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作者：Junying Yua, Maxim A. Vodyanika, Ping Hea, Igor I. Slukvina,b,c, James A. Thomsona,b,d,e作者单位：a Wisconsin National Primate Research Center, University of Wisconsin, Madison, Wisconsin, USA;b WiCell Research Institute, Madison, Wisconsin, USA;c Departments of Pathology and Laboratory Medicine,
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【摘要】; |# B& S% O; N1 L
Here, we examine the ability of undifferentiated human embryonic stem cells (hESCs) to reprogram the nuclei of hESC-derived myeloid precursors following cell¨Ccell fusion. Using an OP9 coculture system, we produced CD45 CD33 myeloperoxidase myeloid precursors from an Oct4¨Cenhanced green fluorescent protein (EGFP) knock-in hESC line and demonstrated that Oct4-EGFP expression was extinguished in these precursors. Upon fusion with undifferentiated hESCs, EGFP expression from the endogenous Oct4 promoter/regulatory region was re-established, ESC-specific surface antigens and marker genes were expressed, and myeloid precursor-specific antigens were no longer detectable. When the hybrid cells were formed into embryoid bodies, upregulation of genes characteristic of the three germ layers and extraembryonic tissues occurred, indicating that the hybrid cells had the potential to differentiate into multiple lineages. Interestingly, the hybrid cells were capable of redifferentiating into myeloid precursors with efficiency comparable with that of diploid hESCs despite their neartetraploid chromosome complement. These results indicate that hESCs are capable of reprogramming nuclei from differentiated cells and that hESC hybrid cells provide a new model system for studying the mechanisms of nuclear reprogramming.
) V& M) d$ r; l; k+ J3 ~$ v. s9 h 【关键词】 Human embryonic stem cells Reprogramming Myeloid precursors Cell fusion Hybrid cells
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The cloning of Dolly dramatically changed the mindset of biologists because it clearly showed that the differentiated state of at least some mammalian cells is reversible . Far from offering proof against transdifferentiation, such examples merely highlight that cell fusion is one mechanism by which transdifferentiation occurs.
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Cell¨Ccell fusion as a model for studying nuclear reprogramming is far from new. In experiments in the 1970s, mouse embryonal carcinoma (EC) cells were fused with thymic cells or friend erythroleukemia cells, and the resulting hybrid cells exhibited developmental properties most similar to those of EC cell components .
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9 E2 v8 t$ V* w* [6 e4 a0 a7 yLike mouse ESCs, human ESCs (hESCs) are capable of indefinite self-renewal and appear to have the ability to differentiate into all cell types of the body . However, in spite of these recent improvements, oocyte availability remains a concern, and understanding the basic mechanisms of nuclear reprogramming may eventually eliminate the need for oocytes. More importantly, a fundamental understanding about the reversibility of distinct developmental states could well lead to novel regenerative therapies that act on endogenous cells in the absence of transplantation.
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$ E- A8 E) P% B' q0 RHere, we tested whether hESCs can reprogram differentiated somatic cells in the hematopoietic lineage, as a first step in identifying the factors that mediate reprogramming. We derived CD45 CD33 myeloperoxidase (MPO ) myeloid precursors from hESCs and fused them with undifferentiated hESCs. In the resulting hybrid cells, an Oct4-EGFP knock-in transgene was reactivated, antigens normally expressed in myeloid precursors were silenced, and ESC-specific genes were expressed. Moreover, the hybrid cells had the ability to differentiate to multiple lineages. These data indicate that transacting factors present in hESCs are sufficient to reprogram nuclei from hematopoietic cells.
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6 H+ Z2 L5 p' {( l7 A0 w1 lMATERIALS AND METHODS
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hESC Culture$ x" l! R# J6 [% C1 Z
! ~5 r& x. R+ g5 |% `, Y5 uhESC line H1 was maintained on irradiated mouse embryonic fibroblasts in Dulbecco¡¯s modified Eagle¡¯s medium/F12 culture medium supplemented with 20% Knockout serum replacer, 1% nonessential amino acids, 1 mM L-glutamine, 0.1 mM ß-mercaptoethanol, and 4 ng/ml basic fibroblast growth factor (bFGF) (all from Invitrogen Corporation, Carlsbad, CA, http://www.invitrogen.com), as previously described . This line was maintained under neomycin selection (Geneticin 100 µg/ml; Invitrogen Corporation). Human H1 hygromycin-resistant ESC line was established through transfection (Fugene 6; Roche Applied Science, Indianapolis, IN, http://www.roche-applied-science.co), with plasmid pPWL512, whose hygromycin cassette was driven by the PGK (phosphoglycerate kinase) promoter (constructed by Peter W. Laird). This cell line was maintained under hygromycin selection (50 µg/ml; Invitrogen Corporation).
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Differentiation of hESCs Toward Myeloid Precursors
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Myeloid precursors were obtained through coculture of human H1 Oct4 knock-in ESCs with mouse OP9 bone marrow stromal cells (Fig. 1A). The OP9 cells were maintained on the gelatin-coated 10-cm plastic dishes (BD Biosciences) in medium (10 ml/dish) consisting of ¨Cminimum essential medium (-MEM; Invitrogen Corporation) supplemented with 20% non¨Cheat-inactivated defined fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, http://www.hyclone.com). The OP9 cultures were split every 4 days at a ratio of 1:7. For hESC differentiation, after OP9 cells reach confluence on the fourth day, half of the medium was changed and the cells were cultured for additional 4 days.
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) y8 Q% r* @' b F" |2 I2 uFigure 1. Differentiation of human H1 Oct4 knock-in embryonic stem cells toward myeloid precursors. (A): Schematics of myeloid precursor derivation and purification from hESCs. (B): Phenotypic analysis of myeloid precursors after Percoll separation. Gray line: isotype control; black line: antibody staining. Abbreviations: hESC, human embryonic stem cell; MPO, myeloperoxidase; pHEMA, poly(2-hydroxyethyl methacrylate). u' A- n4 m& D% \- u4 [* V
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To differentiate hESCs toward myeloid precursors, the human H1 Oct4 knock-in ESCs (p76) were added to the OP9 monolayer (1.5 x 106 per 10-cm dish) in 20 ml of -MEM supplemented with 10% FBS (HyClone Laboratories) and 100 µM monothioglycerol (MTG; Sigma, St. Louis, http://www.sigmaaldrich.com). The hESC/OP9 cell coculture was incubated for 9 days with changes of half of the medium on days 4, 6, and 8. After incubation, the coculture was dispersed into individual cells by collagenase IV treatment (1 mg/ml in -MEM; Invitrogen Corporation) for 20 minutes at 37¡ãC, followed by trypsin treatment (0.05% Trypsin/0.5 mM EDTA; Invitrogen Corporation) for 15 minutes at 37¡ãC. Cells were washed twice with medium, and resuspended at 2 x 106 per ml in -MEM supplemented with 10% FBS, 100 µ M MTG, and 100 ng/ml GM-CSF (Leukine; Berlex Laboratories Inc., Montville, NJ, http://www.berlex.com). Cells were further cultured in the flasks coated with poly(2-hydroxyethyl methacrylate) (pHEMA; Sigma) for 10 days with changes of half of the medium every 3 days. During pHEMA adhesion-preventive culture, adherent cells formed floating aggregates, while myeloid precursors grew as individual cells in suspension. Large cell aggregates were removed by filtration through 100-µM cell strainers (BD Biosciences), while small aggregates and dead cells were removed by centrifugation through 25% Percoll (Sigma). The myeloid precursors recovered from the cell pellet were of greater than 95% purity (CD45 ).. b9 l6 v* k% @& u! m6 f
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Cell Fusion5 o! r+ o8 b C8 y9 K" T
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Human H1 hygromycin-resistant ESCs (p50) were dispersed into individual cells by trypsin treatment (0.05% Trypsin/0.5 mM EDTA) for 10 minutes at 37¡ãC. After washing once with Dulbecco¡¯s phosphate-buffered saline (DPBS; Invitrogen Corporation), H1 ESCs were resuspended in DPBS (1.3 x 107) and mixed with myeloid precursors (4.7 x 107). Fusion was carried out with 50% polyethylene glycol 1500 (PEG 1500; Roche) as described in the product protocol. Selection for hybrid cells (hygromycin 50 µg/ml, Geneticin 100 µg/ml) was applied 48 hours after PEG treatments. After 2¨C3 weeks of drug selection, individual colonies were selected and expanded for further analysis.8 l. @4 [ F; V* v( k2 n
) w5 A; e# F+ t$ fFlow Cytometry
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Adherent cells were individualized by trypsin treatment (0.05% Trypsin/0.5 mM EDTA) and fixed in 2% paraformaldehyde for 20 minutes at room temperature (r.t.). The cells were filtered through a 40-µm mesh and resuspended in fluorescence-activated cell sorter (FACS) buffer (phosphate-buffered saline containing 2% FBS and 0.1% sodium azide). Cells grown in suspension were stained in the FACS buffer supplemented with 1 mM EDTA and 1% normal mouse serum (Sigma). Intracellular MPO staining was performed using Fix&Perm reagents (Caltag Laboratories, Burlingame, CA, http://www.caltag.com). Approximately 100 µl of cell suspension containing 5 x 105 cells was used in each labeling. Both primary and secondary antibody incubation (where applied) were carried out at r.t. for 30 minutes. Control samples were stained with isotype-matched control antibodies. After washing, the cells were resuspended in 300¨C500 µl of the FACS buffer and analyzed on a FACSCalibur flow cytometer (BD Immunocytometry Systems, San Jose, CA, http://www.bd.com) using the Cellquest acquisition and analysis software (BD Immunocytometry Systems). In total, 20,000 events were acquired. All the antibodies used are listed in Table 1. The final data and graphs were analyzed and prepared in FlowJo software (Tree Star, Inc., Ashland, OR, http://www.treestar.com).
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Table 1. Antibodies used in the flow cytometry analysis
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! Q5 Y% r+ E1 t; _0 }' T/ M {Chromosome Counting
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! b( [, h p" J) iWhen hESCs and hybrid cells reached 70%¨C80% confluence in one well of a 6-well plate, 1 ml colcemid solution (10 µg/ml in Hanks¡¯ balanced saline solution; Invitrogen Corporation) was added to each well, and the cells were further incubated for 4 to 6 hours at 37¡ãC. The cells were individualized by trypsin treatment and collected by centrifugation. Cells grown in suspension were directly collected by centrifugation after colcemid treatment. The colcemid-treated cells were then incubated in the prewarmed hypotonic solution (0.075 M KCl; Sigma) at 37¡ãC for 10 minutes, followed by fixation (three times in 3:1 methanol:glacial acetic acid, freshly prepared; Fisher Scientific International Inc., Pittsburgh, PA, http://www.fishersci.com). To prepare the metaphase-spread slides, the cell density for each sample was adjusted to nearly 1 x 106 cells per ml in fixative. The cells were then placed (2 or 3 drops per slide, using a 1-ml pipette tip) near the frosted end of slides (precleaned Superfrost/plus Microscopic slides, 25 x 75 x 1.0 mm, catalog no. 12-550-15; Fisher), which were tilted to facilitate cell spreading. The slides were left to dry at r.t. overnight. After making slides, 2 ml of fixative was added to each sample prior to short-term storage at 4¡ãC or long-term storage at ¨C20¡ãC./ o5 N5 X4 x8 ]: }2 w/ l, J- S% J
/ P2 f: ^% u% a8 L5 k& c# mSlides were stained with Hoechst33342 (5 µg/ml in DPBS; Molecular Probes, Inc., Eugene, OR, http://probes.invitrogen.com) for 30 minutes at r.t. in the dark. After washing in DPBS three times (5 minutes each), they were mounted in the Vector-shield solution (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and imaged with a fluorescence microscope (40x; Leica Microsystems Inc., Bannockburn, IL, http://www.leica-microsystem.com).1 Q8 \/ x# b( q: X4 Q
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Reverse Transcription¨CPolymerase Chain Reaction8 h- T7 B, V" M. L
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Differentiation of hESCs and hybrid cells through embryoid body (EB) formation was carried out as described previously ). The PCR reactions were performed using PCR MasterMix (Promega Corporation, Madison, WI, http://www.promega.com) under the following conditions: 94¡ãC for 5 minutes; various number of cycles of 94¡ãC for 30 seconds, 55¡ãC for 30 seconds, and 72¡ãC for 45 seconds; and 72¡ãC for 5 minutes. All the primers and their related information are shown in Table 2. The amount of template used for each sample was normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers. Briefly, PCR reactions were carried out using GAPDH primers with dilution series of the cDNA for each sample as templates. The dilution factor of the cDNA for each sample was selected so that its GAPDH PCR product would fall in the log region of its increase as a product of the amount of the input template and have levels similar to those of other samples.- }9 r0 p1 \; O. M7 O: f! c( e
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Table 2. Primer sets for reverse transcription¨Cpolymerase chain reaction
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+ D5 w% Z' I7 J; _) sDerivation of Myeloid Precursors from hESCs/ i$ @% x5 s4 A7 |! R$ _
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The H1 Oct4 knock-in ESC line was established using homologous recombination as previously described . In this cell line, both EGFP and neomycin phosphotransferase are expressed from the endogenous Oct4 promoter/regulatory region using dual internal ribosome-entry sites. Thus, the expression of EGFP and neomycin phosphotransferase indicates an active endogenous Oct4 promoter/regulatory region.
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9 _, s' k8 d9 K: X, N* OSuspension culture of cells recovered from day 9 hESC/OP9 coculture plated) (Fig. 1A). As shown in Table 3 and Figure 1B, the cells recovered from the GM-CSF suspension culture expressed panhematopoietic markers CD45 and CD43 and were positive for CD33 and MPO, early markers for myeloid lineage. The majority of these cells were also positive for more mature myeloid markers CD11b and CD11c (Fig. 1B), but negative for mature granulocyte-specific marker CD66b or lymphocyte-specific marker terminal deoxynucleotidyl transferase (data not shown). Most of the CD45¨C cells present in this culture system (
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Table 3. Phenotype of myeloid precursors derived from human H1 Oct4 knock-in embryonic stem cells (ESCs) and ESxCD45 hybrid cells
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Figure 2. EGFP and cell-specific antigen expression in ESxCD45 hybrid cells. (A): BF images of ESxCD45 hybrid cells in ES cell culture medium and PBS (magnification, 20x). EGFP image was taken in PBS to reduce the background fluorescence. (B): Flow cytometry analysis of EGFP expression in H1 Oct4 knock-in ES cell¨Cderived myeloid precursors and ESxCD45 hybrid cells. (C): Expression of human ES cell¨Cspecific antigens SSEA-4 and Tra-1-60 in ES cells and ESxCD45 hybrid cells. (D): Expression of myeloid precursor-specific antigens CD45, CD43, and CD33 in ESxCD45 hybrid cells. Gray line: isotype control; black line: antibody staining. Abbreviations: BF, bright field; EGFP, enhanced green fluorescent protein; ES, embryonic stem; PBS, phosphate-buffered saline.
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( `9 y) Z' g$ J9 z! ~Reprogramming of Myeloid Precursors Through Fusion with hESCs
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H1 ESCs were fused with the myeloid precursors derived from H1 Oct4 knock-in ESCs. Two or three weeks after double drug selection for hybrid cells (ESxCD45), clones with morphology similar to that of the hESCs appeared (efficiency: ~1:1.5 x 105 ESCs, comparable with that of ESxES hybrid cells when individualized hESCs were fused in suspension with PEG ) (Fig. 2A). Importantly, no colonies formed after neomycin selection alone when the myeloid precursors derived from H1 Oct4 knock-in ESCs were plated together with wild-type human H1 ESCs in the absence of PEG treatment, demonstrating that there were no residual ESCs contaminating the myeloid population. Control experiments, in which the myeloid precursors were plated directly on either mouse fibroblast feeder layer or Matrigel without drug selection, also yielded no ESC-like colonies or green cells (data not shown). These data suggested that the hybrid cells did not arise from fusion between ESCs. The following evidence also argued against the involvement of cells of nonmyeloid lineages. First, when most of the CD45¨C cells were depleted with antibody against CD235 (see above), the CD45-enriched cell population (>99%) showed similar reprogramming efficiency upon fusion with ESCs (data not shown). Second, purified myeloid precursors with antibody against CD15 (>98%) also showed similar reprogramming efficiency upon fusion with ESCs (data not shown). Third, we failed to obtain hybrid cells between hESCs and endothelial cells/erythroblasts derived from H1 Oct4 knock-in ESCs.6 s7 m. l8 ?* p" ^
( b3 s2 x/ \" f7 _8 OSix clones were randomly selected for the following experiments. However, because these clones showed similar phenotypes, only the data from one clone were shown here. The hybrid cells were near tetraploid, with an average chromosome number of 85.8 (n = 55, SD = 0.60). Chromosome loss during clonal expansion has frequently been reported in previous cell¨Ccell fusion experiments . All the clones expressed EGFP (Figs. 2A, 2B), indicating that the silenced Oct4 promoter derived from the myeloid precursors was reactivated in these ESxCD45 hybrid cells. Similar to diploid hESCs, the ESxCD45 hybrid cells showed expression of ESC-specific antigens SSEA-4 and Tra-1-60 (Fig. 2C). Moreover, the hybrid cells also expressed ESC-specific genes, including Oct4, Nanog, and TERT, at levels comparable with diploid ESCs normalized to GAPDH expression (lanes 1 and 3 in Figure 3). No or very low level expression of antigens specific to the myeloid precursors (Fig. 2D) and other lineage-specific marker genes (with the exception of Vimentin; lane 1, 3 in Figure 3) was detected in the ESxCD45 hybrid cells. The expression level of Vimentin varied between different clones of hybrid cells, the significance of which is not known, as diploid hESCs and ESxCD45 hybrid cells showed similar plating efficiency during passaging (data not shown).! W' s; r5 T( b- b3 B' Q2 n& o: J
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Figure 3. Gene expression analysis by reverse transcription¨Cpolymerase chain reaction. Lane 1: human H1 hygromycin-resistant ES cells (p50) prior to differentiation; lane 2: 10-day-old EBs from human H1 hygromycin-resistant ES cells; lane 3: ESxCD45 hybrid cells (p23) prior to differentiation; lane 4: 10-day-old EBs from ESxCD45 hybrid cells (p23). Abbreviations: EB, embryoid body; ES, embryonic stem; hESC, human embryonic stem cell.9 Q1 V0 Q; L* p! U/ r$ o) ~" Y
! v1 C' t+ M& `6 v$ l ]- J/ IPluripotency of the ESxCD45 Hybrid Cells
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We formed EBs to test the developmental potential of ESxCD45 hybrid cells. After 10 days of differentiation, ESC-specific genes such as Nanog, Oct4, and TERT were downregulated, and marker genes characteristic of the three germ layers (Pax6 and Vimentin for ESxCD45 hybrid cells prior to differentiation). The myeloid precursors derived from the ES-xCD45 hybrid cells showed similar antigen expression patterns as those derived from H1 Oct4 knock-in ESCs (Fig. 4B and Table 3). The lower percentage of myeloid precursors derived from ESxCD45 hybrid cells expressing CD11b and CD11c suggested that the differentiation of ESxCD45 hybrid cells was slightly delayed compared with the diploid hESCs.( v% q' w, m2 e X+ G4 l+ {, X
1 V( ~5 A1 i2 T8 zFigure 4. Differentiation of ESxCD45 hybrid cells to myeloid precursors. (A): Bright-field images of myeloid precursors derived from H1 ES cells and ESxCD45 hybrid cells. (B): Flow cytometry analysis of antigen expression in ESxCD45 hybrid cell¨Cderived myeloid precursors. Gray line: isotype control; black line: antibody staining. Abbreviations: ES, embryonic stem; MPO, myeloperoxidase.3 D& d5 \1 a& V0 l1 p/ M7 J
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DISCUSSION
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" |1 ~. `( R- c2 B9 f* L$ cIn this report, we examined the ability of hESCs to reprogram differentiated somatic cells through cell¨Ccell fusion. We chose to focus on cells in the hematopoietic lineage for several reasons. First, in previous mouse EC cell, ESC, and EG cell experiments, cells in the hematopoietic lineage were reprogrammed in hybrid cells. Second, a well-characterized set of antibodies for hematopoietic cells is available to characterize starting populations. Third, we have previously developed culture conditions that allow efficient production of hematopoietic cells from ESCs , which will enable us to track the epigenetic status of tagged genes either during differentiation or during reprogramming. And finally, because hematopoietic cells are easily obtained from the adult, a fundamental understanding of reprogramming cells in this lineage would have direct clinical implications.7 d6 z0 m- F3 P3 Z( d( c$ q! p
! X' t: v8 m. lThe in vitro OP9 differentiation system described here allowed us to obtain large quantities of myeloid precursors, which were capable of differentiating into granulocytes, macrophages, and dendritic cells .
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4 X" ]) |! `9 PUnlike some other vertebrates, mammals have a relatively limited ability to regenerate many damaged tissues, and understanding the reasons for this species-specific difference could have profound implications for the treatment of degenerative diseases. Because differentiation is dictated by a series of epigenetic rather than genetic events (i.e., somatic cells and germ cells in mammals generally contain the same genetic material), the differentiated states are, in principle, reversible. The oocyte cytoplasm can reprogram the nuclei of several types of differentiated cells to an undifferentiated state, although it appears to be more difficult to reprogram more differentiated cells ./ T% ]' t* \6 ?' m$ ~$ W
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There nonetheless could be significant species-specific differences between reprogramming human cells and cells from other model organisms. The differences between mouse ESCs and hESCs illustrate how unexpectedly significant species-specific differences can be, even within mammals. For example, the factors that support the self-renewal of hESCs and mouse ESCs appear to be distinct. The LIF/STAT3 (leukemia inhibitory factor/signal transducer and activator of transcription 3) pathway, which is key to the proliferation of mouse ESCs, does not support hESCs and appears to be inactive in conditions that do support hESCs . Thus, if the goal of reprogramming is to convert a differentiated human cell to an ESC, the outcome could well depend on how well these species-specific differences are understood, and hESC¨Csomatic cell hybrid cells provide a new model for exploring those differences.
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ACKNOWLEDGMENTS
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The authors thank Dr. Tenneille Ludwig, Jenny Frane, and Marian Piekarczyk for providing the H1 Oct4 knock-in cell line; and Dr. Takashi Kameda and Deborah J. Faupel for critical reading of this manuscript.: Q6 x. V, J8 t# n w3 U
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This research was supported in part by NIH grant P20 GM069981, DARPA grant DRP5-UWM, and P51 RR000167 to the Wisconsin National Primate Research Center, University of Wisconsin-Madison.
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DISCLOSURES
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J.T. owns stock in and has served on the Board of Cellular Dynamics International within the last 2 years.+ [9 j: c7 C6 p1 \2 C
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