Co-culture of hematopoietic stem/progenitor cells with human osteblasts favours mono/macrophage differentiation at the expense of the erythroid lineage

Hematopoietic stem cells (HSCs) are located in the bone marrow in a specific microenvironment referred as the hematopoietic stem cell niche, where HSCs interact with a variety of stromal cells. Though several components of the stem cell niche have been identified, the regulatory mechanisms through which such components regulate the stem cell fate are still unknown. In order to address this issue, we investigated how osteoblasts (OBs) can affect the molecular and functional phenotype of Hematopoietic Stem/Progenitor Cells (HSPCs) and vice versa. For this purpose, human CD34+ cells were cultured in direct contact with primary human OBs. Our data showed that CD34+ cells cultured with OBs give rise to higher total cell numbers, produce more CFUs and maintain a higher percentage of CD34+CD38- cells compared to control culture. Moreover, clonogenic assay and long-term culture results showed that co-culture with OBs induces a strong increase in mono/macrophage precursors coupled to a decrease in the erythroid ones. Finally, gene expression profiling (GEP) allowed us to study which signalling pathways were activated in the hematopoietic cell fraction and in the stromal cell compartment after coculture. Such analysis allowed us to identify several cytokine-receptor networks, such as WNT pathway, and transcription factors, as TWIST1 and FOXC1, that could be activated by co-culture with OBs and could be responsible for the biological effects reported above. Altogether our results indicate that OBs are able to affect HPSCs on 2 different levels: on one side, they increase the immature progenitor pool in vitro, on the other side, they favor the expansion of the mono/macrophage precursors at the expense of the erythroid lineage

Eosinophils, but not neutrophils, exibit an efficient DNA repair machinary and high nucleolar activity

BACKGROUND AND OBJECTIVES: Traditionally eosinophils have been considered terminally differentiated cells that play a role in host protection against parasites. However, there is some evidence showing that eosinophils are, in fact, multifunctional leukocytes involved in inflammatory responses, as well as in tissue homeostasis. We characterized the transcriptome profile of human eosinophils, and, for the purpose of comparison, the transcriptome profile of neutrophils, monocytes and hematopoietic progenitor cells. Moreover, we studied the activation of selected cellular processes for which a significant differential expression was demonstrated. DESIGN AND METHODS: We profiled gene expression using Affymetrix GeneChips. DNA repair capacity was tested using the comet assay. Nucleoli and their activity were characterized by transmission electron microscopy analysis, silver staining of nucleolus regions (AgNOR) and RNA staining. RESULTS: Gene expression profiling showed that eosinophils appear hierarchically closer to monocytes than to neutrophils. Gene ontology mapping of differentially expressed genes revealed that eosinophils express categories very similar to those expressed by monocytes, related to DNA repair and nucleolar functions. Moreover, our data show that eosinophils and monocytes maintain the ability to repair both double and single strand DNA breaks, whereas neutrophils lack this capacity. Furthermore, eosinophils exhibit nucleolar activity, which is lacking in neutrophils, but resembles that in monocytes. INTERPRETATION AND CONCLUSIONS: The presence of large, active nucleoli in eosinophils, coupled to marked activity of DNA repair systems, suggests that eosinophils are not terminally differentiated cells. Indeed, their transcriptome profile and functional properties are more similar to those of non-terminally differentiated cells such as monocytes, rather than to neutrophils

SOX2 Is a Univocal Marker for Human Oral Mucosa Epithelium Useful in Post-COMET Patient Characterization

Total bilateral Limbal Stem Cells Deficiency is a pathologic condition of the ocular surface due to loss or impairment of corneal stem cell function, altering homeostasis of the corneal epithelium. Cultivated Oral Mucosa Epithelial Transplantation (COMET) is the only autologous treatment for this pathology. During the follow-up, a proper characterization of the transplanted oral mucosa on the ocular surface supports understanding the regenerative process. The previously proposed markers for oral mucosa identification (e.g., keratins 3 and 13) are co-expressed by corneal and conjunctival epithelia. Here, we propose a new specific marker to distinguish human oral mucosa from the epithelia of the ocular surface. We compared the transcriptome of holoclones (stem cells) from the human oral mucosa, limbal and conjunctival cultures by microarray assay. High expression of SOX2 identified the oral mucosa vs. cornea and conjunctiva, while PAX6 was highly expressed in corneal and conjunctival epithelia. The transcripts were validated by qPCR, and immunological methods identified the related proteins. Finally, the proposed markers were used to analyze a 10-year follow-up aniridic patient treated by COMET. These findings will support the follow-up analysis of COMET treated patients and help to shed light on the mechanism of corneal repair and regeneration

miR-382-5p Controls Hematopoietic Stem Cell Differentiation Through the Downregulation of MXD1

microRNAs are key regulators of gene expression that control stem cell fate by posttranscriptional downregulation of hundreds of target genes through seed pairing in their 3' untranslated region. In fact, miRNAs tightly regulate fundamental stem cell processes, like self-renewal, proliferation, and differentiation; therefore, miRNA deregulation may contribute to the development of solid tumors and hematological malignancies. miR-382-5p has been found to be upregulated in patients with myeloid neoplasms, but its role in normal hematopoiesis is still unknown. In this study, we demonstrated that miR-382-5p overexpression in CD34(+) hematopoietic stem/progenitor cells (HSPCs) leads to a significant decrease of megakaryocyte precursors coupled to increase of granulocyte ones. Furthermore, by means of a computational analysis using different prediction algorithms, we identified several putative mRNA targets of miR-382-5p that are downregulated upon miRNA overexpression (ie, FLI1, GATA2, MAF, MXD1, RUNX1, and SGK1). Among these, we validated MXD1 as real target of miR-382-5p by luciferase reporter assay. Finally, we showed that MXD1 knockdown mimics the effects of miR-382-5p overexpression on granulocyte and megakaryocyte differentiation of CD34(+) cells. Overall, our results demonstrated that miR-382-5p expression favors the expansion of granulocyte lineage and impairs megakaryocyte commitment through MXD1 downregulation. Therefore, our data showed for the first time that the miR-382-5p/MXD1 axis plays a critical role in myelopoiesis by affecting the lineage choice of CD34(+) HSPCs

Role of miR-34a-5p in Hematopoietic Progenitor Cells Proliferation and Fate Decision: Novel Insights into the Pathogenesis of Primary Myelofibrosis

Primary Myelofibrosis (PMF) is a chronic Philadelphia-negative myeloproliferative neoplasm characterized by a skewed megakaryopoiesis and an overproduction of proinflammatory and profibrotic mediators that lead to the development of bone marrow (BM) fibrosis. Since we recently uncovered the upregulation of miR-34a-5p in PMF CD34+ hematopoietic progenitor cells (HPCs), in order to elucidate its role in PMF pathogenesis here we unravelled the effects of miR-34a-5p overexpression in HPCs. We showed that enforced expression of miR-34a-5p partially constrains proliferation and favours the megakaryocyte and monocyte/macrophage commitment of HPCs. Interestingly, we identified lymphoid enhancer-binding factor 1 (LEF1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts as miR-34a-5p-targets downregulated after miR-34a-5p overexpression in HPCs as well as in PMF CD34+ cells. Remarkably, the knockdown of NR4A2 in HPCs mimicked the antiproliferative effects of miR-34a-5p overexpression, while the silencing of LEF1 phenocopied the effects of miR-34a-5p overexpression on HPCs lineage choice, by favouring the megakaryocyte and monocyte/macrophage commitment. Collectively our data unravel the role of miR-34a-5p in HPCs fate decision and suggest that the increased expression of miR-34a-5p in PMF HPCs could be important for the skewing of megakaryopoiesis and the production of monocytes, that are key players in BM fibrosis in PMF patients

Comparison between Cultivated Oral Mucosa and Ocular Surface Epithelia for COMET Patients Follow-Up

Total bilateral Limbal Stem Cell Deficiency is a pathologic condition of the ocular surface due to the loss of corneal stem cells. Cultivated oral mucosa epithelial transplantation (COMET) is the only autologous successful treatment for this pathology in clinical application, although abnormal peripheric corneal vascularization often occurs. Properly characterizing the regenerated ocular surface is needed for a reliable follow-up. So far, the univocal identification of transplanted oral mucosa has been challenging. Previously proposed markers were shown to be co-expressed by different ocular surface epithelia in a homeostatic or perturbated environment. In this study, we compared the transcriptome profile of human oral mucosa, limbal and conjunctival cultured holoclones, identifying Paired Like Homeodomain 2 (PITX2) as a new marker that univocally distinguishes the transplanted oral tissue from the other epithelia. We validated PITX2 at RNA and protein levels to investigate 10-year follow-up corneal samples derived from a COMET-treated aniridic patient. Moreover, we found novel angiogenesis-related factors that were differentially expressed in the three epithelia and instrumental in explaining the neovascularization in COMET-treated patients. These results will support the follow-up analysis of patients transplanted with oral mucosa and provide new tools to understand the regeneration mechanism of transplanted corneas

Valproic acid triggers erythro/megakaryocyte lineage decision through induction of GFI1B and MLLT3 expression

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The isopeptidase inhibitor 2cPE triggers proteotoxic stress and ATM activation in chronic lymphocytic leukemia cells

Relapse after treatment is a common and unresolved problem for patients suffering of the B-cell chronic lymphocytic leukemia (B-CLL). Here we investigated the ability of the isopeptidase inhibitor 2cPE to trigger apoptosis in leukemia cells in comparison with bortezomib, another inhibitor of the ubiquitin-proteasome system (UPS). Both inhibitors trigger apoptosis in CLL B cells and gene expression profiles studies denoted how a substantial part of genes up-regulated by these compounds are elements of adaptive responses, aimed to sustain cell survival. 2cPE treatment elicits the up-regulation of chaperones, proteasomal subunits and elements of the anti-oxidant response. Selective inhibition of these responses augments apoptosis in response to 2cPE treatment. We have also observed that the product of the ataxia telangiectasia mutated gene (ATM) is activated in 2cPE treated cells. Stimulation of ATM signaling is possibly dependent on the alteration of the redox homeostasis. Importantly ATM inhibition, mutations or down-modulation increase cell death in response to 2cPE. Overall this work suggests that 2cPE could offer new opportunities for the treatment of B-CLL

Analytic and Dynamic Secretory Profile of Patient-Derived Cytokine-Induced Killer Cells

Adoptive immunotherapy with cytokine induced killer (CIK) cells has shown antitumor activity against several kinds of cancer in preclinical models and clinical trials. CIK cells are a subset of ex vivo expanded T lymphocytes with T-NK phenotype and MHC-unrestricted antitumor activity. The literature provides scant information on cytokines, chemokines and growth factors secreted by CIK cells. Therefore, we investigated the secretory profile of CIK cells generated from tumor patients. The secretome analysis was performed at specific time points (d 1, d 14 and d 21) of CIK cell expansion. Mature CIK cells (d 21) produce a great variety of interleukins and secreted proteins that can be divided into three groups based on their secretion quantity: high (interleukin [IL]-13, regulated on activation normal T cell expressed and secreted [RANTES] chemokine, MIP-1 alpha and 1 beta), medium (IL-1Ra, IL-5, IL-8, IL-10, IL-17, IP-10, INF-gamma, vascular endothelial growth factor [VEGF] and granulocyte-macrophage colony-stimulating factor [GM-CSF]) and low (IL-1 beta, IL-4, IL-6, IL-7, IL-9, IL-12, IL-15, eotaxin, platelet-derived growth factor-bb, basic fibroblast growth factor, G-CSF and monocyte chemoattractant protein [MCP]-1). Moreover, comparing peripheral blood mononuclear cells (PBMCs) (d 1) and mature CIK cells (d 14 and 21) secretomes, we observed that IL-5, IL-10, IL-13, GM-CSF and VEGF were greatly upregulated, while IL-1 beta, IL-6, IL-8, IL-15, IL-17, eotaxin, MCP-1 and RANTES were downregulated. We also performed a gene expression profile analysis of patient-derived CIK cells, showing that mRNA for the different cytokines and secreted proteins was modulated during PBMC-to-CIK differentiation. We highlight previously unknown secretory properties and provide, for the first time, a comprehensive molecular characterization of CIK cells. Our findings provide a rationale to explore the functional implications and possible therapeutic modulation of CIK secretome