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Original A rticle Developmental Differences in Megakaryocyte Maturation Are Determined by the Microenvironment William B. Slayton,a,b David A. Wainman,b Xiao Miao Li,a Zhongbo Hu,a Anil Jotwani,a Christopher R. Cogle,b Danielle Walker,a Robert C. Fisher,b John R. Wingard,c Edward W. Scott,b Martha C. Solaa University of Florida Department of Pediatrics, Gainesville, Florida, USA; bProgram 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

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Key Words. Megakaryocytopoiesis • Thrombopoiesis • Adult bone marrow stem cells • Umbilical cord blood • Development

Abstract Historically, physicians have attributed delayed platelet engraftment following umbilical cord blood transplant to decreased numbers of stem cells in cord blood compared with adult bone marrow. However, recent studies suggest that delayed platelet engraftment may be caused by an intrinsic inability of neonatal stem cells to produce mature, polyploid megakaryocytes. We tested this hypothesis by transplanting adult bone marrow and newborn liver hematopoietic stem and progenitor cells from transgenic mice expressing green fluorescent protein into myeloablated wild-type recipients and comparing the size and ploidy levels of megakaryocytes that developed in adult transplant recipients. Transplanted stem and progenitor cells, regardless of their source, gave rise to megakaryocytes that were larger than normal adult megakaryocytes as early as 7 days post-transplant. However, megakaryocytes that developed after transplant of neonatal stem

and progenitor cells were significantly smaller than those derived from adult stem and progenitor cells. Furthermore, megakaryocytes derived from neonatal cells had lower ploidy values than megakaryocytes derived from adult cells at 18 days post-transplant, when ploidy could first be reliably measured in the bone marrow. These differences in size and ploidy disappeared by 1 month post-transplant. The largest megakaryocytes developed in the spleen. These results suggest that, in the mouse, the microenvironment is responsible for some of the maturational differences in size and ploidy between neonatal and adult megakaryocytes. Furthermore, neonatal and adult megakaryocyte progenitors also have cell-intrinsic differences in the way they engraft and respond to thrombocytopenic stress. These differences may contribute to the delay in platelet engraftment that frequently complicates cord blood transplants. Stem Cells 2005;23:1400–1408

Introduction

approximately 70 days for cord blood compared with 20 days for mobilized peripheral blood stem cells [3, 4]. Prolonged thrombocytopenia leads to an increased risk of fatal bleeding and the risks associated with multiple transfusions, including anaphylaxis [5], alloimmunization [6, 7], and infection [8, 9]. Several groups have suggested that qualitative differences between neonatal and adult megakaryocytes and their progenitors

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 [1, 2]. Platelet engraftment is particularly slow, taking an average of

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: [email protected] Received December 28, 2004; accepted for publication May 17, 2005. ©AlphaMed Press 1066-5099/2005/$12.00/0 doi: 10.1634/stemcells.2004-0373

Stem Cells 2005;23:1400–1408 www.StemCells.com

Slayton, Wainman, Li et al. may contribute to delayed platelet engraftment after cord blood transplant [10–13]. One specific qualitative difference is that neonatal megakaryocytes are smaller and have lower ploidy than adult megakaryocytes [11, 12, 14]. Smaller megakaryocytes with lower ploidy produce fewer platelets in vitro [11]. 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 [11, 13, 15]. 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 [10, 11] and delayed expression of the thrombopoietin receptor in neonatal cells [13]. 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. [16] to track donor-derived platelets post-transplant, using transgenic mice that express green fluorescent protein (GFP) as donors [17]. 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.

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Flow Cytometric Analysis of Transplanted Cells We used flow cytometry to measure the relative numbers of hematopoietic stem and progenitor cells in each cell suspension. Specifically, we incubated hemolyzed fractions of whole bone marrow or neonatal liver cells with phycoerythrin conjugates of B220, GR-1, CD8, Ter-119, and CD11b. Cells were also incubated with the antibodies against the stem cell markers c-kit (allophyocyanine conjugate) and Sca-1 (fluorescein isothiocyanate [FITC] conjugate). One million cells were analyzed per sample, and a total of four neonatal liver and adult bone marrow samples were analyzed.

Transplants At the time of transplant, 2- to 4-month-old recipient animals were irradiated with 950 cGy from a cesium137 source. Recipient animals were anesthetized with isofluorane and infused retroorbitally with 1.5 × 106 bone marrow or neonatal liver cells from GFP donors. Mice were maintained on water treated with trimethoprim/sulfasoxazole for 1 month after transplant.

Cohorts Data represent five separate experiments and a total of 15 animals per time point. Platelet and megakaryocyte engraftment levels were determined on days 7, 14, 28, and 112 post-transplant. A total of five irradiated control animals and 15 healthy GFP animals was analyzed as controls for these experiments.

Materials and Methods Cell Counts and Platelet Analysis Mice We purchased C57/B6 recipient mice and GFP donor mice from the Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). GFP mice (Tg(GFPu)5Nagy/J) [17] were bred onto the C57/BL6 background at our facility. These studies were approved by the University of Florida Institutional Animal Care and Use Committee.

Preparation of Cell Suspensions for Transplant Bone marrow was flushed from the tibiae and femora of 2- to 4month-old GFP animals. Alternatively, livers from 1- to 2-dayold newborn mice were drawn through serially smaller needles and then filtered to produce a single-cell suspension. Cells were lysed with ammonium chloride, counted, and washed in phosphate-buffered saline (PBS).

Culture of Megakaryocyte Progenitors One × 105 neonatal liver or adult bone marrow cells were cultured in a serum-free collagen-based system (MegaCult; StemCell Technologies, Vancouver, British Columbia, Canada, http://www. stemcell.com) in the presence of 10 ng/ml recombinant murine interleukin-3 and 50 ng/ml recombinant human thrombopoietin. After 5 days, cultures were dehydrated, fixed, and histochemically stained for the presence of acetylcholinesterase. Megakaryocyte colonies were then scored using standard methods.

After anesthesia, a 100-μl blood sample was drawn from the retroorbital sinus into a heparinized capillary tube and dispensed into a silicon tube containing 20 μl of anticoagulant citrate dextrose solution (Baxter International, Inc., Deerfield, IL, http://www.baxter. com). Complete blood counts were measured on an automated cell counter (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Platelet-rich plasma was prepared by adding 400 μl of prewarmed PBS containing 0.33% EDTA with 0.5% paraformaldehyde to each blood sample prior to centrifugation at 90g for 10 minutes. Platelet-rich plasma was then incubated with an anti-CD61-PE (Pharmingen, Franklin Lakes, NJ, http://www. bdbiosciences.com/pharmingen). CD61 is glycoprotein IIIa and is expressed on platelets as well as megakaryocytes, endothelium, and macrophages. We identified donor-derived platelets by flow cytometry using standard techniques, based on characteristic size and granularity profile (platelets are smaller and more granular than the macrophages, megakaryocytes, and endothelium), as well as CD61 expression and green fluorescence [16]. We compared the fluorescence level of transplanted animals with 15 healthy C57/Bl6 and GFP controls. Full engraftment was defined as the percentage of platelets that fluoresced green in healthy GFP control animals. Data are expressed as the percentage of green fluorescence in transplanted animals divided by the percentage of fluorescence in healthy GFP controls.

Developmental Differences in Megakaryopoiesis

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Relative Role of Spleen and Bone Marrow To further determine the relative roles of the spleen and bone marrow after transplant, we measured the expansion or contraction of hematopoiesis in the bone marrow and the spleen. Changes in spleen size were determined by establishing the weight of the spleen in milligrams per gram of body weight at each time point after transplant. Bone marrow leukocyte counts were obtained at each time point from a single femur per animal flushed with PBS.

Measurement of Megakaryocyte Density and Diameter We immunohistochemically stained 6-micron sections of spleens or femurs with an anti–von Willebrand factor antibody (DakoCytomation, Carpinteria, CA, http://www.dakocytomation.us). To calculate megakaryocyte volume, we measured the diameters from up to 70 megakaryocytes per tissue section in 15 animals (up to 750 megakaryocytes/time point/cohort), taking care to measure every megakaryocyte in each section. Measurements were performed in a blinded fashion, without the observer knowing the time after transplant or the source of the transplanted cells. We then calculated megakaryocyte volumes from these diameters, assuming a spherical shape. A power analysis determined that 20 megakaryocyte measurements from 10 animals would detect a difference of 200 femtoliters (half the difference between the volume of neonatal and adult megakaryocytes) with an accuracy of 95%. The same measurements were performed in normal fetal liver and adult bone marrow megakaryocytes from healthy GFP mice to establish whether murine neonatal megakaryocytes were indeed smaller than adult megakaryocytes, thus resembling the developmental differences previously described in humans. In addition, we measured the megakaryocyte size in liver sections obtained from 7-day-old mice to establish the effect of newborn liver growth and development on megakaryocyte size.

Ploidy Analysis of Bone Marrow by Flow Cytometry DNA content of bone marrow from normal and transplanted animals was measured by flow cytometry. Flushed bone marrow was treated with hypotonic citrate for 30 minutes as previously described [18]. Bone marrow cells were incubated with purified, unconjugated rat anti-mouse CD41a antibody (Pharmingen), washed, and incubated with anti-rat Cy-5 (Molecular Probes, Inc., Eugene, OR, http://probes.invitrogen.com). CD41a is the glycoprotein IIb expressed specifically on megakaryocytes, platelets, and rare progenitor cells, and together with glycoprotein IIIa makes up the receptor for von Willebrand factor and other stromal proteins. The cells were treated for 30 minutes with propidium iodide (PI) (50 μg/ml) and RNAse. Megakaryocytes were identified based on size, granularity, and CD41 expression. Ploidy of megakaryocytes was measured by PI fluorescence. Lymphocytes from lymph nodes were used as 2N controls.

Ploidy Analysis of Spleen and Newborn Liver by Feulgen Staining Because autofluorescence made identification of splenic megakaryocytes impossible by flow cytometry, we used a commercially available Feulgen staining kit to determine megakaryocyte ploidy in the spleen after transplant (ChromaVision, San Juan Capistrano, CA, http://www.chromavision.com) [19]. The decalcification of the bones caused degradation of DNA, and as a result, Feulgen staining was not a reliable measure of ploidy in the bone marrow. The DNA content of cells morphologically identified as megakaryocytes was calculated by measuring the staining intensity of their nuclei using a cellular analysis system (ChromaVision). The staining intensity of 10 lymphocyte nuclei was also measured in each slide as diploid standards, against which the ploidy of megakaryocytes was calculated. Mean and median ploidy levels were calculated for each time point, and the expressed median ploidy was rounded upward to the nearest higher ploidy. This assumes that the calculated ploidy level will never be higher than the actual ploidy, but may be lower based on what part of the megakaryocyte nucleus is sectioned.

Statistical Analysis We analyzed our data using Excel (Microsoft, Redmond, WA, http://www.microsoft.com). Means and SEM were determined for all time points. We used the two-tailed Student’s t-test to determine significance of differences, with a level of significance set at p < .05.

Results 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

Slayton, Wainman, Li et al. 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.

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).

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-weekold 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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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 × 10 6 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). Donorderived 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 4month 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 (n). Controls consisted of platelet counts from healthy animals (broken line, p) and animals that were irradiated but received no donor stem cells (×). (B): Donorderived 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.

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Developmental Differences in Megakaryopoiesis

Changes in Marrow and Splenic Hematopoiesis

Megakaryocyte Size

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 posttransplant (Fig. 3B).

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.

Megakaryocyte DNA Content

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 (×) and irradiated controls that did not receive transplanted cells (p). (B): Changes in bone marrow cellularity from a single flushed femur.

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). 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 posttransplant, 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).

Slayton, Wainman, Li et al.

Discussion In this study, we tested the previously proposed hypothesis that neonatal megakaryocytes have a cell-intrinsic deficiency in their ability to endoreduplicate and achieve the large size and ploidy levels seen in adults [10–13, 20–22]. These developmental dif-

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 (n) or megakaryocytes from the newborn liver (p). (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 (p). Error bars denote SEM. Abbreviations: ABM, adult bone marrow; NL, neonatal liver.

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ferences have been suggested to contribute to the delayed platelet engraftment that is often seen after cord blood transplantation in humans. Our results suggest that the microenvironment plays a major role in determining the ultimate size and ploidy of megakaryocytes post-transplant. In fact, neonatal stem cells that made small megakaryocytes with low DNA content in the newborn liver were capable of making megakaryocytes that were adult-

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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sized and had adult ploidy levels when transplanted into an adult environment. Neonatal megakaryocyte progenitors that matured in the adult spleen achieved even larger sizes, suggesting that organ-specific microenvironments vary in their ability to support megakarocyte maturation. Thus, we have demonstrated that the relative small size and low ploidy of newborn megakaryocytes is related to a large extent to microenvironmental differences between the neonate and the adult. It is likely that the balance between factors that inhibit megakaryopoiesis, such as transforming growth factor-β and platelet factor-4, and factors that stimulate megakaryopoiesis, such as granulocyte-macrophage colony stimulating factor, thrombopoietin, interleukin-6, or interleukin-11, is tightly regulated and different in each organ and at each stage of development, leading to diverse microenvironments and contributing to the observed changes in megakaryocyte size and ploidy during development. More recently, bone marrow sinusoidal endothelium has been shown to provide microenvironmental signals to megakaryocyte progenitors that promote maturation, providing a niche for megakaryocytes to mature [23]. In these studies, intravenously administered hematopoietic chemokines, such as stromalderived factor-1 (SDF-1) and fibroblast growth factor-4 (FGF4), drew megakaryocyte progenitors to the sinusoidal vascular niche, where interaction with the endothelium promoted megakaryocyte maturation. Treatment with these chemokines also restored platelet production in thrombopoietin-deficient mice and ameliorated thrombocytopenia after treatment with the myelosuppressive agent 5-fluorouracil. As in mice, SDF-1 promoted megakaryocyte maturation in human CD34pos cells, a subset that includes endothelial progenitors as well as hematopoietic stem and progenitor cells [24]. During bone marrow transplant, conditioning radiation induced increased expression of SDF-1 by the bone marrow sinusoids and osteoblasts, resulting in enhanced migration and retention of hematopoietic stem and progenitor cells in the marrow microenvironment [25]. It is certainly possible that enhanced expression of SDF-1, FGF-4, or other factors related to the sinusoidal niche leads to the rapid increase in size of megakaryocytes that occurs after transplant. Previous observations in our laboratory suggest that human neonates do not increase their megakaryocyte size in response to thrombocytopenia [26], and preliminary findings in a murine model of neonatal consumptive thrombocytopenia suggest that the same is true in the murine fetus (Sola, personal communication). Thus, our findings in the transplant model suggest that, although neonatal megakaryocytes have a developmental limitation in their ability to increase size and ploidy, the adult microenvironment is significantly more conducive than the neonatal microenvironment to the generation of large, mature megakaryocytes, particularly in cases of increased demand.

Developmental Differences in Megakaryopoiesis While neonatal cells were capable of increasing size and ploidy levels to that of the adult when making megakaryocytes in the adult microenvironment, these newborn cells had some cell-intrinsic limitations in their ability to mature compared with their adult counterparts. This was particularly true in the bone marrow 7 days post-transplant, in which the neonatal megakaryocytes were significantly smaller than the adult megakaryocytes. These differences were less apparent in the bone marrow throughout the rest of the time course. This may be due to rapid maturation of the neonatal stem cell that is suggested by the increasing size of megakaryocytes in the newborn liver during the first 7 days of life. The differences in megakaryocyte size may also be related to developmental programs set at the progenitor level and may be the result of preprogrammed progenitors unable to respond to the maturational cues in the adult microenvironment. The net effect of such programs would be that the size of megakaryocytes derived from neonatal cells would be lower than those from adult cells early (day 7 post-transplant), but that these differences would decrease when stem cells begin to be active (day 14). Finally, it may be due to cell-intrinsic differences between neonatal and adult stem and progenitor cells to produce large, polyploid megakaryocytes. Such cell-intrinsic differences in neonatal and adult megakaryocyte progenitor activity have been previously described extensively using human cord blood and adult bone marrow in in vitro systems [10–13, 20–22]. Such systems do not test the activity of hematopoietic stem cells. Further limiting dilution studies and head-to-head comparisons of sorted hematopoietic stem and progenitor cells comparing neonatal and adult cells’ ability to reconstitute mice will be necessary to fully understand the effect of developmental changes on the ability of neonatal and adult stem cells to engraft and produce platelets post-transplant. We chose to use newborn liver hematopoietic stem and progenitor cells from the mouse to model the behavior of human cord blood cells because of the abundance of hematopoietic stem and progenitor cells in the liver and the paucity in the blood and bone marrow of the newborn mouse [27]. Whereas some have predicted little hematopoietic stem cell activity in the neonatal liver based on trends extrapolated from midgestation [28], direct studies of stem cell activity in the neonatal liver demonstrate abundant hematopoietic stem and progenitor cell activity and show that the liver is still the major site of hematopoiesis [27, 29]. Our data confirmed the presence of significant hematopoietic stem cell activity in the murine neonatal liver 1–2 days after birth. Like human neonatal bone marrow and cord blood megakaryocyte progenitors, the megakaryocyte progenitors in the neonatal mouse liver produced megakaryocytes that were smaller, had lower ploidy, and were more proliferative than their adult counterparts (Fig. 1). However, as the pups grow during the first week of life, the size differences between the megakaryocytes in the liver and the adult bone marrow are still present but are less evident. While this may

Slayton, Wainman, Li et al. be related to changes in the ability of the week-old pups’ megakaryocytes to release platelets or microenvironmental changes related to the switch to marrow hematopoiesis, it is also plausible that this increase in size is related to rapid developmental changes in the hematopoietic stem cell. While the significance of our study as it relates to human stem cell transplant is unclear, it does not exclude the possibility that cell-intrinsic developmental differences may be responsible for delayed platelet engraftment after cord blood transplant. This, in addition to the decreased number of stem cells that are present in umbilical cord blood relative to adult bone marrow, may lead to prolonged thrombocytopenia [27, 30, 31]. Other factors that may also contribute to delayed engraftment include decreased numbers of suppressor “accessory” T cells [32], as there are fewer of these cells present in umbilical cord blood than in adult bone marrow. Recent studies in mice show that the net effect of these factors can be more important than any individual effect [33]. Potentially viable solutions to improving platelet engraftment after cord blood transplant include approaches that increase stem cell num-

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Conclusion 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 This study was supported by National Institutes of Health grants HL69990 (M.C.S.), CA72769 (E.W.S.), and HL03962 (W.B.S.).

Disclosures The authors indicate no potential conflicts of interest.

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