Umbilical Cord Blood Produces Small Megakaryocytes After Transplantation

Introduction 

Umbilical cord blood (CB) is an important stem cell source for patients who lack other suitable donors. However, slower platelet engraftment is a major drawback of CB transplantation [1]. Platelet engraftment takes an average of approximately 70 days for CB recipients, versus 20 days for mobilized peripheral blood cells derived from adult donors [2, 3]. Posttransplantation thrombocytopenia prolongs the time to transfusion independence and exposes patients to complications of transfusions [4, 5, 6, 7].

Current theories attribute delayed engraftment after CB transplantation to decreased numbers of stem cells compared with other sources, and decreased stem cell number clearly plays a role in delayed engraftment [8, 9]. However, in children, in whom low numbers of stem cells is less of a factor because of lower body weight, CB transplants still have delayed engraftment compared with mobilized peripheral blood or bone marrow (BM) transplants from adult sources [9]. Some have suggested that in addition to the differences in numbers of stem cells between the CB and mobilized peripheral blood or BM products, intrinsic differences in stem cells from neonatal and older donors contribute to the delayed platelet engraftment after CB transplantation [10, 11, 12, 13].

Megakaryocytes of neonates are smaller and have lower ploidy than those of adults [10, 11, 12, 13, 14, 15, 16]. Megakaryocytes achieve adult size at approximately age 1 year, similar to the switch from fetal to adult hemoglobin [17]. Because evidence suggests that small megakaryocytes produce fewer platelets than large megakaryocytes [11, 18], it has been hypothesized that an inability to increase megakaryocyte size and ploidy in response to increased platelet consumption might underlie the predisposition of sick neonates to thrombocytopenia [19]. Applying this logic to the setting of CB transplantation, and based on observations in vitro and in mice [10, 11, 12, 13, 14, 15, 16], we hypothesized that transplanted neonatal stem and progenitor cells would produce smaller megakaryocytes after hematopoietic stem cell transplantation compared with the megakaryocytes produced by adult stem and progenitor cells. We tested this hypothesis by comparing the size, megakaryocyte concentration, and maturational stage of megakaryocytes in the BM of children who received CB transplants with those who received mobilized peripheral blood or BM transplants.

Patients and methods 

Results 

A total of 36 patients (22 with CB and 14 with BM) had achieved platelet engraftment and were eligible for this study. The time to platelet engraftment was significantly longer in the CB group than in the BM group (median, 47 vs 21.5 days, respectively; P < .001). A total of 19 (10 CB, 9 BM) of these patients had a posttransplantation BM biopsy specimen that contained identifiable megakaryocytes. Megakaryocyte concentration, size, and developmental stage were evaluated in engrafted patients who had a BM biopsy specimen that revealed megakaryocytes. The megakaryocyte concentration adjusted for cellularity was similar in the 2 groups (2.28 vs 2.18 megakaryocytes/mm2; P = .85).

In contrast, the megakaryocytes derived from CB were significantly smaller than those derived from adult sources (Figure 1A–C). Megakaryocytes tended to be small and to vary little in size in patients receiving CB transplant (Figure 1A). In contrast, megakaryocytes tended to be larger and more variable in size in patients receiving mobilized peripheral blood or BM (Figure 1B). The median diameter of megakaryocytes was significantly smaller in the CB group than in the BM group (16.7 vs 22.0 μm; P < .001; Figure 1C).

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Figure 1. Representative BM megakaryocytes stained with antibody to CD61 followed by diaminobenzidine (megakaryocytes are brown). (A) Megakaryocytes in a 4-year-old patient 1 month after CB transplantation. Megakaryocytes are small and of relatively uniform size. These megakaryocytes have a single or bilobed nucleus, typical of micromegakaryocytes [40]. (B) Megakaryocytes in a 1-year-old patient 1 month after mobilized peripheral blood transplantation from an adult donor. Megakaryocytes are larger and have more size heterogeneity. (C) Mean megakaryocyte diameter is significantly smaller after CB transplantation than after transplantation of hematopoietic cells from adult sources 1–3 months posttransplantation. (Line = median; box = 25%–75% quartiles; bars = range of individual medians; n = 10 CB transplants; n = 9 BM transplants.)

To determine the combined effect of size and concentration on platelet production, we calculated megakaryocyte mass [25] from each patient and calculated the difference in platelet production per mm2 from CB and adult BM assuming a mean platelet volume of 10 fL. Patients receiving mobilized peripheral blood or BM produced platelets at nearly 3 times the rate as CB recipients (median, 2559 platelets/mm2 vs 893 platelets/mm2; P = .069; Table 1).

Table 1. Megakaryocyte Mass and Platelet Production

	Stem Cell Source	Megakaryocyte Concentration (Megakaryocytes/ mm2)	Megakaryocyte Mass (Megakaryocyte Concentration × Megakaryocyte Volume, P =.069)	Platelets Produced per Square Millimeter (Assuming a Mean Platelet Volume of 10 fL)
	CB	2.28	8933fL/mm2	893
	Mobilized peripheral blood or BM	2.18	25,596fL/mm2	2559

Despite the size differences, there were no significant differences between CB and BM recipients in maturity level of megakaryocytes according to morphological staging (Figure 2). However, CB cells showed a trend toward producing a lower percentage of stage III megakaryocytes, which in normal adults compose the largest megakaryocyte maturational class (25% stage III megakaryocytes in the CB group vs 44% in the BM group; P = .059). In contrast, the percentage of stage IV mature megakaryocytes was higher in the CB group than in the BM group (36% vs 27% stage IV megakaryocytes; P = .19). When stage III and IV megakaryocytes were combined, the percentages of “mature” megakaryocytes were similar.

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Figure 2. Hematoxylin and eosin–stained sections showing individual megakaryocytes assigned a maturational stage based on size, nuclear lobulation, ratio of nucleus to cytoplasm, and degree of cytoplasmic maturation. CB stem and progenitor cells produced a lower percentage of stage III megakaryocytes (25% CB vs 44% BM; P = .059), which in normal adults is the largest megakaryocyte maturational class. In contrast, CB cells produced a higher percentage of stage IV mature megakaryocytes. When stage III and IV (mature) megakaryocytes are considered together, there is little difference between CB and BM transplants.

Discussion 

The results of this study suggest that neonatal and adult hematopoietic stem and megakaryocyte progenitors have intrinsic differences in their ability to produce large megakaryocytes after transplantation. These differences had been demonstrated in tissue culture [10, 11, 12, 13, 14, 15, 16] and in a mouse model of stem cell transplantation, [13], but this is the first time that small megakaryocyte size has been reported posttransplantation in human subjects. The tendency of neonatal stem cells to produce smaller megakaryocytes may contribute to the delayed platelet engraftment after CB transplantation.

In our previous study focusing on the differences in megakaryocyte size after transplantation of stem and progenitor cells from newborn murine liver and BM, we found a rapid increase in the size of megakaryocytes to adult size after transplantation of neonatal cells into the adult environment. However, these megakaryocytes were still significantly smaller than those derived from BM and also had lower ploidy levels. This suggests that both cell intrinsic and microenvironmental factors play a role in megakaryocyte size after transplantation.

Megakaryocyte mature along BM sinusoids, termed the vascular niche, which provide substances that promote megakaryocyte maturation [26]. In contrast to the mouse model, megakaryocyte size increases more slowly after transplantation of neonatal CB cells in humans. Because the current study was performed in decalcified samples, we were unable to perform ploidy analysis to determine whether ploidy differed between the CB and BM/mobilized peripheral blood cohorts.

The age at which megakaryocytes achieve an adult mean megakaryocyte diameter size in humans is unknown. At the time of this study, few adult patients had undergone CB transplantation, and no BM samples from engrafted adults were available for analysis. However, in our study of pediatric transplant recipients, megakaryocyte size was highly correlated with the source of the transplant, but not with the age of the recipient. The youngest patient to receive a mobilized peripheral blood stem cell transplant was age 1 year and had a megakaryocyte size well within the normal range for an adult (21.7 μm). In contrast, the oldest patient to receive CB was age 10 years and had smaller megakaryocytes than any patients who received hematopoietic stem cells from adult sources (mean megakaryocyte diameter, 18.7 μm). Sola and Rimzsa, using the same methods that we used in the current study, demonstrated that the mean megakaryocyte diameter was significantly higher in adults than in neonates (19.4 vs 15.3 μm). The median megakaryocyte diameters seen in patients after CB transplantation lie between those of neonatal megakaryocytes and adult megakaryocytes, suggesting that CB megakaryocyte progenitors respond to environmental cues to increase megakaryocyte size, although this maturational response is blunted compared with more mature stem and progenitor cell sources. In contrast, and using these same benchmarks, adult megakaryocytes 1–3 months posttransplantation into children were larger than megakaryocytes in healthy adults.

Performing megakaryocyte maturational analysis was difficult on archived BM specimens. The best method for analyzing ploidy level is by flow cytometry, measuring DNA content with propidium iodide staining, but this was not possible in fixed, archived samples. Another potential method for measuring DNA content would be through Feulgen staining [14, 27], but this was not possible because the process of decalcifying BM biopsy specimens denatures DNA. Thus, we asked a hematopathologist to morphologically assess maturational stage, based on the size of each megakaryocyte, using a method that incorporates the megakaryocyte size, ratio of nucleus to cytoplasm, and degree of lobulation of the nucleus [20, 21]. Using this method, we found that megakaryopoiesis in the CB group resembled that of the neonate; small megakaryocytes with mature appearing nuclei and cytoplasm (termed “mature micromegakaryocytes”) have been described in CB [19]. These small megakaryocytes have mature-appearing cytoplasm and lobulated nuclei or compacted nuclei and also have a lower DNA content than adult megakaryocytes. Prospectively collecting marrow samples and measuring megakaryocyte ploidy would be a better method for detecting differences in ploidy level after transplantation. In the mouse, such differences were apparent up to 1 month posttransplantation, with megakaryocytes derived from neonatal cells showing lower ploidy levels than megakaryocytes derived from adults [13].

Because children achieve adult megakaryocyte size at age 1 year, which coincides with the time at which hemoglobin switches from fetal to adult forms, a relationship between these 2 phenomena has been suggested [19]. There is evidence that fetal hemoglobin is produced at high levels for several months in patients receiving CB transplants, supporting the idea that a cell intrinsic neonatal erythroid phenotype persists for several months posttransplantation [28, 29, 30, 31]. Megakaryocytes and erythrocytes share common molecular pathways [32, 33, 34, 35, 36], and our results suggest that a neonatal megakaryocytic phenotype also persists posttransplantation. However, the molecular mechanisms that might underlie the switch from a neonatal to an adult megakaryocyte phenotype are poorly understood. Neonatal and adult megakaryocyte progenitors respond differently to the cytokine thrombopoietin in vitro, with neonatal cells predominantly proliferating, remaining small and diploid, and adult cells endoreduplicating, becoming polyploid, and enlarging [37, 38]. At a molecular level, neonatal megakaryocyte precursors had decreased expression of cyclins E and A, which are important regulators of endomitosis [10]. Furthermore, delays in the cellular expression of another mediator of endomitosis, cyclin D3, were evident in CB-derived megakaryocytes precursors compared with those from adult peripheral blood [11]. CB-derived megakaryocytes have been shown to have delayed expression of important megakaryocytic proteins, such as GP IIb/IIIa [12, 37], the thrombopoietin receptor c-mpl [37], platelet factor 4, and β-thromboglobulin, compared with adult-derived megakaryocytes [11]. These molecular differences cause CB cells to produce smaller megakaryocytes and have been associated with lower levels of platelet release in vitro [11]. Recent work by our group shows that neonatal and adult hematopoietic stem and progenitor cells proliferate and mature very differently in response to thrombopoietin plus adult conditioned medium derived from BM stromal cells [39]. Our current study suggests that these developmental differences persist after CB transplantation and could play a role in delayed platelet engraftment.

Although this study demonstrates that megakaryocytes derived from CB are smaller posttransplantation than those derived from BM or mobilized peripheral blood, it does not establish the relationship between small megakaryocyte size and time to platelet recovery. This small study demonstrates the need for a larger prospective study to evaluate the role of small megakaryocyte size in delayed platelet engraftment. That study could evaluate the size of megakaryocytes at 1-month intervals posttransplantation until adult megakaryocyte size was reached, and perform parallel evaluations of hemoglobin electrophoresis to determine the timing of hemoglobin switching. The study could include ploidy analysis, megakaryocyte colony assays, measurements of proliferative and maturational response to thrombopoietin [39], and platelet production assays at each time point [11]. Such a study also could assess the effect of cord size, graft-versus-host disease, and the use of more than 1 donor on megakaryocyte size. Finally, assays to measure activity of molecules involved in developmental differences in endoreduplication, such as cyclins D3, E, A, and B, could be performed to determine whether and how long these molecular differences persist in vivo after transplantation [10, 11].

In summary, the megakaryocytes derived from CB are significantly smaller than those derived from mobilized peripheral blood or BM, at least when measured 1–3 months posttransplantation in pediatric patients. Smaller megakaryocytes may contribute to the delayed platelet engraftment seen after CB transplantation compared with transplants from adult hematopoietic stem cell sources. Understanding the molecular pathways that lead to small megakaryocyte size might provide new pharmacologic targets for treating thrombocytopenia in neonates and after CB transplantation.