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a Center for Hematologic Malignancies, Division of Hematology and Medical Oncology, Oregon Health & Science University, Portland, Oregon, USA;
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b Departments of Medicine and Pathology, University of Utah School of Medicine, Salt Lake City, Utah, USA;
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c Department of Pediatrics, Emory University, Atlanta, Georgia, USA# h' O0 g& O r+ w" ^8 {
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Key Words. Hematopoietic stem cells ? EGFP ? Transgenic mouse ? Bone marrow transplantation ? Hematopoiesis" m- c: a$ j1 U/ H- N
3 y' t/ @; m. ~' B2 u) FCorrespondence: William H. Fleming, M.D., Ph.D., Center for Hematologic Malignancies, Division of Hematology and Medical Oncology, Department of Medicine, Oregon Health & Science University (UHN73C), 3181 SW Sam Jackson Park Rd., Eugene, OR 97239 USA. Telephone: 503-494-1554; Fax: 503-494-2770; e-mail: flemingw@ohsu.edu
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ABSTRACT
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$ j1 z" E* _2 K! R$ A& B" |( o+ IThe common leukocyte antigen (CD45, Ly5) is expressed on all nucleated cells in the peripheral blood of mammals , and mice congenic for Ly5 have long been used to track the differentiation potential of hematopoietic progenitor cell and stem cell subsets . The absence of allelic exclusion at the Ly5 locus allows the generation of double congenic mice expressing both Ly5.1 and Ly5.2, an approach that allows the tracking of two different donor cell populations. Although it is very useful for studying normal hematopoietic development, CD45 expression is not informative in certain hematologic malignancies nor for tracking nonhematopoietic donor cell outcomes .
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1 Z# Q* G7 ~/ }/ H* W* y- [& ?Enhanced green fluorescent protein (EGFP) is a transgenic marker increasingly used to track donor cell progeny. The C57BL/6-TgN(ACTBEGFP)1Osb (1Osb) mouse is reported to "ubiquitously" express EGFP in all cell types except adipocytes and red blood cells . Although this model is widely used to track stem cell fate , many of these studies are controversial . Transgenic marking with EGFP has been associated with false-positive outcomes due to autofluorescence and with as-yet-unexplained false-negative results . We now show that the unmanipulated 1Osb mouse exhibits significant heterogeneity of EGFP expression throughout the hematopoietic stem cell (HSC) compartment. A subpopulation of phenotypically defined EGFP , c-Kit , Sca-1 , lineage– (KSL) HSCs has been identified that generates T cells, B cells, and myelomonocytic cells that no longer express EGFP. Furthermore, the activation status of a cell and the transgene integration site are important determinants of EGFP expression. These results suggest that studies using a single transgenic marker should be interpreted with caution.
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MATERIALS AND METHODS; J8 }1 A& C: m' H8 F
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To evaluate the stability of the donor-cell phenotype in the progeny of genetically marked stem cells, the expression of the EGFP in the widely used 1Osb EGFP mouse was evaluated during steady-state hematopoiesis. A mean of 81% of nucleated BM cells and 94% of the nucleated blood cells showed the EGFP phenotype. EGFP– cells were readily detected in all the major hematopoietic lineages in the peripheral blood (Fig. 1; Table 1) and were also found in the thymus, spleen, and lymph nodes (Table 1). These results indicate that a significant number of mature blood cells do not express EGFP during steady-state hematopoiesis.
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3 K! Z( p3 K4 v+ N: KFigure 1. Heterogeneity of enhanced green fluorescent protein (EGFP) expression during steady-state hematopoiesis. Hematopoietic lineage analysis of peripheral blood of 8- to 12-week-old EGFP 1Osb mice. Coexpression of EGFP and B220 (B cells), CD3 (T cells), and Mac-1,Gr-1 (myelomonocytic cells) was evaluated by fluorescence-activated cell sorting.
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Table 1. Tissue-specific expression (mean ± SEM) of enhanced green fluorescent protein (EGFP) in hematopoietic cells from 1Osb EGFP mice
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9 R% f& Q( ^4 |, Z+ |( mAnalysis of EGFP expression in KSL cells revealed two distinct cell populations. Sixty percent of HSCs expressed high levels of EGFP (EGFPHi), while 40% of HSCs demonstrated medium levels of this donor marker protein (EGFPMed; Fig. 2A). The hematopoietic potential of these two EGFP-expressing HSC subsets was evaluated using an independent donor-cell marker (CD45.2). Either EGFPMed CD45.2 or EGFPHi CD45.2 HSCs were transplanted into lethally irradiated EGFP– CD45.1 recipients. Remarkably, only 73% of the blood cell progeny derived from transplanted EGFPMed HSCs exhibited the anticipated EGFP CD45.2 phenotype (Fig. 2B, left panel). By contrast, more than 98% of the EGFPHi HSC progeny expressed EGFP (Fig. 2C, left panel). To determine if these aberrant EGFP– cell phenotypes were restricted to specific hematopoietic lineages, donor-derived T cells, B cells, and myelomonocytic cells were examined. An absence of EGFP expression was found in distinct populations of donor cells in all major hematopoietic lineages. EGFPHi HSCs gave rise to 10- to 20-fold fewer EGFP– B cells, T cells, and myelomonocytic cells than did the progeny of EGFPMed HSCs (Fig. 2). Serial transplantation revealed similar populations of EGFP– progeny in all hematopoietic lineages for up to 6 months, indicating that the downregulation of EGFP expression occurs at the level of the self-renewing stem cell (Table 2)./ i+ c; Y$ }$ }9 G8 Y' v, f3 A% ^
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Figure 2. Distinct populations of enhanced green fluorescent protein (EGFP) expression in hematopoietic stem cells (HSCs) and their progeny. (A): Lineage marker– bone marrow cells (left panel) were sorted for c-Kit and Sca-1 expression (KSL; middle panel). Two distinct EGFP-expressing HSC subpopulations were identified in 1Osb mice: EGFPMed and EGFPHi (right panel). The percentage of KSL cells is indicated. Based on a 500-HSC dose equivalent, either 200 EGFPMed or 300 EGFPHi KSL cells were transplanted into lethally irradiated CD45.1 wild-type mice along with 1 x 105 host-type (CD45.1 EGFP–) carrier bone marrow cells. Peripheral blood from recipient mice was analyzed 6 months after transplant. Host CD45.1 cells were excluded from further analysis. (B): EGFPMed HSC–derived progeny were evaluated for the coexpression of CD45.2 and EGFP (left panel). Lineage analysis of EGFPMed donor-derived B cells (B220), T cells (CD3), and myelomonocytic cells (Mac-1) in the peripheral blood is shown (right panel). (C): Evaluation of EGFPHi HSC-derived progeny for coexpression of CD45.2 and EGFP (left panel). Lineage analysis of peripheral blood from EGFPHi recipients is shown (right panel). Combined results from three independent experiments for each group are shown. Error bars represent SEM; white bars, EGFP progeny; black bars, EGFP– progeny. Abbreviation: SSC, side scatter.) @5 @& _4 b& |; @8 R+ H
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Table 2. Enhanced green fluorescent protein (EGFP) expression (%; mean ± SEM) in circulating blood cells following transplantation
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8 F9 y2 t6 v& E1 W" pThe functional activity of EGFPMed and EGFPHi HSC populations appeared similar. Both populations generated equivalent numbers of hematopoietic colonies in methylcellulose and in long-term BM stromal assays (data not shown). Transplantation of EGFPMed or EGFPHi HSCs into irradiated recipients gave rise to approximately 80% donor-derived cells in the peripheral blood. This frequency is comparable to that observed with wild-type KSL cells . Multilineage analysis using CD45.2 identified a similar number of T cells, B cells, and myelomonocytic cell progeny derived from both EGFPMed and EGFPHi HSC subsets.3 E, F. z M$ H9 g+ \) f; H
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The majority of thymocytes are T-cell progenitors that are destined to undergo apoptosis . In contrast to mature T cells, only 7% of thymocytes showed high levels of EGFP (Fig. 3A). To determine if activation of the actin promoter enhances EGFP expression, thymocytes were treated with PMA and ionomycin. This polyclonal stimulation produced a greater than 10-fold increase in the frequency of thymocytes expressing high levels of EGFP within 24 hours (Fig. 3B, 3D). This finding demonstrates that the activation state of a thymocyte regulates the expression of the EGFP transgene in this mouse strain." J; T# T' b8 R
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Figure 3. Enhanced green fluorescent protein (EGFP) expression in mitogen-stimulated thymocytes. Thymocytes from 1Osb mice were cultured for 24 hours in (A) Dulbecco’s modified Eagle’s medium alone (control) or (B) media containing phorbol myristate acetate and ionomycin (PMA/I) then analyzed for the level of EGFP expression by flow cytometry. The percentages of thymocytes from the 1Osb EGFP mouse with high levels of EGFP (Hi), low/medium levels of EGFP (Low), and undetectable levels of EGFP (Neg) are shown. Representative plots and percentages are shown. (C): Control wild-type (WT) thymocytes (EGFPNeg) stimulated with PMA/I for 24 hours. (D): Frequency of EGFP-expressing thymocytes from three combined experiments. Mean ± SEM is shown.
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* j) g1 H! |( _% L; vTo evaluate whether the downregulation of EGFP expression in multiple hematopoietic lineages was specific to the 1Osb EGFP mouse, a second EGFP transgenic line was examined. Osb-Y01 mice, produced by the same laboratory, using the identical EGFP construct under the control of the chicken ?-actin promoter elements, have a different integration site . In contrast to the 1Osb line, all phenotypically defined HSCs from Y01 mice exhibited uniformly high levels of EGFP expression (Fig. 4A). This high level of EGFP marker expression was maintained in all mature nucleated peripheral blood cells from Y01 mice. Continued expression of high levels of EGFP was found in the peripheral blood of all recipients transplanted with Y01 HSC (Fig. 4B). Analysis of long-term hematopoietic reconstitution (6 months) did not reveal any subset of B cells, T cells, and myelomonocytic cells that exhibited downregulation of the EGFP transgene.& F" Z; H) N- Z* U0 }" ` p, K
" o" t( l2 T4 R. lFigure 4. c-Kit , Sca-1 , lineage marker– hematopoietic stem cell (KSL HSC) progeny from Y01 enhanced green fluorescent protein (EGFP) mice display uniformly high levels of EGFP expression. (A): Lineage marker– bone marrow cells (SSC; left panel) were sorted for c-Kit Sca-1 (middle panel). High levels of EGFP expression were observed in all KSL cells (right panel). KSL cells were transplanted into irradiated congenic CD45.1 wild-type recipients, and peripheral blood was analyzed 6 months after transplant. (B): Lineage analysis of donor-derived B cells (B220; left panel), T cells (CD3; middle panel), and myelomonocytic cells (Mac-1/Gr-1; right panel) in the peripheral blood. Representative plots and percentages are shown. Host (CD45.1 ) cells were excluded from analysis. Abbreviation: SSC, side scatter.
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D.A.A. and Y.W. contributed equally to this work. This research was supported by NIH grants HL069133 and HL077818 to W.H.F.' x) L7 |0 B, Y& u. W
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发表于 2009-3-5 10:50
发表于 2009-3-31 07:55
