% ¡À 0.2% ) (yellow co-expression). Pax2 immunofluorescent staining (E) showed the mid-hindbrain characteristic of the MS5 culture, with very little overlap between eGFP and Pax2, as anticipated based on the different temporal expression of TH and Pax2 during normal development. (F): There were few DBH  cells generated in either PA6- or MS5-based protocols, and the DBH staining did not appear to overlap with eGFP expression. Bar graphs depicting overlap between GFP and TuJ1 expression (G) and eGFP and TH expression (H) in cells differentiated using PA6-based (white bars) or MS5-based (black bars) protocol for 9 days (*, p

Transplantation and In Vivo Analysis of eGFP-Sorted Cells
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In vitro differentiated 9-kb TH-eGFP cells were FACS sorted for eGFP expression and transplanted. The short-term transplantation to mice showed the presence of eGFP  cells and large numbers of TH neurons in all grafts (Fig. 3A¨C3D). Most TH  neurons appeared to co-express eGFP (Fig. 3C, 3D). However, some cells expressed only eGFP or only TH (Fig. 3C, 3D). Further analysis of the dopamine phenotype of the eGFP  neurons showed that many eGFP  cells expressed aromatic L-amino acid decarboxylase (AADC) (Fig. 3E¨C3H), as well as GIRK2 (Fig. 3I¨C3L), which is relatively enriched in A9 compared with other midbrain dopamine neurons  (Fig. 3M¨C3O). Unexpectedly, during the long-term transplantation of eGFP  cells to 6-hydroxydopamine-lesioned rats, a number of animals started displaying behavioral deficits suggesting teratoma formation and were therefore perfused before the behavioral study was completed. Only animals surviving the entire 10 weeks of testing were included in the behavioral analysis (n = 8). When the number of rotations was analyzed, there was no significant difference between the vehicle and the cell-transplanted groups at any time point (p > .05, ANOVA; supplemental online Fig. 1A). The eight cell-grafted animals that survived until the 10-week time point had a 21% average reduction in the number of rotations (supplemental online Fig. 1B). Graft analysis in four of the surviving animals with reduced number of rotations showed the presence of large numbers of TH  neurons: 12,567, 92,945, 102,515, and 165,326, respectively (supplemental online Fig. 1B). These numbers were directly correlated with the size of the grafts (data not shown) but not with the improvement in the number of rotations. The animal with the largest number of TH  neurons (Fig. 4A¨C4E) had a 70% decrease in the number of rotations (supplemental online Fig. 1B), and this graft contained some TH  neurons, which extended neurites into the host striatum (Fig. 4A, 4C). Approximately 60% of the TH  neurons were also eGFP  (Fig. 4D, 4E). The grafts also contained cells of neuronal morphology, which were either eGFP /TH¨C or eGFP¨C/TH  (Fig. 4D). Further analysis of the dopamine phenotype of the grafted cells showed that most of the eGFP  neurons expressed AADC (Fig. 4F¨C4H) and Pitx3 (Fig. 4I¨C4K). All three germ layers, as visualized by cytokeratin (ectoderm), myosin (mesoderm), and villin (endoderm) staining, were present in the grafts (Fig. 4L¨C4N). No colocalization between eGFP and the germ layer markers was identified.
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Figure 3. Short-term transplantation and in vivo analysis of eGFP-sorted cells. (A¨CO): Naïve mice were transplanted with cells sorted for eGFP expression. Grafts were analyzed 4 weeks post-transplantation. (A): Low-power microphotograph of a graft, showing large numbers of TH  neurons (the nickel-enhanced 3,3'-diaminobenzidine products appear grayish black; the boxed area is shown enlarged in ). Abbreviations: AADC, aromatic L-amino acid decarboxylase; GFP, green fluorescent protein; GIRK2, G protein-activated inwardly rectifying potassium channel 2; Pitx3, paired-like homeodomain transcription factor 3; TH, tyrosine hydroxylase.; E+ v- E* ]- D/ U

Figure 4. Long-term xenotransplantation and in vivo analysis of eGFP-sorted cells. (A¨CN): eGFP  cell transplants into 6-hydroxydopamine-lesioned rats were analyzed 10 weeks post-transplantation. (A): Low-power microphotograph of a graft, showing large numbers of TH  neurons (the nickel-enhanced DAB products appear grayish black; the boxed area to the left is shown enlarged in ). The two groups were not significantly different (p > .05, paired t test) due to the high variability in overlap between TH and eGFP between different grafts. Many of the eGFP  neurons displayed characteristics of midbrain dopamine neurons, as shown by the overlapping expression with AADC (F¨CH) and Pitx3 (I¨CK). Grafts contained all three germ layers, as visualized by staining for cytokeratin (L), myosin (M), and villin (N), but no overlapping expression with eGFP was detected. Scale bars = 200 µm (A), 50 µm (B, C, F¨CN), and 25 µm (D). Abbreviations: AADC, aromatic L-amino acid decarboxylase; eGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; Pitx3, paired-like homeodomain transcription factor 3; TH, tyrosine hydroxylase.+ p& m1 @0 R5 H! \

Identification of Proliferative eGFP  Cells in the Grafts+ e4 }! `( G4 }0 [

Further analysis showed that in addition to eGFP  cells of a neuronal phenotype, grafts contained clusters of immature eGFP /SSEA-1  cells (Fig. 5A¨C5I). All grafts analyzed contained proliferative cells, as demonstrated by Ki67 staining (Fig. 5E¨C5I). The majority of Ki67  cells in the grafts were not eGFP . However, a large proportion of the eGFP  cells showing a non-neuronal morphology were Ki67  and some of these eGFP /Ki67  cells also expressed SSEA-1 on their surface (Fig. 5E¨C5I). There were large nestin  areas in the grafts surrounding the GFP  cells of immature morphology, but very few, if any, cells were GFP /nestin  (Fig. 5J¨C5L).6 y+ p1 T- e" m+ a5 a6 p
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Figure 5. Identification of proliferative eGFP  cells in the grafts. (A¨CL): Analysis of mouse grafts 4 weeks post-transplantation showed that there were cluster of SSEA-1 /eGFP  cells in some grafts (). Abbreviations: GFP, green fluorescent protein; SSEA, stage-specific embryonic antigen.2 S6 y0 b$ C" D: `0 ?
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Purification of Neurons from In Vitro Differentiated TH-eGFP Cells by Negative Selection for SSEA-1( \# F) e0 P5 f) ?# Z* |4 w
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Immunofluorescent analysis of differentiated TH-eGFP mES cells showed that there were SSEA-1  cells present in the cultures. Most individual colonies consisted of many eGFP  cells of neuronal morphology with few or no SSEA-1  cells. In such colonies, few or no cells were eGFP /SSEA-1  (Fig. 6A¨C6D). Some colonies, however, contained a majority of SSEA-1  cells (Fig. 6B, 6E, 6F), and in some instances, these cells were eGFP  (Figs 6E, 6F). These eGFP /SSEA-1  cells had a non-neuronal morphology and a lower expression of eGFP than the eGFP  cells with neuronal morphology (Fig. 6E, 6F). Some of these eGFP /SSEA-1  cells also stained for Oct-4 (supplemental online Fig. 2). The expression of SSEA-1 was not correlated with the size of the colony, since adjacent colonies of the same size were sometimes entirely SSEA-1  or contained only eGFP  cells of neuronal morphology (Fig. 6B). Dissociation and replating of the TH-eGFP cultures after 9 days of differentiation, as if the cells were to be processed for FACS, surprisingly showed that most cells surviving this procedure were SSEA-1  cells of a non-neuronal morphology (Fig. 6G). This experiment also showed that some SSEA-1  cells expressed eGFP at a high intensity after dissociation (Fig. 6G). Live staining and FACS of differentiated TH-eGFP cells for SSEA-1 showed that the original D3 mES cell clone (n = 3) used for the genetic manipulations contained fewer SSEA-1  events (cells) after differentiation, compared with both the empty vector (EV-1) (n = 3) and 9-kb TH-eGFP clone (n = 3) that were derived from this mES cell line (Fig. 6H). Karyotypic analysis did not reveal any acquired chromosomal abnormalities in our 9-kb TH-eGFP clone compared with the D3 mES cells (supplemental online Fig. 3), which could have explained the increase in SSEA-1  events. To remove cells with proliferative capacity and acquire eGFP  neurons only from differentiated TH-eGFP cells, we FACS sorted eGFP /SSEA-1¨C cells. A very small fraction of cells that were present after the trypsin dissociation and the FACS procedure were eGFP /SSEA-1¨C (Fig. 7A). The majority of cells sorted for eGFP alone were SSEA1  and had a non-neuronal morphology (Fig. 7B). However, a double sort for the fraction of cells that were eGFP /SSEA-1¨C (1.4% of the parental population, n = 6) resulted in a population of cells with mostly neuronal morphology (Fig. 7C, 7D). Approximately 60% of these eGFP  neurons stained for TH (Fig. 7D).
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Figure 6. In vitro analysis of SSEA-1 expression in differentiated mouse embryonic stem (mES) cell cultures. (A¨CH): Analysis of in vitro-differentiated 9-kilobase (kb) TH-eGFP cells showed that most colonies contained many eGFP  neurons and few SSEA-1  cells and no or very few cells that were eGFP /SSEA-1  (), and in some instances, these cells were SSEA-1 /eGFP  (E, F). The eGFP /SSEA-1  cells appeared to have a lower expression of eGFP than the eGFP  cells with neuronal morphology before dissociation (C, F). The expression of SSEA-1 did not appear to be correlated with the size of the colony (B). Dissociation of cultures followed by replating showed that most cell surviving this procedure were SSEA-1  (G). Fluorescence-activated cell sorting analysis showed that 9-kb TH-eGFP cells, as well as the EV1-transfected mES cells, contained a higher number of SSEA-1  cells than the original D3 mES cells used for the genetic manipulations (H). Scale bars = 100 µm (A¨CC, E) and 50 µm (D, F, G). Abbreviations: k, kilobase; EV1, empty vector; GFP, green fluorescent protein; SSEA, stage-specific embryonic antigen; TH, tyrosine hydroxylase.
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Figure 7. Purification of neurons from in vitro-differentiated TH-eGFP cultures by negative selection for SSEA-1. Live stain and fluorescence-activated cell sorting (FACS) for eGFP  and SSEA-1, after in vitro differentiation on MS5 for 9 days, showed that most eGFP  cells were SSEA1 , although a small population of cells that were eGFP /SSEA-1¨C could be isolated (A). The EV-1-transfected mouse embryonic stem cell clone, differentiated for the same length of time as the 9-kb TH-eGFP clone, was used as a negative control for fluorescence (A). Plating of cells sorted only for eGFP fluorescence resulted in a population consisting mainly of SSEA-1  cells of a non-neuronal morphology (2 days in vitro post-FACS) (B). Cells negatively sorted for SSEA-1 and positively for eGFP gave a population of high neuronal purity (A, C, D). Some of the eGFP  neurons from this double sort also expressed TH (2 days in vitro post-FACS) (D). Abbreviations: Ab, antibody; eGFP, enhanced green fluorescent protein; EV-1, empty vector; GFP, green fluorescent protein; kb, kilobase; SSEA, stage-specific embryonic antigen; TH, tyrosine hydroxylase.

DISCUSSION

Genetic engineering of mES cells using transcription factors such as Nurr1 , which could partly explain the presence of eGFP /TH¨C neurons of a dopamine cell morphology. Once the promoter region is without activation, TH expression will fade away faster than the eGFP expression.# J4 J$ b: z9 F, C$ o2 m+ z

Transplantation of 9-kb TH-eGFP-sorted cells generated grafts with large numbers of TH neurons with an appropriate midbrain phenotype. However, our grafts contained up to 135-fold more TH neurons than expected, based on previous analyses of FACS-sorted primary embryonic TH neurons . This shows that even neurons that have extended long processes in vivo and/or in vitro can survive dissociation.* {6 Q/ h8 Q; L' d5 ?

Despite the large number of TH neurons in the grafts, some of which extended fibers into the host, there was no general significant behavioral improvement. There were TH neurons in the grafts that extended neurites into the host, although most TH neurons did not. Improvement in amphetamine-induced rotational behavior can, however, be seen without morphological integration of grafted cells . The reason we do not see improvement is likely due to the size and disruptive nature of the grafts. The lack of correlation between the total number of TH neurons in the grafts and improvement in the number of amphetamine-induced rotations supports this hypothesis. In addition, the presence of other cell types in the grafts makes it very difficult to interpret the behavior.
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eGFP  cells to be used for transplantation were sorted using a high purity mask to avoid the inclusion of eGFP¨C cells in our positive fraction. Reanalysis of sorted cells did, however, reveal the presence of GFP¨C cells in the eGFP  sorted fraction (90% of purified cells expressed eGFP; data not shown), perhaps partly due to bleaching or cell damage. The extent of eGFP¨C cells in the eGFP  fraction was similar to a previous study where teratoma formation after transplantation of 200,000 cells was avoided . ES cell cultures are never completely synchronized, and cells of many developmental stages are therefore present simultaneously. Although the integration site of our construct could influence the expression of eGFP, the transient expression of TH in multiple cell types during development could also explain the presence of eGFP  cell with proliferative potential. Any approach using TH to label dopamine neurons from ES cells, whether different lengths of the promoter are used or a knock-in strategy, will generate a mixed population of cells unless the ES culture systems can be completely synchronized in time and designed to specifically generate only one cell type.

Normal transient expression of TH during development could result in the generation of eGFP  cells of multiple cell lineages, as previously discussed. However, the expression of eGFP in a context of SSEA-1 in our cells could be due to misexpression related to transformation of the mES cells during clonal expansion. SSEA-1, a cell surface carbohydrate antigen (CD15 or Lewisx antigen), is typically expressed in preimplantation mouse embryos beginning at the eight-cell stage, in teratocarcinoma stem cells, ES cells, and adult CNS stem cells, but not in their differentiated derivatives . In that study, no further characterization of these eGFP /TH¨C cells was reported, but it seems possible that some of these cells could have been SSEA-1 . In our study, we further purified the population of GFP /SSEA-1¨C cells by FACS. This population had high neuronal purity and contained many cells with co-expression of eGFP and TH. The efficiency of FACS for these neuronal eGFP  cells (SSEA-1¨C) was, however, too low to enable a meaningful transplantation study for in vivo analysis.7 n8 `' E1 Z/ W) P
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In conclusion, our study illustrates the complexity of using a single marker for selection of a specific cell population in ES cell cultures, which are not synchronized in time or specific enough to generate only one cell type. Positive selection for eGFP combined with a negative selection of an immature marker could provide an enriched neuronal population for transplantation in our context. Similar combinatorial strategies might be necessary also when other cellular markers for selection of a mature specific cell type are used.7 h- U3 k5 f* R1 m) |5 p$ y
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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The authors indicate no potential conflicts of interest.

Acknowledgments2 e$ U0 h/ K6 j4 k9 m1 h

This work was supported by Udall Parkinson's Disease Center of Excellence grant P50 NS39793 and the Orchard, Anti-Aging, and Stern foundations. E.H. was supported by a fellowship from the Swedish Brain Foundation.. s" G( v4 C2 n! z
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