Ariane Tormin

CD146 expression on primary nonhematopoietic bone marrow stem cells is correlated with in situ localization

Nonhematopoietic bone marrow mesenchymal stem cells (BM-MSCs) are of central importance for bone marrow stroma and the hematopoietic environment. However, the exact phenotype and anatomical distribution of specified MSC populations in the marrow are unknown. We characterized the phenotype of primary human BM-MSCs and found that all assayable colony-forming units-fibroblast (CFU-Fs) were highly and exclusively enriched not only in the lin⁻/CD271⁺/CD45⁻/CD146⁺ stem-cell fraction, but also in lin⁻/CD271⁺/CD45⁻/CD146(⁻/low) cells. Both populations, regardless of CD146 expression, shared a similar phenotype and genotype, gave rise to typical cultured stromal cells, and formed bone and hematopoietic stroma in vivo. Interestingly, CD146 was up-regulated in normoxia and down-regulated in hypoxia. This was correlated with in situ localization differences, with CD146 coexpressing reticular cells located in perivascular regions, whereas bone-lining MSCs expressed CD271 alone. In both regions, CD34⁺ hematopoietic stem/progenitor cells were located in close proximity to MSCs. These novel findings show that the expression of CD146 differentiates between perivascular versus endosteal localization of non-hematopoietic BM-MSC populations, which may be useful for the study of the hematopoietic environment.

Bone Marrow Multipotent Mesenchymal Stroma Cells Act as Pericyte-like Migratory Vehicles in Experimental Gliomas.

Bone marrow-derived multipotent mesenchymal stroma cells (MSCs) have emerged as cellular vectors for gene therapy of solid cancers. We implanted enhanced green fluorescent protein-expressing rat MSCs directly into rat malignant gliomas to address their migratory capacity, phenotype, and effects on tumor neovascularization and animal survival. A single intratumoral injection of MSCs infiltrated the majority of invasive glioma extensions (72 +/- 14%) and a substantial fraction of distant tumor microsatellites (32 +/- 6%). MSC migration was highly specific for tumor tissue. Grafted MSCs integrated into tumor vessel walls and expressed pericyte markers alpha-smooth muscle actin, neuron-glia 2, and platelet-derived growth factor receptor-beta but not endothelial cell markers. The pericyte marker expression profile and perivascular location of grafted MSCs indicate that these cells act as pericytes within tumors. MSC grafting did not influence tumor microvessel density or survival of tumor-bearing animals. The antiangiogenic drug Sunitinib markedly reduced the numbers of grafted MSCs migrating within tumors. We found no MSCs within gliomas following intravenous (i.v.) injections. Thus, MSCs should be administered by intratumoral implantations rather than by i.v. injections. Intratumorally grafted pericyte-like MSCs might represent a particularly well-suited vector system for delivering molecules to affect tumor angiogenesis and for targeting cancer stem cells within the perivascular niche.

Characterization of bone marrow-derived mesenchymal stromal cells (MSC) based on gene expression profiling of functionally defined MSC subsets.

BACKGROUND AIMS Human mesenchymal stromal cells (MSC) are promising candidates for cell therapy because of their intriguing properties (high proliferation and differentiation capacity, microenvironmental function and immune modulation). However, MSC are heterogeneous and a better understanding of the heterogeneity of the cells that form the MSC cultures is critical. METHODS Human MSC were generated in standard cultures and stained with carboxyfluorescein succinimidyl ester (CFSE) for cell division tracking. Gene expression profiling of MSC that were sorted based on functional parameters (i.e. proliferation characteristics) was utilized to characterize potential MSC subpopulations (progenitor content and differentiation capacity) and identify potential MSC subpopulation markers. RESULTS The majority of MSC had undergone more than two cell divisions (79.7+/-2.0%) after 10 days of culture, whereas 3.5+/-0.9% of MSC had not divided. MSC were then sorted into rapidly dividing cells (RDC) and slowly/non-dividing cells (SDC/NDC). Colony-forming unit-fibroblast (CFU-F) frequencies were lowest in NDC and highest in RDC with low forward-/side-scatter properties (RDC(lolo)). Comparative microarray analysis of NDC versus RDC identified 102 differentially expressed genes. Two of these genes (FMOD and VCAM1) corresponded to cell-surface molecules that enabled the prospective identification of a VCAM1(+)/FMOD(+) MSC subpopulation, which increased with passage and showed very low progenitor activity and limited differentiation potential. CONCLUSIONS These data clearly demonstrate functional differences within MSC cultures. Furthermore, this study shows that cell sorting based on proliferation characteristics and gene expression profiling can be utilized to identify surface markers for the characterization of MSC subpopulations.

Mesenchymal stromal cells from primary osteosarcoma are non-malignant and strikingly similar to their bone marrow counterparts.

Mesenchymal stromal cells (MSC) are multipotent cells that can be isolated from a number of human tissues. In cancer, MSC have been implicated with tumor growth, invasion, metastasis, drug resistance and were even suggested as possible tumor‐initiating cells in osteosarcoma (OS). However, MSC from OS and their possible tumor origin have not yet been thoroughly investigated. Therefore, primary OS mesenchymal progenitors and OS‐derived MSC were studied. OS samples contained very high frequencies of mesenchymal progenitor cells as measured by the colony‐forming unit fibroblast (CFU‐F) assay (median: 1,117 colonies per 105 cells, range: 133–3,000, n = 6). This is considerably higher compared to other human tissues such as normal bone marrow (BM) (1.3 ± 0.2 colonies per 105 cells, n = 8). OS‐derived MSC (OS‐MSC) showed normal MSC morphology and expressed the typical MSC surface marker profile (CD105/CD73/CD90/CD44/HLA‐classI/CD166 positive, CD45/CD34/CD14/CD19/HLA‐DR/CD31 negative). Furthermore, all OS‐MSC samples could be differentiated into the osteogenic lineage, and all but one sample into adipocytes and chondrocytes. Genetic analysis of OS‐MSC as well as OS‐derived spheres showed no tumor‐related chromosomal aberrations. OS‐MSC expression of markers related to tumor‐associated fibroblasts (fibroblast surface protein, alpha‐smooth muscle actin, vimentin) was comparable to BM‐MSC and OS‐MSC growth was considerably affected by tyrosine kinase inhibitors. Taken together, our results demonstrate that normal, non‐malignant mesenchymal stroma cells are isolated from OS when MSC culture techniques are applied. OS‐MSC represent a major constituent of the tumor microenvironment, and they share many properties with BM‐derived MSC.

Human Embryonic Stem Cell-Derived Mesenchymal Stroma Cells (hES-MSCs) Engraft In Vivo and Support Hematopoiesis without Suppressing Immune Function: Implications for Off-The Shelf ES-MSC Therapies

Mesenchymal stroma cells (MSCs) have a high potential for novel cell therapy approaches in clinical transplantation. Commonly used bone marrow-derived MSCs (BM-MSCs), however, have a restricted proliferative capacity and cultures are difficult to standardize. Recently developed human embryonic stem cell-derived mesenchymal stroma cells (hES-MSCs) might represent an alternative and unlimited source of hMSCs. We therefore compared human ES-cell-derived MSCs (hES-MP002.5 cells) to normal human bone marrow-derived MSCs (BM-MSCs). hES-MP002.5 cells had lower yet reasonable CFU-F capacity compared with BM-MSC (8±3 versus 29±13 CFU-F per 100 cells). Both cell types showed similar immunophenotypic properties, i.e. cells were positive for CD105, CD73, CD166, HLA-ABC, CD44, CD146, CD90, and negative for CD45, CD34, CD14, CD31, CD117, CD19, CD 271, SSEA-4 and HLA-DR. hES-MP002.5 cells, like BM-MSCs, could be differentiated into adipocytes, osteoblasts and chondrocytes in vitro. Neither hES-MP002.5 cells nor BM-MSCs homed to the bone marrow of immune-deficient NSG mice following intravenous transplantation, whereas intra-femoral transplantation into NSG mice resulted in engraftment for both cell types. In vitro long-term culture-initiating cell assays and in vivo co-transplantation experiments with cord blood CD34+ hematopoietic cells demonstrated furthermore that hES-MP002.5 cells, like BM-MSCs, possess potent stroma support function. In contrast to BM-MSCs, however, hES-MP002.5 cells showed no or only little activity in mixed lymphocyte cultures and phytohemagglutinin (PHA) lymphocyte stimulation assays. In summary, ES-cell derived MSCs might be an attractive unlimited source for stroma transplantation approaches without suppressing immune function.

Rat Multipotent Mesenchymal Stromal Cells Lack Long-Distance Tropism to 3 Different Rat Glioma Models

BACKGROUND Viral gene therapy of malignant brain tumors has been restricted by the limited vector distribution within the tumors. Multipotent mesenchymal stromal cells (MSCs) and other precursor cells have shown tropism for gliomas, and these cells are currently being explored as potential vehicles for gene delivery in glioma gene therapy. OBJECTIVE To investigate MSC migration in detail after intratumoral and extratumoral implantation through syngeneic and orthotopic glioma models. METHODS Adult rat bone marrow-derived MSCs were transduced to express enhanced green fluorescent protein and implanted either directly into or at a distance from rat gliomas. RESULTS We found no evidence of long-distance MSC migration through the intact striatum toward syngeneic D74(RG2), N32, and N29 gliomas in the ipsilateral hemisphere or across the corpus callosum to gliomas located in the contralateral hemisphere. After intratumoral injection, MSCs migrated extensively, specifically within N32 gliomas. The MSCs did not proliferate within tumors, suggesting a low risk of malignant transformation of in vivo grafted cell vectors. Using a model for surgical glioma resection, we found that intratumorally grafted MSCs migrate efficiently within glioma remnants after partial surgical resection. CONCLUSION The findings point to limitations for the use of MSCs as vectors in glioma gene therapy, although intratumoral MSC implantation provides a dense and tumor-specific vector distribution.

Characterization of human ESC cell-derived MSC compared to bone marrow-derived MSC indicates that hES-MSC might be a possible alternative and unlimited cell source for clinical off-the-shelf MSC therapies

Hematopoietic Cell Transplantation Comorbitdity Index (HCT-CI), is a score validated by Sorror et al. to evaluate transplant Transplant-Related Mortality (TRM) incidence, in patients undergoing Allogeneic Transplant. The aim of our study was to assess TRM and the reliability of this score in AML elderly patients undergoing Autologous Transplant. We have treated 101 elderly AML patients aged over 60 years, diagnosed between june 1999 and September 2009. All patients received an induction and consolidation treatment including HD-ARA-C and Idarubicin, with a 30% reduction dose in over 70 years patients; those obtaining a successful mobilization (CD34 + cell > 3 × 106/kg) underwent Autologous Transplant, 12 after Busulfan plus Melfalan (BUMEL) and 9 after BEAM conditioning. The remaining patients received alternative consolidation treatment. We obtained a 71% CR, 49 patients were in CCR and eligible to a second consolidation; 21 of these (43%) obtained a successful CD34 mobilization and underwent Autologous Transplant. Six patients had a HCT-CI 0 score, 11 had a score between 1 and 2, 4 patients had a > 3 score. One of the latter patients had a previous cancer, 2 had an heart valve disease and 1 had diabetes, peptic ulcer requiring treatments and previous severe infectious complications. Five out 21 patients died of transplant, with a 25% cumulative 1-year TRM incidence. Surprisingly no patients with a > 3 HCT-CI score died, vs. one patient in the 1–2 score group (9%) and 4 patients in the 0 score group (67%) (P = 0.02). Age < and > 70 years did not affect the TRM rate, but all the fi ve deaths occurred after the administration of Busulfan plus Melfalan (BUMEL) with an higher TRM rate in this group (42%, P = 0.04) in comparison with the patients receiving BEAM. In our small series all 6 patients with a 0 HCT-CI score received BUMEL as conditioning (P = 0.01), suggesting that the higher TRM rate in this group might be related to the administration of a more toxic conditioning regimen. Elderly patients generally have several comorbidities and HCTCI score might be an insuffi cient tool in assessing their frailty. The high TRM rate in elderly AML patients suggests that some of these patients should receive less toxic conditioning or alternative consolidation regimen. Larger retrospective and multicentric studies are therefore needed in order to better validate this score in this setting.

Transendothelial migration capacity of human culture-derived mesenchymal stromal cells and primary CD271+bone marrow cells

Fanconi anemia (FA) is an autosomal recessive chromosomal instability disorder with a very high risk of head and neck squamous cell carcinoma (HNSCC), notably after hematopoietic stem cell transplantation (HSCT). Risk factors of HNSCC are well known: HSCT conditioning with chemotherapy and radiotherapy, prolonged immunosuppressive treatment and chronic graft versus host disease (GvHD). However, HNSCC characteristics in FA patients after HSCT are not well described. We thus analyzed 13 cases of HNSCC in FA patients who underwent HSCT in Saint-Louis Hospital, between 1976 and 2007. Patients’ age at time of transplantation ranged from 4.5 to 19.2 years (median: 9.7 years). All patients received bone marrow as source of stem cells and 10 patients were transplanted from related donors. Concerning the conditioning all patients received irradiation. All patients developed extensive chronic GvHD with lichen of oral mucosa. HNSCC was diagnosed at a median time of 10 years after HSCT. There were diverse tumors’ sites: tongue (n=8), palate (n=5), gingival (n=2), jugal (n=2), hypopharynx (n=1), oropharynx (n=1). Three patients had 2 tumors’ sites. The TNM classification was: T1 (n=6), T2 (n=2), T3 (n=2), T4 (n= 3). Four patients were lymph nodes positives, 6 were N0. Concerning the treatment, 10 patients had surgery, with clear margins for seven. Neck dissection was carried out for 6 patients. Three patients had irradiation; 1 patient had chemotherapy, and one patient had cryotherapy. Chemotherapy and radiotherapy were associated with toxicity. Five patients presented progressive disease during or immediately after the treatment and five patients relapsed within a median of 5.9 months from diagnosis. For those 10 patients, death occurred with a median time of 6.4 months after diagnosis. At the end of the study, 3 patients were alive without any evidence of recurrence or metastasis. The time of follow up after diagnosis for these patients was: 2.5 months, 3.5 months and 16.5 months, respectively. HNSCC in FA patients after HSCT is aggressive and related with very poor prognosis. A systematic oral follow-up is essential to allow early surgery which remains the only curative treatment. It is very important to attempt to prevent this cancer by reducing chronic GvHD and using conditioning without irradiation. P414 Idarubicin containing regimen for HSCT prolongs mucosal toxicity A.H.E. Herbers*, J.P. Donnelly, N.M.A. Blijlevens Radboud University Nijmegen Medical Cent (Nijmegen, NL)