Developmental Differences in Megakaryocyte Maturation Are Determined by the Microenvironment
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《干细胞学杂志》
a University of Florida Department of Pediatrics, Gainesville, Florida, USA;
b Program in Stem Cell Biology and Regenerative Medicine, University of Florida Shands Cancer Center, Gainesville, Florida, USA;
c The Blood and Marrow Transplant Program, University of Florida, Gainesville, Florida, USA
Key Words. Megakaryocytopoiesis ? Thrombopoiesis ? Adult bone marrow stem cells ? Umbilical cord blood ? Development
Correspondence: William B. Slayton, M.D., J. Hillis Miller Health Center, Box 100296, Gainesville, Florida 32610, USA. Telephone: 352-392-5633; Fax: 352-392-2875; e-mail: slaytwb@peds.ufl.edu
ABSTRACT
Umbilical cord blood contains stem cells that can be used for hematopoietic stem cell transplantation in patients who lack a sibling or matched unrelated bone marrow donor. Slow engraftment and graft rejection account for the majority of transplant-related morbidity and mortality after cord blood transplantation . Platelet engraftment is particularly slow, taking an average of approximately 70 days for cord blood compared with 20 days for mobilized peripheral blood stem cells . Prolonged thrombocytopenia leads to an increased risk of fatal bleeding and the risks associated with multiple transfusions, including anaphylaxis , alloimmunization , and infection .
Several groups have suggested that qualitative differences between neonatal and adult megakaryocytes and their progenitors may contribute to delayed platelet engraftment after cord blood transplant . One specific qualitative difference is that neonatal megakaryocytes are smaller and have lower ploidy than adult megakaryocytes . Smaller megakaryocytes with lower ploidy produce fewer platelets in vitro . A second qualitative difference is that megakaryocyte progenitors from human cord blood proliferate more in culture than progenitors derived from adult peripheral blood or bone marrow . Proposed mechanisms to explain the increased proliferation and decreased maturation of neonatal megakaryocyte progenitors include differences in the expression of cell cycle proteins that control endomitosis and delayed expression of the thrombopoietin receptor in neonatal cells . Studies that defined these molecular differences were performed in culture in response to recombinant thrombopoietin. These in vitro studies do not, however, reliably reproduce the complex bone marrow or splenic microenvironment (in the mouse) in which megakaryocytes develop after transplant.
We used a method developed by Nakorn et al. to track donor-derived platelets post-transplant, using transgenic mice that express green fluorescent protein (GFP) as donors . We used this model to test the hypothesis that neonatal stem and progenitor cells have an intrinsic tendency to produce small megakaryocytes with low DNA content, and that these small megakaryocytes lead to slower platelet engraftment after transplant.
MATERIALS AND METHODS
Megakaryopoiesis in Newborn and Adult Mice
We first sought to determine whether neonatal murine liver megakaryocytes and progenitors were phenotypically similar in size, ploidy, and proliferative capacity to their human counterparts. Specifically, we sought to determine whether neonatal megakaryocytes were smaller and had lower DNA content than those from adult mice. The number of megakaryocytes in the liver of newborn animals was much higher than in the spleen, where megakaryocytes were rarely found, or in the bone marrow, which had small marrow spaces and contained few megakaryocytes. As in human fetuses and neonates, neonatal murine liver megakaryocytes (from GFP animals) were smaller (p .00005; Fig. 1A) and of lower ploidy (p < .00001; Fig. 1B) than megakaryocytes in the adult bone marrow or spleen. Adult splenic megakarocytes were significantly larger than those in the adult marrow (p = .03). As the pups grew and matured, the liver megakaryocytes increased in size but were still significantly smaller than adult bone marrow megakaryocytes at 7 days of life (p = .02). Furthermore, the neonatal cells proliferated more than adult cells, as shown by the increased proportion of burst-forming unit-megakaryocyte (BFU-meg) cultured from neonatal liver cells (Fig. 1C). These experiments established the similarities between neonatal mouse and neonatal human megakaryocytes in terms of size, ploidy, and proliferative capacity and suggested that our system would be useful to study whether these differences persist after transplant.
Figure 1. Comparison of newborn and adult megakaryocytes in different organs. (A): Volume of megakaryocytes in the newborn liver, newborn spleen, 1-week-old liver, adult bone marrow, and adult spleen. Megakaryocyte volumes were calculated from their diameter, assuming a spherical shape. Error bars denote SEM (n = 5–15 mice per cohort, 25–100 megakaryocytes per mouse). One-week-old liver megakaryocytes were significantly larger than newborn megakaryocytes but smaller than adult bone marrow megakaryocytes (p = .02) (B): Ploidy analysis of newborn and adult animals. Megakaryocyte ploidy was measured in the newborn liver and adult bone marrow, and compared with 2N controls. (C): Megakaryocyte colony formation. The ability of neonatal (black bar) and adult (gray bar) cells to produce megakaryocyte colonies in collagen in response to interleukin-3 and thrombopoietin was compared (n = total of 10 mice per cohort, and represents the combined data from three separate experiments). Abbreviations: BFU-meg, burst-forming unit-megakaryocyte; CFU-meg, colony-forming unit-megakaryocyte; PI, propidium iodide.
Stem and Progenitor Cell Numbers
We compared the number of stem and progenitor cells in neonatal liver versus adult bone marrow by measuring the percentage of total cells that did not express mature lineage markers (Linneg) and expressed the stem cell markers c-kit and Sca-1 (SKL cells). We found that the percentage of SKL cells per total nucleated cells was remarkably constant and constituted approximately 0.5% of the total nucleated cells in both the newborn liver and adult bone marrow cell suspensions (n = four neonatal livers and adult bone marrows analyzed).
Platelet Engraftment Kinetics
We then transplanted equal numbers of unfractionated, hemolyzed neonatal liver and adult bone marrow cells from transgenic mice expressing GFP into lethally irradiated adult C57/B6 mice and followed platelet engraftment kinetics. We saw no difference in the peripheral blood platelet engraftment kinetics between animals receiving newborn liver or adult bone marrow cells. In contrast to irradiated controls, animals transplanted with 1.5 x 106 donor cells had a rapid increase in platelet counts between days 7 and 14, regardless of whether they received adult or neonatal cells. Platelet counts in transplanted animals reached the levels of healthy controls 4 weeks post-transplant (Fig. 2A). Donor-derived platelets were detected as early as 7 days after transplant, using flow cytometry for green fluorescence, and reached levels of healthy GFP control animals by 2 weeks post-transplant (Fig. 2B). Platelet engraftment was sustained at the 1-month and 4-month time points, regardless of whether animals received neonatal or adult donor cells.
Figure 2. Platelet engraftment and chimerism. (A): Platelet engraftment after transplantation of stem and progenitor cells derived from neonatal liver () or adult bone marrow (). Controls consisted of platelet counts from healthy animals (broken line, ) and animals that were irradiated but received no donor stem cells (x). (B): Donor-derived platelets from newborn liver (black) and adult bone marrow (gray) as identified by green fluorescence. Control consisted of platelets from healthy green fluorescent protein transgenic mice.
Changes in Marrow and Splenic Hematopoiesis
To understand the ability of our transplanted neonatal cells and adult cells to support post-transplant hematopoiesis, we measured the relative changes in cellularity within the bone marrow and spleen. Hematopoiesis increased dramatically in the spleen during the first 2 weeks after transplant, effacing normal splenic architecture. In fact, spleens nearly doubled in weight relative to healthy controls 7 days post-transplant due to hematopoietic expansion (Fig. 3A). Bone marrow cellularity was similar regardless of stem cell source. In stark contrast to the spleen, marrow cellularity, measured as leukocyte counts per single femur, was 17% of the cellularity of healthy controls 7 days post-transplant but was four times higher than irradiated controls. Marrow cellularity approached healthy control levels 4 weeks post-transplant, only to decrease by 4 months post-transplant (Fig. 3B).
Figure 3. Changes in hematopoietic activity in the spleen and liver post-transplant. (A): Changes in splenic cellularity were determined by measuring the spleen weight relative to body weight in animals that received neonatal (black) and adult (gray) cells. Controls consisted of healthy C57/B6 animals (x) and irradiated controls that did not receive transplanted cells (). (B): Changes in bone marrow cellularity from a single flushed femur.
Megakaryocyte Size
We then measured the diameter of megakaryocytes at each time point post-transplant. At 7 and 14 days post-transplant, both adult bone marrow and newborn liver cells gave rise to megakaryocytes that were larger than normal adult megakaryocytes. However, megakaryocytes derived from newborn cells were significantly smaller than those derived from adult bone marrow cells (p = .056) 7 days post-transplant. This difference was less apparent throughout the rest of the time course. Regardless of the source of transplanted cells, the largest megakaryocytes were found in the spleen. In fact, 1 week post-transplant, newborn liver cells produced splenic megakaryocytes that were six times larger than normal newborn liver megakaryocytes and nearly three times larger in volume than normal adult bone marrow megakaryocytes (Figs. 4A–4C). Megakaryocyte size decreased to control levels by 4 months post-transplant.
Figure 4. Changes in megakaryocyte size. (A): Perivascular, small megakaryocytes in the newborn liver. (B): Perivascular megakaryocytes in the spleen 7 days after transplant of NL cells. These are considerably larger than NL megakaryocytes. (C): Mean megakaryocyte volume in the bone marrow in animals transplanted with NL (black) or ABM (gray) cells. Controls consisted of bone marrow megakaryocytes from healthy adult animals () or megakaryocytes from the newborn liver (). (D): Mean megakaryocyte volume in the spleen in animals transplanted with NL or ABM cells. Controls consisted of splenic megakaryocytes from healthy adult animals () or from newborn liver (). Error bars denote SEM. Abbreviations: ABM, adult bone marrow; NL, neonatal liver.
Megakaryocyte DNA Content
Marrow megakaryocyte ploidy analysis was performed by flow cytometry using flushed bone marrow cells treated with hypotonic citrate, as previously described. On post-transplant days 7 and 14, the small number of megakaryocytes in the bone marrow precluded the measurement of ploidy levels. Similar to our size observations, however, at post-transplant day 18, megakaryocytes derived from adult bone marrow cells reached higher ploidy levels than megakaryocytes derived from newborn liver cells. Specifically, megakaryocytes derived from newborn liver exhibited a ploidy distribution that was remarkably similar to that of megakaryocytes in healthy adult bone marrow, with a median ploidy of 16N. In contrast, megakaryocytes from animals receiving adult bone marrow displayed higher than normal ploidy levels, with more cells reaching 32N (Fig. 5A). This result did not vary from animal to animal based on degree of thrombocytopenia, but rather seemed to be fixed based on the developmental state of the donor cells. By 1 month post-transplant, ploidy levels from newborn liver– and adult bone marrow–derived megakaryocytes were almost identical and were further shifted toward 32N. By 4 months post-transplant, ploidy in both cohorts had reverted to the levels of healthy adult controls (Fig. 5A).
Figure 5. Ploidy analysis of megakaryocytes from neonatal and adult donors. (A): Changes in megakaryocyte ploidy in the bone marrow after transplant by flow cytometry. (B): Changes in mean ploidy of splenic megakaryocytes after transplant by Feulgen staining. Abbreviation: PI, propidium iodide.
Ploidy analysis was also performed in the post-transplant spleen using Feulgen staining, and the results were compared with both healthy adult spleen and neonatal liver. At 7 days post-transplant, newborn liver–derived megakaryocytes had a mean ploidy of 17N (median of 16N) compared with 7N (median 8N) in normal newborn liver. Megakaryocytes derived from adult bone marrow cells also exhibited a higher mean ploidy (15 N, median 16N) than normal adult splenic megakaryocytes (mean 10N, median 16N). These differences were not statistically significant. Megakaryocyte ploidy decreased in parallel with the gradual decrease in megakaryocyte size over the 4-month observation period (Fig. 5B).
DISCUSSION
In summary, we have shown that neonatal stem and progenitor cells are capable of producing adult-sized megakaryocytes when placed in an adult microenvironment in the mouse. This study suggests that the small size and lower DNA content of neonatal megakaryocytes is due to both microenvironmental and cell-intrinsic factors. Understanding these factors may lead to improvements in platelet and overall engraftment after cord blood transplant.
ACKNOWLEDGMENTS
Gluckman E, Rocha V, Chevret S. Results of unrelated umbilical cord blood hematopoietic stem cell transplant. Transfus Clin Biol 2001;8:146–154.
Laughlin MJ, Barker J, Bambach B et al. Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors. N Engl J Med 2001;344:1815–1822.
Couban S, Simpson DR, Barnett MJ et al. A randomized multicenter comparison of bone marrow and peripheral blood in recipients of matched sibling allogeneic transplants for myeloid malignancies. Blood 2002;100:1525–1531.
Sanz GF, Saavedra S, Planelles D et al. Standardized, unrelated donor cord blood transplantation in adults with hematologic malignancies. Blood 2001;98:2332–2338.
Domen RE, Hoeltge GA. Allergic transfusion reactions: an evaluation of 273 consecutive reactions. Arch Pathol Lab Med 2003;127:316–320.
Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. The Trial to Reduce Alloimmunization to Platelets Study Group. N Engl J Med 1997;337:1861–1869.
Rozman P. Platelet antigens. The role of human platelet alloantigens (HPA) in blood transfusion and transplantation. Transpl Immunol 2002;10:165–181.
Wagner SJ, Friedman LI, Dodd RY. Transfusion-associated bacterial sepsis. Clin Microbiol Rev 1994;7:290–302.
Dutta U, Raina V, Garg PK et al. A prospective study on the incidence of hepatitis B & C infections amongst patients with lymphoproliferative disorders. Indian J Med Res 1998;107:78–82.
Bornstein R, Garcia-Vela J, Gilsanz F et al. Cord blood megakaryocytes do not complete maturation, as indicated by impaired establishment of endomitosis and low expression of G1/S cyclins upon thrombopoietin-induced differentiation. Br J Haematol 2001;114:458–465.
Mattia G, Vulcano F, Milazzo L et al. Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release. Blood 2002;99:888–897.
Schipper LF, Brand A, Reniers N et al. Differential maturation of megakaryocyte progenitor cells from cord blood and mobilized peripheral blood. Exp Hematol 2003;31:324–330.
Miyazaki R, Ogata H, Iguchi T et al. Comparative analyses of megakaryocytes derived from cord blood and bone marrow. Br J Haematol 2000;108:602–609.
Sola MC, Rimsza LM. Mechanisms underlying thrombocytopenia in the neonatal intensive care unit. Acta Paediatr Suppl 2002;91:66–73.
Sola MC, Calhoun DA, Hutson AD et al. Plasma thrombopoietin concentrations in thrombocytopenic and non-thrombocytopenic patients in a neonatal intensive care unit. Br J Haematol 1999;104:90–92.
Nakorn TN, Miyamoto T, Weissman IL. Characterization of mouse clonogenic megakaryocyte progenitors. Proc Natl Acad Sci U S A 2003;100:205–210.
Hadjantonakis AK, Gertsenstein M, Ikawa M et al. Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech Dev 1998;76:79–90.
Jackson CW, Brown LK, Somerville BC et al. Two-color flow cytometric measurement of DNA distributions of rat megakaryocytes in unfixed, unfractionated marrow cell suspensions. Blood 1984;63:768–778.
de Alarcon PA, Graeve JL. Analysis of megakaryocyte ploidy in fetal bone marrow biopsies using a new adaptation of the feulgen technique to measure DNA content and estimate megakaryocyte ploidy from biopsy specimens. Pediatr Res 1996;39:166–170.
Debili N, Issaad C, Masse JM et al. Expression of CD34 and platelet glycoproteins during human megakaryocytic differentiation. Blood 1992;80:3022–3035.
Kuwaki T, Hagiwara T, Yuki C et al. Quantitative analysis of thrombopoietin receptors on human megakaryocytes. FEBS Lett 1998;427:46–50.
van den Oudenrijn S, von dem Borne AE, de Haas M. Differences in megakaryocyte expansion potential between CD34(+) stem cells derived from cord blood, peripheral blood, and bone marrow from adults and children. Exp Hematol 2000;28:1054–1061.
Avecilla ST, Hattori K, Heissig B et al. Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis. Nat Med 2004;10:64–71.
Guerriero R, Mattia G, Testa U et al. Stromal cell-derived factor 1alpha increases polyploidization of megakaryocytes generated by human hematopoietic progenitor cells. Blood 2001;97:2587–2595.
Ponomaryov T, Peled A, Petit I et al. Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function. J Clin Invest 2000;106:1331–1339.
Sola MC, Christensen RD, Du Y et al. Neonates fail to increase their marrow megakaryocyte mass in response to thrombocytopenia. Pediatr Res 2002;51:242a.
Harrison DE, Astle CM. Short- and long-term multilineage repopulating hematopoietic stem cells in late fetal and newborn mice: models for human umbilical cord blood. Blood 1997;90:174–181.
Morrison SJ, Hemmati HD, Wandycz AM et al. The purification and characterization of fetal liver hematopoietic stem cells. Proc Natl Acad Sci U S A 1995;92:10302–10306.
Wolf NS, Bertoncello I, Jiang D et al. Developmental hematopoiesis from prenatal to young-adult life in the mouse model. Exp Hematol 1995;23:142–146.
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Adams GB, Chabner KT, Foxall RB et al. Heterologous cells cooperate to augment stem cell migration, homing, and engraftment. Blood 2003;101:45–51.
Szilvassy SJ, Meyerrose TE, Ragland PL et al. Differential homing and engraftment properties of hematopoietic progenitor cells from murine bone marrow, mobilized peripheral blood, and fetal liver. Blood 2001;98:2108–2115.
Barker JN, Weisdorf DJ, Wagner JE. Creation of a double chimera after the transplantation of umbilical-cord blood from two partially matched unrelated donors. N Engl J Med 2001;344:1870–1871.
Christopherson KW 2nd, Hangoc G, Broxmeyer HE. Cell surface peptidase CD26/dipeptidylpeptidase IV regulates CXCL12/stromal cell-derived factor-1 alpha-mediated chemotaxis of human cord blood CD34+ progenitor cells. J Immunol 2002;169:7000–7008.(William B. Slaytona,b, Da)
b Program in Stem Cell Biology and Regenerative Medicine, University of Florida Shands Cancer Center, Gainesville, Florida, USA;
c The Blood and Marrow Transplant Program, University of Florida, Gainesville, Florida, USA
Key Words. Megakaryocytopoiesis ? Thrombopoiesis ? Adult bone marrow stem cells ? Umbilical cord blood ? Development
Correspondence: William B. Slayton, M.D., J. Hillis Miller Health Center, Box 100296, Gainesville, Florida 32610, USA. Telephone: 352-392-5633; Fax: 352-392-2875; e-mail: slaytwb@peds.ufl.edu
ABSTRACT
Umbilical cord blood contains stem cells that can be used for hematopoietic stem cell transplantation in patients who lack a sibling or matched unrelated bone marrow donor. Slow engraftment and graft rejection account for the majority of transplant-related morbidity and mortality after cord blood transplantation . Platelet engraftment is particularly slow, taking an average of approximately 70 days for cord blood compared with 20 days for mobilized peripheral blood stem cells . Prolonged thrombocytopenia leads to an increased risk of fatal bleeding and the risks associated with multiple transfusions, including anaphylaxis , alloimmunization , and infection .
Several groups have suggested that qualitative differences between neonatal and adult megakaryocytes and their progenitors may contribute to delayed platelet engraftment after cord blood transplant . One specific qualitative difference is that neonatal megakaryocytes are smaller and have lower ploidy than adult megakaryocytes . Smaller megakaryocytes with lower ploidy produce fewer platelets in vitro . A second qualitative difference is that megakaryocyte progenitors from human cord blood proliferate more in culture than progenitors derived from adult peripheral blood or bone marrow . Proposed mechanisms to explain the increased proliferation and decreased maturation of neonatal megakaryocyte progenitors include differences in the expression of cell cycle proteins that control endomitosis and delayed expression of the thrombopoietin receptor in neonatal cells . Studies that defined these molecular differences were performed in culture in response to recombinant thrombopoietin. These in vitro studies do not, however, reliably reproduce the complex bone marrow or splenic microenvironment (in the mouse) in which megakaryocytes develop after transplant.
We used a method developed by Nakorn et al. to track donor-derived platelets post-transplant, using transgenic mice that express green fluorescent protein (GFP) as donors . We used this model to test the hypothesis that neonatal stem and progenitor cells have an intrinsic tendency to produce small megakaryocytes with low DNA content, and that these small megakaryocytes lead to slower platelet engraftment after transplant.
MATERIALS AND METHODS
Megakaryopoiesis in Newborn and Adult Mice
We first sought to determine whether neonatal murine liver megakaryocytes and progenitors were phenotypically similar in size, ploidy, and proliferative capacity to their human counterparts. Specifically, we sought to determine whether neonatal megakaryocytes were smaller and had lower DNA content than those from adult mice. The number of megakaryocytes in the liver of newborn animals was much higher than in the spleen, where megakaryocytes were rarely found, or in the bone marrow, which had small marrow spaces and contained few megakaryocytes. As in human fetuses and neonates, neonatal murine liver megakaryocytes (from GFP animals) were smaller (p .00005; Fig. 1A) and of lower ploidy (p < .00001; Fig. 1B) than megakaryocytes in the adult bone marrow or spleen. Adult splenic megakarocytes were significantly larger than those in the adult marrow (p = .03). As the pups grew and matured, the liver megakaryocytes increased in size but were still significantly smaller than adult bone marrow megakaryocytes at 7 days of life (p = .02). Furthermore, the neonatal cells proliferated more than adult cells, as shown by the increased proportion of burst-forming unit-megakaryocyte (BFU-meg) cultured from neonatal liver cells (Fig. 1C). These experiments established the similarities between neonatal mouse and neonatal human megakaryocytes in terms of size, ploidy, and proliferative capacity and suggested that our system would be useful to study whether these differences persist after transplant.
Figure 1. Comparison of newborn and adult megakaryocytes in different organs. (A): Volume of megakaryocytes in the newborn liver, newborn spleen, 1-week-old liver, adult bone marrow, and adult spleen. Megakaryocyte volumes were calculated from their diameter, assuming a spherical shape. Error bars denote SEM (n = 5–15 mice per cohort, 25–100 megakaryocytes per mouse). One-week-old liver megakaryocytes were significantly larger than newborn megakaryocytes but smaller than adult bone marrow megakaryocytes (p = .02) (B): Ploidy analysis of newborn and adult animals. Megakaryocyte ploidy was measured in the newborn liver and adult bone marrow, and compared with 2N controls. (C): Megakaryocyte colony formation. The ability of neonatal (black bar) and adult (gray bar) cells to produce megakaryocyte colonies in collagen in response to interleukin-3 and thrombopoietin was compared (n = total of 10 mice per cohort, and represents the combined data from three separate experiments). Abbreviations: BFU-meg, burst-forming unit-megakaryocyte; CFU-meg, colony-forming unit-megakaryocyte; PI, propidium iodide.
Stem and Progenitor Cell Numbers
We compared the number of stem and progenitor cells in neonatal liver versus adult bone marrow by measuring the percentage of total cells that did not express mature lineage markers (Linneg) and expressed the stem cell markers c-kit and Sca-1 (SKL cells). We found that the percentage of SKL cells per total nucleated cells was remarkably constant and constituted approximately 0.5% of the total nucleated cells in both the newborn liver and adult bone marrow cell suspensions (n = four neonatal livers and adult bone marrows analyzed).
Platelet Engraftment Kinetics
We then transplanted equal numbers of unfractionated, hemolyzed neonatal liver and adult bone marrow cells from transgenic mice expressing GFP into lethally irradiated adult C57/B6 mice and followed platelet engraftment kinetics. We saw no difference in the peripheral blood platelet engraftment kinetics between animals receiving newborn liver or adult bone marrow cells. In contrast to irradiated controls, animals transplanted with 1.5 x 106 donor cells had a rapid increase in platelet counts between days 7 and 14, regardless of whether they received adult or neonatal cells. Platelet counts in transplanted animals reached the levels of healthy controls 4 weeks post-transplant (Fig. 2A). Donor-derived platelets were detected as early as 7 days after transplant, using flow cytometry for green fluorescence, and reached levels of healthy GFP control animals by 2 weeks post-transplant (Fig. 2B). Platelet engraftment was sustained at the 1-month and 4-month time points, regardless of whether animals received neonatal or adult donor cells.
Figure 2. Platelet engraftment and chimerism. (A): Platelet engraftment after transplantation of stem and progenitor cells derived from neonatal liver () or adult bone marrow (). Controls consisted of platelet counts from healthy animals (broken line, ) and animals that were irradiated but received no donor stem cells (x). (B): Donor-derived platelets from newborn liver (black) and adult bone marrow (gray) as identified by green fluorescence. Control consisted of platelets from healthy green fluorescent protein transgenic mice.
Changes in Marrow and Splenic Hematopoiesis
To understand the ability of our transplanted neonatal cells and adult cells to support post-transplant hematopoiesis, we measured the relative changes in cellularity within the bone marrow and spleen. Hematopoiesis increased dramatically in the spleen during the first 2 weeks after transplant, effacing normal splenic architecture. In fact, spleens nearly doubled in weight relative to healthy controls 7 days post-transplant due to hematopoietic expansion (Fig. 3A). Bone marrow cellularity was similar regardless of stem cell source. In stark contrast to the spleen, marrow cellularity, measured as leukocyte counts per single femur, was 17% of the cellularity of healthy controls 7 days post-transplant but was four times higher than irradiated controls. Marrow cellularity approached healthy control levels 4 weeks post-transplant, only to decrease by 4 months post-transplant (Fig. 3B).
Figure 3. Changes in hematopoietic activity in the spleen and liver post-transplant. (A): Changes in splenic cellularity were determined by measuring the spleen weight relative to body weight in animals that received neonatal (black) and adult (gray) cells. Controls consisted of healthy C57/B6 animals (x) and irradiated controls that did not receive transplanted cells (). (B): Changes in bone marrow cellularity from a single flushed femur.
Megakaryocyte Size
We then measured the diameter of megakaryocytes at each time point post-transplant. At 7 and 14 days post-transplant, both adult bone marrow and newborn liver cells gave rise to megakaryocytes that were larger than normal adult megakaryocytes. However, megakaryocytes derived from newborn cells were significantly smaller than those derived from adult bone marrow cells (p = .056) 7 days post-transplant. This difference was less apparent throughout the rest of the time course. Regardless of the source of transplanted cells, the largest megakaryocytes were found in the spleen. In fact, 1 week post-transplant, newborn liver cells produced splenic megakaryocytes that were six times larger than normal newborn liver megakaryocytes and nearly three times larger in volume than normal adult bone marrow megakaryocytes (Figs. 4A–4C). Megakaryocyte size decreased to control levels by 4 months post-transplant.
Figure 4. Changes in megakaryocyte size. (A): Perivascular, small megakaryocytes in the newborn liver. (B): Perivascular megakaryocytes in the spleen 7 days after transplant of NL cells. These are considerably larger than NL megakaryocytes. (C): Mean megakaryocyte volume in the bone marrow in animals transplanted with NL (black) or ABM (gray) cells. Controls consisted of bone marrow megakaryocytes from healthy adult animals () or megakaryocytes from the newborn liver (). (D): Mean megakaryocyte volume in the spleen in animals transplanted with NL or ABM cells. Controls consisted of splenic megakaryocytes from healthy adult animals () or from newborn liver (). Error bars denote SEM. Abbreviations: ABM, adult bone marrow; NL, neonatal liver.
Megakaryocyte DNA Content
Marrow megakaryocyte ploidy analysis was performed by flow cytometry using flushed bone marrow cells treated with hypotonic citrate, as previously described. On post-transplant days 7 and 14, the small number of megakaryocytes in the bone marrow precluded the measurement of ploidy levels. Similar to our size observations, however, at post-transplant day 18, megakaryocytes derived from adult bone marrow cells reached higher ploidy levels than megakaryocytes derived from newborn liver cells. Specifically, megakaryocytes derived from newborn liver exhibited a ploidy distribution that was remarkably similar to that of megakaryocytes in healthy adult bone marrow, with a median ploidy of 16N. In contrast, megakaryocytes from animals receiving adult bone marrow displayed higher than normal ploidy levels, with more cells reaching 32N (Fig. 5A). This result did not vary from animal to animal based on degree of thrombocytopenia, but rather seemed to be fixed based on the developmental state of the donor cells. By 1 month post-transplant, ploidy levels from newborn liver– and adult bone marrow–derived megakaryocytes were almost identical and were further shifted toward 32N. By 4 months post-transplant, ploidy in both cohorts had reverted to the levels of healthy adult controls (Fig. 5A).
Figure 5. Ploidy analysis of megakaryocytes from neonatal and adult donors. (A): Changes in megakaryocyte ploidy in the bone marrow after transplant by flow cytometry. (B): Changes in mean ploidy of splenic megakaryocytes after transplant by Feulgen staining. Abbreviation: PI, propidium iodide.
Ploidy analysis was also performed in the post-transplant spleen using Feulgen staining, and the results were compared with both healthy adult spleen and neonatal liver. At 7 days post-transplant, newborn liver–derived megakaryocytes had a mean ploidy of 17N (median of 16N) compared with 7N (median 8N) in normal newborn liver. Megakaryocytes derived from adult bone marrow cells also exhibited a higher mean ploidy (15 N, median 16N) than normal adult splenic megakaryocytes (mean 10N, median 16N). These differences were not statistically significant. Megakaryocyte ploidy decreased in parallel with the gradual decrease in megakaryocyte size over the 4-month observation period (Fig. 5B).
DISCUSSION
In summary, we have shown that neonatal stem and progenitor cells are capable of producing adult-sized megakaryocytes when placed in an adult microenvironment in the mouse. This study suggests that the small size and lower DNA content of neonatal megakaryocytes is due to both microenvironmental and cell-intrinsic factors. Understanding these factors may lead to improvements in platelet and overall engraftment after cord blood transplant.
ACKNOWLEDGMENTS
Gluckman E, Rocha V, Chevret S. Results of unrelated umbilical cord blood hematopoietic stem cell transplant. Transfus Clin Biol 2001;8:146–154.
Laughlin MJ, Barker J, Bambach B et al. Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors. N Engl J Med 2001;344:1815–1822.
Couban S, Simpson DR, Barnett MJ et al. A randomized multicenter comparison of bone marrow and peripheral blood in recipients of matched sibling allogeneic transplants for myeloid malignancies. Blood 2002;100:1525–1531.
Sanz GF, Saavedra S, Planelles D et al. Standardized, unrelated donor cord blood transplantation in adults with hematologic malignancies. Blood 2001;98:2332–2338.
Domen RE, Hoeltge GA. Allergic transfusion reactions: an evaluation of 273 consecutive reactions. Arch Pathol Lab Med 2003;127:316–320.
Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. The Trial to Reduce Alloimmunization to Platelets Study Group. N Engl J Med 1997;337:1861–1869.
Rozman P. Platelet antigens. The role of human platelet alloantigens (HPA) in blood transfusion and transplantation. Transpl Immunol 2002;10:165–181.
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