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Stem Cells 2005;23:638-643 www.StemCells.com
© 2005 AlphaMed Press

Donor Marker Infidelity in Transgenic Hematopoietic Stem Cells

Daniel A. Andersona, Yanna Wua, Shuguang Jianga, Xingqi Zhanga, Philip R. Streetera, Gerald J. Spangrudeb, David R. Archerc, William H. Fleminga

a Center for Hematologic Malignancies, Division of Hematology and Medical Oncology, Oregon Health & Science University, Portland, Oregon, USA;
b Departments of Medicine and Pathology, University of Utah School of Medicine, Salt Lake City, Utah, USA;
c Department of Pediatrics, Emory University, Atlanta, Georgia, USA

Key Words. Hematopoietic stem cells • EGFP • Transgenic mouse • Bone marrow transplantation • Hematopoiesis

Correspondence: 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{at}ohsu.edu


   ABSTRACT
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transgenic marking approaches are increasingly used to evaluate the developmental potential of stem cells. However, cell fate mapping studies using different transgenic marking systems have produced conflicting results. These disparate findings may be due in part to the infidelity of donor marker gene expression. Analysis of hematopoietic stem cells (c-Kit+, Sca-1+, lineage marker– [KSL]) from a transgenic mouse (1Osb) engineered to ubiquitously express the enhanced green fluorescent protein (EGFP) reveals two distinct populations. Forty percent of KSL cells demonstrate intermediate levels of EGFP fluorescence and differentiate into subpopulations of B cells, T cells, and myeloid cells that do not express EGFP. By contrast, progeny of the remaining 60% of KSL cells are almost exclusively EGFP bright. Long-term multilineage hematopoietic reconstitution and serial transplantation experiments show that these differences in EGFP are a property of self-renewing stem cells. Furthermore, both the transgene integration site and the activation status of a cell are important determinants of EGFP expression. These results indicate that a combination of donor cell markers is required to reliably track the full differentiation potential of transgenic stem cells.


   INTRODUCTION
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The common leukocyte antigen (CD45, Ly5) is expressed on all nucleated cells in the peripheral blood of mammals [1], and mice congenic for Ly5 have long been used to track the differentiation potential of hematopoietic progenitor cell and stem cell subsets [2]. 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 [3] nor for tracking nonhematopoietic donor cell outcomes [4].

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 [5]. Although this model is widely used to track stem cell fate [6–15], many of these studies are controversial [16]. Transgenic marking with EGFP has been associated with false-positive outcomes due to autofluorescence [17] and with as-yet-unexplained false-negative results [18]. 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.


   MATERIALS AND METHODS
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Hemizygous C57BL/6-TgN(ACTBEGFP)1Osb mice (Jackson Laboratory Bar Harbor, ME, http://www.jax.org), C57BL/6 TgN(act-EGFP)OsbY01 mice (provided by Dr. Masaru Okabe, Osaka University, Osaka, Japan), and C57BL/6-Ly-5.2 mice were maintained in the animal care facility at the Oregon Health & Science University (OHSU). All procedures were approved by the institutional animal care committee at OHSU.

Lineage Analysis of HSCs
Peripheral blood was depleted of erythrocytes, and single-cell suspensions were prepared from bone marrow (BM), spleen, thymus, and lymph nodes. Dead cells were excluded by a combination of scatter gates and propidium-iodide staining. The expression of the cell-surface antigens CD45.2-PE (phycoerythrin) or CD45.1-PE and markers for T cells (CD3-APC [allophycocyanin]), B cells (B220-APC), or myelomonocytic cells (Mac-1-APC and Gr-1-APC) was determined using a FACScan II cytometer (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, CA, www.bdbiosciences.com) [19].

Isolation of EGFPHi and EGFPMed HSCs (KSL)
BM cells were labeled with antibodies to c-Kit-APC, Ly6AE-B SA-PharRed (Sca-1), and a PE-conjugated lineage mixture (B220, CD3, CD4, CD5, CD8, Mac-1, Gr-1, Ter119). KSL cells were enriched to homogeneity by double sorting using a FACS-Vantage cell sorter, as described previously [4, 20].

Transplantation and Hematopoietic Engraftment
Congenic recipient mice (CD45.1) received 1,200 cGy of lethal irradiation in two doses of 600 cGy delivered 3 hours apart [21]. Either 200 EGFPMed or 300 EGFPHi KSL donor cells (CD45.2), equivalent to their relative proportion in the 1Osb mouse, were combined with 1 x 105 host-type carrier BM cells and injected i.v. into irradiated recipients. Blood was processed as described above and analyzed by flow cytometry for multilineage engraftment of donor-derived EGFP+ cells.

Mitogen Activation of Thymocytes
Single-cell suspensions were made from the thymus of EGFP 1Osb and wild-type C57BL/6 mice. Thymocytes were cultured in either Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum alone or in the presence of phorbol myristate acetate (PMA; 50 ng/ml) and ionomycin (1 µg/ml) for 24 hours at 37°C. EGFP expression was evaluated by fluorescence-activated cell sorting.


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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 [5] 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. 1Go; Table 1Go) and were also found in the thymus, spleen, and lymph nodes (Table 1Go). These results indicate that a significant number of mature blood cells do not express EGFP during steady-state hematopoiesis.



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  		Figure 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
 
Analysis 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. 2AGo). 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. 2BGo, left panel). By contrast, more than 98% of the EGFPHi HSC progeny expressed EGFP (Fig. 2CGo, 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. 2Go). 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 2Go).



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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.

 

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  		Table 2. Enhanced green fluorescent protein (EGFP) expression (%; mean ± SEM) in circulating blood cells following transplantation
 
The 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 [22]. 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.

The majority of thymocytes are T-cell progenitors that are destined to undergo apoptosis [23]. In contrast to mature T cells, only 7% of thymocytes showed high levels of EGFP (Fig. 3AGo). To determine if activation of the actin promoter [24] 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, 3DGo). This finding demonstrates that the activation state of a thymocyte regulates the expression of the EGFP transgene in this mouse strain.



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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.

 
To 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 ß-actin promoter elements, have a different integration site [25]. In contrast to the 1Osb line, all phenotypically defined HSCs from Y01 mice exhibited uniformly high levels of EGFP expression (Fig. 4AGo). 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. 4BGo). 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.



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  		Figure 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.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several genetically based marking systems have been used in cell fate mapping studies. Donor-recipient chimeras using a congenic difference at the Ly5 (CD45) locus have been used widely for determining donor-cell outcomes in the hematopoietic system [2, 20, 26]. The expression of these Ly5 congenic markers is restricted to the hematopoietic system, thereby precluding the use of this marker system in most somatic cell types. Sex-mismatched donors are useful for determining donor-cell outcomes by karyotype analysis or fluorescence in situ hybridization (FISH) [27]. However, evaluation and quantification of donor-cell outcomes by this method is limited to nonviable cells and requires significant tissue processing. The development of transgenic marking systems, particularly EGFP, has promised ubiquitous marking and provides for rapid analysis of donor markers using multiple techniques. Most important, this approach permits prospective isolation of stem cell populations and tracking of their progeny in vivo [13, 28]. Here we show that the utility of EGFP transgenic lines has important limitations.

The apparent simplicity of identifying donor-cell outcomes using EGFP as a donor marker is deceptive. In the skeletal muscle, Jackson et al. [17] report that skeletal muscle fiber–specific autofluorescence can be misinterpreted as EGFP-marked donor-cell progeny. Conversely, retroviral EGFP marking of somatic cells can lead to the downregulation of transgenes. HSCs that displayed stable retroviral integration in the progeny of long-term reconstituted primary recipients were silenced upon secondary transfer [29]. In this context, the donor-cell outcomes would be underestimated; if the donor-cell outcomes are only present at a low frequency, they may go undetected. In agreement with this finding, our data show that significant populations of donor-cell outcomes will be overlooked if EGFP 1Osb is used as the only donor marker.

Using EGFP may also pose other challenges in interpreting cell fate mapping studies. The EGFP protein can induce expression of other signaling pathways. For example, it has been shown that EGFP can induce expression of HSP70 in vivo [30]. The upregulation of HSP70 could have downstream effects on prostaglandin E2 production, which may have hemodynamic consequences by inducing vasodilatation in these mice. Tissue-specific toxicity may be associated with EGFP expression. Huang et al. [31] have reported that the expression of EGFP in a dose-dependent manner can result in cardiomyopathy in transgenic mice. We have also observed cardiomegaly in the EGFP partner of a parabiosed pair of C57B16 mice (data not shown). It remains to be determined if the observed cardiotoxicity occurs due to the additional hemodynamic stress associated with the anastomosis of two independent circulatory systems.

Our data show that EGFP expression in the thymus is associated with the activation status of the cell. Polyclonal mitogen stimulation of immature T cells results in a significant increase in the levels of EGFP protein prior to the first wave of cell proliferation. Whether this is due to increased transcription, enhanced stability of the EGFP protein, or both remains to be determined. In nucleated mature blood cells, there is a similar frequency of EGFP– cells in all the major hematopoietic lineages. EGFPLo KSL cells exclusively give rise to EGFPLo or EGFP– progeny, while EGFPHi KSL cells give rise to EGFPHi and rare EGFPLo outcomes on both primary and secondary transplantation. This result demonstrates EGFP regulation at the level of the stem cell.

Although transgenic makers are increasingly used to identify donor-cell outcomes, the results from several studies remain controversial [32–34]. A combination of criteria—including tissue-specific morphology, cell lineage–specific marker expression, and the absence of the panhematopoietic marker CD45—is necessary for identifying nonhematopoietic cell outcomes. In addition, the functional activity of the differentiated cell progeny should be carefully evaluated.

Recent reports of neuronal cell outcomes are controversial [34, 35]. Downregulated transgene expression may be responsible for an apparent absence of donor-derived cells. Castro et al. [35] used the Rosa26 mouse to evaluate the potential of side population cells to produce neural cell progeny in vivo. This system revealed no donor-derived neural cell outcomes in the brains of any of the mice analyzed. By contrast, Mezey et al. [18] showed that transplanting sex-mismatched EGFP BM cells did not produce donor outcomes when assayed for EGFP fluorescence; donor cells were readily identifiable when interphase FISH was used to identify Y chromosomes. These results, along with our own in the hematopoietic system, suggest that the findings of stem cell fate studies using transgenic mice need to be confirmed using a second independent donor marker system.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received November 22, 2004; accepted for publication January 18, 2005.



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