Myocardial infarction induces bone marrow megakaryocyte proliferation, maturation and platelet production.

Platelet, apart from its classic role of homeostasis, serves also as a crucial immune cell component that contributes to the aggravation of atherosclerosis. It has been reported that myocardial infarction (MI) triggers leukocytosis in the bone marrow and spleen, which accelerates post-MI atherosclerosis. However, it remains unclear whether thrombopoiesis is also enhanced after MI. Here, using flow cytometry and bone marrow whole-mount immunofluorescence staining combined with three-dimensional (3D) reconstruction, we for the first time demonstrated an enhanced thrombopoiesis and megakaryopoiesis in a mouse model of coronary artery ligation as a mimic of MI. We showed that MI leads to increasing number of peripheral platelets, as well as elevating number and larger size of bone marrow MKs. We also observed more proplatelets and fragmented MKs, and a closer spatial distribution of MK populations to the bone marrow vascular niche after MI. This study provides direct evidence that MI induces bone marrow megakaryocyte proliferation, maturation and platelet production. It opens a new scope that targeting platelet production might become a novel therapeutic approach for attenuating post-MI atherosclerosis.

SDF-1 dynamically mediates megakaryocyte niche occupancy and thrombopoiesis at steady state and following radiation injury.

Megakaryocyte (MK) development in the bone marrow progresses spatially from the endosteal niche, which promotes MK progenitor proliferation, to the sinusoidal vascular niche, the site of terminal maturation and thrombopoiesis. The chemokine stromal cell-derived factor-1 (SDF-1), signaling through CXCR4, is implicated in the maturational chemotaxis of MKs toward sinusoidal vessels. Here, we demonstrate that both IV administration of SDF-1 and stabilization of endogenous SDF-1 acutely increase MK-vasculature association and thrombopoiesis with no change in MK number. In the setting of radiation injury, we find dynamic fluctuations in marrow SDF-1 distribution that spatially and temporally correlate with variations in MK niche occupancy. Stabilization of altered SDF-1 gradients directly affects MK location. Importantly, these SDF-1-mediated changes have functional consequences for platelet production, as the movement of MKs away from the vasculature decreases circulating platelets, while MK association with the vasculature increases circulating platelets. Finally, we demonstrate that manipulation of SDF-1 gradients can improve radiation-induced thrombocytopenia in a manner additive with earlier TPO treatment. Taken together, our data support the concept that SDF-1 regulates the spatial distribution of MKs in the marrow and consequently circulating platelet numbers. This knowledge of the microenvironmental regulation of the MK lineage could lead to improved therapeutic strategies for thrombocytopenia.

C1galt1-deficient mice exhibit thrombocytopenia due to abnormal terminal differentiation of megakaryocytes.

C1galt1 is essential for synthesis of the core 1 structure of mucin-type O-glycans. To clarify the physiological role of O-glycans in adult hematopoiesis, we exploited the interferon-inducible Mx1-Cre transgene to conditionally ablate the C1galt(flox) allele (Mx1-C1). Mx1-C1 mice exhibit severe thrombocytopenia, giant platelets, and prolonged bleeding times. Both the number and DNA ploidy of megakaryocytes in Mx1-C1 bone marrow were similar to those in wild-type (WT) mice. However, there were few proplatelets in Mx1-C1 primary megakaryocytes. Conversely, bone marrow transplanted from Mx1-C1 to WT and splenectomized Mx1-C1 mice gave rise to observations similar to those described above. The expression of GPIbα messenger RNA was unchanged in Mx1-C1 bone marrow, whereas flow cytometric and western blot analyses using megakaryocytes and platelets revealed that the expression of GPIbα protein was significantly reduced in Mx1-C1 mice. Moreover, circulating Mx1-C1 platelets exhibited an increase in the number of microtubule coils, despite normal levels of α- and ß-tubulin. Our observations suggest that O-glycan is required for terminal megakaryocyte differentiation and platelet production and that the decrease in GPIbα in cells lacking O-glycan might be caused by increased proteolysis.

Megakaryocyte-specific deletion of the protein-tyrosine phosphatases Shp1 and Shp2 causes abnormal megakaryocyte development, platelet production, and function.

The SH2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2 have been implicated in regulating signaling from a variety of platelet and megakaryocyte receptors. In this study, we investigate the functions of Shp1 and Shp2 in megakaryocytes and platelets. Megakaryocyte/platelet (MP)-specific deletion of Shp1 in mice resulted in platelets being less responsive to collagen-related peptide due to reduced GPVI expression and signaling via the Src family kinase (SFK)-Syk-PLCγ2 pathway, and fibrinogen due to reduced SFK activity. By contrast, deletion of Shp2 in the MP lineage resulted in macrothrombocytopenia and platelets being hyper-responsive to anti-CLEC-2 antibody and fibrinogen. Shp1- and Shp2-deficient megakaryocytes had partial blocks at 2N/4N ploidy; however, only the latter exhibited reduced proplatelet formation, thrombopoietin, and integrin signaling. Mice deficient in both Shp1 and Shp2 were severely macrothrombocytopenic and had reduced platelet surface glycoprotein expression, including GPVI, αIIbß3, and GPIbα. Megakaryocytes from these mice were blocked at 2N/4N ploidy and did not survive ex vivo. Deletion of the immunoreceptor tyrosine-based inhibition motif-containing receptor G6b-B in the MP lineage phenocopied multiple features of Shp1/2-deficient mice, suggesting G6b-B is a critical regulator of Shp1 and Shp2. This study establishes Shp1 and Shp2 as major regulators of megakaryocyte development, platelet production, and function.

Establishment of erythroleukemic GAK14 cells and characterization of GATA1 N-terminal domain.

GATA1 is a transcription factor essential for erythropoiesis and megakaryopoiesis. It has been found that Gata1 gene knockdown heterozygous female (Gata1(G1.05/+)) mice spontaneously develop erythroblastic leukemias. In this study, we have generated a novel Gata1 knockdown erythroblastic cell line, designated GAK14, from the leukemia cells in the Gata1(G1.05/+) mice. Although GAK14 cells maintain immature phenotype on OP9 stromal cells in the presence of erythropoietin and stem cell factor, the cells produce Gr-1-, Mac1-, B220-, CD3e- or CD49b-positive hematopoietic cells when co-cultured with DAS104-8 feeder cells. However, GAK14 cells did not produce erythroid and megakaryocytic lineages, perhaps due to the absence of GATA1. Indeed, GAK14 cells became capable of differentiating into mature erythroid cells when complemented with full-length GATA1 and co-cultured with fetal liver-derived FLS5 stromal cells. This differentiation potential was impaired when GATA1 lacking the N-terminal domain was complemented. The N-terminal domain is known to contribute to the pathogenesis of transient abnormal myelopoiesis and acute megakaryoblastic leukemia related to Down syndrome. These results thus showed that GAK14 cells will serve as a powerful tool for dissecting domain function of GATA1 and that the GATA1 N-terminal domain is essential for the erythroid differentiation of GAK14 cells.

Quebec platelet disorder is linked to the urokinase plasminogen activator gene (PLAU) and increases expression of the linked allele in megakaryocytes.

Quebec platelet disorder (QPD) is an autosomal dominant disorder with high penetrance that is associated with increased risks for bleeding. The hallmark of QPD is a gain-of-function defect in fibrinolysis due to increased platelet content of urokinase plasminogen activator (uPA) without systemic fibrinolysis. We hypothesized that increased expression of uPA by differentiating QPD megakaryocytes is linked to PLAU. Genetic marker analyses indicated that QPD was significantly linked to a 2-Mb region on chromosome 10q containing PLAU with a maximum multipoint logarithm of the odds (LOD) score of +11 between markers D10S1432 and D10S1136. Analysis of PLAU by sequencing and Southern blotting excluded mutations within PLAU and its known regulatory elements as the cause of QPD. Analyses of uPA mRNA indicated that QPD distinctly increased transcript levels of the linked PLAU allele with megakaryocyte differentiation. These findings implicate a mutation in an uncharacterized cis element near PLAU as the cause of QPD.

Survivin is not required for the endomitotic cell cycle of megakaryocytes.

Survivin is a member of the chromosome passenger complex, which plays an important role in chromosome alignment, separation, and cytokinesis. Although survivin is required for the proliferation and survival of hematopoietic stem and progenitor cells, the extent to which it is necessary for endomitosis of megakaryocytes remains controversial. To determine whether survivin is required for polyploidization, we analyzed mice with a megakaryocyte-specific deletion. PF4-Cre/survivin(fl/fl) mice harbored normal platelet counts with megakaryocytes that reached ploidy states comparable with those of control littermates. The CD41(+) cells within these animals showed little excision but increased annexin V staining, implying that survivin is required for survival of megakaryocyte progenitors in vivo. In contrast, megakaryocytes in which survivin was excised ex vivo showed robust excision and an increased degree of polyploidization. These results demonstrate that survivin is necessary for survival of megakaryocyte progenitors, but is not required for polyploidization of committed megakaryocytes.

Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation.

Quebec platelet disorder (QPD) is an inherited bleeding disorder associated with increased urokinase plasminogen activator (uPA) in platelets but not in plasma, intraplatelet plasmin generation, and alpha-granule protein degradation. These abnormalities led us to investigate uPA expression by QPD CD34(+) progenitors, cultured megakaryocytes, and platelets, and whether uPA was stored in QPD alpha-granules. Although QPD CD34(+) progenitors expressed normal amounts of uPA, their differentiation into megakaryocytes abnormally increased expression of the uPA gene but not the flanking genes for vinculin or calcium/calmodulin-dependent protein kinase IIgamma on chromosome 10. The increased uPA production by cultured QPD megakaryocytes mirrored their production of alpha-granule proteins, which was normal. uPA was localized to QPD alpha-granules and it showed extensive colocalization with alpha-granule proteins in both cultured QPD megakaryocytes and platelets, and with plasminogen in QPD platelets. In QPD megakaryocytes, cultured without or with plasma as a source of plasminogen, alpha-granule proteins were stored undegraded and this was associated with much less uPA-plasminogen colocalization than in QPD platelets. Our studies indicate that the overexpression of uPA in QPD emerges with megakaryocyte differentiation, without altering the expression of flanking genes, and that uPA is costored with alpha-granule proteins prior to their proteolysis in QPD.

Effects of CXCR1 and CXCR2 inhibition on expansion and differentiation of umbilical cord blood CD133(+) cells into megakaryocyte progenitor cells.

OBJECTIVE: There have been various reports on the roles of CXC receptors (CXCR) in modulation of hematopoiesis. In the present study, we investigated the effects of CXCR1 and/or CXCR2 inhibition on expansion and differentiation of umbilical cord blood (UCB) CD133(+) cells into megakaryocytic progenitors. MATERIALS AND METHODS: Purified UCB CD133(+) cells were cultured in a serum-free liquid culture either in the presence or absence of neutralizing anti-CXCR1 and/or anti-CXCR2 antibodies in combination with a conventional cytokine cocktail for up to 14days. Expression of megakaryocytic lineage markers (CD41 and CD61) and determination of ploidy level were determined by flowcytometry. In addition, colony-forming unit assay was performed using CD133(+) cultures in serum-free collagen-based medium containing the cytokine cocktail plus neutralizing CXCR1 and -R2 antibodies. Colony forming unit-megakaryocyte (CFU-MKs) and non-MKs were counted after immunocytochemistry staining on day 12. RESULTS: We show that while simultaneous inhibition of both CXCR1 and -R2 causes a significant reduction in the fold expansion of UCB CD133(+) cells, it also leads to an increase in percentages of CD61(+), CD41(+), and CFU-MK populations. CONCLUSION: CXCR1 and CXCR2 play significant roles in the suppression of megakaryopoiesis. We demonstrate that blocking of this suppressive effect by a simultaneous inhibition of both receptors can enhance the differentiation of UCB CD133(+) cells into megakayocytic progenitors.

Megakaryocyte progenitor cell function is enhanced upon aging despite the functional decline of aged hematopoietic stem cells.

Age-related morbidity is associated with a decline in hematopoietic stem cell (HSC) function, but the mechanisms of HSC aging remain unclear. We performed heterochronic HSC transplants followed by quantitative analysis of cell reconstitution. Although young HSCs outperformed old HSCs in young recipients, young HSCs unexpectedly failed to outcompete the old HSCs of aged recipients. Interestingly, despite substantial enrichment of megakaryocyte progenitors (MkPs) in old mice in situ and reported platelet (Plt) priming with age, transplanted old HSCs were deficient in reconstitution of all lineages, including MkPs and Plts. We therefore performed functional analysis of young and old MkPs. Surprisingly, old MkPs displayed unmistakably greater regenerative capacity compared with young MkPs. Transcriptome analysis revealed putative molecular regulators of old MkP expansion. Collectively, these data demonstrated that aging affects HSCs and megakaryopoiesis in fundamentally different ways: whereas old HSCs functionally decline, MkPs gain expansion capacity upon aging.

Valproic acid and all trans retinoic acid differentially induce megakaryopoiesis and platelet-like particle formation from the megakaryoblastic cell line MEG-01.

Both, the activity of transcription factors as well as epigenetic alterations in defined DNA regions regulate cellular differentiation processes. Hence, neuronal differentiation from neural progenitor cells is promoted by the transcription factor all trans retinoic acid (ATRA) and the histone deacetylase inhibitor valproic acid (VPA). VPA has also been shown to be involved in differentiation of tumor cells and to greatly improve the reprogramming of human somatic cells to induced pluripotent stem cells. Here we have investigated the impact of ATRA and VPA on the differentiation of megakaryoctes and platelets from the megakaryocyte progenitor cell line MEG-01. Our results show that treatment with ATRA (10⁻¹¹ M) and VPA (2 × 10⁻³ M) induces megakaryopoiesis of MEG-01 cells as estimated by polyploidy, formation of characteristic proplatelets and elevated expression of the megakaryocytic markers CD41 and CD61. The resulting megakaryocytes stayed viable for more than 3 weeks and shed platelet-like particles positive for CD41, CD61 and CD42b into the supernatant. Platelet-like particles responded to thrombin receptor activating peptide (TRAP-6) with increased externalization of P-selectin. Thus, ATRA and VPA proved to be efficient agents for the gentle induction of megakaryopoiesis and thrombopoiesis of MEG-01 cells providing the possibility to study molecular events underlying megakaryopoiesis and human platelet production over longer time periods.

PKC plays a crucial roles in c-mpl gene expression in megakaryoblastic cells.

Thrombopoietin is the cytokine involved in megakaryopoiesis and its receptor (c-Mpl) is considered to regulate development of megakaryocyte. In this research, to elucidate the underlying mechanisms of c-mpl gene expression in megakaryoblastic cells, we investigated the effect of a protein kinase C (PKC) on c-mpl promoter activity in a time-dependent manner. PKC is a member of a family of serine/threonine protein kinases in the cytosol involved in cell growth and differentiation. Phorbol 12-myristate 13-acetate (PMA) is known as PKC activator, significantly enhanced the c-mpl promoter activity and PKC inhibitor, 2-methylpiperazine dihydrochloride (H-7) suppressed the up-regulation of PMA-induced promoter activity and this effect decreased in a time-dependent manner. These results clearly suggest that in megakaryoblastic cells, PKC plays the crucial role in the initiation of up-regulation of PMA-induced c-mpl promoter activity.

Aspirin-Dependent Effects on Purinergic P2Y1 Receptor Expression.

Chronic treatment with aspirin in healthy volunteers (HVs) is associated with recovery of adenosine diphosphate (ADP)-induced platelet activation. The purinergic P2Y1 receptor exerts its effects via a Gq-protein, which is the same biochemical pathway activated by thromboxane-A2 receptor. We hypothesized that recovery of ADP-induced platelet activation could be attributed to increased P2Y1 expression induced by chronic aspirin exposure. We performed a multi-phase investigation which embraced both in vitro and in vivo experiments conducted in (1) human megakaryoblastic DAMI cells, (2) human megakaryocytic progenitor cell cultures, (3) platelets obtained from HVs treated with aspirin and (4) platelets obtained from aspirin-treated patients. DAMI cells treated with aspirin or WY14643 (PPARα agonist) had a significant up-regulation of P2Y1 mRNA, which was shown to be a PPARα-dependent process. In human megakaryocytic progenitors, in the presence of aspirin or WY14643, P2Y1 mRNA expression was higher than in mock culture. P2Y1 expression increased in platelets obtained from HVs treated with aspirin for 8 weeks. Platelets obtained from patients who were on aspirin for more than 2 months had increased P2Y1 expression and ADP-induced aggregation compared with patients on aspirin treatment for less than a month. Overall, our results suggest that aspirin induces genomic changes in megakaryocytes leading to P2Y1 up-regulation and that PPARα is the nuclear receptor involved in this regulation. Since P2Y1 is coupled to the same Gq-protein of thromboxane-A2 receptor, platelet adaptation in response to pharmacological inhibition seems not to be receptor specific, but may involve other receptors with the same biochemical pathway.

Evolving insights into the synergy between erythropoietin and thrombopoietin and the bipotent erythroid/megakaryocytic progenitor cell.

Although the synergy between erythropoietin and thrombopoietin has previously been pointed out, the clonal demonstration of a human bipotent erythroid/megakaryocytic progenitor (MEP) was first published in Experimental Hematology (Papayannopoulou T, Brice M, Farrer D, Kaushansky K. Exp Hematol. 1996;24:660-669) and later in the same year in Blood (Debili N, Coulombel L, Croisille L, et al. Blood. 1996;88:1284-1296). This demonstration, and the fact that both bipotent and monopotent erythroid or megakaryocytic progenitors co-express markers of both lineages and respond to both lineage-specific transcription factors, has provided a background for the extensive use of MEP assessment by fluorescence-activated cell sorting in many subsequent studies. Beyond this, the demonstration of shared regulatory elements and the presence of single mutations affecting both lineages have inspired further studies to decipher how the shift in transcription factor networks occurs from one lineage to the other. Furthermore, in addition to shared effects, erythropoietin and thrombopoietin have additional independent effects. Most notable for thrombopoietin is its effect on hematopoietic stem cells illustrated by in vitro and in vivo approaches.

Radioprotectors and Radiomitigators for Improving Radiation Therapy: The Small Business Innovation Research (SBIR) Gateway for Accelerating Clinical Translation.

Although radiation therapy is an important cancer treatment modality, patients may experience adverse effects. The use of a radiation-effect modulator may help improve the outcome and health-related quality of life (HRQOL) of patients undergoing radiation therapy either by enhancing tumor cell killing or by protecting normal tissues. Historically, the successful translation of radiation-effect modulators to the clinic has been hindered due to the lack of focused collaboration between academia, pharmaceutical companies and the clinic, along with limited availability of support for such ventures. The U.S. Government has been developing medical countermeasures against accidental and intentional radiation exposures to mitigate the risk and/or severity of acute radiation syndrome (ARS) and the delayed effects of acute radiation exposures (DEARE), and there is now a drug development pipeline established. Some of these medical countermeasures could potentially be repurposed for improving the outcome of radiation therapy and HRQOL of cancer patients. With the objective of developing radiation-effect modulators to improve radiotherapy, the Small Business Innovation Research (SBIR) Development Center at the National Cancer Institute (NCI), supported by the Radiation Research Program (RRP), provided funding to companies from 2011 to 2014 through the SBIR contracts mechanism. Although radiation-effect modulators collectively refer to radioprotectors, radiomitigators and radiosensitizers, the focus of this article is on radioprotection and mitigation of radiation injury. This specific SBIR contract opportunity strengthened existing partnerships and facilitated new collaborations between academia and industry. In this commentary, we assess the impact of this funding opportunity, outline the review process, highlight the organ/site-specific disease needs in the clinic for the development of radiation-effect modulators, provide a general understanding of a framework for gathering preclinical and clinical evidence to obtain regulatory approval and provide a basis for broader venture capital needs and support from pharmaceutical companies to fully capitalize on the advances made thus far in this field.

Inflammation-Induced Emergency Megakaryopoiesis Driven by Hematopoietic Stem Cell-like Megakaryocyte Progenitors.

Infections are associated with extensive platelet consumption, representing a high risk for health. However, the mechanism coordinating the rapid regeneration of the platelet pool during such stress conditions remains unclear. Here, we report that the phenotypic hematopoietic stem cell (HSC) compartment contains stem-like megakaryocyte-committed progenitors (SL-MkPs), a cell population that shares many features with multipotent HSCs and serves as a lineage-restricted emergency pool for inflammatory insults. During homeostasis, SL-MkPs are maintained in a primed but quiescent state, thus contributing little to steady-state megakaryopoiesis. Even though lineage-specific megakaryocyte transcripts are expressed, protein synthesis is suppressed. In response to acute inflammation, SL-MkPs become activated, resulting in megakaryocyte protein production from pre-existing transcripts and a maturation of SL-MkPs and other megakaryocyte progenitors. This results in an efficient replenishment of platelets that are lost during inflammatory insult. Thus, our study reveals an emergency machinery that counteracts life-threatening platelet depletions during acute inflammation.

Role of mTOR1 and mTOR2 complexes in MEG-01 cell physiology.

The function of the mammalian target of rapamycin (mTOR) is upregulated in response to cell stimulation with growing and differentiating factors. Active mTOR controls cell proliferation, differentiation and death. Since mTOR associates with different proteins to form two functional macromolecular complexes, we aimed to investigate the role of the mTOR1 and mTOR2 complexes in MEG-01 cell physiology in response to thrombopoietin (TPO). By using mTOR antagonists and overexpressing FKBP38, we have explored the role of both mTOR complexes in proliferation, apoptosis, maturation-like mechanisms, endoplasmic reticulum-stress and the intracellular location of both active mTOR complexes during MEG-01 cell stimulation with TPO. The results demonstrate that mTOR1 and mTOR2 complexes play different roles in the physiology of MEG-01 cells and in the maturation-like mechanisms; hence, these findings might help to understand the mechanism underlying generation of platelets.

Histone arginine methylation keeps RUNX1 target genes in an intermediate state.

The coordinated recruitment of epigenetic regulators of gene expression by transcription factors such as RUNX1 (AML1, acute myeloid leukemia 1) is crucial for hematopoietic differentiation. Here, we identify protein arginine methyltransferase 6 (PRMT6) as a central functional component of a RUNX1 corepressor complex containing Sin3a and HDAC1 in human hematopoietic progenitor cells. PRMT6 is recruited by RUNX1 and mediates asymmetric histone H3 arginine-2 dimethylation (H3R2me2a) at megakaryocytic genes in progenitor cells. H3R2me2a keeps RUNX1 target genes in an intermediate state with concomitant H3K27me3 and H3K4me2 but not H3K4me3. Upon megakaryocytic differentiation PRMT6 binding is lost, the H3R2me2a mark decreases and a coactivator complex containing WDR5/MLL and p300/pCAF is recruited. This leads to an increase of H3K4me3 and H3K9ac, which result in augmented gene expression. Our results provide novel mechanistic insight into how RUNX1 activity in hematopoietic progenitor cells maintains differentiation genes in a suppressed state but poised for rapid transcriptional activation.

SH2-inositol phosphatase 1 negatively influences early megakaryocyte progenitors.

BACKGROUND: The SH2-containing-5'inositol phosphatase-1 (SHIP) influences signals downstream of cytokine/chemokine receptors that play a role in megakaryocytopoiesis, including thrombopoietin, stromal-cell-derived-Factor-1/CXCL-12 and interleukin-3. We hypothesize that SHIP might control megakaryocytopoiesis through effects on proliferation of megakaryocyte progenitors (MKP) and megakaryocytes (MK). METHODOLOGY AND PRINCIPAL FINDINGS: Herein, we report the megakaryocytic phenotype and MK functional assays of hematopoietic organs of two strains of SHIP deficient mice with deletion of the SHIP promoter/first exon or the inositol phosphatase domain. Both SHIP deficient strains exhibit a profound increase in MKP numbers in bone marrow (BM), spleen and blood as analyzed by flow cytometry (Lin(-)c-Kit+CD41+) and functional assays (CFU-MK). SHIP deficient MKP display increased phosphorylation of Signal Transducers and Activators of Transcription 3 (STAT-3), protein kinase B (PKB/AKT) and extracellular signal-regulated kinases (ERKs). Despite increased MKP content, total body number of mature MK (Lin(-)c-kit(-)CD41+) are not significantly changed as SHIP deficient BM contains reduced MK while spleen MK numbers are increased. Reduction of CXCR-4 expression in SHIP deficient MK may influence MK localization to the spleen instead of the BM. Endomitosis, process involved in MK maturation, was preserved in SHIP deficient MK. Circulating platelets and red blood cells are also reduced in SHIP deficient mice. CONCLUSIONS/SIGNIFICANCE: SHIP may play an important role in regulation of essential signaling pathways that control early megakaryocytopoiesis in vivo.