scholarly journals Human NOTCH4 Is a Key Target of RUNX1 in Megakaryocytic Differentiation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 425-425
Author(s):  
Yueying Li ◽  
Chen Jin ◽  
Hao Bai ◽  
Shu Sun ◽  
Paul P. Liu ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Target Genes ◽  
Ex Vivo ◽  
Hematopoietic Progenitor Cells ◽  
Rna Seq ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Progenitor ◽  
Megakaryocytic Differentiation ◽  
Human Ipscs

Abstract Megakaryocytes (MK), which produce platelets, play important roles in blood coagulation and hemostasis. The master transcription factor RUNX1 regulates lineage-specific transcriptional targets and key signaling pathways, and is known to be essential for megakaryopoiesis. Mono-allelic RUNX1 mutations lead to familial platelet disorder (FPD), which is characterized by thrombocytopenia and abnormal platelet functions. A high percentage (~50%) of these FPD patients later develop myelodysplastic syndromes and acute myeloid leukemia. The exact mechanisms underlying deregulated megakaryopoiesis in FPD remain unclear, partially due to the lack of an adequate experimental model mimicking the human disease. For example, engineered laboratory mice and zebrafish with only one copy of the Runx1 gene do not develop bleeding disorders or leukemia. Using an in vitro hematopoietic differentiation system, we found that megakaryocytic differentiation from FPD-derived induced pluripotent stem cells (iPSCs) were defective (Connelly et al., 2014). Targeted correction of the mutated RUNX1 allele by genome editing restored the MK production and functions, validating the central role of RUNX1 in megakaryopoiesis (Connelly et al., 2014). In this new study, we pursued the hypothesis that direct target genes regulated by RUNX1 play important roles in human megakaryopoiesis. We first performed RNA-Seq analysis on differentiated hematopoietic cells from FPD-iPSCs (harboring a mono-allelic RUNX1 mutation) and RUNX1-corrected isogenic iPSCs. Seventy-nine genes were expressed at a significantly higher level (p<0.01, FDR<0.05) while 93 genes were expressed at a significantly lower level (p<0.01, FDR<0.05) in the RUNX1-corrected cells as compared to the FPD-iPSCs. To determine whether these differentially expressed genes (DEGs) are the direct targets of RUNX1, we additionally performed genome-wide location analysis of RUNX1 by ChIP-Seq using the same hematopoietic cell population differentiated from the RUNX1-corrected isogenic iPSCs. We detected 5266 (FDR<0.05) binding sites in 4526 gene loci. Combined with the DEG data from RNA-Seq analyses, we further identified 37 up-regulated genes (such as ITGB3 and PF4) and 27 down-regulated genes with RUNX1 binding to the gene's proximity. Among the 64 differentially expressed genes with RUNX1 binding, Gene Ontology (GO) analysis revealed that only 13 genes including PF4 have been reported to be relevant to megakaryopoiesis. In order to verify the roles of these RUNX1 target genes in hematopoiesis and megakaryopoiesis, we carried out gene knockout (KO) experiments by CRISPR-Cas9 in normal human iPSCs followed by in vitro hematopoietic differentiation assays. We first focused on the "down-regulated" genes by RUNX1 binding, with the hypothesis that their KO may enhance hematopoiesis and/or megakaryopoiesis from normal iPSCs. One of such genes is NOTCH4, a member of NOTCH receptor family that plays important roles in development and cell fate determination. A previous study showed that NOTCH signaling specifies MK development from mouse hematopoietic progenitor cells (Mercher et al., 2008), while we have not seen publications on the NOTCH4 in human MK development. Using the improved CRISPR technology, we successfully achieved KO of one copy of NOTCH4 in the wildtype iPSCs. We found that heterozygous KO of NOTCH4 increased MK (progenitor) production by 95% (p<0.05), while the production of CD34+ multipotent hematopoietic progenitor cells were not affected. To further verify its function, we inhibited NOTCH4 signaling with a gamma-secretase inhibitor. Notably, inhibition of NOTCH4 signal starting at day 2 of hematopoietic differentiation improved the efficiency of MK progenitor production by 50% (p<0.05) and more mature MK production by 70% (p<0.05). Taken together, we conclude that NOTCH4, a newly discovered RUNX1 target gene, negatively regulates megakaryopoiesis in a developmental-stage specific manner. Unlocking this inhibitory effect by small molecule inhibitors can promote MK production ex vivo. The described approach will enable us to discover additional novel genes that influence human hematopoiesis and megakaryopoiesis, which in turn will help to promote ex vivo generation of MKs from human iPSCs or postnatal hematopoietic stem/progenitor cells. Disclosures No relevant conflicts of interest to declare.

FDA-Approved Pharmacological Agents, Romiplostium and Oprelvekin, Synergistically Promote Megakaryocytic Differentiation From Human iPSCs In a Chemically Defined System

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1208-1208
Author(s):  
Yanfeng Liu ◽  
Yongxing Gao ◽  
Sid Shah ◽  
Lewis Becker ◽  
Linzhao Cheng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Embryoid Bodies ◽  
Hematopoietic Progenitor Cells ◽  
Patient Specific ◽  
Hematopoietic Progenitor ◽  
Free System ◽  
Serum Free ◽  
Megakaryocytic Differentiation

Abstract Platelets, anucleate cells derived from megakaryocytes (MKs) that are generated within the bone marrow, play an important role in the process of physiological hemostasis and in vascular repair. Low platelets in the blood stream result in bleeding risk in thrombocytopenic patients with liver failure, leukemia, or undergoing chemotherapy. Platelet transfusions remain the mainstay of treatment and require a constant supply of platelets. Because platelets from donor blood have a short life-span (only few days in storage), platelets are always in a short supply. In vitro generation of MKs and platelets from human induced pluripotent stem cells (hiPSCs) would provide a patient-specific renewable cell source of MKs and platelets to treat thrombocytopenic patients at risk of hemorrhage. We derived integration-free hiPSCs from peripheral blood cells of more than 20 individuals, and examined two methods of in vitro differentiation into MKs: i) co-culture on 10T1/2 cells or OP9 cells first developed by Takayama et al. (2010), and ii) a feeder-free and serum-free system by first forming embryoid bodies (EBs) in a chemically defined condition, similar to the recently published method of Pick et al. (2013). Although both methods gave rise with similar efficiency to CD41a+CD42a+ MKs with large cell size and high-ploidy DNA, we chose to focus on the feeder-free system that began with EB formation with centrifugal aggregation of hiPSCs (spin-EBs) because it is cheaper, faster, easier to scale up, and represents a chemically defined system. To investigate the effect of growth factors on hiPSC differentiation to MKs, we modified the spin-EB system to three steps: i) mesoderm induction and hematopoietic commitment in the presence of BMP4, VEGF, bFGF and SCF (day 0 to day 11), ii) hematopoietic progenitor and MK differentiation by adding TPO (day 11 to 14), and iii) MK maturation (day 14 to 19). To assess whether the FDA-approved pharmacological agent, Romiplostium (Nplate®, TPO analog), has a similar effect to TPO on MK differentiation from hiPSCs, we isolated hematopoietic progenitor cells at day 14, and differentiated them into MKs with Romiplostium or TPO. Our data demonstrated that Romiplostium (50 ng/ml) gave a 3-fold increase of CD41a+CD42a+ MKs, with similar dose-dependent kinetics as TPO. IL-11 has also been reported to enhance MK development. To test whether FDA-approved pharmacological IL-11, Oprelvekin (Neumega®), further stimulated MK differentiation from hiPSCs, we cultured hematopoietic progenitor cells from day 14 in the presence of Romiplostium and Oprelvekin for 5 days. Our data showed that Romiplostium and Oprelvekin synergistically promote megakaryocytic differentiation. In the presence of Romiplostium, 60 to 95 % of cells were CD41a+CD42a+ MKs. Addition of Oprelvekin significantly increased the number of CD41a+CD42a+ MKs, but not the percentage of CD41a+CD42a+ MKs, suggesting that Oprelvekin enhanced a proliferation of MK progenitors. So far, 10 hiPSC lines from several individuals have been tested using the combination of Romiplostium and Oprelvekin in the feeder-free and serum-free differentiation condition. We are currently investigating if the MKs and platelets generated by this defined and scalable system are as fully functional as those generated from bone marrow CD34+ cells from healthy donors. * The first three authors contributed equally; This study is supported in part by an NIH grant U01 HL-107446 and 2012-MSCRFII-0124 (to ZZ Wang). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Single OCT4 gene introduced into human bone-marrow derived mesenchymal stromal cells can generate putative iPS cells with the potential to differentiate into CD34+ hematopoietic progenitor cells ex vivo

2020 ◽  
Author(s):  
Xiaoping Guo ◽  
Sisi Li ◽  
Wenwen Weng ◽  
Xiaojun Xu ◽  
Chan Liao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Ips Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Progenitor ◽  
Plasmid Transfection ◽  
Defined Culture

Abstract Background: The ex vivo production of CD34+ hematopoietic progenitor cells from human bone-marrow mesenchymal stromal cells derived induced pluipotent stem cells (iPSCs) could serve as a feasible way to study patient-specific hematological disease from the perspective of hematopoietic differentiation. Different studies using virus-based or virus-free methods to reprogramming somatic cells into iPSCs by using fewer than four transcription factors, of which have the potential to differentiation in CD34+ hematopoietic progenitor cells. In this study, we demonstrate the generation of putative iPS cells from BMSCs with single OCT4 by plasmid transfection, which can differentiate into hematopoietic progenitor cells in defined culture system.Objective: To generate induced pluripotent stem cells (iPSCs) from bone marrow stromal cells (BMSCs) using a plasmid pcDNA3.1 constructed with a single transcription factor gene OCT4 (pcDNA3.1-OCT4) and to evaluate the hematopoietic differentiation potential of the putative BMSCs-iPSCs.Methods: BMSCs with ectopic high expression of OCT4 (BMSCs-OCT4) previously established by our group were cultured in traditional human ESC medium. Colonies with characteristic embryonic stem (ES) cell morphologies were selected and expanded in vitro. The undifferentiated status of putative BMSCs-iPSCs was confirmed by alkaline phosphatase (ALP) staining, telomerase activity assay, pluripotent marker expression and differentiation in vitro to form EBs and in vivo teratoma formation. The expression of pluripotent markers and ES markers were verified by RT-PCR, flow cytometry (FCM) and cellular immunofluorescence assay (CIFA). The hematopoietic differentiation potential into CD34+ progenitor cells by exposure to a defined culture system supplemented with a cocktail of hematopoietic growth factors was evaluated, of which the expression was confirmed by RT-PCR and FCM.Results: BMSCs were successfully reprogrammed into pluripotent stem cells resembling ESCs by introduction single transcription factor OCT4 gene constructed into the eukaryogenic plasmid pcDNA3.1. The putative BMSC-iPSCs were positive for ALP and telomerase activity, as well as the pluripotent stem cell markers including TRA-1-60, SSEA4, TRA-1-81, SOX2 and NANOG as detected by FCM and CIFA. Moreover, the above MSCs-OCT4 could form EBs ex vivo and express ectoderm (TUBB3+, WNT1+), mesoderm (Brachyury+, TBX20+), and endoderm (SPARC+) genes. By treatment with a cocktail containing BMP4 (50ng/ml), IL-3 (10ng/ml), IL-6 (10ng/ml), Flt-3 Ligand (300ng/ml), SCF (300ng/ml) and G-CSF (50ng/ml), the proportion of CD34+ progenitor cells increased from 0.93±0.46% in untransfected parental BMSCs and 1.58±1.29% in undifferentiated BMSC-iPS cells to 16.16±1.27% and 25.40±3.08% in day 14 and 21 differentiated BMSC-iPS cells, respectively. Moreover, the proportion of CD34+ progenitor cells were higher in the group with diverse concentration of growth factor cocktail induction, the proportion of CD34+ cells reached 31.39±3.60% and 73.68±6.63% in day 14 and 21 differentiated BMSC-iPS cells, respectively.Conclusion: In this study, we have clearly demonstrated the generation of putative iPS cells (or partly reprogrammed iPSCs from BMSCs with ectopic high expression of OCT4 by plasmid transfection). The BMSCs-derived iPSCs display the typical morphology and growth pattern as iPS cells when they are maintained in undifferentiated pluripotent state. Moreover, the putative BMSCs-derived iPSCs can differentiate into hematopoietic progenitor cells in defined culture system containing a cocktail of six or seven growth factors. Our findings provide a feasible way to generate hematopoietic progenitor cells using patient-specific iPSCs generated by plasmid transfection for hematological disease modeling.

scholarly journals Hemgn is a direct transcriptional target of HOXB4 and induces expansion of murine myeloid progenitor cells

Blood ◽  
2010 ◽  
Vol 116 (5) ◽  
pp. 711-719 ◽  
Author(s):  
Jie Jiang ◽  
Hui Yu ◽  
Yan Shou ◽  
Geoffrey Neale ◽  
Sheng Zhou ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Direct Transcriptional Target ◽  
Myeloid Progenitor ◽  
Transcriptional Target

HOXB4, a member of the Homeobox transcription factor family, promotes expansion of hematopoietic stem cells and hematopoietic progenitor cells in vivo and ex vivo when overexpressed. However, the molecular mechanisms underlying this effect are not well understood. To identify direct target genes of HOXB4 in primary murine hematopoietic progenitor cells, we induced HOXB4 function in lineage-negative murine bone marrow cells, using a tamoxifen-inducible HOXB4-ERT2 fusion protein. Using expression microarrays, 77 probe sets were identified with differentially changed expression in early response to HOXB4 induction. Among them, we show that Hemogen (Hemgn), encoding a hematopoietic-specific nuclear protein of unknown function, is a direct transcriptional target of HOXB4. We show that HOXB4 binds to the promoter region of Hemgn both ex vivo and in vivo. When we overexpressed Hemgn in bone marrow cells, we observed that Hemgn promoted cellular expansion in liquid cultures and increased self-renewal of myeloid colony-forming units in culture, partially recapitulating the effect of HOXB4 overexpression. Furthermore, down-regulation of Hemgn using an shRNA strategy proved that Hemgn contributes to HOXB4-mediated expansion in our myeloid progenitor assays. Our results identify a functionally relevant, direct transcriptional target of HOXB4 and identify other target genes that may also participate in the HOXB4 genetic network.

scholarly journals The ability of bone marrow progenitor cells to form colonies in ex vivo culture of MDS patients

2021 ◽  
Vol 4 ◽  
pp. 21-25
Author(s):  
Denys Bilko ◽  
Margaryta Pakharenko ◽  
Nadiia Bilko
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Pathological Process ◽  
Hematopoietic Progenitor Cells ◽  
Refractory Anemia ◽  
Hematopoietic Progenitor ◽  
Ex Vivo Culture

The results of in vitro hematopoiesis studies have provided most of the knowledge about the organization, regulation, and development of the human hematopoietic system over the past three to four decades. However, due to the impossibility of an appropriate assessment of hematopoietic stem cells (HSC) in humans and because of the shortcomings of methodological approaches to determining the role of hematopoietic progenitor cells in the pathogenesis of MDS and to predicting the course of the pathological process, semiliquid agar cultures of bone marrow from patients with myelodysplastic syndrome were used. Myelodysplastic syndrome (MDS) refers to a clinically, morphologically, and genetically heterogeneous group of diseases characterized by clonalism and arising from mutations at the level of hematopoietic progenitor cells. Proliferation of such a mutated stem cell progenitors leads to ineffective maturation of myeloid lineage cells and dysplastic changes in the bone marrow (BM). The aim of the study was to establish the relationship between the functional activity of hematopoietic progenitor cells in the ex vivo culture and the activity of the pathological process in the myelodysplastic syndrome. We studied bone marrow samples from patients with the myelodysplastic syndrome, namely refractory anemia with excess blasts I (MDS RAEB I) and refractory anemia with excess blasts II (MDS RAEB II) and AML under conditions in vitro, as well as their clinical laboratory data. It was found that the percentage of blasts and myeloblasts in the samples of patients with AML and MDS RAEB II increased, compared to the samples of patients with MDS RAEB I (63.5±3.9 %, 18.05±1.01 % and 9.49±1.53 % respectively). An increase in the number of erythrocytes and hemoglobin content was noted in the group of patients with MDS RAEB I compared with MDS RAEB II (2.9±1.4×1012 / l and 105.04±3.6 g / l versus 9±0.8×1012 / l and 84.5±4.8 g / l, respectively). The analysis of the results of BM studies of patients with MDS in in vitro culture indicated a significant lag in the formation of cell aggregates during cultivation and a pronounced inhibition of the colony-forming ability of progenitor cells, compared to the control. A noticeable decrease in the colony-forming ability was observed in patients with MDS RAEB I, MDS RAEB II and AML in this sequence – 4.1±1.2 per 1×105 explanted cells, 3.2±0.9 per 1×105 explanted cells and 2.0±0.6 per 1×105 explanted cells, respectively. The analysis of hematological parameters and the results of BM cells cultivation at different stages of MDS indicates that the colony-forming ability of progenitor cells correlates with the depth of the pathological process.

scholarly journals Gata2 Expression and Function in PML-RARA-driven Preleukemia and APL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 534-534
Author(s):  
Casey D.S. Katerndahl ◽  
Timothy J Ley ◽  
Michelle A Cai ◽  
Timothy P Rooney ◽  
Sai Mukund Ramakrishnan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Target Genes ◽  
Hematopoietic Progenitor Cells ◽  
Rna Seq ◽  
Hematopoietic Progenitor ◽  
Self Renewal ◽  
Similar Frequency ◽  
Exon 2 ◽  
Empty Vector

Self-renewal is a key feature of the cells that initiate acute myeloid leukemia. To identify the mechanisms involved in PML-RARA (PR)-driven self-renewal, we made use of Ctsg-PR mice, which have PR knocked into the UTR of the first exon of Ctsg. Ctsg-PR drives the expression of PR in myeloid progenitor cells and gives these cells the ability to serially replate in methylcellulose-based colony assays. Most Ctsg-PR mice develop acute promyelocytic leukemia (APL) with an average latency of ~300 days. To identify target genes regulated by Ctsg-PR, we performed single cell RNA-seq (scRNA-seq) on whole bone marrow from young, preleukemic Ctsg-PR mice or age-matched littermates. We identified 959 differentially expressed genes (DEGs) within myeloid progenitors (546 upregulated by PR, and 413 downregulated). Gata2 was identified as a DEG in this analysis, and we confirmed this phenotype with bulk RNA-seq of purified promyelocytes from young, preleukemic WT vs. Ctsg-PR mice. All APLs derived from Ctsg-PR mice also expressed high levels of Gata2. To identify the immediate-early target genes of PR, we transduced human CD34+ cord blood cells with MSCV-IRES-GFP retroviruses containing PR, a mutant PR with a RARA DNA binding mutation (C88A, known to abolish PR replating), or an empty vector. ScRNA-seq analysis of these cells after 7 days of ex vivo culture identified 1815 DEGs (1301 upregulated and 514 downregulated by PR) in GFP+ cells expressing PR, compared to GFP+ cells transduced with PRC88A or empty vector. Among the DEGs, Gata2 was upregulated 5-fold in the PR GFP+ cells. Identical short-term retroviral overexpression studies with mouse marrow revealed that PR expression caused an expansion of hematopoietic progenitor cells that overexpressed Gata2. Finally, although normal human promyelocytes do not express GATA2, virtually all primary human APL samples do. Combined, these studies strongly suggest that GATA2 is a target gene of PR, and may therefore play a role in the development of APL. Based on our expression data, we hypothesized that Gata2 inactivation would reduce PR-driven self-renewal. To test this hypothesis, we bred Ctsg-PR mice to Rosa26-Cas9 mice, which ubiquitously express Cas9. Marrow from the resulting Ctsg-PR x Cas9 mice was electroporated with guide RNAs (gRNAs) targeting the zinc finger 1 (ZF1) domain or exon 2 of Gata2, or control loci (Actb intron 5 or Rosa26 intron 1) and serially replated in methylcellulose with SCF, IL-3, and IL-6. CRISPR-Cas9 efficiently induced a wide array of indel mutations at all gRNA target sites. Two days after electroporation, the frequency of Gata2 alleles with indels at the target site ranged from 64% to 93% (n=4); hundreds of different Gata2 indels were generated in each experiment. To our surprise, the Gata2 targeted Ctsg-PR cells replated with dramatically higher efficiency than control locus targeted cells (Figure 1). The enhanced replating efficiency was dependent upon PR, since Gata2 targeted Rosa26-Cas9 bone marrow did not serially replate. Further, cells with Gata2 indels were positively selected for over time. For example, in one experiment, the frequency of a 12 bp deletion in Gata2 (that caused an in-frame deletion in ZF1) rose from an initial variant allele frequency (VAF) of 3% to 70% after 8 weeks of replating. A larger Gata2 deletion was co-selected at a similar frequency, and resulted in the deletion of Gata2 exon 4, which encodes 90% of ZF1. Virtually all of the positively selected cells contained Gata2 indels, demonstrating the competitive advantage of Gata2 loss-of-function mutations in this setting. Control gRNAs did not lead to significant changes in plating efficiency, nor were any indels selected for. Additionally, Ctsg-PR x Cas9 cells with Gata2 indels shifted from a neutrophilic phenotype (Gr1+ CD11b+) to a monocytic one (CD11b+ Gr1-) at late passages. To further investigate the role of Gata2 in Ctsg-PR induced APL, we sequenced the genomes of 16 mouse APLs and found that 2 samples had spontaneous mutations in Gata2 (R330L and N363fs, with VAFs of 74% and 39% respectively). In summary, these data provide evidence that PR positively regulates Gata2, and that Gata2 in turn promotes neutrophilic differentiation of committed, "reprogrammed" myelomonocytic progenitors. Surprisingly, Gata2 appears to contribute to the lineage fate and proliferative capacity of PR-expressing hematopoietic progenitor cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Human Cytomegalovirus US28 Is Important for Latent Infection of Hematopoietic Progenitor Cells

2015 ◽  
Vol 90 (6) ◽  
pp. 2959-2970 ◽  
Author(s):  
Monica S. Humby ◽  
Christine M. O'Connor
Keyword(s):  
Progenitor Cells ◽  
Human Cytomegalovirus ◽  
Latent Infection ◽  
Ex Vivo ◽  
Artificial Chromosome ◽  
Hematopoietic Progenitor Cells ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Recombinant Viruses

ABSTRACTHuman cytomegalovirus (HCMV) resides latently in hematopoietic progenitor cells (HPCs). During latency, only a subset of HCMV genes is transcribed, including one of the four virus-encoded G protein-coupled receptors (GPCRs), US28. Although US28 is a multifunctional lytic protein, its function during latency has remained undefined. We generated a panel of US28 recombinant viruses in the bacterial artificial chromosome (BAC)-derived clinical HCMV strain TB40/E-mCherry. We deleted the entire US28 open reading frame (ORF), deleted all four of the viral GPCR ORFs, or deleted three of the HCMV GPCRs but not the US28 wild-type protein. Using these recombinant viruses, we assessed the requirement for US28 during latency in the Kasumi-3in vitrolatency model system and in primaryex vivo-cultured CD34+HPCs. Our data suggest that US28 is required for latency as infection with viruses lacking the US28 ORF alone or in combination with the remaining HCMV-encoded GPCR results in transcription from the major immediate early promoter, the production of extracellular virions, and the production of infectious virus capable of infecting naive fibroblasts. The other HCMV GPCRs are not required for this phenotype as a virus expressing only US28 but not the remaining virus-encoded GPCRs is phenotypically similar to that of wild-type latent infection. Finally, we found that US28 copurifies with mature virions and is expressed in HPCs upon virus entry although its expression at the time of infection does not complement the US28 deletion latency phenotype. This work suggests that US28 protein functions to promote a latent state within hematopoietic progenitor cells.IMPORTANCEHuman cytomegalovirus (HCMV) is a widespread pathogen that, once acquired, remains with its host for life. HCMV remains latent, or quiescent, in cells of the hematopoietic compartment and upon immune challenge can reactivate to cause disease. HCMV-encoded US28 is one of several genes expressed during latency although its biological function during this phase of infection has remained undefined. Here, we show that US28 aids in promoting experimental latency in tissue culture.

scholarly journals Effects of recombinant alpha and gamma interferons on the in vitro growth of circulating hematopoietic progenitor cells (CFU-GEMM, CFU-Mk, BFU-E, and CFU-GM) from patients with myelofibrosis with myeloid metaplasia

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1014-1019 ◽  
Author(s):  
C Carlo-Stella ◽  
M Cazzola ◽  
A Gasner ◽  
G Barosi ◽  
L Dezza ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Progenitor Cell ◽  
Hematopoietic Progenitor Cells ◽  
Definitive Treatment ◽  
Hematopoietic Progenitor Cell ◽  
In Vitro Growth ◽  
Hematopoietic Progenitor ◽  
Myeloid Metaplasia ◽  
Myelofibrosis With Myeloid Metaplasia

Myelofibrosis with myeloid metaplasia (MMM) is a chronic myeloproliferative disorder due to clonal expansion of a pluripotent hematopoietic progenitor cell with secondary marrow fibrosis. No definitive treatment has as yet been devised for this condition, which shows a marked variability in clinical course. To evaluate whether excessive hematopoietic progenitor cell proliferation could be controlled by recombinant human interferon alpha (rIFN-alpha) and gamma (rIFN-gamma), we studied the effects of these agents on the in vitro growth of pluripotent and lineage-restricted circulating hematopoietic progenitor cells in 18 patients with MMM. A significant increase in the growth (mean +/- 1 SEM) per milliliter of peripheral blood of CFU-GEMM (594 +/- 253), CFU-Mk (1,033 +/- 410), BFU-E (4,799 +/- 2,020) and CFU- GM (5,438 +/- 2,505) was found in patients as compared with normal controls. Both rIFN-alpha and rIFN-gamma (10 to 10(4) U/mL) produced a significant dose-dependent suppression of CFU-GEMM, CFU-Mk, BFU-E, and CFU-GM growth. Concentrations of rIFN-alpha and rIFN-gamma causing 50% inhibition of colony formation were 37 and 163 U/mL for CFU-GEMM, 16 and 69 U/mL for CFU-Mk, 53 and 146 U/mL for BFU-E, and 36 and 187 U/mL for CFU-GM, respectively. A marked synergistic effect was found between rIFN-alpha and rIFN-gamma: combination of the two agents produced inhibitory effects greater than or equivalent to those of 10- to 100- fold higher concentrations of single agents. These studies (a) confirm that circulating hematopoietic progenitors are markedly increased in MMM, (b) indicate that these presumably abnormal progenitors are normally responsive to rIFNs in vitro, and (c) show that IFNs act in a synergistic manner when used in combination. Because rIFN-gamma can downregulate collagen synthesis in vivo, this lymphokine could be particularly useful in the treatment of patients with MMM.

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