Antonella Poloni

Human Dedifferentiated Adipocytes Show Similar Properties to Bone Marrow‐Derived Mesenchymal Stem Cells

Mature adipocytes are generally considered terminally differentiated because they have lost their proliferative abilities. Here, we studied the gene expression and functional properties of mature adipocytes isolated from human omental and subcutaneous fat tissues. We also focused on dedifferentiated adipocytes in culture and their morphologies and functional changes with respect to mature adipocytes, stromal‐vascular fraction (SVF)‐derived mesenchymal stem cells (MSCs) and bone marrow (BM)‐derived MSCs. Isolated mature adipocytes expressed stem cell and reprogramming genes. They replicated in culture after assuming a fibroblast‐like shape and expanded similarly to SVF‐ and BM‐derived MSCs. During the dedifferentiation process, mature adipocytes lost their lineage gene expression profile, assumed the typical mesenchymal morphology and immunophenotype, expressed stem cell genes and differentiated into multilineage cells. Moreover, during the dedifferentiation process, we showed changes in the epigenetic status of mature adipocytes, which led dedifferentiated adipocytes to display a similar DNA methylation condition to BM‐derived MSCs. Like SVF‐ and BM‐derived MSCs, dedifferentiated adipocytes were able to inhibit the proliferation of stimulated lymphocytes in coculture while mature adipocytes stimulated their growth. Furthermore, dedifferentiated adipocytes maintained the survival and complete differentiation characteristic of hematopoietic stem cells. This is the first study that in addition to characterizing isolated and dedifferentiated adipocytes also reports on the immunoregulatory and hematopoietic supporting functions of these cells. This structural and functional characterization might have clinical applications of both mature and dedifferentiated adipocytes in such fields, as regenerative medicine. STEM CELLS 2012;30:965–974

Specific molecular signatures predict decitabine response in chronic myelomonocytic leukemia

Myelodysplastic syndromes and chronic myelomonocytic leukemia (CMML) are characterized by mutations in genes encoding epigenetic modifiers and aberrant DNA methylation. DNA methyltransferase inhibitors (DMTis) are used to treat these disorders, but response is highly variable, with few means to predict which patients will benefit. Here, we examined baseline differences in mutations, DNA methylation, and gene expression in 40 CMML patients who were responsive or resistant to decitabine (DAC) in order to develop a molecular means of predicting response at diagnosis. While somatic mutations did not differentiate responders from nonresponders, we identified 167 differentially methylated regions (DMRs) of DNA at baseline that distinguished responders from nonresponders using next-generation sequencing. These DMRs were primarily localized to nonpromoter regions and overlapped with distal regulatory enhancers. Using the methylation profiles, we developed an epigenetic classifier that accurately predicted DAC response at the time of diagnosis. Transcriptional analysis revealed differences in gene expression at diagnosis between responders and nonresponders. In responders, the upregulated genes included those that are associated with the cell cycle, potentially contributing to effective DAC incorporation. Treatment with CXCL4 and CXCL7, which were overexpressed in nonresponders, blocked DAC effects in isolated normal CD34+ and primary CMML cells, suggesting that their upregulation contributes to primary DAC resistance.

Characterization and expansion of mesenchymal progenitor cells from first-trimester chorionic villi of human placenta

BACKGROUND Mesenchymal stromal cells (MSC) have been identified in a variety of fetal and adult tissues, including bone marrow (BM), fetal blood and liver. We report on the isolation, expansion and differentiation in vitro of MSC-like cells from chorionic villi (CV). METHODS We evaluated 10 samples of CV collected at the first trimester (gestational age 11-13 weeks). We only used cells taken from back-up culture after a successful karyotype analysis. CV cells were characterized by morphologic, immunophenotypic and molecular analysis. The differentiation ability of mesenchymal and neural lineages was detected using specific culture conditions. Cell expansion was assessed after plating cells at different densities in different media, supplemented with animal and human serum. RESULTS CV cells showed a homogeneous population of spindle-shaped cells after the first passage. Cells expressed CD90, CD105, CD73, CD44, CD29 and CD13 but not CD45, CD14, CD34 and CD117. They expressed Oct-4, Rex-1, GATA-4 and nestin, which characterize the undifferentiated stem cell state. They differentiated into osteocytes, adipocytes, chondrocytes and neuronal cells. Cell expansion was greater than that of adult BM-derived MSC, 9 logs with fetal bovine serum and 6 logs with human serum. Despite their high proliferative capacity, we did not observe any karyotypic abnormalities after culture. DISCUSSION Our study shows that CV cells have better potential for expansion than adult stem cells. They can proliferate in a medium with human allogeneic serum and can differentiate into mesenchymal and neural lineages. CV cells may be an excellent cell source for therapeutic applications.

Human Mesenchymal Stem Cells from Chorionic Villi and Amniotic Fluid are not Susceptible to Transformation after Extensive in Vitro Expansion

Mesenchymal stem cells (MSCs) are promising candidates for cell therapy and tissue engineering. Increasing evidence suggests that MSCs isolated from fetal tissues are more plastic and grow faster than adult MSCs. In this study, we characterized human mesenchymal progenitor cells from chorionic villi (CV) and amniotic fluid (AF) isolated during the first and second trimesters, respectively, and compared them with adult bone marrow-derived MSCs (BM). We evaluated 10 CV, 10 AF, and 6 BM samples expanded until the MSCs reached senescence. We used discarded cells from prenatal analyses for all the experiments. To evaluate the replicative stability of these cells, we studied the telomerase activity, hTERT gene transcription, and telomere length in these cells. Spontaneous chromosomal alterations were excluded by cytogenetic analysis. We studied the expression of c-myc and p53, tumor-associated genes, at different passage in culture and the capacity of these cells to grow in an anchorage-independent manner by using soft agar assay. We isolated homogeneous populations of spindle-shaped CV, AF, and BM cells expressing mesenchymal immunophenotypic markers throughout the period of expansion. CV cells achieved 14 ± 0.9 logs of expansion in 118 days and AF cells achieved 21 ± 0.9 logs in 118 days, while BM cells achieved 11 × 0.4 logs in 84 days. Despite their high proliferation capacity, fetal MSCs showed no telomerase activity, no hTERT and c-myc transcriptions, and maintained long, stable telomeres. A constant expression level of p53 and a normal karyotype were preserved throughout long-term expansion, suggesting the safety of fetal MSCs. In conclusion, our results indicate that fetal MSCs could be an alternative, more accessible resource for cell therapy and regenerative medicine.

Selection of CD271 cells and human AB serum allows a large expansion of mesenchymal stromal cells from human bone marrow

BACKGROUND Mesenchymal stromal cells (MSC) are promising candidates for cell therapy and tissue engineering and may be used to treat acute graft-versus-host disease (GvHD). However, major obstacles for their clinical use are the required cell dose and the biosafety and potential immunogenicity of fetal bovine serum (FBS), which is a crucial supplement of all media currently used for the culture of MSC. METHODS In this study MSC were successfully expanded after selection of CD271 cells from human bone marrow (BM) mononuclear cells in medium supplemented with 10% pooled allogeneic human serum. RESULTS We isolated MSC from 10 healthy donor BM by plastic adherence and immunomagnetic selection of the CD271(+) fraction and expanded MSC in medium supplemented with pooled human allogeneic serum and animal serum. We isolated a homogeneous multipotent population by CD271(+) selection with a proliferation rate that was higher than MSC isolated by plastic adherence, 6.8+/-1.57 compared with 2.07+/-1.40 logs. Similar to cells generated in animal serum medium, MSC from allogeneic human serum were positive for mesenchymal markers and negative for hematopoietic markers; moreover they expressed embryonic stem cell genes. A normal karyotype and differentiation capacity into adipogenic, osteogenic and chondrogenic lineages and neurosphere-like structures were preserved throughout long-term culture. DISCUSSION Expansion of MSC is both feasible and large with a CD271-selected population in medium supplemented with 10% pooled allogeneic human serum, without loss of multipotent differentiation capacity or karyotype alterations.

Long-term outcome and prospective validation of NIH response criteria in 39 patients receiving imatinib for steroid-refractory chronic GVHD

Forty adults aged 28 to 73 years were entered into a prospective trial of imatinib for the treatment of steroid-refractory chronic graft-versus-host disease (SR-cGVHD). After 6 months, intention-to-treat (ITT) analysis of 39 patients who received the drug, regardless of the duration of treatment, revealed 14 partial responses (PR), 4 minor responses (MR) with relevant steroid sparing (46%) according to Couriel criteria, and 20 ≥ PR (51.3%), as per the National Institutes of Health (NIH) criteria and NIH severity score changes. The best responses were seen in the lungs, gut, and skin (35%, 50%, and 32%, respectively). After a median follow-up of 40 months, 28 patients were alive, with a 3-year overall survival (OS) and event-free survival of 72% and 46%, respectively. The 3-year OS was 94% for patients responding at 6 months and 58% for nonresponders according to NIH response, suggesting that these criteria represent a reliable tool for predicting OS after second-line treatment. Monitoring of anti-platelet-derived growth factor receptor (PDGF-R) antibodies showed a significant decrease in PDGF-R stimulatory activity in 7 responders, whereas it remained high in 4 nonresponders. This study confirms the efficacy of imatinib against SR-cGVHD and suggests that the response at 6 months significantly predicts long-term survival.

Salvage therapy with an outpatient DHAP schedule followed by PBSC transplantation in 79 lymphoma patients: an intention to mobilize and transplant analysis

Chemotherapy followed by autologous transplantation may be an efficient salvage treatment in malignant lymphomas. We investigated the feasibility, tolerability and efficacy of an outpatient schedule of dexamethasone, cytarabine and cisplatin (DHAP), followed by peripheral blood progenitor cell autografting as salvage treatment in patients with high grade (HG), low grade (LG) non‐Hodgkins lymphoma (NHL) and Hodgkins Disease (HD). A total of 159 DHAP courses (median: 2, range: 1–5), was administered on outpatient basis to 79 patients (31 LG‐NHL, 28 HG‐NHL and 20 HD), with the intention to mobilize and to transplant. A successful collection was not achieved in 40% LG‐NHL, 10% HD and 20% HG‐NHL patients. The risk to fail the collection was significantly related to the number of previous chemotherapy courses (>6) (P = 0.005, RR = 1.4), to the pretransplant status (P = 0.04, RR = 13.5) and to the previous fludarabine administration (P = 0.01, RR = 20). High dose therapy (HDT) was feasible in 60 patients (76%). The overall treatment related mortality was 3.8%. The overall response rate (ORR) was 81% with a 57.6% overall survival (OS) at 62 months (95% CI: 45–69.3%) and a progression free survival (PFS) of 42% at 74 months (95% CI: 26.7–58%). The diagnosis of HG‐NHL and the non‐response to DHAP resulted to reduce respectively the OS (P = 0.007, RR = 2.8) and PFS probability (P = 0.01, RR = 4.1). In conclusion this outpatient schedule of DHAP is a well tolerated, efficient salvage and mobilizing regimen not only in HG‐NHL, but also in LG‐NHL and in HD. Randomized studies are needed to better define the role of DHAP in LG‐NHL and HD patients.

Engraftment capacity of mesenchymal cells following hematopoietic stem cell transplantation in patients receiving reduced-intensity conditioning regimen

The engraftment ability of mesenchymal cells was investigated in 26 patients receiving allogeneic transplantation from HLA-identical siblings with reduced-intensity conditioning (RIC). The stem cell source was bone marrow (BM) in eight patients and G-CSF-mobilized peripheral blood hematopoietic cells in 18 cases. A total of 32 patients engrafted very quickly and the chimerism evaluation (both on myeloid and on lymphoid subsets) showed that they were full donor by day 60. At the time of the study they were in complete hematological remission and displayed a full donor hematopoiesis. Two patients showed early disease progression while one did not engraft. Forty-eight out-marrow samples harvested from the 26 patients generated a marrow stromal layer adequate for the chimerism evaluation. Monocyte-macrophage contamination of marrow stromal layers was always reduced below 2% by repeated trypsinizations and treatment with the leucyl-leucine (leu-leu) methyl ester. The chimerism evaluation was performed by PCR analysis of STRs microsatellites and the amelogenin locus, by using capillary electrophoresis (CE) and by FISH analysis in case of the sex mismatch. In eight patients, a partial donor origin of stromal cells was shown (7–86% cells of donor). The source of hematopoietic cells was BM in three patients and mobilized peripheral blood in the other five.

Long-term hematologic reconstitution after autologous peripheral blood progenitor cell transplantation: a comparison between controlled-rate freezing and uncontrolled-rate freezing at 80°C

BACKGROUND : The most widely used system for peripheral blood progenitor cell (PBPC) cryopreservation is controlled‐rate freezing (CRF). Uncontrolled‐rate freezing (URF) at –80°C has also been used, but its clinical impact has not been studied sufficiently yet.

Molecular and functional characterization of human bone marrow adipocytes

Adipocytes are a cell population largely located in the human bone marrow cavity. In this specific microenvironment where adipocytes can interact with a variety of different cells, the role of fat is mainly unknown. To our knowledge, this report is the first to characterize mature adipocytes isolated from human bone marrow (BM-A) molecularly and functionally to better understand their roles into the hematopoietic microenvironment. Healthy BM-A were isolated after collagenase digestion and filtration. We studied the morphology of BM-A, their gene expression and immunophenotypic profile and their functional ability in the hematopoietic microenvironment, comparing them with adipocytes derived from adipose tissue (AT-A). BM-A showed a unilocular lipid morphology similar to AT-A and did not lose their morphology in culture; they showed a comparable pattern of stem cell-surface antigens to AT-A. In line with these observations, molecular data showed that BM-A expressed some embryonic stem cells genes, such as Oct4, KLf4, c-myc, Gata4, Tbx1, and Sox17, whereas they did not express the stem cell markers Sox2 and Nanog. Moreover, BM-A had long telomeres that were similar to bone marrow mesenchymal stem cells. Notably, BM-A supported the survival and differentiation of hematopoietic stem cells in long-term cultures. These results showed that BM-A are stromal cells with a gene expression pattern that distinguished them from AT-A. BM-A showed stem cell properties through their hematopoietic supporting function, which was certainly linked to their role in the maintenance of the bone marrow microenvironment. Depending on specific demands, BM-A may acquire different functions based on their local environment.