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a University of Florida Department of Pediatrics, Gainesville, Florida, USA;
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/ n, r8 b, R9 s' g. T: N1 u7 Zb Program in Stem Cell Biology and Regenerative Medicine, University of Florida Shands Cancer Center, Gainesville, Florida, USA;) B& A5 r5 b$ x" |7 }
, W0 h" ]5 r: z4 Jc The Blood and Marrow Transplant Program, University of Florida, Gainesville, Florida, USA" M6 |# g" Q- g
2 x0 m- _& l) T8 R- U7 E5 A, }Key Words. Megakaryocytopoiesis ? Thrombopoiesis ? Adult bone marrow stem cells ? Umbilical cord blood ? Development6 E% _; L' e6 U3 S& ~2 m) x0 @
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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& ?5 Q9 ^" o/ ^3 j) X
! D. g4 n* q5 cABSTRACT
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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 .& k; @ N$ h& o
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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.4 k* \4 T% y+ b$ B
) n6 U/ Q7 f5 Y9 E9 n: pWe 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.7 ~2 w6 `7 |! _" H$ N0 u6 {
7 J; ^! d" {9 J' O1 N! vMATERIALS AND METHODS1 D) v- x3 v! G0 K5 v" m0 S. c. N9 F
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Megakaryopoiesis in Newborn and Adult Mice
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# p+ K/ @4 T2 E) rWe 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
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* Q. N) ~7 |+ `0 FFigure 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.
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: t5 b+ y/ ^9 K6 Y- r4 wStem and Progenitor Cell Numbers
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" J0 C) E& ~; p# P: x, b; {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).' L2 d) H" W, p+ s/ ] P
: m5 c4 T) T u* Z5 EPlatelet Engraftment Kinetics
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# k+ X3 v Y9 E) zWe 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.
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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.% ~5 c6 a' P' g. k5 Y! e
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Changes in Marrow and Splenic Hematopoiesis
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) R3 Z; t8 M* J4 A3 jTo 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).
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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.
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Megakaryocyte Size. {& j4 h6 U: I7 e) Y( `% o
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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.
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/ Z' A; i" i7 Z0 zFigure 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.
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Megakaryocyte DNA Content
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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).9 l- \$ b% |$ v9 G; ]- H
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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.
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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).& l! C8 |8 [1 K/ F) @
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DISCUSSION
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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.+ @% V7 Z3 B: ^% n- c
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