Donor Marker Infidelity in Transgenic Hematopoietic Stem Cells
http://www.100md.com
《干细胞学杂志》
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@ohsu.edu
ABSTRACT
The 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 .
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.
MATERIALS AND METHODS
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.
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.
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. 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).
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.
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 . 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 . 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.
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 ?-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.
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
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
Tung JS, Saga Y, Boyse EA. Structural features of Ly-5 glycoproteins of the mouse and counterparts in other mammals. Immunogenetics 1988;28:271–277.
Boyse EA. The increasing value of congenic mice in biomedical research. Lab Anim Sci 1977;27:771–781.
Bataille R, Robillard N, Pellat-Deceunynck C et al. A cellular model for myeloma cell growth and maturation based on an intraclonal CD45 hierarchy. Immunol Rev 2003;194:105–111.
Bailey AS, Jiang S, Afentoulis M et al. Transplanted adult hematopoietic stem cells differentiate into functional endothelial cells. Blood 2004;103:13–19.
Okabe M, Ikawa M, Kominami K et al. "Green mice" as a source of ubiquitous green cells. FEBS Lett 1997;407:313–319.
Takakura N, Watanabe T, Suenobu S et al. A role for hematopoietic stem cells in promoting angiogenesis. Cell 2000;102:199–209.
Sata M, Saiura A, Kunisato A et al. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med 2002;8:403–409.
Polleux F, Morrow T, Ghosh A. Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature 2000;404:567–573.
Obata Y, Kono T, Hatada I. Gene silencing: maturation of mouse fetal germ cells in vitro. Nature 2002;418:497.
Mezey E, Chandross KJ, Harta G et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000;290:1779–1782.
McGavern DB, Christen U, Oldstone MB. Molecular anatomy of antigen-specific CD8+ T cell engagement and synapse formation in vivo. Nat Immunol 2002;3:918–925.
LaBarge MA, Blau HM. Biological progression from adult bone marrow to mononucleate muscle stem cell to multinucleate muscle fiber in response to injury. Cell 2002;111:589–601.
Grant MB, May WS, Caballero S et al. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med 2002;8:607–612.
Camargo FD, Green R, Capetenaki Y et al. Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates. Nat Med 2003;9:1520–1527.
Brazelton TR, Rossi FM, Keshet GI et al. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 2000;290:1775–1779.
Holden C, Vogel G. Stem cells. Plasticity: time for a reappraisal? Science 2002;296:2126–2129.
Jackson KA, Snyder DS, Goodell MA. Skeletal muscle fiber-specific green autofluorescence: potential for stem cell engraftment artifacts. STEM CELLS 2004;22:180–187.
Mezey E, Nagy A, Szalayova I et al. Comment on "Failure of bone marrow cells to transdifferentiate into neural cells in vivo." Science 2003;299:1184.
Montfort MJ, Olivares CR, Mulcahy JM et al. Adult blood vessels restore host hematopoiesis following lethal irradiation. Exp Hematol 2002;30:950–956.
Baumann CI, Bailey AS, Li W et al. PECAM-1 is expressed on hematopoietic stem cells throughout ontogeny and identifies a population of erythroid progenitors. Blood 2004;104:1010–1016.
Streeter PR, Dudley LZ, Fleming WH. Activation of the G-CSF and Flt-3 receptors protects hematopoietic stem cells from lethal irradiation. Exp Hematol 2003;31:1119–1125.
Fleming WH, Alpern EJ, Uchida N et al. Steel factor influences the distribution and activity of murine hematopoietic stem cell in vivo. Proc Natl Acad Sci U S A 1993;90:3760–3764.
Starr TK, Jameson SC, Hogquist KA. Positive and negative selection of T cells. Annu Rev Immunol 2003;21:139–176.
Furue M, Katz SI, Kawakami Y et al. Coordinate expression of src family protooncogenes in T cell activation and its modulation by cyclosporine. J Immunol 1990;144:736–739.
Nakanishi T, Kuroiwa A, Yamada S et al. FISH analysis of 142 EGFP transgene integration sites into the mouse genome. Genomics 2002;80:564–574.
Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science 1988;241:58–62.
Jiang S, Walker L, Afentoulis M et al. Transplanted human bone marrow contributes to vascular endothelium. Proc Natl Acad Sci U S A 2004;101:16891–16896.
Willenbring H, Bailey AS, Foster M et al. Myelomonocytic cells are sufficient for therapeutic cell fusion in liver. Nat Med 2004;10:744–748.
Klug CA, Cheshier S, Weissman IL. Inactivation of a EGFP retrovirus occurs at multiple levels in long-term repopulating stem cells and their differentiated progeny. Blood 2000;96:894–901.
Zhang F, Hackett NR, Lam G et al. Green fluorescent protein selectively induces HSP70-mediated up-regulation of COX-2 expression in endothelial cells. Blood 2003;102:2115–2121.
Huang WY, Aramburu J, Douglas PS et al. Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy. Nat Med 2000;6:482–483.
Anderson DJ, Gage FH, Weissman IL. Can stem cells cross lineage boundaries? Nat Med 2001;7:393–395.
Vogel G. Stem cell research: studies cast doubt on plasticity of adult cells. Science 2002;295:1989–1991.
Wagers AJ, Sherwood RI, Christensen JL et al. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 2002;297:2256–2259.
Castro RF, Jackson KA, Goodell MA et al. Failure of bone marrow cells to transdifferentiate into neural cells in vivo. Science 2002;297:1299.(Daniel A. Andersona, Yann)
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@ohsu.edu
ABSTRACT
The 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 .
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.
MATERIALS AND METHODS
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.
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.
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. 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).
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.
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 . 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 . 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.
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 ?-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.
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
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
Tung JS, Saga Y, Boyse EA. Structural features of Ly-5 glycoproteins of the mouse and counterparts in other mammals. Immunogenetics 1988;28:271–277.
Boyse EA. The increasing value of congenic mice in biomedical research. Lab Anim Sci 1977;27:771–781.
Bataille R, Robillard N, Pellat-Deceunynck C et al. A cellular model for myeloma cell growth and maturation based on an intraclonal CD45 hierarchy. Immunol Rev 2003;194:105–111.
Bailey AS, Jiang S, Afentoulis M et al. Transplanted adult hematopoietic stem cells differentiate into functional endothelial cells. Blood 2004;103:13–19.
Okabe M, Ikawa M, Kominami K et al. "Green mice" as a source of ubiquitous green cells. FEBS Lett 1997;407:313–319.
Takakura N, Watanabe T, Suenobu S et al. A role for hematopoietic stem cells in promoting angiogenesis. Cell 2000;102:199–209.
Sata M, Saiura A, Kunisato A et al. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med 2002;8:403–409.
Polleux F, Morrow T, Ghosh A. Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature 2000;404:567–573.
Obata Y, Kono T, Hatada I. Gene silencing: maturation of mouse fetal germ cells in vitro. Nature 2002;418:497.
Mezey E, Chandross KJ, Harta G et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000;290:1779–1782.
McGavern DB, Christen U, Oldstone MB. Molecular anatomy of antigen-specific CD8+ T cell engagement and synapse formation in vivo. Nat Immunol 2002;3:918–925.
LaBarge MA, Blau HM. Biological progression from adult bone marrow to mononucleate muscle stem cell to multinucleate muscle fiber in response to injury. Cell 2002;111:589–601.
Grant MB, May WS, Caballero S et al. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med 2002;8:607–612.
Camargo FD, Green R, Capetenaki Y et al. Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates. Nat Med 2003;9:1520–1527.
Brazelton TR, Rossi FM, Keshet GI et al. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 2000;290:1775–1779.
Holden C, Vogel G. Stem cells. Plasticity: time for a reappraisal? Science 2002;296:2126–2129.
Jackson KA, Snyder DS, Goodell MA. Skeletal muscle fiber-specific green autofluorescence: potential for stem cell engraftment artifacts. STEM CELLS 2004;22:180–187.
Mezey E, Nagy A, Szalayova I et al. Comment on "Failure of bone marrow cells to transdifferentiate into neural cells in vivo." Science 2003;299:1184.
Montfort MJ, Olivares CR, Mulcahy JM et al. Adult blood vessels restore host hematopoiesis following lethal irradiation. Exp Hematol 2002;30:950–956.
Baumann CI, Bailey AS, Li W et al. PECAM-1 is expressed on hematopoietic stem cells throughout ontogeny and identifies a population of erythroid progenitors. Blood 2004;104:1010–1016.
Streeter PR, Dudley LZ, Fleming WH. Activation of the G-CSF and Flt-3 receptors protects hematopoietic stem cells from lethal irradiation. Exp Hematol 2003;31:1119–1125.
Fleming WH, Alpern EJ, Uchida N et al. Steel factor influences the distribution and activity of murine hematopoietic stem cell in vivo. Proc Natl Acad Sci U S A 1993;90:3760–3764.
Starr TK, Jameson SC, Hogquist KA. Positive and negative selection of T cells. Annu Rev Immunol 2003;21:139–176.
Furue M, Katz SI, Kawakami Y et al. Coordinate expression of src family protooncogenes in T cell activation and its modulation by cyclosporine. J Immunol 1990;144:736–739.
Nakanishi T, Kuroiwa A, Yamada S et al. FISH analysis of 142 EGFP transgene integration sites into the mouse genome. Genomics 2002;80:564–574.
Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science 1988;241:58–62.
Jiang S, Walker L, Afentoulis M et al. Transplanted human bone marrow contributes to vascular endothelium. Proc Natl Acad Sci U S A 2004;101:16891–16896.
Willenbring H, Bailey AS, Foster M et al. Myelomonocytic cells are sufficient for therapeutic cell fusion in liver. Nat Med 2004;10:744–748.
Klug CA, Cheshier S, Weissman IL. Inactivation of a EGFP retrovirus occurs at multiple levels in long-term repopulating stem cells and their differentiated progeny. Blood 2000;96:894–901.
Zhang F, Hackett NR, Lam G et al. Green fluorescent protein selectively induces HSP70-mediated up-regulation of COX-2 expression in endothelial cells. Blood 2003;102:2115–2121.
Huang WY, Aramburu J, Douglas PS et al. Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy. Nat Med 2000;6:482–483.
Anderson DJ, Gage FH, Weissman IL. Can stem cells cross lineage boundaries? Nat Med 2001;7:393–395.
Vogel G. Stem cell research: studies cast doubt on plasticity of adult cells. Science 2002;295:1989–1991.
Wagers AJ, Sherwood RI, Christensen JL et al. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 2002;297:2256–2259.
Castro RF, Jackson KA, Goodell MA et al. Failure of bone marrow cells to transdifferentiate into neural cells in vivo. Science 2002;297:1299.(Daniel A. Andersona, Yann)