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Megakaryocyte ontogeny: Clinical and molecular significance

  • Kamaleldin E. Elagib
    Correspondence
    Offprint requests to:Department of Pathology, University of Virginia School of Medicine, P.O. Box 800904, Charlottesville, VA 22908;
    Affiliations
    Department of Pathology, University of Virginia School of Medicine, Charlottesville, VA, USA
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  • Ashton T. Brock
    Affiliations
    Department of Pathology, University of Virginia School of Medicine, Charlottesville, VA, USA
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  • Adam N. Goldfarb
    Correspondence
    Offprint requests to:Department of Pathology, University of Virginia School of Medicine, P.O. Box 800904, Charlottesville, VA 22908;
    Affiliations
    Department of Pathology, University of Virginia School of Medicine, Charlottesville, VA, USA
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Open ArchivePublished:February 28, 2018DOI:https://doi.org/10.1016/j.exphem.2018.02.003

      Highlights

      • 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 [
      • Weissman I.L.
      • Anderson D.J.
      • Gage F.
      Stem and progenitor cells: origins, phenotypes, lineage commitments, and transdifferentiations.
      ]. 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 [
      • Machlus K.R.
      • Italiano Jr, J.E.
      The incredible journey: from megakaryocyte development to platelet formation.
      ]. 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 [
      • Allen Graeve J.L.
      • de Alarcon P.A.
      Megakaryocytopoiesis in the human fetus.
      ]. 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) [
      • Ma D.C.
      • Sun Y.H.
      • Chang K.Z.
      • Zuo W.
      Developmental change of megakaryocyte maturation and DNA ploidy in human fetus.
      ]. 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 [
      • Fuchs D.A.
      • McGinn S.G.
      • Cantu C.L.
      • Klein R.R.
      • Sola-Visner M.C.
      • Rimsza L.M.
      Developmental differences in megakaryocyte size in infants and children.
      ]. Although most studies have focused on healthy individuals, the size difference between fetal and adult Mks has also been observed in thrombocytopenic subjects [
      • Sola-Visner M.C.
      • Christensen R.D.
      • Hutson A.D.
      • Rimsza L.M.
      Megakaryocyte size and concentration in the bone marrow of thrombocytopenic and nonthrombocytopenic neonates.
      ].
      Table 1Phenotypic characteristics of fetal and adult Mks
      ParameterFetal/Neonatal MksAdult MksReferences
      SizeSmallerLarger
      • Allen Graeve J.L.
      • de Alarcon P.A.
      Megakaryocytopoiesis in the human fetus.
      ,
      • Ma D.C.
      • Sun Y.H.
      • Chang K.Z.
      • Zuo W.
      Developmental change of megakaryocyte maturation and DNA ploidy in human fetus.
      ,
      • Fuchs D.A.
      • McGinn S.G.
      • Cantu C.L.
      • Klein R.R.
      • Sola-Visner M.C.
      • Rimsza L.M.
      Developmental differences in megakaryocyte size in infants and children.
      ,
      • Sola-Visner M.C.
      • Christensen R.D.
      • Hutson A.D.
      • Rimsza L.M.
      Megakaryocyte size and concentration in the bone marrow of thrombocytopenic and nonthrombocytopenic neonates.
      PolyploidizationLess polyploidMore polyploid
      • Ma D.C.
      • Sun Y.H.
      • Chang K.Z.
      • Zuo W.
      Developmental change of megakaryocyte maturation and DNA ploidy in human fetus.
      ,
      • de Alarcon P.A.
      • Graeve J.L.
      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.
      ,
      • 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.
      ProliferationHyperproliferative in ex vivo cultureLess proliferative in ex vivo culture
      • 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.
      ,
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
      MaturationExpress Mk maturation markersExpress Mk maturation markers
      • 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.
      ,
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
      Proplatelet FormationForm fewer proplateletsForm more proplatelets
      • 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.
      ,
      • Ignatz M.
      • Sola-Visner M.
      • Rimsza L.M.
      • et al.
      Umbilical cord blood produces small megakaryocytes after transplantation.
      Concurrent with smaller size, fetal Mks also have a lower ploidy that increases with ontogenic stage [
      • de Alarcon P.A.
      • Graeve J.L.
      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.
      ]. 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 [
      • Ma D.C.
      • Sun Y.H.
      • Chang K.Z.
      • Zuo W.
      Developmental change of megakaryocyte maturation and DNA ploidy in human fetus.
      ]. Similar findings have been reported with ex vivo culture-derived Mks from cord blood (CB) versus adult peripheral blood (PB) progenitors [
      • 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.
      ]. Specifically, ~ 80% of CB-derived Mks had 2N ploidy and 2.6% were 8N, whereas 40% of the PB-derived Mks had ≥ 8N ploidy [
      • 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.
      ].
      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 [
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
      ]. In another similarly performed study, CB progenitors expanded 60-fold and PB cells underwent only a 10-fold amplification [
      • 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.
      ]. 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 [
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
      ].
      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) [
      • Kato A.
      • Kawamata N.
      • Tamayose K.
      • et al.
      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.
      ]. Importantly, the CD42 complex represents a marker of late-stage Mk differentiation [
      • Ruggeri Z.M.
      • De Marco L.
      • Gatti L.
      • Bader R.
      • Montgomery R.R.
      Platelets have more than one binding site for von Willebrand factor.
      ]. CB progenitor-derived Mks also demonstrate abundant expression of the platelet proteins von Willebrand factor and P-selectin [
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
      ]. 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 [
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
      ].
      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 [
      • 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.
      ]. 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 [
      • Ferrer-Marin F.
      • Stanworth S.
      • Josephson C.
      • Sola-Visner M.
      Distinct differences in platelet production and function between neonates and adults: implications for platelet transfusion practice.
      ]. The propensity for thrombocytopenia is inversely proportional to gestational age and results from cell-intrinsic defects in Mk morphogenesis (i.e., enlargement and polyploidization) [
      • Ferrer-Marin F.
      • Stanworth S.
      • Josephson C.
      • Sola-Visner M.
      Distinct differences in platelet production and function between neonates and adults: implications for platelet transfusion practice.
      ]. 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 [
      • Ree I.M.C.
      • Fustolo-Gunnink S.F.
      • Bekker V.
      • Fijnvandraat K.J.
      • Steggerda S.J.
      • Lopriore E.
      Thrombocytopenia in neonatal sepsis: Incidence, severity and risk factors.
      ]. 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 [
      • Sparger K.A.
      • Assmann S.F.
      • Granger S.
      • et al.
      Platelet transfusion practices among very-low-birth-weight infants.
      ]. 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 [
      • Pastos K.M.
      • Slayton W.B.
      • Rimsza L.M.
      • Young L.
      • Sola-Visner M.C.
      Differential effects of recombinant thrombopoietin and bone marrow stromal-conditioned media on neonatal versus adult megakaryocytes.
      ].
      Table 2Clinical significance of fetal Mks and challenges of ex vivo platelet formation
      Clinical and Therapeutic Impact of Mk OntogenyReferences
      Neonatal thrombocytopenia
      • Ferrer-Marin F.
      • Stanworth S.
      • Josephson C.
      • Sola-Visner M.
      Distinct differences in platelet production and function between neonates and adults: implications for platelet transfusion practice.
      ,
      • Ree I.M.C.
      • Fustolo-Gunnink S.F.
      • Bekker V.
      • Fijnvandraat K.J.
      • Steggerda S.J.
      • Lopriore E.
      Thrombocytopenia in neonatal sepsis: Incidence, severity and risk factors.
       Premature neonates
       Neonates with sepsis
      Delayed platelet recovery after CB-HSC transplantation
      • Ignatz M.
      • Sola-Visner M.
      • Rimsza L.M.
      • et al.
      Umbilical cord blood produces small megakaryocytes after transplantation.
      ,
      • Solh M.
      • Brunstein C.
      • Morgan S.
      • Weisdorf D.
      Platelet and red blood cell utilization and transfusion independence in umbilical cord blood and allogeneic peripheral blood hematopoietic cell transplants.
       Need for more frequent platelet transfusions after CB-SC transplantations
      Megakaryoblastic neoplasia
      • Arber D.A.
      • Brunning R.D.
      • Orazi A.
      • Porwit A.
      • Peterson L.
      • Thiele J.
      Acute myeloid leukaemia with recurrent genetic abnormalities.
      ,
      • Nikolaev S.I.
      • Santoni F.
      • Vannier A.
      • et al.
      Exome sequencing identifies putative drivers of progression of transient myeloproliferative disorder to AMKL in infants with Down syndrome.
      ,
      • de Rooij J.D.
      • Branstetter C.
      • Ma J.
      • et al.
      Pediatric non-Down syndrome acute megakaryoblastic leukemia is characterized by distinct genomic subsets with varying outcomes.
       Down syndrome transient myeloproliferative disorder
       Acute megakaryoblastic leukemia with the RBM15-MKL1 fusion
      Challenges of ex vivo platelet production as a source for platelets
      • Gollomp K.
      • Lambert M.P.
      • Poncz M.
      Current status of blood “pharming”: megakaryoctye transfusions as a source of platelets.
      ,
      • Thon J.N.
      • Mazutis L.
      • Wu S.
      • et al.
      Platelet bioreactor-on-a-chip.
       Poor proliferative capacity of adult Mks
       Infantile nature of CB-derived and iPSC-derived Mks
       Inefficient platelet formation ex vivo
      The second problem consists of delayed platelet recovery in umbilical CB hematopoietic stem cell (CB-HSC) transplantation recipients [
      • Ignatz M.
      • Sola-Visner M.
      • Rimsza L.M.
      • et al.
      Umbilical cord blood produces small megakaryocytes after transplantation.
      ]. For transplantation, CB-HSCs offer several advantages over adult HSCs and may represent the only curative option for hard-to-match patients with lethal diseases [
      • Gluckman E.
      History of cord blood transplantation.
      ]. 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 [
      • Solh M.
      • Brunstein C.
      • Morgan S.
      • Weisdorf D.
      Platelet and red blood cell utilization and transfusion independence in umbilical cord blood and allogeneic peripheral blood hematopoietic cell transplants.
      ]. 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 [
      • Ignatz M.
      • Sola-Visner M.
      • Rimsza L.M.
      • et al.
      Umbilical cord blood produces small megakaryocytes after transplantation.
      ]. 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) [
      • Arber D.A.
      • Brunning R.D.
      • Orazi A.
      • Porwit A.
      • Peterson L.
      • Thiele J.
      Acute myeloid leukaemia with recurrent genetic abnormalities.
      ]. 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 [
      • Arber D.A.
      • Brunning R.D.
      • Orazi A.
      • Porwit A.
      • Peterson L.
      • Thiele J.
      Acute myeloid leukaemia with recurrent genetic abnormalities.
      ]. 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 [
      • Nikolaev S.I.
      • Santoni F.
      • Vannier A.
      • et al.
      Exome sequencing identifies putative drivers of progression of transient myeloproliferative disorder to AMKL in infants with Down syndrome.
      ]. 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 [
      • Yoshida K.
      • Toki T.
      • Okuno Y.
      • et al.
      The landscape of somatic mutations in Down syndrome-related myeloid disorders.
      ]. Similarly, AMKL R-M displays a strikingly sparse mutational landscape compared with other classes of non-Down syndrome AMKL [
      • de Rooij J.D.
      • Branstetter C.
      • Ma J.
      • et al.
      Pediatric non-Down syndrome acute megakaryoblastic leukemia is characterized by distinct genomic subsets with varying outcomes.
      ]. Murine models also highlight the importance of ontogenic stage, with knockins for both GATA1s and RBM15-MKL1 displaying Mk abnormalities that are largely restricted to the fetal liver period [
      • Li Z.
      • Godinho F.J.
      • Klusmann J.H.
      • Garriga-Canut M.
      • Yu C.
      • Orkin S.H.
      Developmental stage-selective effect of somatically mutated leukemogenic transcription factor GATA1.
      ,
      • Mercher T.
      • Raffel G.D.
      • Moore S.A.
      • et al.
      The OTT-MAL fusion oncogene activates RBPJ-mediated transcription and induces acute megakaryoblastic leukemia in a knockin mouse model.
      ].
      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 [
      • Gollomp K.
      • Lambert M.P.
      • Poncz M.
      Current status of blood “pharming”: megakaryoctye transfusions as a source of platelets.
      ]. Recent refinements in ex vivo culture of Mks derived from a variety of sources have enhanced feasibility of producing bioactive platelets or Mks for transfusion [
      • Gollomp K.
      • Lambert M.P.
      • Poncz M.
      Current status of blood “pharming”: megakaryoctye transfusions as a source of platelets.
      ,
      • Thon J.N.
      • Mazutis L.
      • Wu S.
      • et al.
      Platelet bioreactor-on-a-chip.
      ]. 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 [
      • Takayama N.
      • Nishimura S.
      • Nakamura S.
      • et al.
      Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells.
      ]. However, the infantile nature of iPSC-derived Mks greatly restricts the efficiency of platelet production in this system [
      • Bluteau O.
      • Langlois T.
      • Rivera-Munoz P.
      • et al.
      Developmental changes in human megakaryopoiesis.
      ]. 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).
      Figure 1
      Figure 1Schematic model of fetal versus adult Mks generated in ex vivo culture. The graph depicts some of the features of Mks derived from fetal progenitors such as enhanced proliferation and impaired morphogenesis. In contrast, Mks derived from adult progenitors show enhanced morphogenesis and diminished proliferation. Listed are intrinsic and extrinsic factors that determine these differences. Ontogenic modulators represent pharmacologic agents that promote phenotypic switching through targeting of intrinsic and extrinsic determinants.

      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 [
      • Notta F.
      • Zandi S.
      • Takayama N.
      • et al.
      Distinct routes of lineage development reshape the human blood hierarchy across ontogeny.
      ]. 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
      ParameterPredicted Effects in MegakaryopoiesisReferences
      Progenitor Origin
       Fetal Mks arise from HSC and committed progenitors downstream of HSCsHeterogeneous origin
      • Notta F.
      • Zandi S.
      • Takayama N.
      • et al.
      Distinct routes of lineage development reshape the human blood hierarchy across ontogeny.
       Adult Mks originate directly from HSCsHomogenous origin
      • Notta F.
      • Zandi S.
      • Takayama N.
      • et al.
      Distinct routes of lineage development reshape the human blood hierarchy across ontogeny.
      Erythroid Gene Expression
       Fetal Mks express erythroid antigensIncomplete lineage consolidation
      • Woo A.J.
      • Wieland K.
      • Huang H.
      • et al.
      Developmental differences in IFN signaling affect GATA1s-induced megakaryocyte hyperproliferation.
      ,
      • Elagib K.E.
      • Lu C.H.
      • Mosoyan G.
      • et al.
      Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.
       Adult Mks devoid of erythroid antigensComplete lineage consolidation
      • Woo A.J.
      • Wieland K.
      • Huang H.
      • et al.
      Developmental differences in IFN signaling affect GATA1s-induced megakaryocyte hyperproliferation.
      ,
      • Elagib K.E.
      • Lu C.H.
      • Mosoyan G.
      • et al.
      Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.
      Cytokine and Cytokine Receptor Expression
       Tpo higher in neonates than adultsPositively regulates megakaryopoiesis
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
      ,
      • Walka M.M.
      • Sonntag J.
      • Dudenhausen J.W.
      • Obladen M.
      Thrombopoietin concentration in umbilical cord blood of healthy term newborns is higher than in adult controls.
       Tpo receptor (C-MPL) upregulated in fetal MksPositively regulates megakaryopoiesis
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
       SDF1 receptor (CXCR-4) downregulated in fetal MksNegatively regulates megakaryopoiesis
      • Bluteau O.
      • Langlois T.
      • Rivera-Munoz P.
      • et al.
      Developmental changes in human megakaryopoiesis.
      ,
      • Riviere C.
      • Subra F.
      • Cohen-Solal K.
      • et al.
      Phenotypic and functional evidence for the expression of CXCR4 receptor during megakaryocytopoiesis.
      ,
      • Mazharian A.
      • Watson S.P.
      • Severin S.
      Critical role for ERK1/2 in bone marrow and fetal liver-derived primary megakaryocyte differentiation, motility, and proplatelet formation.
      ,
      • Ferrer-Marin F.
      • Gutti R.
      • Liu Z.J.
      • Sola-Visner M.
      MiR-9 contributes to the developmental differences in CXCR-4 expression in human megakaryocytes.
       TGFβ receptor upregulated in fetal MksNegatively regulates megakaryopoiesis
      • Bluteau O.
      • Langlois T.
      • Rivera-Munoz P.
      • et al.
      Developmental changes in human megakaryopoiesis.
      Signaling Pathways
       IGF/mTOR pathway hyperactivated in fetal MksIncreases Mk proliferation
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
      ,
      • Klusmann J.H.
      • Godinho F.J.
      • Heitmann K.
      • et al.
      Developmental stage-specific interplay of GATA1 and IGF signaling in fetal megakaryopoiesis and leukemogenesis.
       JAK2 pathway hyperactivated in fetal MksIncreases Mk proliferation
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
       Cell cycle, DNA replication, and mitosis components are enriched in adult MksIncreases Mk polyploidization
      • Bluteau O.
      • Langlois T.
      • Rivera-Munoz P.
      • et al.
      Developmental changes in human megakaryopoiesis.
       p21 is downregulated in fetal MksIncreases proliferation and reduces polyploidization
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
      ,
      • Raslova H.
      • Baccini V.
      • Loussaief L.
      • et al.
      Mammalian target of rapamycin (mTOR) regulates both proliferation of megakaryocyte progenitors and late stages of megakaryocyte differentiation.
       TGFβ signaling is hyperactivated in fetal MksNegatively regulates Mk polyploidization
      • Bluteau O.
      • Langlois T.
      • Rivera-Munoz P.
      • et al.
      Developmental changes in human megakaryopoiesis.
      Transcription Factors and Transcription
       GATA-1 is upregulated in fetal MksPermits differentiation
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
       P-TEFb is less active in fetal MksDecreases Mk morphogenesis and suppression of erythroid genes
      • Elagib K.E.
      • Lu C.H.
      • Mosoyan G.
      • et al.
      Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.
      RNA Binding Factors
       IGF2BP3 is expressed in fetal MksDecreases P-TEFb activity, Mk morphogenesis, and suppression of erythroid genes
      • Elagib K.E.
      • Lu C.H.
      • Mosoyan G.
      • et al.
      Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.
       Lin28B is expressed in fetal MksPositively regulates the expression of erythroid genes
      • Bluteau O.
      • Langlois T.
      • Rivera-Munoz P.
      • et al.
      Developmental changes in human megakaryopoiesis.
      ,
      • Elagib K.E.
      • Lu C.H.
      • Mosoyan G.
      • et al.
      Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.
       HMGA1 is expressed in fetal MkEnhances megakaryopoiesis
      • Elagib K.E.
      • Lu C.H.
      • Mosoyan G.
      • et al.
      Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.
      MicroRNA (miR)
       miR-9 and miR-224 are upregulated in fetal MksPredicted to inhibit megakaryopoiesis
      • Bluteau O.
      • Langlois T.
      • Rivera-Munoz P.
      • et al.
      Developmental changes in human megakaryopoiesis.
      ,
      • Ferrer-Marin F.
      • Gutti R.
      • Liu Z.J.
      • Sola-Visner M.
      MiR-9 contributes to the developmental differences in CXCR-4 expression in human megakaryocytes.
       miR-99a is upregulated in fetal MksPredicted to enhance Mk proliferation
      • Kandi R.
      • Gutti U.
      • Undi R.
      • Sahu I.
      • Gutti R.K.
      Understanding thrombocytopenia: physiological role of microRNA in survival of neonatal megakaryocytes.
       miR-181a is downregulated in fetal MksPredicted to enhance erythroid gene expression
      • Li X.
      • Zhang J.
      • Gao L.
      • et al.
      MiR-181 mediates cell differentiation by interrupting the Lin28 and let-7 feedback circuit.
       Let-7 miRs are downregulated in fetal MksPredicted to enhance erythroid gene expression
      • Bluteau O.
      • Langlois T.
      • Rivera-Munoz P.
      • et al.
      Developmental changes in human megakaryopoiesis.
      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 [
      • Woo A.J.
      • Wieland K.
      • Huang H.
      • et al.
      Developmental differences in IFN signaling affect GATA1s-induced megakaryocyte hyperproliferation.
      ]. Recently, we confirmed these findings in human progenitors, with Mks derived from CB but not adult progenitors showing partial expression of the erythroid antigen glycophorin A (GPA) [
      • Elagib K.E.
      • Lu C.H.
      • Mosoyan G.
      • et al.
      Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.
      ]. 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 [
      • Psaila B.
      • Barkas N.
      • Iskander D.
      • et al.
      Single-cell profiling of human megakaryocyte-erythroid progenitors identifies distinct megakaryocyte and erythroid differentiation pathways.
      ].
      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 [
      • Walka M.M.
      • Sonntag J.
      • Dudenhausen J.W.
      • Obladen M.
      Thrombopoietin concentration in umbilical cord blood of healthy term newborns is higher than in adult controls.
      ]. 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 [
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
      ]. Consistent with their enhanced Tpo sensitivity, CB Mks expressed elevated levels of TpoR and of mTOR downstream targets S6K and p-4E-BP-1 [
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
      ]. 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 [
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
      ,
      • Raslova H.
      • Baccini V.
      • Loussaief L.
      • et al.
      Mammalian target of rapamycin (mTOR) regulates both proliferation of megakaryocyte progenitors and late stages of megakaryocyte differentiation.
      ]. Therefore, CB Mk hyperactivation of mTOR combined with p21 deficiency might uncouple Mk maturation from polyploidization and promote proliferative expansion during development [
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
      ]. In this context, the recent implication of mTORC1 in activating erythroid genes (through inducing mitochondrial biogenesis [
      • Liu X.
      • Zhang Y.
      • Ni M.
      • et al.
      Regulation of mitochondrial biogenesis in erythropoiesis by mTORC1-mediated protein translation.
      ]) 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 [
      • Guerriero R.
      • Mattia G.
      • Testa U.
      • et al.
      Stromal cell-derived factor 1alpha increases polyploidization of megakaryocytes generated by human hematopoietic progenitor cells.
      ,
      • Avecilla S.T.
      • Hattori K.
      • Heissig B.
      • et al.
      Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis.
      ]. Interestingly, the SDF-1 receptor CXCR4 was found to be deficient in human fetal Mks [
      • Bluteau O.
      • Langlois T.
      • Rivera-Munoz P.
      • et al.
      Developmental changes in human megakaryopoiesis.
      ,
      • Riviere C.
      • Subra F.
      • Cohen-Solal K.
      • et al.
      Phenotypic and functional evidence for the expression of CXCR4 receptor during megakaryocytopoiesis.
      ,
      • Mazharian A.
      • Watson S.P.
      • Severin S.
      Critical role for ERK1/2 in bone marrow and fetal liver-derived primary megakaryocyte differentiation, motility, and proplatelet formation.
      ,
      • Ferrer-Marin F.
      • Gutti R.
      • Liu Z.J.
      • Sola-Visner M.
      MiR-9 contributes to the developmental differences in CXCR-4 expression in human megakaryocytes.
      ]. This deficiency was correlated with fetal-specific expression of the microRNAs (miRs) miR-9 and miR-224, which target CXCR4 transcripts [
      • Ferrer-Marin F.
      • Gutti R.
      • Liu Z.J.
      • Sola-Visner M.
      MiR-9 contributes to the developmental differences in CXCR-4 expression in human megakaryocytes.
      ]. Therefore, differential expression of CXCR4 may also contribute to ontogenic differences in Mk phenotype.
      Characterization of the Mk transcriptome at the fetal, neonatal, and adult stages revealed enrichment in adult Mks of transcripts related to differentiation, platelet formation, and cell cycle [
      • Bluteau O.
      • Langlois T.
      • Rivera-Munoz P.
      • et al.
      Developmental changes in human megakaryopoiesis.
      ]. 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 [
      • Liu Z.J.
      • Italiano Jr, J.
      • Ferrer-Marin F.
      • et al.
      Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes.
      ].
      Several miRs have been implicated in regulating megakaryopoiesis [
      • Garzon R.
      • Pichiorri F.
      • Palumbo T.
      • et al.
      MicroRNA fingerprints during human megakaryocytopoiesis.
      ,
      • Edelstein L.C.
      • Bray P.F.
      MicroRNAs in platelet production and activation.
      ,
      • Emmrich S.
      • Henke K.
      • Hegermann J.
      • Ochs M.
      • Reinhardt D.
      • Klusmann J.H.
      miRNAs can increase the efficiency of ex vivo platelet generation.
      ,
      • Qu M.
      • Fang F.
      • Zou X.
      • et al.
      miR-125b modulates megakaryocyte maturation by targeting the cell-cycle inhibitor p19INK4D.
      ]. Thirty-two miRs have been found to differ between fetal-like Mks (from embryonic stem cells) and adult Mks [
      • Bluteau O.
      • Langlois T.
      • Rivera-Munoz P.
      • et al.
      Developmental changes in human megakaryopoiesis.
      ]. Among those enriched in the fetal-like Mks were miR-9 and miR-224, both of which target CXCR4 [
      • Ferrer-Marin F.
      • Gutti R.
      • Liu Z.J.
      • Sola-Visner M.
      MiR-9 contributes to the developmental differences in CXCR-4 expression in human megakaryocytes.
      ]. Also enriched in fetal Mks is miR99a [
      • Kandi R.
      • Gutti U.
      • Undi R.
      • Sahu I.
      • Gutti R.K.
      Understanding thrombocytopenia: physiological role of microRNA in survival of neonatal megakaryocytes.
      ], 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 [
      • Kandi R.
      • Gutti U.
      • Undi R.
      • Sahu I.
      • Gutti R.K.
      Understanding thrombocytopenia: physiological role of microRNA in survival of neonatal megakaryocytes.
      ]. These data thus suggest that miR-99a contributes to the hyperproliferative phenotype in fetal Mks by promoting cell cycle transition through the classic G1–S checkpoint.
      A miR found to be downregulated in fetal Mks is miR-181a, which putatively targets LIN28B, encoding an oncofetal RNA-binding factor [
      • Li X.
      • Zhang J.
      • Gao L.
      • et al.
      MiR-181 mediates cell differentiation by interrupting the Lin28 and let-7 feedback circuit.
      ]. Lin28b displays selective expression in fetal HSCs and Mks and likely contributes to the decreased levels of Let-7 miRs found in fetal progenitors [
      • Bluteau O.
      • Langlois T.
      • Rivera-Munoz P.
      • et al.
      Developmental changes in human megakaryopoiesis.
      ,
      • Elagib K.E.
      • Lu C.H.
      • Mosoyan G.
      • et al.
      Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.
      ,
      • Copley M.R.
      • Babovic S.
      • Benz C.
      • et al.
      The Lin28b-let-7-Hmga2 axis determines the higher self-renewal potential of fetal haematopoietic stem cells.
      ]. Lin28b has been implicated in ontogenic programming of HSCs, erythroid lineages, and lymphoid lineages [
      • Copley M.R.
      • Babovic S.
      • Benz C.
      • et al.
      The Lin28b-let-7-Hmga2 axis determines the higher self-renewal potential of fetal haematopoietic stem cells.
      ,
      • Lee Y.T.
      • de Vasconcellos J.F.
      • Yuan J.
      • et al.
      LIN28B-mediated expression of fetal hemoglobin and production of fetal-like erythrocytes from adult human erythroblasts ex vivo.
      ,
      • Yuan J.
      • Nguyen C.K.
      • Liu X.
      • Kanellopoulou C.
      • Muljo S.A.
      Lin28b reprograms adult bone marrow hematopoietic progenitors to mediate fetal-like lymphopoiesis.
      ], 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 [
      • Undi R.B.
      • Gutti U.
      • Gutti R.K.
      Role of let-7b/Fzd4 axis in mitochondrial biogenesis through wnt signaling: In neonatal and adult megakaryocytes.
      ]. 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 [
      • Macaulay I.C.
      • Thon J.N.
      • Tijssen M.R.
      • et al.
      Canonical Wnt signaling in megakaryocytes regulates proplatelet formation.
      ]. 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 [
      • Elagib K.E.
      • Rubinstein J.D.
      • Delehanty L.L.
      • et al.
      Calpain 2 activation of P-TEFb drives megakaryocyte morphogenesis and is disrupted by leukemogenic GATA1 mutation.
      ]. 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 [
      • Elagib K.E.
      • Rubinstein J.D.
      • Delehanty L.L.
      • et al.
      Calpain 2 activation of P-TEFb drives megakaryocyte morphogenesis and is disrupted by leukemogenic GATA1 mutation.
      ,
      • Elagib K.E.
      • Goldfarb A.N.
      Megakaryocytic irreversible P-TEFb activation.
      ]. 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).
      Figure 2
      Figure 2Model of ontogenic regulation of megakaryocyte morphogenesis. In adult megakaryopoiesis (left arrow), downregulation of LARP7 and proteolysis of MePCE destabilize 7SK snRNA, leading to unopposed P-TEFb activation. This mode of P-TEFb activation promotes upregulation of megakaryocyte morphogenesis factors, most notably MKL1, as well as upregulation of HEXIM1 and lineage consolidation, via erythroid repression. In fetal megakaryopoiesis (right arrow), expression of IGF2BP3 stabilizes 7SK snRNA despite downregulation of LARP7 and MePCE. Persistence of 7SK allows for feedback inhibition of P-TEFb, dampening both the upregulation of megakaryocyte morphogenesis factors such as MKL1 and lineage consolidation via erythroid repression. (Reproduced with permission from Elagib et al.
      [
      • Elagib K.E.
      • Lu C.H.
      • Mosoyan G.
      • et al.
      Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.
      ]
      ).
      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 [
      • Elagib K.E.
      • Lu C.H.
      • Mosoyan G.
      • et al.
      Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.
      ]. 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 [
      • Cheng E.C.
      • Luo Q.
      • Bruscia E.M.
      • et al.
      Role for MKL1 in megakaryocytic maturation.
      ,
      • Halene S.
      • Gao Y.
      • Hahn K.
      • et al.
      Serum response factor is an essential transcription factor in megakaryocytic maturation.
      ,
      • Smith E.C.
      • Thon J.N.
      • Devine M.T.
      • et al.
      MKL1 and MKL2 play redundant and crucial roles in megakaryocyte maturation and platelet formation.
      ]. ACTN1 knockdown blocks adult Mk morphogenesis [
      • Elagib K.E.
      • Rubinstein J.D.
      • Delehanty L.L.
      • et al.
      Calpain 2 activation of P-TEFb drives megakaryocyte morphogenesis and is disrupted by leukemogenic GATA1 mutation.
      ] and germline human mutations have been identified in rare cases of autosomal-dominant macrothrombocytopenia [
      • Kunishima S.
      • Okuno Y.
      • Yoshida K.
      • et al.
      ACTN1 mutations cause congenital macrothrombocytopenia.
      ,
      • Gueguen P.
      • Rouault K.
      • Chen J.M.
      • et al.
      A missense mutation in the alpha-actinin 1 gene (ACTN1) is the cause of autosomal dominant macrothrombocytopenia in a large French family.
      ]. Similarly, FlnA loss of function impairs Mk polyploidization in culture and causes macrothrombocytopenia in vivo in mice and humans [
      • Nurden P.
      • Debili N.
      • Coupry I.
      • et al.
      Thrombocytopenia resulting from mutations in filamin A can be expressed as an isolated syndrome.
      ,
      • Jurak Begonja A.
      • Hoffmeister K.M.
      • Hartwig J.H.
      • Falet H.
      FlnA-null megakaryocytes prematurely release large and fragile platelets that circulate poorly.
      ].
      Despite manifesting a failure in the Mk pathway of P-TEFb activation, fetal Mks execute the key initiating steps in this pathway: downregulation of MePCE and LARP7 [
      • Elagib K.E.
      • Lu C.H.
      • Mosoyan G.
      • et al.
      Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.
      ]. 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 [
      • Elagib K.E.
      • Lu C.H.
      • Mosoyan G.
      • et al.
      Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.
      ,
      • Dutta A.
      • Hutchison R.E.
      • Mohi G.
      Hmga2 promotes the development of myelofibrosis in Jak2V617F knockin mice by enhancing TGF-beta1 and Cxcl12 pathways.
      ]. Therefore, ectopic expression of IGF2BP3 in adult progenitors induces a fetal phenotypic shift of Mks in both human cell culture and murine marrow transplantation models [
      • Elagib K.E.
      • Lu C.H.
      • Mosoyan G.
      • et al.
      Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.
      ]. These findings may partly explain the diminished platelet counts observed in mice stably engrafted with HSCs expressing IGF2BP3 [
      • Palanichamy J.K.
      • Tran T.M.
      • Howard J.M.
      • et al.
      RNA-binding protein IGF2BP3 targeting of oncogenic transcripts promotes hematopoietic progenitor proliferation.
      ]. 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 [
      • Elagib K.E.
      • Lu C.H.
      • Mosoyan G.
      • et al.
      Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.
      ]. 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.

      Microenvironmental influences

      A role for the microenvironment in influencing Mk ontogenic phenotype has been suggested in mouse transplantation experiments [
      • Slayton W.B.
      • Wainman D.A.
      • Li X.M.
      • et al.
      Developmental differences in megakaryocyte maturation are determined by the microenvironment.
      ]. 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 [
      • Woo A.J.
      • Wieland K.
      • Huang H.
      • et al.
      Developmental differences in IFN signaling affect GATA1s-induced megakaryocyte hyperproliferation.
      ]. 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 [
      • Heazlewood S.Y.
      • Neaves R.J.
      • Williams B.
      • Haylock D.N.
      • Adams T.E.
      • Nilsson S.K.
      Megakaryocytes co-localise with hemopoietic stem cells and release cytokines that up-regulate stem cell proliferation.
      ]. Klusmann et al. demonstrated that stromal IGF2 promotes proliferation of fetal but not adult Mks through the activation of an IGFR1/mTOR/E2F signaling pathway [
      • Klusmann J.H.
      • Godinho F.J.
      • Heitmann K.
      • et al.
      Developmental stage-specific interplay of GATA1 and IGF signaling in fetal megakaryopoiesis and leukemogenesis.
      ]. 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 [
      • Elagib K.E.
      • Lu C.H.
      • Mosoyan G.
      • et al.
      Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.
      ] 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 [
      • Thompson N.L.
      • Flanders K.C.
      • Smith J.M.
      • Ellingsworth L.R.
      • Roberts A.B.
      • Sporn M.B.
      Expression of transforming growth factor-beta 1 in specific cells and tissues of adult and neonatal mice.
      ,
      • Sugiyama D.
      • Kulkeaw K.
      • Mizuochi C.
      TGF-beta-1 up-regulates extra-cellular matrix production in mouse hepatoblasts.
      ]. Fetal Mks show enhanced expression of TGFβR/BMP and extracellular matrix factors induced by TGFβ [
      • Bluteau O.
      • Langlois T.
      • Rivera-Munoz P.
      • et al.
      Developmental changes in human megakaryopoiesis.
      ]. Phenotypic consequences of Mk exposure to TGFβ include impaired enlargement and polyploidization [
      • Kuter D.J.
      • Gminski D.M.
      • Rosenberg R.D.
      Transforming growth factor beta inhibits megakaryocyte growth and endomitosis.
      ,
      • Huang N.
      • Lou M.
      • Liu H.
      • Avila C.
      • Ma Y.
      Identification of a potent small molecule capable of regulating polyploidization, megakaryocyte maturation, and platelet production.
      ], implicating this cytokine as a morphogenic modulator. However, TGFβ signaling also mediates features of adult-type megakaryopoiesis by promoting extension of proplatelet processes [
      • Badalucco S.
      • Di Buduo C.A.
      • Campanelli R.
      • et al.
      Involvement of TGFbeta1 in autocrine regulation of proplatelet formation in healthy subjects and patients with primary myelofibrosis.
      ]. Therefore, the effects of this pathway are complex and may depend on differentiation stage.

      Conclusion

      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 [
      • Elagib K.E.
      • Lu C.H.
      • Mosoyan G.
      • et al.
      Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.
      ,
      • Huang N.
      • Lou M.
      • Liu H.
      • Avila C.
      • Ma Y.
      Identification of a potent small molecule capable of regulating polyploidization, megakaryocyte maturation, and platelet production.
      ]. Future studies will pave the way toward enjoying the best of both ontogenic worlds: scalability and productivity.

      Acknowledgments

      This work was supported by grants from the National Institutes of Health ( DK090926 and HL130550 ).

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