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Differentiation of embryonic stem cells in adult bone marrow
JOURNAL OF
GENETICS AND GENOMICS J. Genet. Genomics 37 (2010) 431−439 www.jgenetgenomics.org
Differentiation of embryonic stem cells in adult bone marrow Yueying Li a, b, d, Jing He b, Fengchao Wang b, Zhenyu Ju c, d, Sheng Liu b, Yu Zhang b, Zhaohui Kou b, Yanfeng Liu a, d, Tao Cheng a, d, *, Shaorong Gao b, d, * a
State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China b
c
National Institute of Biological Sciences, Beijing 102206, China
Max-Planck-Partner Group on Stem Cell Aging, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing 100021, China d
Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China. Received for publication 6 May 2010; revised 30 May 2010; accepted 1 June 2010
Abstract Embryonic stem cells (ESCs) are a potential source of generating transplantable hematopoietic stem and progenitor cells, which in turn can serve as “seed” cells for hematopoietic regeneration. In this study, we aimed to gauge the ability of mouse ESCs directly differentiating into hematopoietic cells in adult bone marrow (BM). To this end, we first derived a new mouse ESC line that constitutively expressed the green fluorescent protein (GFP) and then injected the ESCs into syngeneic BM via intra-tibia. The progeny of the transplanted ESCs were then analyzed at different time points after transplantation. Notably, however, most injected ESCs differentiated into non-hematopoietic cells in the BM whereas only a minority of the cells acquired hematopoietic cell surface markers. This study provides a strategy for evaluating the differentiation potential of ESCs in the BM micro-environment, thereby having important implications for the physiological maintenance and potential therapeutic applications of ESCs. Keywords: embryonic stem cells; differentiation; bone marrow; transplantation
Introduction It is generally accepted that two major cell lineages could be distinguished in the adult bone marrow (BM) which include the hematopoietic cell lineage and non-hematopoietic stroma cells. The stroma cells provide distinct hematopoietic microenvironment that regulates * Corresponding author. Tel: +86-10-8072 8967, Fax: +86-10-8072 7535 (S. Gao); Tel & Fax: +86-22-2390 9156 (T. Cheng). E-mail address: [email protected] (S. Gao); [email protected] (T. Cheng) DOI: 10.1016/S1673-8527(09)60062-X
hematopoietic stem cells (HSCs) maintenance and facilitates hematopoiesis. HSCs are the only stem cells found in BM that could differentiate into hematopoietic cells. Therefore, bone marrow transplantation has been widely used to cure blood diseases including leukemia. However, very rare number of HSCs could be retrieved and moreover, histocompatibility of the cells is the other major limitation for HSCs transplantation. Subsequently, embryonic stem cells (ESCs) or induced pluripotent stem (iPS) cells could overcome most of the limitations and be potentially used to generate tissues of value for regenerative medicine (Paris and Stout, 2010). Since BM is a well-studied
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microenvironment, it provides an attractive engraftment site for cell transplantation, which is not limited to HSCs transplantation. Pancreatic islet transplantation has ever been performed in BM because of its protected and extravascular (but well-vascularized) microenvironment (Cantarelli et al., 2009). ESCs, derived from the inner cell mass of preimplantation blastocyst, possess two important characteristics: self-renewal and differentiation into cells belonging to all three germ layers, and this plasticity is unlimited. These important characteristics make ESCs a great candidate in regenerative medicine and future treatments for a wide variety of diseases, including cardiovascular (Cao et al., 2006), neurodegenerative (Keirstead et al., 2005), and endocrine (Fujikawa et al., 2005) disorders, etc. However, some obstacles, including ethical issues, immunorejection and tumorigenesis, need to be overcome before the potential benefits of ESCs can be translated to a clinical application. Traditionally, ESCs, used to treat a myriad of hematopoietic malignancies and disorders by transplantation, need to be pre-differentiated into HSCs by embryoid body (EB) and coculture and expansion on OP9 stromal cells in the presence of hematopoietic cytokines. The ESC-derived HSCs can reconstitute and rescue the lethally irradiated mice (McKinney-Freeman and Daley, 2007). Since ESCs demonstrated greater pluripotency than HSCs or MSCs, they can be used as a potential source of generating transplantable HSCs or MSCs in vitro. However, it remains undetermined if ESCs can survive in the BM and if so, how ESCs differentiate in the BM microenvironment. A more recent study has clearly shown that direct transplanting human ESCs into the hippocampus of athymic nude rats can rescue the cognitive impairment caused by irradiation (Acharya et al., 2009). In contrast to the previous studies that have pre-differentiated ESCs into HSCs before transplantation, we chose to transplant ESCs directly into the BM to view how the ESCs survive and differentiate in the BM. To fulfill this goal, we derived a new ESC line which carried the chicken beta-actin-GFP transgene. The transplanted cells were visualized at different time points post-transplantation. Moreover, we further characterized the properties of the progenies of transplanted ESCs after a long time surviving in the BM.
Materials and methods This study was performed in accordance with the guidelines of the Animal Care and Use Committee of the National Institute of Biological Sciences and with the Guide for the Care and Use of Laboratory Animals.
ESC derivation and culture aGFP+ ESCs were derived from available inbred mouse strains: 129/Sv × CAG/EGFP transgenic C57BL/6-Tg mice, in which green fluorescent protein (GFP) expression was under the control of a chicken beta-actin promoter and cytomegalovirus enhancer. Blastocyst-stage mouse embryos (3.5-day-old) were collected and plated individually on a 96-well dish with mitomycin C-arrested mouse embryonic fibroblast monolayer (feeder) in ESC Derivation Medium: Knockout Dulbecco’s modified Eagle’s medium (DMEM) with 15% Knockout serum replacement, 1,000 U/mL mouse leukemia inhibitory factor (LIF), 50 μg/mL Pen/Strep, 10−4 mol/L nonessential amino acids, 2 mmol/L L-glutamine, and 10−4 mol/L 2-mercaptoethanol. After 5−6 days in culture, the inner cell mass outgrowth was removed into another 96-well dish with feeder. Two days later, the inner cell mass outgrowth was trypsinized with 0.05% trypsin/EDTA (Invitrogen, USA) on a 96-well dish with feeder. The ESCs were moved every 2 days onto larger dishes with freshly prepared feeder layers, and were fed every day with new ESC Culture Medium: DMEM with 15% fetal bovine serum, 1000 U/mL mouse LIF, 50 μg/mL Pen/Strep, 10−4 mol/L Nonessential amino acids, 2 mmol/L L-glutamine, and 10−4 mol/L 2-mercaptoethanol. Culture dishes were kept at 37oC in a humidified atmosphere with 5% CO2.
Reverse transcription polymerase chain reaction (RT-PCR) analysis Total RNA was extracted from the ESCs using Trizol reagent (Invitrogen) following the manufacturer’s instructions. The RNA was then converted into cDNA using oligo-dT and the M-MLV Reverse Transcriptase Kit (Promega, USA). Polymerase chain reactions (PCR) were carried out for 30 cycles (94°C, 30 s; 60°C, 30 s; 72°C, 30 s). The following primer sequences were used: Oct3/4 F: 5′-GAAGCAGAAGAGGATCACCTTG-3′, R: 5′-TTCTTAAGGCTGAGCTGCAAG-3′;
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Nanog F: 5′-CCTCAGCCTCCAGCAGATGC-3′, R: 5′-CCGCTTGCACTTCACCCTTTG-3′; Sox2 F: 5′-GCGGAGTGGAAACTTTTGTCC-3′, R: 5′-CGGGAAGCGTGTACTTATCCTT-3′; Klf4 F: 5′-TGATGGTGCTTGGTGAGTTG-3′, R: 5′- TTGCACATCTGAAACCACAG-3′; c-myc F: 5′-TCTCCATCCTATGTTGCGGTC-3′, R: 5′-TCCAAGTAACTCGGTCATCATCT-3′; GAPDH F: 5′-CGGAGTCAACGGATTTGGTCGTAT-3′, R: 5′- AGCCTTCTCCATGGTGGTGAAGAC-3′.
Alkaline phosphatase staining and immunocytochemical analysis The cells were fixed with 4% paraformaldehyde for 2 min at room temperature. Alkaline phosphatase activity was detected using a kit from Chemicon (USA) and following protocols provided by the manufacturer. For immunocytochemical staining, colonies were fixed for 2 h at room temperature with 4% paraformaldehyde and then incubated at room temperature for 15 min with 1% Triton X-100/phosphate-buffered saline (PBS). Cells were washed three times in PBS and blocked at 37oC for over 3 h with 4% normal goat serum (Chemicon). Subsequently, cells were incubated at 4°C overnight with primary antibody to Oct4 (1:500, Santa Cruz Biotechnology, USA), SSEA-1 (1:500, Chemicon), Nanog (1:500, Cosmobio, USA), or Sox2 (1:500, Abcam, UK). Cells were washed three times in PBS and incubated at 37oC for 2 h with goat anti-rabbit Alexa-Flour 594-conjugated and goat anti-mouse Alexa-Fluor IgG or IgM 633-conjugated (Invitrogen) secondary antibodies (1:500 in 1% normal goat serum in PBS). Unbound secondary antibodies were removed in three washes with PBS. Nuclei were identified by DAPI (Invitrogen) staining at a dilution of 1:10,000 at room temperature for 5 min. Images were acquired using a confocal laser scanning microscope, LSM 510 META (Carl Zeiss, Germany).
In vitro and in vivo differentiation In vitro differentiation was performed using three methods: the formation of embryoid body (EB), the formation of teratomas and the generation of chimeras. In the EB formation method, ESCs were dissociated into single cells and plated at 2 × 105 cells/mL in suspension culture in the
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absence of LIF using Iscove’s modified Eagle’s medium supplemented with 15% fetal bovine serum, 2 mmol/L L-glutamine, and 0.1 mmol/L nonessential amino acids on a Ultra Low Attachment 6-well plate. In the teratoma method, 2 × 106 cells in 200 µL PBS were injected under the inguinal skin of Severe Combined Immunodeficiency (SCID) mice. After 2 weeks, the teratomas were excised, fixed in 10% paraformaldehyde, and subjected to histological examination with hematoxylin and eosin staining. In the chimera method, chimeric mice were obtained through the microinjection of ESCs into 3.5-day-old blastocysts isolated from CD1 mice and reimplantation of the microinjected blastocysts into the uterine horns of 2.5-day pseudopregnant CD1 foster mothers. The chimeric offspring were identified by coat color. Germline transmission of the ESC genome was then tested by crossing high-percentage chimeras with CD1 mice to establish the ESC-line-derived coat color in F1 offspring.
Karyotype analysis Two days after ESCs were seeded, they were exposed to 0.25 μg/mL colcemid for 3.5 h, digested, collected, and exposed to hypotonic solution for 6 min. Cells were dropped onto a cold glass slide after being fixed with methanol/acetic acid (3:1) twice for 1 h in total. The karyotype was determined by microscopic examination after conventional Giemsa staining and G-banding analysis. More than twenty chromosomal spreads were counted per population.
ESCs transplantation and recovery of GFP+ cells from bone marrow Recipient C57BL/6 female mice at 4−6 weeks of age were treated with sublethal 700 cGy via Co60 irradiator one day prior to transplantation. ESCs were digested and adhered to the untreated tissue culture dish for 30 min to deplete feeder cells. Then the ESCs were passed through a 70 μm nylon mesh to remove clumps and kept on ice before transplantation. The mice were anesthetized with Avertin and the right tibia was gently drilled with a 26-gauge needle into the bone marrow cavity. Intra-bone-marrow injection was carried out using a microsyringe. Cells (1 × 106) in 50 μL total volume were transplanted into the right tibia of each recipient mouse. At various time points post-transplantation of aGFP+ ESCs, bone marrow cells were flushed from the tibias and femurs of
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the transplanted mice. Single-cell suspension from the bone marrow of transplanted mice was cultured on 35 mm or 60 mm dishes in ESC culture medium. Then we kept observing the GFP+ cell under fluorescent microscope every day.
Flow cytometric analysis At various time points post-transplantation of aGFP+ ESCs, 100 µL of peripheral blood was withdrawn from each recipient. Bone marrow cells were flushed from the tibias of the recipients. Cell suspension was treated with red cell lysis buffer and passed through a 70 μm nylon mesh before flow cytometric analysis. Bone marrow mononuclear cells were stained with PerCP-Cy5.5conjugated CD45.2 antibody (eBioscience, USA) and APC-Cy7 conjugated lineage antibodies (a mixture of anti-CD3, CD4, CD8, B220, Gr-1, Mac-1, and Ter-119 antibodies; eBioscience).
Colony-forming unit-fibroblast (CFU-F) assay and hematopoietic differentiation Isolation of cells from the grafted bones of recipients transplanted with aGFP+ ESCs was performed by either marrow flushing or bone digesting. Cells were cultured with MesenCult® Medium (STEMCELL Technologies, Canada). The CFU-F assay was carried out following manufacturer’s instructions. For in vitro hematopoietic differentiation assay, after red cell lysis and passing through a 70 μm nylon mesh, bone marrow cells were cultured with hematopoietic differentiation medium: Iscove’s Modified Dulbecco Medium with 1% methylcellulose, 10% plasma-derived serum, 5% protein-free hybridoma medium, 100 ng/mL Stem Cell Factor, 5 ng/mL mouse thrombopoietin, 2 U/mL human erythropoietin, 25 ng/mL mouse IL-11, 30 ng/mL IL-3, 30 ng/mL mouse GM-CSF, 30 ng/mL mouse G-CSF, and 5 ng/mL mouse IL-6 (Shen and Qu, 2008). After being cultured for 10 days at 37oC in a humidified atmosphere with 5% CO2, colonies were counted using a reverse fluorescence microscope.
Results Establishment of a new embryonic stem cell line used in our study To track the injected ESCs after transplantation, we de-
rived a new kind of mouse ESC line aGFP+ ESCs, and the GFP expression of the progeny was independent of their differentiation status. The ESC line exhibited green colonies typical of undifferentiated ESCs (Fig. 1A). aGFP+ ESCs expressed undifferentiated cell-specific genes such as Oct4, Nanog, Sox2 and other genes such as Klf4 and c-myc, similar to normal R1 ESCs (Fig. 1B). This ESC line displayed high levels of alkaline phosphatase activity and was positive for Oct4, Nanog, Sox2 and SSEA-1 (Fig. 1, C and D). aGFP+ ESCs also formed normal embryoid bodies (Fig. 1E). Teratomas were harvested 2 weeks after the subcutaneous injection of aGFP+ ESCs into SCID mice. Histological examination showed that the teratomas contained various tissues of the three germ layers (Fig. 1F). This ESC line resulted in chimeric offspring and most of these chimeras transmitted the ESC genome to their offspring (Fig. 1G). Karyotyping of aGFP+ ESCs showed that at least 95% of the cells were diploid (Fig. 1H). Therefore, this newly-derived ESC line was qualified to serve the purposes of our current study.
In-tibia injecting of aGFP+ ESCs into bone marrow To evaluate the differentiation ability of ESCs in the adult bone marrow, we transplanted the ESCs directly into bone marrow cavities of the mice to minimize the peripheral loss by intravenous administration. We transplanted 1 × 106 aGFP+ ESCs into the right tibia of each sublethally irradiated recipient mouse. Most mice that received the ESCs could survive normally for a long term with the maximum of nearly two years. Although 26 mice (25% in the hosts) have developed tumors in a total of 103 sublethally irradiated recipients, we intended to focus on the transplanted animals in which no tumor was formed. At various time points post-transplantation, we recovered the engrafted GFP+ cells from the bone marrow mononuclear cells of recipients. We cultured the cells in ESC culture medium the same as the status before transplantation. We observed that GFP+ cells were spread and proliferated like mesenchymal cells and astrocyte-, endothelial-, fibroblast-like cells 2−3 days later (Fig. 2). The results suggested that after transplantation of ESCs into bone marrow, ESCs have a tendency to differentiate into non-hematopoietic cells.
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Fig. 1. Characterization of aGFP+ embryonic stem cells. The aGFP+ ESCs, whose GFP expression was under the control of a chicken beta-actin promoter, were derived from heterozygous transgenic mouse C57BL/6-Tg (CAG/EGFP) × 129/Sv. A: a phase-contrast image of aGFP+ ESCs. Scale bar stands for 50 μm. B: RT-PCR of pluripotent markers. R1 ESCs, mouse embryonic fibroblast (MEF) and template-free PCR systems (Tm-) were used as controls. C: alkaline phosphatase staining. Scale bar stands for 50 μm. D: fluorescent immunostaining of pluripotent markers. Scale bar stands for 50 μm. E: embryonic body formation. Scale bar stands for 200 μm. F: teratoma formation. Teratomas were harvested 4 weeks after subcutaneous injection of aGFP+ ESCs into SCID mice. Ectodermal epidermis (a), mesodermal immature cartilage (b), and endodermal intestinal tract epithelium (c) were detected by hematoxylin and eosin staining. G: chimeric mouse and the F1 offspring. H: karyotype (40, XY).
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Fig. 2. Observation of recovered GFP+ cells after transplantation. At various time points post-transplantation of aGFP+ ESCs, we recovered the engrafted GFP+ cells from bone marrow and observed the cells everyday with fluorescent microscopy. GFP+ cells were proliferated as A: astrocyte-like cells; B: mesenchymal cells; C: endothelial- like cells; D: fibroblast-like cells. Scale bar stands for 50 μm.
Flow cytometric analysis of progeny of engrafted ESCs To analyze the differentiation potential of ESCs after transplantation in bone marrow, we examined the engrafted cells by flow cytometric analysis of the bone marrow and peripheral blood of the recipients. Flow cytometric analysis and fluorescent microscopy analysis showed that GFP+ cells were detectable in the peripheral blood of 62.5% of the recipients, though the proportion of green cells was very low (~0.01%). In the bone marrow of grafted bones, only 3% of GFP+ cells could be found in the subsets of hematopoietic cell populations (CD45+, lineage+) (Fig. 3). However, using fluorescent microscopy, we found that many GFP+ cells still adhere to the bone after repeated flushing of the bone marrow and scraping the bone, suggesting that only a small amount of the GFP+ cells could be acquired for flow cytometric analysis.
Differentiation of ESCs in bone marrow after transplantation In order to collect GFP+ cells as much as possible and
analyze the property of engrafted GFP+ cells after a long time survival in bone marrow, we isolated cells by both marrow flushing and bone digesting and directly cultured the cells with MesenCult® Medium or hematopoietic differentiation medium. The CFU-F assay showed that many green colonies formed in the MesenCult® Medium (Fig. 4). In contrast, no green colonies formed in the hematopoietic differentiation medium, further confirming the limited differentiation potential of engrafted aGFP+ ESCs toward hematopoietic lineage.
Discussion In this study, we derived a new ESC line, aGFP+ ESCs that express the GFP regardless its differentiation status. The cell line, expressing pluripotent ESC markers and demonstrating germline transmission ability, are pluripotent and are able to differentiate normally after transplantation. We further investigated how ESCs differentiate by directly injecting the ESCs into BM. This model established a system through which the progenies of ESCs could be further investigated. Through observation under
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Fig. 3. Flow cytometric analysis of progeny of transplanted aGFP+ ESCs. At various time points post-transplantation of aGFP+ ESCs, bone marrow cells were flushed from the tibias of the sublethally irradiated recipients and control (untransplanted) mice. Bone marrow mononuclear cells were stained with CD45 and a combination of lineage antibodies (Lin).
Fig. 4. Colony-forming unit-fibroblast assay of progeny of transplanted aGFP+ ESCs. Isolation of cells from marrow plug and bone fragments by marrow flushing, bone crushing and digesting. Cells were cultured by MesenCult® Medium for over 10 days at 37°C in 5% CO2. Green colonies were formed. A, C: colonies from bright field images. B, D: green colonies from fluorescent images. Scale bar stands for 50 μm.
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fluorescent microscopy and analysis by flow cytometric analysis and in vitro culture, we concluded that injected ESCs in BM tend to differentiated into non-hematopoietic cells whereas very few cells acquired hematopoietic cell surface markers. Adult stem cells offer tremendous potential for accelerating and hold many promises for future clinical applications and regenerative medicine. HSCs are the best-characterized somatic stem cells so far, but in vitro expansion has been unsuccessful, limiting the future therapeutic potential of these cells (Wilson and Trumpp, 2006). ESCs may have greater protective power and pluripotency than adult stem cells. ESC paracrine protective mechanisms in surgical ischemia are superior to those of adult stem cells (Crisostomo et al., 2008). ESCs serve as a source for generation of unlimited quantities of cardiomyocytes for myocardial repair, and the initial success to repair and improve myocardial function in experimental models of heart disease has been quite promising (Zhang and Pasumarthi, 2008). Moreover, a recent study has clearly shown that direct transplanting human ESCs into the hippocampus of athymic nude rats can rescue the cognitive impairment caused by irradiation (Acharya et al., 2009). Therefore, ESCs are of great value as a potential and remarkable source of transplantable stem cells. The adult BM are composed of two main and distinct lineages: the hematopoietic tissue and a group of mesenchymal-derived stroma cells (Anjos-Afonso and Bonnet, 2007). Cells that constitute the bone marrow stroma are fibroblasts, macrophages, adipocytes, osteoblasts, osteoclasts, and endothelial cells. Interestingly, BM has been identified as an organ containing HSCs as well as non-hematopoietic stem cells, such as mesenchmal stem cells (MSCs) (Friedenstein et al., 1970), multipotent adult progenitor cells (Jiang et al., 2002), marrow-isolated adult multilineage inducible cells (D’Ippolito et al., 2004), and very small embryonic-like stem cells (Kucia et al., 2006). These non-hematopoietic stem cells have shown the features similar with ESCs, which suggested that BM could be a potential site for ESC residents and differentiation. However, to our surprise, most injected ESCs in BM differentiated into non-hematopoietic cells whereas few cells acquired hematopoietic cell surface markers, which will prompt us to further investigate the relationship between ESCs and BM microenvironment. Although these results are encouraging, we should also be cautious about the transplantation of ESCs or
ESC-derivatives. In our study, teratomas could be detected in 25% of the recipients. On the other hand, although ESCs represent one of the most promising cell types for tissue transplantation and regeneration and cellular therapy, tumorigenesis has to be solved before they can be used for clinical applications. In conclusion, our present study demonstrates that most injected ESCs in BM differentiated into non-hematopoietic cells whereas a minority acquired hematopoietic cell surface markers. Therefore, our study has important implications for the understanding of embryonic stem cell development and physiological maintenance in bone marrow, as well as for the safety evaluation of therapeutic use of ESCs and ESC-derivatives in BM.
Acknowledgements The work was supported by the grants from Tianjin Government (Nos. 07JCZDJC10600, 08ZCKFSF03200 and 09ZCZDSF03800) and the Ministry of Science and Technology of China (Nos. 2008AA1011005, 2008AA022311, 2009CB521803, 2009CB918900, 2010CB944900 and 2010DFB30270). We thank members of our laboratories for helpful comments on the manuscript.
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ischemia via paracrine actions. Am. J. Physiol. Heart Circ. Physiol. 295: H1726−1735. D’Ippolito, G., Diabira, S., Howard, G.A., Menei, P., Roos, B.A., and Schiller, P.C. (2004). Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential. J. Cell Sci. 117: 2971−2981. Friedenstein, A.J., Chailakhjan, R.K., and Lalykina, K.S. (1970). The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 3: 393−403. Fujikawa, T., Oh, S.H., Pi, L., Hatch, H.M., Shupe, T., and Petersen, B.E. (2005). Teratoma formation leads to failure of treatment for type I diabetes using embryonic stem cell-derived insulin-producing cells. Am. J. Pathol. 166: 1781−1791. Jiang, Y., Jahagirdar, B.N., Reinhardt, R.L., Schwartz, R.E., Keene, C.D., Ortiz-Gonzalez, X.R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M., Du, J., Aldrich, S., Lisberg, A., Low, W.C., Largaespada, D.A., and Verfaillie, C.M. (2002). Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418: 41−49.
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GENETICS AND GENOMICS J. Genet. Genomics 37 (2010) 431−439 www.jgenetgenomics.org
Differentiation of embryonic stem cells in adult bone marrow Yueying Li a, b, d, Jing He b, Fengchao Wang b, Zhenyu Ju c, d, Sheng Liu b, Yu Zhang b, Zhaohui Kou b, Yanfeng Liu a, d, Tao Cheng a, d, *, Shaorong Gao b, d, * a
State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China b
c
National Institute of Biological Sciences, Beijing 102206, China
Max-Planck-Partner Group on Stem Cell Aging, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing 100021, China d
Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China. Received for publication 6 May 2010; revised 30 May 2010; accepted 1 June 2010
Abstract Embryonic stem cells (ESCs) are a potential source of generating transplantable hematopoietic stem and progenitor cells, which in turn can serve as “seed” cells for hematopoietic regeneration. In this study, we aimed to gauge the ability of mouse ESCs directly differentiating into hematopoietic cells in adult bone marrow (BM). To this end, we first derived a new mouse ESC line that constitutively expressed the green fluorescent protein (GFP) and then injected the ESCs into syngeneic BM via intra-tibia. The progeny of the transplanted ESCs were then analyzed at different time points after transplantation. Notably, however, most injected ESCs differentiated into non-hematopoietic cells in the BM whereas only a minority of the cells acquired hematopoietic cell surface markers. This study provides a strategy for evaluating the differentiation potential of ESCs in the BM micro-environment, thereby having important implications for the physiological maintenance and potential therapeutic applications of ESCs. Keywords: embryonic stem cells; differentiation; bone marrow; transplantation
Introduction It is generally accepted that two major cell lineages could be distinguished in the adult bone marrow (BM) which include the hematopoietic cell lineage and non-hematopoietic stroma cells. The stroma cells provide distinct hematopoietic microenvironment that regulates * Corresponding author. Tel: +86-10-8072 8967, Fax: +86-10-8072 7535 (S. Gao); Tel & Fax: +86-22-2390 9156 (T. Cheng). E-mail address: [email protected] (S. Gao); [email protected] (T. Cheng) DOI: 10.1016/S1673-8527(09)60062-X
hematopoietic stem cells (HSCs) maintenance and facilitates hematopoiesis. HSCs are the only stem cells found in BM that could differentiate into hematopoietic cells. Therefore, bone marrow transplantation has been widely used to cure blood diseases including leukemia. However, very rare number of HSCs could be retrieved and moreover, histocompatibility of the cells is the other major limitation for HSCs transplantation. Subsequently, embryonic stem cells (ESCs) or induced pluripotent stem (iPS) cells could overcome most of the limitations and be potentially used to generate tissues of value for regenerative medicine (Paris and Stout, 2010). Since BM is a well-studied
432
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microenvironment, it provides an attractive engraftment site for cell transplantation, which is not limited to HSCs transplantation. Pancreatic islet transplantation has ever been performed in BM because of its protected and extravascular (but well-vascularized) microenvironment (Cantarelli et al., 2009). ESCs, derived from the inner cell mass of preimplantation blastocyst, possess two important characteristics: self-renewal and differentiation into cells belonging to all three germ layers, and this plasticity is unlimited. These important characteristics make ESCs a great candidate in regenerative medicine and future treatments for a wide variety of diseases, including cardiovascular (Cao et al., 2006), neurodegenerative (Keirstead et al., 2005), and endocrine (Fujikawa et al., 2005) disorders, etc. However, some obstacles, including ethical issues, immunorejection and tumorigenesis, need to be overcome before the potential benefits of ESCs can be translated to a clinical application. Traditionally, ESCs, used to treat a myriad of hematopoietic malignancies and disorders by transplantation, need to be pre-differentiated into HSCs by embryoid body (EB) and coculture and expansion on OP9 stromal cells in the presence of hematopoietic cytokines. The ESC-derived HSCs can reconstitute and rescue the lethally irradiated mice (McKinney-Freeman and Daley, 2007). Since ESCs demonstrated greater pluripotency than HSCs or MSCs, they can be used as a potential source of generating transplantable HSCs or MSCs in vitro. However, it remains undetermined if ESCs can survive in the BM and if so, how ESCs differentiate in the BM microenvironment. A more recent study has clearly shown that direct transplanting human ESCs into the hippocampus of athymic nude rats can rescue the cognitive impairment caused by irradiation (Acharya et al., 2009). In contrast to the previous studies that have pre-differentiated ESCs into HSCs before transplantation, we chose to transplant ESCs directly into the BM to view how the ESCs survive and differentiate in the BM. To fulfill this goal, we derived a new ESC line which carried the chicken beta-actin-GFP transgene. The transplanted cells were visualized at different time points post-transplantation. Moreover, we further characterized the properties of the progenies of transplanted ESCs after a long time surviving in the BM.
Materials and methods This study was performed in accordance with the guidelines of the Animal Care and Use Committee of the National Institute of Biological Sciences and with the Guide for the Care and Use of Laboratory Animals.
ESC derivation and culture aGFP+ ESCs were derived from available inbred mouse strains: 129/Sv × CAG/EGFP transgenic C57BL/6-Tg mice, in which green fluorescent protein (GFP) expression was under the control of a chicken beta-actin promoter and cytomegalovirus enhancer. Blastocyst-stage mouse embryos (3.5-day-old) were collected and plated individually on a 96-well dish with mitomycin C-arrested mouse embryonic fibroblast monolayer (feeder) in ESC Derivation Medium: Knockout Dulbecco’s modified Eagle’s medium (DMEM) with 15% Knockout serum replacement, 1,000 U/mL mouse leukemia inhibitory factor (LIF), 50 μg/mL Pen/Strep, 10−4 mol/L nonessential amino acids, 2 mmol/L L-glutamine, and 10−4 mol/L 2-mercaptoethanol. After 5−6 days in culture, the inner cell mass outgrowth was removed into another 96-well dish with feeder. Two days later, the inner cell mass outgrowth was trypsinized with 0.05% trypsin/EDTA (Invitrogen, USA) on a 96-well dish with feeder. The ESCs were moved every 2 days onto larger dishes with freshly prepared feeder layers, and were fed every day with new ESC Culture Medium: DMEM with 15% fetal bovine serum, 1000 U/mL mouse LIF, 50 μg/mL Pen/Strep, 10−4 mol/L Nonessential amino acids, 2 mmol/L L-glutamine, and 10−4 mol/L 2-mercaptoethanol. Culture dishes were kept at 37oC in a humidified atmosphere with 5% CO2.
Reverse transcription polymerase chain reaction (RT-PCR) analysis Total RNA was extracted from the ESCs using Trizol reagent (Invitrogen) following the manufacturer’s instructions. The RNA was then converted into cDNA using oligo-dT and the M-MLV Reverse Transcriptase Kit (Promega, USA). Polymerase chain reactions (PCR) were carried out for 30 cycles (94°C, 30 s; 60°C, 30 s; 72°C, 30 s). The following primer sequences were used: Oct3/4 F: 5′-GAAGCAGAAGAGGATCACCTTG-3′, R: 5′-TTCTTAAGGCTGAGCTGCAAG-3′;
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Nanog F: 5′-CCTCAGCCTCCAGCAGATGC-3′, R: 5′-CCGCTTGCACTTCACCCTTTG-3′; Sox2 F: 5′-GCGGAGTGGAAACTTTTGTCC-3′, R: 5′-CGGGAAGCGTGTACTTATCCTT-3′; Klf4 F: 5′-TGATGGTGCTTGGTGAGTTG-3′, R: 5′- TTGCACATCTGAAACCACAG-3′; c-myc F: 5′-TCTCCATCCTATGTTGCGGTC-3′, R: 5′-TCCAAGTAACTCGGTCATCATCT-3′; GAPDH F: 5′-CGGAGTCAACGGATTTGGTCGTAT-3′, R: 5′- AGCCTTCTCCATGGTGGTGAAGAC-3′.
Alkaline phosphatase staining and immunocytochemical analysis The cells were fixed with 4% paraformaldehyde for 2 min at room temperature. Alkaline phosphatase activity was detected using a kit from Chemicon (USA) and following protocols provided by the manufacturer. For immunocytochemical staining, colonies were fixed for 2 h at room temperature with 4% paraformaldehyde and then incubated at room temperature for 15 min with 1% Triton X-100/phosphate-buffered saline (PBS). Cells were washed three times in PBS and blocked at 37oC for over 3 h with 4% normal goat serum (Chemicon). Subsequently, cells were incubated at 4°C overnight with primary antibody to Oct4 (1:500, Santa Cruz Biotechnology, USA), SSEA-1 (1:500, Chemicon), Nanog (1:500, Cosmobio, USA), or Sox2 (1:500, Abcam, UK). Cells were washed three times in PBS and incubated at 37oC for 2 h with goat anti-rabbit Alexa-Flour 594-conjugated and goat anti-mouse Alexa-Fluor IgG or IgM 633-conjugated (Invitrogen) secondary antibodies (1:500 in 1% normal goat serum in PBS). Unbound secondary antibodies were removed in three washes with PBS. Nuclei were identified by DAPI (Invitrogen) staining at a dilution of 1:10,000 at room temperature for 5 min. Images were acquired using a confocal laser scanning microscope, LSM 510 META (Carl Zeiss, Germany).
In vitro and in vivo differentiation In vitro differentiation was performed using three methods: the formation of embryoid body (EB), the formation of teratomas and the generation of chimeras. In the EB formation method, ESCs were dissociated into single cells and plated at 2 × 105 cells/mL in suspension culture in the
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absence of LIF using Iscove’s modified Eagle’s medium supplemented with 15% fetal bovine serum, 2 mmol/L L-glutamine, and 0.1 mmol/L nonessential amino acids on a Ultra Low Attachment 6-well plate. In the teratoma method, 2 × 106 cells in 200 µL PBS were injected under the inguinal skin of Severe Combined Immunodeficiency (SCID) mice. After 2 weeks, the teratomas were excised, fixed in 10% paraformaldehyde, and subjected to histological examination with hematoxylin and eosin staining. In the chimera method, chimeric mice were obtained through the microinjection of ESCs into 3.5-day-old blastocysts isolated from CD1 mice and reimplantation of the microinjected blastocysts into the uterine horns of 2.5-day pseudopregnant CD1 foster mothers. The chimeric offspring were identified by coat color. Germline transmission of the ESC genome was then tested by crossing high-percentage chimeras with CD1 mice to establish the ESC-line-derived coat color in F1 offspring.
Karyotype analysis Two days after ESCs were seeded, they were exposed to 0.25 μg/mL colcemid for 3.5 h, digested, collected, and exposed to hypotonic solution for 6 min. Cells were dropped onto a cold glass slide after being fixed with methanol/acetic acid (3:1) twice for 1 h in total. The karyotype was determined by microscopic examination after conventional Giemsa staining and G-banding analysis. More than twenty chromosomal spreads were counted per population.
ESCs transplantation and recovery of GFP+ cells from bone marrow Recipient C57BL/6 female mice at 4−6 weeks of age were treated with sublethal 700 cGy via Co60 irradiator one day prior to transplantation. ESCs were digested and adhered to the untreated tissue culture dish for 30 min to deplete feeder cells. Then the ESCs were passed through a 70 μm nylon mesh to remove clumps and kept on ice before transplantation. The mice were anesthetized with Avertin and the right tibia was gently drilled with a 26-gauge needle into the bone marrow cavity. Intra-bone-marrow injection was carried out using a microsyringe. Cells (1 × 106) in 50 μL total volume were transplanted into the right tibia of each recipient mouse. At various time points post-transplantation of aGFP+ ESCs, bone marrow cells were flushed from the tibias and femurs of
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the transplanted mice. Single-cell suspension from the bone marrow of transplanted mice was cultured on 35 mm or 60 mm dishes in ESC culture medium. Then we kept observing the GFP+ cell under fluorescent microscope every day.
Flow cytometric analysis At various time points post-transplantation of aGFP+ ESCs, 100 µL of peripheral blood was withdrawn from each recipient. Bone marrow cells were flushed from the tibias of the recipients. Cell suspension was treated with red cell lysis buffer and passed through a 70 μm nylon mesh before flow cytometric analysis. Bone marrow mononuclear cells were stained with PerCP-Cy5.5conjugated CD45.2 antibody (eBioscience, USA) and APC-Cy7 conjugated lineage antibodies (a mixture of anti-CD3, CD4, CD8, B220, Gr-1, Mac-1, and Ter-119 antibodies; eBioscience).
Colony-forming unit-fibroblast (CFU-F) assay and hematopoietic differentiation Isolation of cells from the grafted bones of recipients transplanted with aGFP+ ESCs was performed by either marrow flushing or bone digesting. Cells were cultured with MesenCult® Medium (STEMCELL Technologies, Canada). The CFU-F assay was carried out following manufacturer’s instructions. For in vitro hematopoietic differentiation assay, after red cell lysis and passing through a 70 μm nylon mesh, bone marrow cells were cultured with hematopoietic differentiation medium: Iscove’s Modified Dulbecco Medium with 1% methylcellulose, 10% plasma-derived serum, 5% protein-free hybridoma medium, 100 ng/mL Stem Cell Factor, 5 ng/mL mouse thrombopoietin, 2 U/mL human erythropoietin, 25 ng/mL mouse IL-11, 30 ng/mL IL-3, 30 ng/mL mouse GM-CSF, 30 ng/mL mouse G-CSF, and 5 ng/mL mouse IL-6 (Shen and Qu, 2008). After being cultured for 10 days at 37oC in a humidified atmosphere with 5% CO2, colonies were counted using a reverse fluorescence microscope.
Results Establishment of a new embryonic stem cell line used in our study To track the injected ESCs after transplantation, we de-
rived a new kind of mouse ESC line aGFP+ ESCs, and the GFP expression of the progeny was independent of their differentiation status. The ESC line exhibited green colonies typical of undifferentiated ESCs (Fig. 1A). aGFP+ ESCs expressed undifferentiated cell-specific genes such as Oct4, Nanog, Sox2 and other genes such as Klf4 and c-myc, similar to normal R1 ESCs (Fig. 1B). This ESC line displayed high levels of alkaline phosphatase activity and was positive for Oct4, Nanog, Sox2 and SSEA-1 (Fig. 1, C and D). aGFP+ ESCs also formed normal embryoid bodies (Fig. 1E). Teratomas were harvested 2 weeks after the subcutaneous injection of aGFP+ ESCs into SCID mice. Histological examination showed that the teratomas contained various tissues of the three germ layers (Fig. 1F). This ESC line resulted in chimeric offspring and most of these chimeras transmitted the ESC genome to their offspring (Fig. 1G). Karyotyping of aGFP+ ESCs showed that at least 95% of the cells were diploid (Fig. 1H). Therefore, this newly-derived ESC line was qualified to serve the purposes of our current study.
In-tibia injecting of aGFP+ ESCs into bone marrow To evaluate the differentiation ability of ESCs in the adult bone marrow, we transplanted the ESCs directly into bone marrow cavities of the mice to minimize the peripheral loss by intravenous administration. We transplanted 1 × 106 aGFP+ ESCs into the right tibia of each sublethally irradiated recipient mouse. Most mice that received the ESCs could survive normally for a long term with the maximum of nearly two years. Although 26 mice (25% in the hosts) have developed tumors in a total of 103 sublethally irradiated recipients, we intended to focus on the transplanted animals in which no tumor was formed. At various time points post-transplantation, we recovered the engrafted GFP+ cells from the bone marrow mononuclear cells of recipients. We cultured the cells in ESC culture medium the same as the status before transplantation. We observed that GFP+ cells were spread and proliferated like mesenchymal cells and astrocyte-, endothelial-, fibroblast-like cells 2−3 days later (Fig. 2). The results suggested that after transplantation of ESCs into bone marrow, ESCs have a tendency to differentiate into non-hematopoietic cells.
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Fig. 1. Characterization of aGFP+ embryonic stem cells. The aGFP+ ESCs, whose GFP expression was under the control of a chicken beta-actin promoter, were derived from heterozygous transgenic mouse C57BL/6-Tg (CAG/EGFP) × 129/Sv. A: a phase-contrast image of aGFP+ ESCs. Scale bar stands for 50 μm. B: RT-PCR of pluripotent markers. R1 ESCs, mouse embryonic fibroblast (MEF) and template-free PCR systems (Tm-) were used as controls. C: alkaline phosphatase staining. Scale bar stands for 50 μm. D: fluorescent immunostaining of pluripotent markers. Scale bar stands for 50 μm. E: embryonic body formation. Scale bar stands for 200 μm. F: teratoma formation. Teratomas were harvested 4 weeks after subcutaneous injection of aGFP+ ESCs into SCID mice. Ectodermal epidermis (a), mesodermal immature cartilage (b), and endodermal intestinal tract epithelium (c) were detected by hematoxylin and eosin staining. G: chimeric mouse and the F1 offspring. H: karyotype (40, XY).
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Fig. 2. Observation of recovered GFP+ cells after transplantation. At various time points post-transplantation of aGFP+ ESCs, we recovered the engrafted GFP+ cells from bone marrow and observed the cells everyday with fluorescent microscopy. GFP+ cells were proliferated as A: astrocyte-like cells; B: mesenchymal cells; C: endothelial- like cells; D: fibroblast-like cells. Scale bar stands for 50 μm.
Flow cytometric analysis of progeny of engrafted ESCs To analyze the differentiation potential of ESCs after transplantation in bone marrow, we examined the engrafted cells by flow cytometric analysis of the bone marrow and peripheral blood of the recipients. Flow cytometric analysis and fluorescent microscopy analysis showed that GFP+ cells were detectable in the peripheral blood of 62.5% of the recipients, though the proportion of green cells was very low (~0.01%). In the bone marrow of grafted bones, only 3% of GFP+ cells could be found in the subsets of hematopoietic cell populations (CD45+, lineage+) (Fig. 3). However, using fluorescent microscopy, we found that many GFP+ cells still adhere to the bone after repeated flushing of the bone marrow and scraping the bone, suggesting that only a small amount of the GFP+ cells could be acquired for flow cytometric analysis.
Differentiation of ESCs in bone marrow after transplantation In order to collect GFP+ cells as much as possible and
analyze the property of engrafted GFP+ cells after a long time survival in bone marrow, we isolated cells by both marrow flushing and bone digesting and directly cultured the cells with MesenCult® Medium or hematopoietic differentiation medium. The CFU-F assay showed that many green colonies formed in the MesenCult® Medium (Fig. 4). In contrast, no green colonies formed in the hematopoietic differentiation medium, further confirming the limited differentiation potential of engrafted aGFP+ ESCs toward hematopoietic lineage.
Discussion In this study, we derived a new ESC line, aGFP+ ESCs that express the GFP regardless its differentiation status. The cell line, expressing pluripotent ESC markers and demonstrating germline transmission ability, are pluripotent and are able to differentiate normally after transplantation. We further investigated how ESCs differentiate by directly injecting the ESCs into BM. This model established a system through which the progenies of ESCs could be further investigated. Through observation under
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Fig. 3. Flow cytometric analysis of progeny of transplanted aGFP+ ESCs. At various time points post-transplantation of aGFP+ ESCs, bone marrow cells were flushed from the tibias of the sublethally irradiated recipients and control (untransplanted) mice. Bone marrow mononuclear cells were stained with CD45 and a combination of lineage antibodies (Lin).
Fig. 4. Colony-forming unit-fibroblast assay of progeny of transplanted aGFP+ ESCs. Isolation of cells from marrow plug and bone fragments by marrow flushing, bone crushing and digesting. Cells were cultured by MesenCult® Medium for over 10 days at 37°C in 5% CO2. Green colonies were formed. A, C: colonies from bright field images. B, D: green colonies from fluorescent images. Scale bar stands for 50 μm.
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fluorescent microscopy and analysis by flow cytometric analysis and in vitro culture, we concluded that injected ESCs in BM tend to differentiated into non-hematopoietic cells whereas very few cells acquired hematopoietic cell surface markers. Adult stem cells offer tremendous potential for accelerating and hold many promises for future clinical applications and regenerative medicine. HSCs are the best-characterized somatic stem cells so far, but in vitro expansion has been unsuccessful, limiting the future therapeutic potential of these cells (Wilson and Trumpp, 2006). ESCs may have greater protective power and pluripotency than adult stem cells. ESC paracrine protective mechanisms in surgical ischemia are superior to those of adult stem cells (Crisostomo et al., 2008). ESCs serve as a source for generation of unlimited quantities of cardiomyocytes for myocardial repair, and the initial success to repair and improve myocardial function in experimental models of heart disease has been quite promising (Zhang and Pasumarthi, 2008). Moreover, a recent study has clearly shown that direct transplanting human ESCs into the hippocampus of athymic nude rats can rescue the cognitive impairment caused by irradiation (Acharya et al., 2009). Therefore, ESCs are of great value as a potential and remarkable source of transplantable stem cells. The adult BM are composed of two main and distinct lineages: the hematopoietic tissue and a group of mesenchymal-derived stroma cells (Anjos-Afonso and Bonnet, 2007). Cells that constitute the bone marrow stroma are fibroblasts, macrophages, adipocytes, osteoblasts, osteoclasts, and endothelial cells. Interestingly, BM has been identified as an organ containing HSCs as well as non-hematopoietic stem cells, such as mesenchmal stem cells (MSCs) (Friedenstein et al., 1970), multipotent adult progenitor cells (Jiang et al., 2002), marrow-isolated adult multilineage inducible cells (D’Ippolito et al., 2004), and very small embryonic-like stem cells (Kucia et al., 2006). These non-hematopoietic stem cells have shown the features similar with ESCs, which suggested that BM could be a potential site for ESC residents and differentiation. However, to our surprise, most injected ESCs in BM differentiated into non-hematopoietic cells whereas few cells acquired hematopoietic cell surface markers, which will prompt us to further investigate the relationship between ESCs and BM microenvironment. Although these results are encouraging, we should also be cautious about the transplantation of ESCs or
ESC-derivatives. In our study, teratomas could be detected in 25% of the recipients. On the other hand, although ESCs represent one of the most promising cell types for tissue transplantation and regeneration and cellular therapy, tumorigenesis has to be solved before they can be used for clinical applications. In conclusion, our present study demonstrates that most injected ESCs in BM differentiated into non-hematopoietic cells whereas a minority acquired hematopoietic cell surface markers. Therefore, our study has important implications for the understanding of embryonic stem cell development and physiological maintenance in bone marrow, as well as for the safety evaluation of therapeutic use of ESCs and ESC-derivatives in BM.
Acknowledgements The work was supported by the grants from Tianjin Government (Nos. 07JCZDJC10600, 08ZCKFSF03200 and 09ZCZDSF03800) and the Ministry of Science and Technology of China (Nos. 2008AA1011005, 2008AA022311, 2009CB521803, 2009CB918900, 2010CB944900 and 2010DFB30270). We thank members of our laboratories for helpful comments on the manuscript.
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