Mesangiogenic Progenitor Cells Derived from One Novel CD64brightCD31brightCD14neg Population in Human Adult Bone Marrow

Mesenchymal Stromal Cells (MSCs) have been the object of extensive research for decades, due to their intrinsic clinical value. Nonetheless, the unambiguous identification of a unique in vivo MSC progenitor is still lacking, and the hypothesis that these multipotent cells could possibly arise from different in vivo precursors has been gaining consensus in the last years. We identified a novel multipotent cell population in human adult bone marrow that we firstly named Mesodermal Progenitor Cells (MPCs) for the ability to differentiate toward the mesenchymal lineage while still retaining angiogenic potential. Despite extensive characterization, MPCs positioning within the differentiation pathway and whether they can be ascribed as possible distinctive progenitor of the MSC lineage is still unclear. Here we describe the ex vivo isolation of one novel bone marrow sub-population (Pop#8) with the ability to generate MPCs. Multicolor flow cytometry in combination with either FACS or MACS cell sorting were applied to characterize Pop#8 as CD64brightCD31brightCD14neg. We defined Pop#8 properties in culture, including the potential of Pop#8-derived MPCs to differentiate into MSCs. Gene expression data were suggestive of Pop#8 in vivo involvement in HSC niche constitution/maintenance. Pop#8 resulted over three logs more frequent than other putative MSC progenitors, corroborating the idea that most of the controversies regarding culture expanded MSCs could be the consequence of different culture conditions which select or promote particular sub-populations of precursors

ED-B-Containing Isoform of Fibronectin in Tumor Microenvironment of Thymomas:A Target for a Theragnostic Approach

Simple Summary The extra-domain B fibronectin (ED-B FN) is highly expressed in thymic epithelial tumors (TETs), as demonstrated by in vivo targeting using 131I-labeled L19 small immunoprotein (131I-L19-SIP) and immunohistochemistry with a predominant expression by stromal cells of a thymoma microenvironment rather than epithelial cells. Such high expression derived from the induction of stromal cells shifts FN production to the ED-B subtype. Our results suggest that Radretumab radioimmunotherapy (R-RIT) inefficacy is not related to low TET ED-B expression but to multifactorial aspects including patients' inherent characteristics, the pattern expression of the target, the biological characteristics of the tumor, and the format of the target agent, which contribute to the resistance of tumor cells to treatment. Aim: to exploit tissue-specific interactions among thymic epithelial tumor (TETs) cells and extra-domain B fibronectin (ED-B FN). Material and methods: The stromal pattern of ED-B FN expression was investigated through tumor specimen collection and molecular profiling in 11 patients with recurrent TETs enrolled in prospective theragnostic phase I/II trials with Radretumab, an ED-B FN specific recombinant human antibody. Radretumab radioimmunotherapy (R-RIT) was offered to patients who exhibited the target expression. Experiments included immunochemical analysis (ICH), cell cultures, immunophenotypic analysis, Western blot, slot-blot assay, and quantitative RT-PCR of two primary thymoma cultures we obtained from patients' samples and in the Ty82 cell line. Results: The in vivo scintigraphic demonstration of ED-B FN expression resulted in R-RIT eligibility in 8/11 patients, of which seven were treated. The best observed response was disease stabilization (n = 5/7) with a duration of 4.3 months (range 3-5 months). IHC data confirmed high ED-B FN expression in the peripherical microenvironment rather than in the center of the tumor, which was more abundant in B3 thymomas. Further, there was a predominant expression of ED-B FN by the stromal cells of the thymoma microenvironment rather than the epithelial cells. Conclusions: Our data support the hypothesis that thymomas induce stromal cells to shift FN production to the ED-B subtype, likely representing a favorable hallmark for tumor progression and metastasis. Collectively, results derived from clinical experience and molecular insights of the in vitro experiments suggested that R-RIT inefficacy is unlikely related to low target expression in TET, being the mechanism of R-RIT resistance eventually related to patients' susceptibility (i.e., inherent characteristics), the pattern expression of the target (i.e., at periphery), the biological characteristics of the tumor (i.e., aggressive and resistant phenotypes), and/or to format of the target agent (i.e., 131I-L19-SIP)

Constitutive Expression of Pluripotency-Associated Genes in Mesodermal Progenitor Cells (MPCs)

Background: We recently characterized a progenitor of mesodermal lineage (MPCs) from the human bone marrow of adults or umbilical cord blood. These cells are progenitors able to differentiate toward mesenchymal, endothelial and cardiomyogenic lineages. Here we present an extensive molecular characterization of MPCs, from bone marrow samples, including 39 genes involved in stem cell machinery, differentiation and cell cycle regulation. Methodology/Principal Findings: MPCs are cytofluorimetrically characterized and quantitative RT-PCR was performed to evaluate the gene expression profile, comparing it with MSCs and hESCs lines. Immunofluorescence and dot-blot analysis confirm qRT-PCR data. MPCs exhibit an increased expression of OCT4, NANOG, SALL4, FBX15, SPP1 and to a lesser extent c-MYC and KLF4, but lack LIN28 and SOX2. MPCs highly express SOX15. Conclusions/Significance: MPCs express many pluripotency-associated genes and show a peculiar Oct-4 molecular circuit. Understanding this unique molecular mechanism could lead to identifying MPCs as feasible, long telomeres, target cells for reprogramming with no up-regulation of the p53 pathway. Furthermore MPCs are easily and inexpensively harvested fro

Nanotopography Induced Human Bone Marrow Mesangiogenic Progenitor Cells (MPCs) to Mesenchymal Stromal Cells (MSCs) Transition

Mesangiogenic progenitor cells (MPCs) are a very peculiar population of cells present in the human adult bone marrow, only recently discovered and characterized. Owing to their differentiation potential, MPCs can be considered progenitors for mesenchymal stromal cells (MSCs), and for this reason they potentially represent a promising cell population to apply for skeletal tissue regeneration applications. Here, we evaluate the effects of surface nanotopography on MPCs, considering the possibility that this specific physical stimulus alone can trigger MPC differentiation toward the mesenchymal lineage. In particular, we exploit nanogratings to deliver a mechanical, directional stimulus by contact interaction to promote cell morphological polarization and stretching. Following this interaction, we study the MPC-MSC transition by i. analyzing the change in cell morphotype by immunostaining of the key cell-adhesion structures and confocal fluorescence microscopy, and ii. quantifying the expression of cell-phenotype characterizing markers by flow cytometry. We demonstrate that the MPC mesengenic differentiation can be induced by the solely interaction with the NGs, in absence of any other external, chemical stimulus. This aspect is of particular interest in the case of multipotent progenitors as MPCs that, retaining both mesengenic and angiogenic potential, possess a high clinical appeal

Mesodermal Progenitor Cells (MPCs) Differentiate into Mesenchymal Stromal Cells (MSCs) by Activation of Wnt5/Calmodulin Signalling Pathway

Mesenchymal Stromal Cells (MSCs) remain poorly characterized because of the absence of manifest physical, phenotypic, and functional properties in cultured cell populations. Despite considerable research on MSCs and their clinical application, the biology of these cells is not fully clarified and data on signalling activation during mesenchymal differentiation and proliferation are controversial. The role of Wnt pathways is still debated, partly due to culture heterogeneity and methodological inconsistencies. Recently, we described a new bone marrow cell population isolated from MSC cultures that we named Mesodermal Progenitor Cells (MPCs) for their mesenchymal and endothelial differentiation potential. An optimized culture method allowed the isolation from human adult bone marrow of a highly pure population of MPCs (more than 97%), that showed the distinctive SSEA-4+CD105+CD90(neg) phenotype and not expressing MSCA-1 antigen. Under these selective culture conditions the percentage of MSCs (SSEA-4(neg)CD105+CD90(bright) and MSCA-1+), in the primary cultures, resulted lower than 2%.We demonstrate that MPCs differentiate to MSCs through an SSEA-4+CD105+CD90(bright) early intermediate precursor. Differentiation paralleled the activation of Wnt5/Calmodulin signalling by autocrine/paracrine intense secretion of Wnt5a and Wnt5b (p<0.05 vs uncondictioned media), which was later silenced in late MSCs (SSEA-4(neg)). We found the inhibition of this pathway by calmidazolium chloride specifically blocked mesenchymal induction (ID₅₀ =  0.5 µM, p<0.01), while endothelial differentiation was unaffected.The present study describes two different putative progenitors (early and late MSCs) that, together with already described MPCs, could be co-isolated and expanded in different percentages depending on the culture conditions. These results suggest that some modifications to the widely accepted MSC nomenclature are required

The Polycomb BMI1 Protein Is Co-expressed With CD26+ in Leukemic Stem Cells of Chronic Myeloid Leukemia

The Polycomb gene BMI1 expression exerts a negative predictive impact on several hematological malignancies, such as acute and chronic myeloid leukemia (CML), myelofibrosis, and follicular lymphoma. As already demonstrated in CML, BMI1 is responsible for the resistance to the tyrosine kinase inhibitors (TKIs) in a BCR-ABL1-independent way. Even if, it is unknown where BMI1 in CML is expressed (in progenitors or more mature cells). We decided, therefore, to evaluate if and where the BMI1 protein is located, focusing mainly on the CD34+/CD38-/CD26+ CML progenitors. To begin we measured, by flow cytometry, the proportion of CD34+/CD26+ cells in 31 bone marrow samples from 20 CML patients, at diagnosis and during treatment with imatinib. After that the bone marrow blood smears were stained with antibodies anti-CD26, BCR-ABL1, and BMI1. These smears were observed by a confocal laser microscope and a 3D reconstruction was then performed. At diagnosis, CD34+/CD26+ cells median value/μL was 0.48; this number increased from diagnosis to the third month of therapy and then reduced during treatment with imatinib. The number and behavior of the CD26+ progenitors were independent from the BCR-ABL1 expression, but they summed up what previously observed about the BMI1 expression modulation. In this work we demonstrate for the first time that in CML the BMI1 protein is co-expressed with BCR-ABL1 only in the cytoplasm of the CD26+ precursors; on the contrary, in other hematological malignancies where BMI1 is commonly expressed (follicular lymphoma, essential thrombocytemia, acute myeloid leukemia), it was not co-localized with CD26 or, obviously, with BCR-ABL1. Once translated into the clinical context, if BMI1 is a marker of stemness, our results would suggest the combination of the BMI1 inhibitors with TKIs as an interesting object of research, and, probably, as a promising way to overcome resistance in CML patients

Isolating Mesangiogenic progenitor cells (MPCs) from human bone marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as “Mesangiogenic Progenitor Cells”

Virtualidad y trabajo colectivo: una experiencia pedagógica durante el aislamiento. Reseña del proyecto interdisciplinar “Conjeturas y aproximaciones: lxs jóvenes pensando la pandemia”

Todavía no había finalizado el primer semestre del 2020, crecía la incertidumbre sobre los procesos pedagógicos, el agotamiento de lxs trabajadores de la educación y el malestar de lxs estudiantes. Frente a este panorama, imaginamos un espacio colectivo de trabajo que nos permitiera mitigar las distancias físicas y acercar lo que la virtualidad estaba individualizando. Conversado entre pares, manifestado por lxs estudiantes en la nuevísima y vertiginosa educación virtual que encaramos, creímos que el objetivo de sostener el vínculo pedagógico como algo más que un acto reproductivo de contenidos aislados debía encararse desde una propuesta que se animase a reflexionar sobre el inmediato presente. Así, el proyecto interdisciplinar “Conjeturas y aproximaciones: lxs jóvenes pensando la pandemia”, se pensó para trabajar entre tres materias: Filosofía, Formación para la Vida y el Trabajo, y Ciudadanía y Política, en las orientaciones tanto de Ciencias Sociales como de Ciencias Naturales de sexto año del Colegio San José, para Nivel Secundario. Aun cuando inicialmente previmos un tiempo acotado en su desarrollo, su recorrido continuó hasta finalizar el ciclo lectivo.Fil: Vanoli, Fernando Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro Experimental de la Vivienda Económica; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Montali, Guido. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Estudios sobre Cultura y Sociedad. Universidad Nacional de Córdoba. Centro de Investigaciones y Estudios sobre Cultura y Sociedad; Argentina. Universidad de Buenos Aires; ArgentinaFil: Sorbera, Pedro Oscar. Universidad Nacional de Villa María; Argentina. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Farga, Gisel Marina. Universidad Nacional de Córdoba. Facultad de Filosofía y Humanidades; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Specific integrin expression is associated with podosome-like structures on mesodermal progenitor cells.

Mesenchymal stromal cells (MSCs) are a heterogeneous cell population capable of differentiating toward several cell lines in vitro and, possibly, in vivo. Within cultured MSCs, we identified and purified a precursor cell population [mesodermal progenitor cells (MPCs)] retaining robust proliferation potential and ability to differentiate into endothelial or mesenchymal cells. MPC-derived MSCs retain the ability to further differentiate into osteoblasts, cartilage, or fat cells. Here we further characterized MPCs and MSCs by evaluating expression of integrins and adhesion molecules showing their ability to assemble the molecular machinery involved in endothelium adhesion. MPCs were shown to interact with activated and nonactivated endothelium, whereas MSCs exhibited activation of focal adhesion complexes, higher cell motility, and reduced or absent adhesiveness onto endothelial cells, suggesting a matrix remodeling vocation. We also reported a consistent expression of CXCR4 on the MPC cell surface, suggesting that the different phenotypic behavior could be related to specific functions of the cell in each differentiation stage