Ann De Becker

Chromatin remodeling agent trichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stem cells derived of adult bone marrow

BackgroundThe capability of human mesenchymal stem cells (hMSC) derived of adult bone marrow to undergo in vitro hepatic differentiation was investigated.ResultsExposure of hMSC to a cocktail of hepatogenic factors [(fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF), insulin-transferrin-sodium-selenite (ITS) and dexamethasone)] failed to induce hepatic differentiation. Sequential exposure to these factors (FGF-4, followed by HGF, followed by HGF+ITS+dexamethasone), however, resembling the order of secretion during liver embryogenesis, induced both glycogen-storage and cytokeratin (CK)18 expression. Additional exposure of the cells to trichostatin A (TSA) considerably improved endodermal differentiation, as evidenced by acquisition of an epithelial morphology, chronological expression of hepatic proteins, including hepatocyte-nuclear factor (HNF)-3β, alpha-fetoprotein (AFP), CK18, albumin (ALB), HNF1α, multidrug resistance-associated protein (MRP)2 and CCAAT-enhancer binding protein (C/EBP)α, and functional maturation, i.e. upregulated ALB secretion, urea production and inducible cytochrome P450 (CYP)-dependent activity.ConclusionhMSC are able to undergo mesenchymal-to-epithelial transition. TSA is hereby essential to promote differentiation of hMSC towards functional hepatocyte-like cells.

Homing and migration of mesenchymal stromal cells: How to improve the efficacy of cell therapy?

Mesenchymal stromal cells (MSCs) are currently being investigated for use in a wide variety of clinical applications. For most of these applications, systemic delivery of the cells is preferred. However, this requires the homing and migration of MSCs to a target tissue. Although MSC homing has been described, this process does not appear to be highly efficacious because only a few cells reach the target tissue and remain there after systemic administration. This has been ascribed to low expression levels of homing molecules, the loss of expression of such molecules during expansion, and the heterogeneity of MSCs in cultures and MSC culture protocols. To overcome these limitations, different methods to improve the homing capacity of MSCs have been examined. Here, we review the current understanding of MSC homing, with a particular focus on homing to bone marrow. In addition, we summarize the strategies that have been developed to improve this process. A better understanding of MSC biology, MSC migration and homing mechanisms will allow us to prepare MSCs with optimal homing capacities. The efficacy of therapeutic applications is dependent on efficient delivery of the cells and can, therefore, only benefit from better insights into the homing mechanisms.

Bone Marrow-Derived Mesenchymal Stromal Cells Are Attracted by Multiple Myeloma Cell-Produced Chemokine CCL25 and Favor Myeloma Cell Growth In Vitro and In Vivo

Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells that are predominantly localized in the bone marrow (BM). Mesenchymal stromal cells (MSCs) give rise to most BM stromal cells that interact with MM cells. However, the direct involvement of MSCs in the pathophysiology of MM has not been well addressed. In this study, in vitro and in vivo migration assays revealed that MSCs have tropism toward MM cells, and CCL25 was identified as a major MM cell‐produced chemoattractant for MSCs. By coculture experiments, we found that MSCs favor the proliferation of stroma‐dependent MM cells through soluble factors and cell to cell contact, which was confirmed by intrafemoral coengraftment experiments. We also demonstrated that MSCs protected MM cells against spontaneous and Bortezomib‐induced apoptosis. The tumor‐promoting effect of MSCs correlated with their capacity to enhance AKT and ERK activities in MM cells, accompanied with increased expression of CyclinD2, CDK4, and Bcl‐XL and decreased cleaved caspase‐3 and poly(ADP‐ribose) polymerase expression. In turn, MM cells upregulated interleukin‐6 (IL‐6), IL‐10, insulin growth factor‐1, vascular endothelial growth factor, and dickkopf homolog 1 expression in MSCs. Finally, infusion of in vitro‐expanded murine MSCs in 5T33MM mice resulted in a significantly shorter survival. MSC infusion is a promising way to support hematopoietic recovery and to control graft versus host disease in patients after allogeneic hematopoietic stem cell transplantation. However, our data suggest that MSC‐based cytotherapy has a potential risk for MM disease progression or relapse and should be considered with caution in MM patients. STEM CELLS 2012; 30:266–279.

An Improved Harvest and in Vitro Expansion Protocol for Murine Bone Marrow-Derived Mesenchymal Stem Cells

Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species, the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest, followed by an immunodepletion step using microbeads coated with CD11b, CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion, a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1), CD90, CD105 and CD73 cell surface markers, but negative for the hematopoietic surface markers CD14, CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic, osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time.

Efficient differentiation of human embryonic stem cells into a homogeneous population of osteoprogenitor-like cells

The use of human embryonic stem cells (hESC) in both research and therapeutic applications requires relatively large homogeneous populations of differentiated cells. The differentiation of three hESC lines into highly homogeneous populations of osteoprogenitor-like (hESC-OPL) cells is reported here. These cells could be expanded in a defined culture system for more than 18 passages, and showed a fibroblast-like morphology and a normal stable karyotype. The cells were strongly positive for the same antigenic markers as mesenchymal stem cells but negative for markers of haematopoetic stem cells. The hESC-OPL cells were able to differentiate into the osteogenic, but not into the chondrogenic or adipogenic, lineage and were positive for markers of early stages of osteogenic differentiation. When cultured in the presence of osteogenic supplements, the cells indicated the capacity to achieve, under inductive conditions, a mature osteoblast phenotype. The differentiation protocol is based on a monolayer approach, and does not require any exogenous factors other than fetal calf serum, or coculture systems of animal or human origin. This method is likely to be amenable to large-scale production of homogeneous osteoprogenitor-like cells and thus overcomes one of the major problems of differentiation of hESC, with important relevance for further cell therapy studies.

Upregulation of miR-135b is involved in the impaired osteogenic differentiation of mesenchymal stem cells derived from multiple myeloma patients.

Previous studies have demonstrated that mesenchymal stem cells from multiple myeloma (MM) patients (MM-hMSCs) display a distinctive gene expression profile, an enhanced production of cytokines and an impaired osteogenic differentiation ability compared to normal donors (ND-hMSCs). However, the underlying molecular mechanisms are unclear. In the present study, we observed that MM-hMSCs exhibited an abnormal upregulation of miR-135b, showing meanwhile an impaired osteogenic differentiation and a decrease of SMAD5 expression, which is the target of miR-135b involved in osteogenesis. By gain and loss of function studies we confirmed that miR-135b negatively regulated hMSCs osteogenesis. We also found that MM cell-produced factors stimulated ND-hMSCs to upregulate the expression of miR-135b. Importantly, treatment with a miR-135b inhibitor promoted osteogenic differentiation in MM-hMSCs. Finally, we observed that MM cell-derived soluble factors could induce an upregulation of miR-135b expression in ND-hMSCs in an indirect coculture system and the miR-135b expression turned to normal level after the removal of MM cells. Collectively, we provide evidence that miR-135b is involved in the impaired osteogenic differentiation of MSCs derived from MM patients and might therefore be a promising target for controlling bone disease.

Effect of the HDAC inhibitor vorinostat on the osteogenic differentiation of mesenchymal stem cells in vitro and bone formation in vivo.

Aim:Vorinostat, a histone deacetylase (HDAC) inhibitor currently in a clinical phase III trial for multiple myeloma (MM) patients, has been reported to cause bone loss. The purpose of this study was to test whether, and to what extent, vorinostat influences the osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro and bone formation in vivo.Methods:Bone marrow-derived MSCs were prepared from both normal donors and MM patients. The MSCs were cultured in an osteogenic differentiation induction medium to induce osteogenic differentiation, which was evaluated by alkaline phosphatase (ALP) staining, Alizarin Red S staining and the mRNA expression of osteogenic markers. Naïve mice were administered vorinostat (100 mg/kg, ip) every other day for 3 weeks. After the mice were sacrificed, bone formation was assessed based on serum osteocalcin level and histomorphometric analysis.Results:Vorinostat inhibited the viability of hMSCs in a concentration-dependent manner (the IC50 value was 15.57 μmol/L). The low concentration of vorinostat (1 μmol/L) did not significantly increase apoptosis in hMSCs, whereas pronounced apoptosis was observed following exposure to higher concentrations of vorinostat (10 and 50 μmol/L). In bone marrow-derived hMSCs from both normal donors and MM patients, vorinostat (1 μmol/L) significantly increased ALP activity, mRNA expression of osteogenic markers, and matrix mineralization. These effects were associated with upregulation of the bone-specifying transcription factor Runx2 and with the epigenetic alterations during normal hMSCs osteogenic differentiation. Importantly, the mice treated with vorinostat did not show any bone loss in response to the optimized treatment regimen.Conclusion:Vorinostat, known as a potent anti-myeloma drug, stimulates MSC osteogenesis in vitro. With the optimized treatment regimen, any decrease in bone formation was not observed in vivo.

In vitro expanded bone marrow-derived murine (C57Bl/KaLwRij) mesenchymal stem cells can acquire CD34 expression and induce sarcoma formation in vivo

Mesenchymal stem cells (MSCs) have currently generated numerous interests in pre-clinical and clinical applications due to their multiple lineages differentiation potential and immunomodulary effects. However, accumulating evidence indicates that MSCs, especially murine MSCs (mMSCs), can undergo spontaneous transformation after long-term in vitro culturing, which might reduce the therapeutic application possibilities of these stem cells. In the present study, we observed that in vitro expanded bone marrow (BM) derived mMSCs from the C57Bl/KaLwRij mouse strain can lose their specific stem cells markers (CD90 and CD105) and acquire CD34 expression, accompanied with an altered morphology and an impaired tri-lineages differentiation capacity. Compared to normal mMSCs, these transformed mMSCs exhibited an increased proliferation rate, an enhanced colony formation and migration ability as well as a higher sensitivity to anti-tumor drugs. Transformed mMSCs were highly tumorigenic in vivo, resulting in aggressive sarcoma formation when transplanted in non-immunocompromised mice. Furthermore, we found that Notch signaling downstream genes (hey1, hey2 and heyL) were significantly upregulated in transformed mMSCs, while Hedgehog signaling downstream genes Gli1 and Ptch1 and the Wnt signaling downstream gene beta-catenin were all decreased. Taken together, we observed that murine in vitro expanded BM-MSCs can transform into CD34 expressing cells that induce sarcoma formation in vivo. We assume that dysregulation of the Notch(+)/Hh(-)/Wnt(-) signaling pathway is associated with the malignant phenotype of the transformed mMSCs.

Symptomatic myelodysplasia with normal blood counts: a diagnostic enigma complicating the management of a patient with severe platelet dysfunction

As a rule, myelodysplastic syndromes (MDS) are accompanied by peripheral cytopenia in one or more cell lineages [1]. Platelet function defects (PFDs) are frequently seen in MDS, but usually only cause mild bleeding symptoms [2,3]. We report for the fi rst time a case of MDS that manifested as a clinically severe and initially unexplained PFD with completely normal blood counts. Th e diagnosis of MDS was made retrospectively, after the patient had gradually progressed toward refractory thrombocytopenia with minimal dysplastic features, and fi nally acute myeloid leukemia (AML). Th is is a case of a 56-year-old man who presented with a 6-year history of recurrent rectal blood loss for which he was admitted to our hospital on several occasions. Multiple colonoscopies and gastroscopies could fi nd no clear cause aside from the presence of internal hemorrhoids, for which he was surgically treated. Th is intervention, however, had very little impact on his symptoms. Th e patient reported a recently developed bleeding trend, presenting as recurrent epistaxis, for which cauterization was needed on two occasions, as well as a tendency to develop cutaneous purpura. Th ere was no family history of a bleeding disorder. In between hemorrhagic episodes, complete blood counts and clotting studies, including von Willebrand factor antigen and activity tests, were unremarkable. Kidney and functional liver tests as well as serum protein electrophoresis revealed no abnormalities. Th ere were no signs of increased fi brinolysis (D-dimers 476 μ g/L). Platelet light transmission aggregometry was disturbed with all tested agonists: the aggregation response to adenosine diphosphate, collagen and ristocetin was 35%, 47% and 60%, respectively; there was a strikingly absent response to arachidonic acid. A possible impact of medication was excluded by repeated analysis under controlled circumstances. Due to the lack of any bleeding diathesis in the patient ’ s history, a genetic defect was unlikely. Any plausible explanation for the fi ndings could not be found at that point, and hence the patient was treated symptomatically with frequent erythrocyte transfusions and tranexamic acid, having very little impact on the bleeding frequency. Six years after the first symptoms appeared, the patient developed an isolated thrombocytopenia: hemoglobin concentration 13.4 g/dL, red cell count 4.6 10 12 /L, mean corpuscular volume 90.7 fL, platelet count 95 10 9 /L, white cell count 11.6 10 9 /L. A blood film showed the presence of giant platelets. Hemorrhagic incidents became more common: over a period of 2 years the patient was hospitalized five times because of massive rectorrhagia for which transfusion of red blood cells was frequently mandatory. Endoscopic and colonoscopic investigations were again unremarkable except for the sporadic detection of small arteriovascular malformations. These lesions were difficult to observe because of persistent bleeding and blood clots in the gastrointestinal tract. Both angiography and Meckel scan were negative. Thrombocytopenia and the appearance of large platelets prompted the diagnosis of idiopathic thrombocytopenic purpura (ITP). A bone marrow aspirate revealed an increased number of megakaryocytes, lacking any other significant abnormalities. This finding supported the diagnosis of ITP. The patient was treated with corticosteroids, inducing a modest but only temporary response of platelet count and bleeding. Tranexamic acid was able to stop the blood loss for a brief period, but the patient quickly relapsed despite increasing the dosage. Ultimately, donor platelets were administered at a regular interval, with a good impact on the symptoms. Th is patient ’ s case was put up for reevaluation when cytogenetic analysis of the bone marrow revealed the presence of trisomy 21 as the sole karyotypic abnormality, present in 15 of 20 analyzed metaphases. Th is very rare acquired chromosomal aberration has sporadically been

Mesenchymal stem cells in multiple myeloma: a therapeutical tool or target?

Multiple myeloma (MM) is a malignant plasma cell (PC) disorder, characterized by a complex interactive network of tumour cells and the bone marrow (BM) stromal microenvironment, contributing to MM cell survival, proliferation and chemoresistance. Mesenchymal stem cells (MSCs) represent the predominant stem cell population of the bone marrow stroma, capable of differentiating into multiple cell lineages, including fibroblasts, adipocytes, chondrocytes and osteoblasts. MSCs can migrate towards primary tumours and metastatic sites, implying that these cells might modulate tumour growth and metastasis. However, this issue remains controversial and is not well understood. Interestingly, several recent studies have shown functional abnormalities of MM patient-derived MSCs indicating that MSCs are not just by-standers in the BM microenvironment but rather active players in the pathophysiology of this disease. It appears that the complex interaction of MSCs and MM cells is critical for MM development and disease outcome. This review will focus on the current understanding of the biological role of MSCs in MM as well as the potential utility of MSC-based therapies in this malignancy.