Domenico Mattiucci

Plasticity of human dedifferentiated adipocytes toward endothelial cells

The process of cellular differentiation in terminally differentiated cells is thought to be irreversible, and these cells are thought to be incapable of differentiating into distinct cell lineages. Our previous study showed that mature adipocytes represent an alternative source of mesenchymal stem cells. Here, results showed the capacity of mature adipocytes to differentiate into endothelial-like cells, using the ability of these cells to revert into an immature phase without any relievable chromosomal alterations. Mature adipocytes were isolated from human omental and subcutaneous fat and were dedifferentiated in vitro. The resulting cells were subcultivated for endothelial differentiation and were analyzed for their expression of specific genes and proteins. Endothelial-like cells were harvested from the differentiation medium and were traditionally cultured to evaluate the endothelial markers and the karyotype. Cells cultured in specific medium formed tube-like structures and expressed several endothelial marker genes and proteins. The endothelial-like cells expressed significantly higher levels of vascular endothelium growth factor receptor 2, vascular endothelial cadherin, Von Willebrand factor, and CD133 than the untreated cells. These cells were positively stained for CD31 and vascular endothelial cadherin, markers of mature endothelial cells. Moreover, the low-density lipoprotein-uptake assay demonstrated a functionally endothelial differentiation of these cells. When these cells were harvested and reseeded in basal medium, they lost the endothelial markers and reacquired the typical mesenchymal stem cell markers and the ability to expand in a short time period. Moreover, karyotype analysis showed that these cells reverted into an immature phase without any karyotype alterations. In conclusion, the results showed that adipocytes exhibited a great plasticity toward the endothelial lineage, suggesting their possible use in cell therapy applications for vascular disease.

Interaction between human mature adipocytes and lymphocytes induces T-cell proliferation

BACKGROUND AIMS Adipose tissue is a critical organ that plays a major role in energy balance regulation and the immune response through intricate signals. METHODS We report on the inter-relation between mature adipocytes and lymphocytes in terms of adipocyte-derived T-cell chemo-attractants and adipocyte metabolic effects on lymphocytes. RESULTS During the culture time, mature adipocytes changed their structural and functional properties into de-differentiated cells. Isolated mature adipocytes expressed significantly higher levels of CIITA, major histocompatibility complex II (human leukocyte antigen [HLA]-DR) and costimulatory signal molecule CD80 compared with adipocytes after the de-differentiation process. Moreover, human leukocyte antigen-G, which may prevent the immune responses of mesenchymal stromal cells, was expressed at lower level in mature adipocytes compared with de-differentiated adipocytes. In line with these molecular data, functional results showed different immunoregulatory properties between adipocytes before and after the de-differentiation process. Mature adipocytes stimulated the proliferation of total lymphocytes and immunoselected cell populations CD3+, CD4+ and CD8+ in a direct contact-dependent way that involved the major histocompatibility complex I and II pathways. Moreover, adipocytes secreted potential chemo-attractant factors, but data showed that adipocyte-derived culture medium was not sufficient to activate lymphocyte proliferation, suggesting that a direct contact between adipocytes and immune cells was needed. However, specific mature adipocyte cytokines enhanced lymphocyte proliferation in a mixed lymphocyte reaction. CONCLUSIONS In conclusion, cross-talk occurs between adipocytes and lymphocytes within adipose tissue involving T-cell chemo-attraction by mature adipocytes. Our findings, together with current observations in the field, provide a rationale to identify adipocyte-lymphocyte cross-talk that instigates adipose inflammation.

Bone marrow adipocytes support haematopoietic stem cell survival

In bone marrow (BM), hematopoietic elements are mingled with adipocytes (BM‐A), which are the most abundant stromal component in the niche. BM‐A progressively increase with aging, eventually occupying up to 50% of BM cavities. In this work, the role played by BM‐A was explored by studying primary human BM‐A isolated from hip surgery patients at the molecular level, through microarray analysis, and at the functional level, by assessing their relationship with primary human hematopoietic stem cells (HSC) by the long‐term culture initiating cell (LTC‐IC) assay. Findings demonstrated that BM‐A are capable of supporting HSC survival in the LTC‐IC assay, since after 5 weeks of co‐culture, HSC were still able to proliferate and differentiate. Furthermore, critical molecules such as C‐X‐C motif chemokine 12 (CXCL12), interleukin (IL)‐8, colony‐stimulating factor 3 (CSF3), and leukaemia inhibitory factor (LIF), were expressed at similar levels in BM‐A and in primary human BM mesenchymal stromal cells (BM‐MSC), whereas IL‐3 was higher in BM‐A. Interestingly, BM‐A displayed a different gene expression profile compared with subcutaneous adipose tissue adipocytes (AT‐A) collected from abdominal surgery patients, especially in terms of regulation of lipid metabolism, stemness genes, and white‐to‐brown differentiation pathways. Accordingly, analysis of the gene pathways involved in hematopoiesis regulation showed that BM‐A are more closely related to BM‐MSC than to AT‐A. The present data suggest that BM‐A play a supporting role in the hematopoietic niche and directly sustain HSC survival.

Glial-Like Differentiation Potential of Human Mature Adipocytes

The potential ability to differentiate dedifferentiated adipocytes into a neural lineage is attracting strong interest as an emerging method of producing model cells for the treatment of a variety of neurological diseases. Here, we describe the efficient conversion of dedifferentiated adipocytes into a neural-like cell population. These cells grew in neurosphere-like structures and expressed a high level of the early neuroectodermal marker Nestin. These neurospheres could proliferate and express stemness genes, suggesting that these cells could be committed to the neural lineage. After neural induction, NeuroD1, Sox1, Double Cortin, and Eno2 were not expressed. Patch clamp data did not reveal different electrophysiological properties, indicating the inability of these cells to differentiate into mature neurons. In contrast, the differentiated cells expressed a high level of CLDN11, as demonstrated using molecular method, and stained positively for the glial cell markers CLDN11 and GFAP, as demonstrated using immunocytochemistry. These data were confirmed by quantitative results for glial cell line-derived neurotrophic factor production, which showed a higher secretion level in neurospheres and the differentiated cells compared with the untreated cells. In conclusion, our data demonstrate morphological, molecular, and immunocytochemical evidence of initial neural differentiation of mature adipocytes, committing to a glial lineage.

Human white adipocytes convert into "rainbow" adipocytes in vitro

White adipocytes are plastic cells able to reversibly transdifferentiate into brown adipocytes and into epithelial glandular cells under physiologic stimuli in vivo. These plastic properties could be used in future for regenerative medicine, but are incompletely explored in their details. Here, we focused on plastic properties of human mature adipocytes (MA) combining gene expression profile through microarray analysis with morphologic data obtained by electron and time lapse microscopy. Primary MA showed the classic morphology and gene expression profile of functional mature adipocytes. Notably, despite their committed status, MA expressed high levels of reprogramming genes. MA from ceiling cultures underwent transdifferentiation toward fibroblast‐like cells with a well‐differentiated morphology and maintaining stem cell gene signatures. The main morphologic aspect of the transdifferentiation process was the secretion of large lipid droplets and the development of organelles necessary for exocrine secretion further supported the liposecretion process. Of note, electron microscope findings suggesting liposecretion phenomena were found also in explants of human fat and rarely in vivo in fat biopsies from obese patients. In conclusion, both MA and post‐liposecretion adipocytes show a well‐differentiated phenotype with stem cell properties in line with the extraordinary plasticity of adipocytes in vivo. J. Cell. Physiol. 232: 2887–2899, 2017.

Overexpression of CDKN2B (p15INK4B) and altered global DNA methylation status in mesenchymal stem cells of high-risk myelodysplastic syndromes

Overexpression of CDKN2B (p15INK4B) and altered global DNA methylation status in mesenchymal stem cells of high-risk myelodysplastic syndromes

Biosafety evidence for human dedifferentiated adipocytes.

Mature adipocytes have shown dynamic plasticity to be converted into fibroblast‐like and lipid‐free cells. After the dedifferentiation process, these cells re‐entered the cell cycle and acquired a high proliferation potential, becoming a valid source of stem cells. However, many aspects of the cellular biosafety about dedifferentiated fat cells remained unclear. This study aimed to elucidate their potential susceptibility to malignant transformation and to ascertain the safety of these cells for clinical use. To evaluate the genomic stability of dedifferentiated adipocytes, telomere length, hTERT gene transcription, the capacity of these cells to grow in an anchorage‐independent manner and the presence of DNA damage by single cell gel electrophoresis assay were studied. Spontaneous chromosomal alterations were excluded by cytogenetic analysis and the expression level of c‐myc and p53, tumor associated genes, were assessed, evaluating also p53 loss of function mutations. Despite the high proliferation capacity of dedifferentiated adipocytes, these cells showed stable telomere length compared with mature adipocytes, no hTERT transcriptions and consequently no telomerase activity, suggesting that both transformation and senescence were avoided. A constant expression level of c‐myc and p53, the inability of dedifferentiated adipocytes to grow in an anchorage‐independent manner, the absence of DNA damage suggested the safety of these cells. Moreover, a normal karyotype was preserved throughout the dedifferentiation process. Data in vivo showed that dedifferentiated adipocytes analyzed for tumorigenicity did not develop tumors. In conclusion, our data indicated that dedifferentiated adipocytes could be a relatively easily accessible resource for cell therapy and regenerative medicine. J. Cell. Physiol. 230: 1525–1533, 2015.

DNA demethylating therapy reverts mesenchymal stromal cells derived from high risk myelodysplastic patients to a normal phenotype.

CV, SOZ, KJG, SC, MLG, MR, PLPR, MTOC, IL, MLP, MS and FC: None. Carolina Villegas Da Ros Mariano Linares Garcia Sebastian Ortiz Zuluaga Karla Javier Gonzalez Sofia Costa Monica Roig Pedro Luis Perez Rodriguez Mayte Orero Castello Irene Luna Maria L opez-Pav ıa Magdalena Sanchez Felix Carbonell Department of Haematology, Hospital General Universitario de Valencia, Valencia, Spain. E-mail: caro.villegas.daros@gmail.com

Aging- and Senescence-associated Changes of Mesenchymal Stromal Cells in Myelodysplastic Syndromes

Hematopoietic stem and progenitor cells reside within the bone marrow (BM) microenvironment. By a well-balanced interplay between self-renewal and differentiation, they ensure a lifelong supply of mature blood cells. Physiologically, multiple different cell types contribute to the regulation of stem and progenitor cells in the BM microenvironment by cell-extrinsic and cell-intrinsic mechanisms. During the last decades, mesenchymal stromal cells (MSCs) have been identified as one of the main cellular components of the BM microenvironment holding an indispensable role for normal hematopoiesis. During aging, MSCs diminish their functional and regenerative capacities and in some cases encounter replicative senescence, promoting inflammation and cancer progression. It is now evident that alterations in specific stromal cells that comprise the BM microenvironment can contribute to hematologic malignancies, and there is growing interest regarding the contribution of MSCs to the pathogenesis of myelodysplastic syndromes (MDSs), a clonal hematological disorder, occurring mostly in the elderly, characterized by ineffective hematopoiesis and increased tendency to acute myeloid leukemia evolution. The pathogenesis of MDS has been associated with specific genetic and epigenetic events occurring both in hematopoietic stem cells (HSCs) and in the whole BM microenvironment with an aberrant cross talk between hematopoietic elements and stromal compartment. This review highlights the role of MSCs in MDS showing functional and molecular alterations such as altered cell-cycle regulation with impaired proliferative potential, dysregulated cytokine secretion, and an abnormal gene expression profile. Here, the current knowledge of impaired functional properties of both aged MSCs and MSCs in MDS have been described with a special focus on inflammation and senescence induced changes in the BM microenvironment. Furthermore, a better understanding of aberrant BM microenvironment could improve future potential therapies.