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Fetal megakaryocytes (Mks) are phenotypically different from adult Mks.
Fetal Mks are matured as adult Mks.
Fetal Mk phenotypic features predispose to certain Mk-related diseases.
Cell-intrinsic and -extrinsic factors contribute to Mk ontogenic differences.
Fetal Mks can be established in adult mode to enhance ex vivo platelet production.
Fetal megakaryocytes (Mks) differ from adult Mks in key parameters that affect their capacity for platelet production. However, despite being smaller, more proliferative, and less polyploid, fetal Mks generally mature in the same manner as adult Mks. The phenotypic features unique to fetal Mks predispose patients to several disease conditions, including infantile thrombocytopenia, infantile megakaryoblastic leukemias, and poor platelet recovery after umbilical cord blood stem cell transplantations. Ontogenic Mk differences also affect new strategies being developed to address global shortages of platelet transfusion units. These donor-independent, ex vivo production platforms are hampered by the limited proliferative capacity of adult-type Mks and the inferior platelet production by fetal-type Mks. Understanding the molecular programs that distinguish fetal versus adult megakaryopoiesis will help in improving approaches to these clinical problems. This review summarizes the phenotypic differences between fetal and adult Mks, the disease states associated with fetal megakaryopoiesis, and recent advances in the understanding of mechanisms that determine ontogenic Mk transitions.
Megakaryocytes (Mks) are specialized mammalian marrow cells responsible for platelet production. They arise from bipotent megakaryocyte-erythroid progenitors [
]. Their differentiation includes a unique program of endomitosis that drives nuclear polyploidization and cellular enlargement. This process is accompanied by lineage consolidation involving downregulation of erythroid genes and upregulation of Mk surface markers, as well as development of cytoplasmic granules, multivesicular bodies, and demarcation membranes. Upon achieving polyploidization and enlargement, Mks undergo cytoskeletal remodeling to induce the formation of proplatelets, preplatelets, and, ultimately, platelets [
]. During mammalian embryogenesis, primitive Mks with limited polyploidization capacity appear early in the yolk sac. The first definitive Mks arise from hematopoietic progenitors in the fetal liver (FL) and are then produced in the bone marrow (BM). In humans, there are clear phenotypic differences between fetal/neonatal (collectively referred to as fetal) and adult Mks and these differences have major health implications.
Phenotypic differences and similarities between fetal and adult mks
The smaller size of fetal Mks has been well established for several decades (Table 1). Using immunohistochemical staining and light microscope morphometry on healthy human tissues, Allen Graeve and de Alarcon estimated diameters of 14.0–15.2 µm for fetal Mks at 12–21 weeks gestation compared with 18.4–20.6 µm for adult Mks [
]. Subsequent studies confirmed the diameters of human fetal Mks at 3 months gestation to range from 12.4–14.8 µm depending on whether marrow or liver was analyzed. Although diameters reached 16.1 µm by 7 months gestation, fetal Mks remained consistently smaller than those produced in human adult BM (21.9 µm in diameter) [
]. A unimodal Gaussian distribution of Mk size persists in neonates and infants until approximately 24 months. At this age, the size distribution becomes bimodal, with subpopulations of smaller and larger Mks, and by age 4, most Mks have transitioned into a larger size range characteristic of adulthood [
]. Using in situ DNA staining of human FL tissue sections, investigators found the percentage of Mks with 8N ploidy to increase from 16% at 3 months gestation to 33% at 6 months. Mks ≥ 16N were seen only after 7–8 months gestation. In BM, only 24% of late fetal Mks had ≥ 64N ploidy compared with 68% of adult BM Mks [
Fetal and adult Mks also differ in mitotic rates, with multiple experiments showing increased proliferation of fetal Mks. In Mk cultures conducted under standardized conditions, Liu et al. described a 70-fold expansion of CB CD34+ cells compared with 5-fold in PB CD34+ cells [
]. The ontogenic differences in progenitor expandability inversely reflect their capacity for polyploidization, suggesting that the diminished proliferation of adult progenitors may result from enhanced transition to endomitosis [
Although fetal Mks undergo incomplete enlargement and polyploidization, they fully upregulate most lineage-specific factors, including the membrane receptors and granule components necessary for platelet formation and function. Therefore, fetal and adult Mks express similar levels of membrane proteins integrin alpha-IIb (CD41), integrin beta-3 (CD61), and glycoprotein Ibα (CD42b) [
Ancient ubiquitous protein 1 binds to the conserved membrane-proximal sequence of the cytoplasmic tail of the integrin alpha subunits that plays a crucial role in the inside-out signaling of alpha IIbbeta 3.
]. CB Mks were also observed to have mature ultrastructural characteristics such as an enlarged cytoplasm, abundant granules, and a well-developed demarcation membrane system. Similar observations have been made with murine neonatal Mks [
The most important phenotypic difference between fetal and adult Mks concerns their platelet-producing efficiency. Despite their mature marker expression, CB-derived Mks produce 3-fold fewer proplatelets and platelets on a per-cell basis compared with PB-derived Mks [
]. This difference likely results from the fetal polyploidization deficit, which limits enlargement and ultimately restricts the allocation of cell mass toward platelet formation. Whether fetal Mks have additional proplatelet formation defects independent of their size remains to be determined.
Clinical significance of the infantile megakaryocyte phenotype
The distinct phenotypic features of fetal Mks may have a variety of clinical consequences (Table 2). Considered below are four problems in which this phenotype has been implicated as a major contributing factor. The first of these problems, neonatal thrombocytopenia, occurs in ~5% of all neonates, 22–35% of neonatal intensive care unit admissions, and ~73% of low-birth-weight infants < 1,000 g [
]. One of the most common inciting features consists of sepsis, most likely due to an increased demand placed on platelet production. In a recent study in The Netherlands, sepsis was identified in 7% of all hospitalized neonates and severe thrombocytopenia (< 50,000 platelets/µL) occurred in 20% of septic patients [
]. The presence of thrombocytopenia in this cohort increased risk of mortality almost 4-fold. Management of neonatal thrombocytopenia remains controversial, with platelet transfusions frequently provided to prevent intraventricular hemorrhage (IVH). A recent clinical trial confirmed that neonatal thrombocytopenia predisposes to IVH but found no correlation between the degree of risk and the degree of thrombocytopenia and could identify no benefit associated with platelet transfusion [
]. Against such a tenuous benefit must be weighed the risks of platelet transfusion, which include bacterial infection, transfusion-mediated lung injury, alloimmunization, and financial cost. Thrombopoietin (Tpo) receptor agonists have gained widespread clinical use in enhancing platelet production in adults. However, compelling in vitro data predict that these agents will lack efficacy in neonates because Tpo stimulation paradoxically exacerbates defects in infantile Mk morphogenesis [
]. A major drawback of CB-HSC transplantation has been inferior platelet recovery. In a study of adult leukemia/lymphoma patients, CB-HSC recipients experienced a 3-fold delay in the time to platelet independence compared with recipients of adult PB-HSCs [
]. This delay translated into a 2-fold increase in the number of platelet transfusions required. In pediatric transplantation recipients, CB-HSCs were associated with a 2.3-fold delay in platelet recovery and marrow morphometry documented equivalent Mk numbers but decreased Mk size in CB-HSC recipients compared with adult HSC recipients [
]. In fact, the differences in Mk size correlated directly with the differences in platelet recovery.
The third clinical problem concerns the leukemic propensity of fetal Mk progenitors. Two distinct Mk neoplasms occur almost exclusively in neonates: (1) Down syndrome-associated transient myeloproliferative disorder (DS-TMD) and (2) acute megakaryoblastic leukemia with the RBM15-MKL1 gene fusion (AMKL R-M) [
]. Epidemiologic profiles suggest that fetal status may constitute an oncogenic “hit” for these entities. Such a concept is supported in DS-TMD by the spontaneous disease regression as neonates age over several months [
]. Recent whole-exome sequencing studies provide further support. One such study has shown that the majority of DS-TMD cases carry no secondary genetic abnormalities beyond the hallmark GATA1s mutations coupled with trisomy 21 [
]. A subsequent study demonstrated that DS-AMKL arises from a TMD clone that acquires additional mutations in multiple genes, including cohesin components, CTCF, epigenetic regulators such as EZH2 and KANSL1, and members of signaling pathways such as the JAK family and RAS pathways [
The fourth problem relates to recent initiatives to develop donor-independent sources of platelets to treat patients with thrombocytopenia. The need for such sources is emerging in developed countries due to steadily rising platelet demands coupled with restricted donor supplies [
]. A major limiting factor in this process is the poor proliferative capacity of adult type Mks in culture despite efficient platelet biogenesis. In contrast, CB Mks proliferate extensively in culture but show limited platelet production. New technology has permitted Mk generation from induced pluripotent stem cells (iPSCs), raising the possibility of personalized platelet cultivation [
]. Therefore, efficient scale-up will require a biphasic system in which Mk expansion is accomplished in fetal mode, followed by induction of an adult program to maximize platelet production (Fig. 1).
Molecular differences between fetal and adult megakaryopoiesis
Multiple signaling and transcriptional programs control megakaryopoiesis. The phenotypic differences between fetal and adult Mks likely arise from ontogenic differences in these programs. Studies from the past three decades have revealed several molecular differences between fetal and adult stage megakaryopoiesis (Table 3). Most of these differences are cell intrinsic, but the microenvironment may also contribute to Mk ontogenic transitions. In addition, recent work suggests that developmental origin may also distinguish fetal from adult Mk progenitors [
]. In particular, adult Mk progenitors appear to originate from the HSC compartment, whereas fetal Mk progenitors also arise from committed progenitors downstream of HSCs. These differences in cell of origin could also contribute to the distinct phenotypic features of fetal and adult Mks.
Table 3Molecular and signaling differences between fetal and adult Mks
Predicted Effects in Megakaryopoiesis
Fetal Mks arise from HSC and committed progenitors downstream of HSCs
Despite complete execution of most aspects of the lineage program, fetal Mks do show a tendency toward “leaky” erythroid gene expression. This feature was initially identified by Woo et al. in comparing gene expression profiles of purified Mk progenitors from murine fetal liver versus adult marrow: seven erythroid transcripts were in the top 122 fetal-upregulated transcripts [
]. The concurrent expression of CD41 and GPA suggests a diminished ability of CB-derived Mks to undergo complete lineage consolidation. Although CD41 expression on progenitors does not exclude the possibility for erythroid development, Psaila et al. observed that CD42 expression does represent full commitment to the Mk lineage with loss of erythroid potential [
Multiple secreted factors regulate megakaryopoiesis at various stages. The main megakaryopoietic cytokine, which acts both at early and late stages, is Tpo. In humans, circulating Tpo levels are significantly higher in neonates than in adults [
]. Tpo engagement of its receptor (TpoR, encoded by MPL) activates multiple signaling cascades including the JAK/STAT, MEK/MAPK, and PI3K/Akt/mTOR pathways. In studies by Liu et al., Tpo-stimulated CB-derived Mks activated MAPK/ERK to a similar degree as adult counterparts, but showed hyperactivation of JAK2 and mTOR pathways [
]. As a functional corollary, inhibition of mTOR by rapamycin reduced proliferation and maturation of CB Mks without affecting polyploidization. In contrast, mTOR inhibition in adult Mks affected all parameters including polyploidization, a difference attributable to higher adult levels of cyclin-dependent kinase inhibitor 1 (p21), which is required for mTOR regulation of polyploidization [
]) might partially explain the leaky expression of erythroid genes in CB Mks.
An additional megakaryopoietic cytokine consists of stromal cell derived factor 1 (SDF-1). SDF-1 has been shown to enhance polyploidization of human Mks and to mediate migration of murine Mks to the marrow vascular niche, a milieu promoting terminal differentiation and platelet release [
]. In fetal Mks, the most highly enriched transcripts were related to angiogenesis, integrins, extracellular matrix, and transforming growth factor β receptor (TGFβR)/bone morphogenetic protein (BMP) signaling. Comparison of mRNAs encoding transcription factors essential for megakaryopoiesis (GABPα, FLI1, RUNX1, GATA1, FOG1, and NF-E2) showed differences only in GATA1 expression, which was highly enhanced in fetal Mks [
], which is predicted to target CTDSPL, encoding a retinoblastoma protein (Rb) phosphatase that promotes E2F binding by Rb. Increased miR-99a in fetal Mks correlates with decreased CTDSPL, increased hyperphosphorylated Rb, and E2F-mediated induction of D-type cyclins [
], but its influence on megakaryopoiesis remains undetermined. One potential effect of Lin28b on megakaryopoiesis could be through alteration of Wnt signaling. Therefore, Lin28b suppression of Let-7b could potentially induce expression of the receptor Frizzled4 because the corresponding transcript is a putative target of this miR [
]. Let-7b suppression also causes downregulation of Wnt3a and upregulation of Wnt5b. Wnt3a has been found to enhance Mk maturation and proplatelet formation, whereas Wnt5a appears to inhibit these processes [
]. Therefore, Lin28b could theoretically contribute to Mk ontogenic regulation through a Let-7–Wnt pathway, but experimental verification of such a pathway is needed.
Molecular differences in the core transcriptional machinery also distinguish adult and fetal Mks. Adult Mk morphogenesis imposes massive transcriptional demands, which are met through a unique program of RNA polymerase II (RNAPII) activation [
]. This program involves global and irreversible activation of the P-TEFb kinase complex (Cdk9/Cyclin T), the functions of which are to release RNAPII from sites of promoter-proximal stalling and accelerate transcriptional elongation [
]. In most cell types, P-TEFb predominantly resides in an inactive reservoir ensconced in a large ribonucleoprotein complex that contains its inhibitor HEXIM1/2 (Fig. 2). This complex, known as the 7SK snRNP, contains the RNA scaffold 7SK and the 7SK-stabilizing proteins MePCE and LARP7. P-TEFb activation in non-Mks occurs through a release mechanism that is target gene localized, reversible, and controlled by feedback inhibition. A unique aspect of P-TEFb activation in Mks consists of its irreversible release due to destruction of the 7SK snRNP (Fig. 2). A key step initiating this process is the upregulation of the active protease calpain 2, which degrades MePCE directly. Inhibition or knockdown of calpain 2 blocks adult Mk morphogenesis. The other 7SK-stabilizing factor, LARP7, also undergoes downregulation during Mk differentiation by a calpain-independent mechanism involving transcript modulation. Loss of MePCE and LARP7 cause 7SK degradation and release of active P-TEFb. This mode of P-TEFb activation is particularly important for induction of a cohort of cytoskeletal factors that drive adult Mk morphogenesis. These factors include Mkl1, Filamin A (FlnA), Hic-5, and α-actinin-1 (ACTN1).
Fetal Mks manifest several molecular defects symptomatic of impaired P-TEFb activation: a failure to induce the P-TEFb-dependent cytoskeletal factors, a deficiency in phosphorylation of the P-TEFb substrates RNAPII and Spt5, and a global decrease in histone H2B K120 monoubiquitination, a P-TEFb-driven epigenetic mark [
]. The failure to induce the P-TEFb-dependent cytoskeletal factors most likely explains the diminished morphogenesis of fetal Mks. Therefore, Mkl1 has been identified as a master regulator of adult Mk morphogenesis, with deficiency in mice compromising polyploidization and platelet production [
]. These steps normally suffice for the destruction of the 7SK RNA and dissolution of the kinase-repressive complex. However, 7SK levels remain high in fetal Mks despite downregulation of stabilizing proteins, suggesting the existence of a fetal-specific 7SK-stabilizing factor(s) that blocks morphogenesis. Functional screening of candidates through enforced expression in adult Mks has ruled out several fetal RNA-binding proteins, including HMGA1 and HMGA2, but has implicated IGF2BP3, an oncofetal mRNA-binding factor [
]. Further support for involvement of IGF2BP3 comes from loss-of-function studies in which knockdown shifts fetal Mks toward an adult phenotype with regard to morphogenesis and platelet formation. Molecular findings validating IGF2BP3 as a fetal-specific 7SK stabilizer include its direct interaction with this RNA target, its regulation of 7SK levels, and its regulation of P-TEFb signaling. Specifically, IGF2BP3 knockdown in fetal Mks decreases 7SK levels, increases phosphorylation of P-TEFb substrates, and upregulates expression of P-TEFb-dependent cytoskeletal factors. These findings indicate that IGF2BP3 serves as a master switch for the Mk ontogenic phenotype (Fig. 2).
Therapeutic targeting of IGF2BP3 activity may prove to be challenging, but its expression in fetal Mks can be modulated by inhibitors of bromo and extra-terminal domain (BET) factors [
]. Specifically, BET inhibitors downregulate IGF2BP3 in fetal Mks and promote the phenotypic and molecular features of an adult program, including enhancement of platelet release. Enforced expression of IGF2BP3 blunts the drug effects, supporting it as a relevant target. These findings provide proof of principle for pharmacologic manipulation of the Mk ontogenic phenotype by circumventing a fetal-specific blockade in P-TEFb signaling.
A role for the microenvironment in influencing Mk ontogenic phenotype has been suggested in mouse transplantation experiments [
]. Engraftment of fetal stem cells in adult mice yields fetal-type Mks 1 week after transplantation, but after 1 month, donor-derived Mks assume adult size and ploidy. A clue to the nature of this influence came from gene expression profiling of fetal and adult Mk progenitors showing type I interferon (IFN-I) response genes to be significantly upregulated in adult versus fetal cells [
]. Furthermore, ex vivo treatment of fetal Mk progenitors with IFN-α significantly blunted their proliferation. Therefore, IFN-producing myeloid cells in the adult marrow may provide an extrinsic cue to modulate the Mk ontogenic phenotype.
An important fetal microenvironmental cue likely comprises insulin-like growth factors (IGFs), which drive tissue growth during embryogenesis and are known to induce HSC proliferation [
]. This pathway is repressed by wild-type GATA1 but not the leukemogenic GATA1s mutant associated with DS-TMD. Molecular determinants of fetal IGF2 expression are the IGF2BP family members, which bind and stabilize IGF transcripts. The abundant expression of IGF2BP3 in human neonatal hematopoietic progenitors [
] suggests that these cells could serve as autocrine and juxtacrine sources of IGF2 within the fetal liver. An additional fetal influence consists of TGFβ1, which is abundantly produced by fetal liver hepatoblasts and sinusoidal endothelium [
]. Therefore, the effects of this pathway are complex and may depend on differentiation stage.
Multiple intrinsic and extrinsic factors contribute to Mk ontogenic differences (Table 3). Fetal factors promote cellular proliferation through a standard mitotic cell cycle, whereas adult factors promote a morphogenesis program that employs an endomitotic cell cycle. A complete understanding of these factors will permit therapeutic manipulation of the ontogenic phenotype. Potential applications include novel treatments for neonatal thrombocytopenia and Mk neoplasms, as well as optimized approaches for ex vivo platelet or Mk production. To optimize ex vivo yield and quality, the hyperproliferative pathways of fetal Mks can be exploited for initial expansion and the morphogenetic circuitry of adult Mks can be harnessed for subsequent platelet release (Fig. 1). Feasibility has already been demonstrated for pharmacologic targeting of fetal factors such as IGF2BP3 and TGFβ to elicit adult properties [
Ancient ubiquitous protein 1 binds to the conserved membrane-proximal sequence of the cytoplasmic tail of the integrin alpha subunits that plays a crucial role in the inside-out signaling of alpha IIbbeta 3.