ABSTRACT
Hematopoietic stem cell transplantation (HSCT) using umbilical cord blood (UCB) progenitors is increasingly being used. One of the problems that may arise after UCB transplantation is an impaired engraftment. Either intrabone (IB) injection of hematopoietic progenitors or mesenchymal stem cell (MSC) coadministration has been proposed among the strategies to improve engraftment. In the current study, we have assessed the effects of both approaches. Thus, NOD/SCID recipients were transplanted with human UCB CD34+ cells administered either intravenously (IV) or IB, receiving or not bone marrow (BM)-derived MSCs also IV or IB (in the right femur). Human HSC engraftment was measured 3 and 6 weeks after transplantation. Injected MSCs were tracked weekly by bioluminescence. Also, lodgment within the BM niche was assessed at the latter time point by immuno-fluorescence. Our study shows regarding HSC engraftment that the number of BM human CD45+ cells detected 3 weeks after transplantation was significantly higher in mice cotransplanted with human MSCs. Moreover, these mice had a higher myeloid (CD13+) engraftment and a faster B-cell (CD19+) chimerism. At the late time point evaluated (6 weeks), human engraftment was higher in the group in which both strategies were employed (IB injection of HSC and MSC coadministration). When assessing human MSC administration route, we were able to track MSCs only in the injected femurs, whereas they lost their signal in the contralateral bones. These human MSCs were mainly located around blood vessels in the subendosteal region. In summary, our study shows that MSC coadministration can enhance HSC engraftment in our xenogenic transplantation model, as well as IB administration of the CD34+ cells does. The combination of both strategies seems to be synergistic. Interestingly, MSCs were detected only where they were IB injected contributing to the vascular niche.
Subject(s)
Fetal Blood/cytology , Hematopoietic Stem Cell Transplantation , Mesenchymal Stem Cell Transplantation , Adult , Animals , Antigens, CD34/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Bone and Bones/physiology , Chimerism , Female , Graft Survival/immunology , HEK293 Cells , Hematopoietic Stem Cells/cytology , Humans , Leukocyte Common Antigens/metabolism , Male , Mesenchymal Stem Cells/cytology , Mice , Mice, Inbred NOD , Mice, SCID , Middle Aged , Transplantation, HeterologousABSTRACT
PRAME is a tumor associated antigen (TAA) of particular interest since it is widely expressed by lymphoid and myeloid malignancies. Several studies have associated high PRAME RNA levels with good prognosis in acute myeloid leukemia (AML). PRAME expression is regulated at the epigenetic level. For this reason inhibitors of DNA methylation, such as 5-azacytidine, can modulate the expression of this TAAs. In the current study we analyzed the effect of 5-azaC on the expression of PRAME in blasts versus CD34+ cells from healthy donors in an attempt to increase its expression, thus inducing a potential target for therapeutic strategies.
Subject(s)
Antigens, CD34/metabolism , Antigens, Neoplasm/genetics , Azacitidine/pharmacology , Leukemia, Myeloid, Acute/genetics , Stem Cells/metabolism , Antigens, Neoplasm/metabolism , Antimetabolites, Antineoplastic/pharmacology , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Blood Donors , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Cells, Cultured , CpG Islands/genetics , Cytogenetic Analysis , Epigenesis, Genetic/drug effects , Epigenesis, Genetic/physiology , Gene Expression Regulation, Leukemic/drug effects , Health , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Stem Cells/drug effects , Stem Cells/physiologyABSTRACT
PURPOSE: The aim of this study was to optimise the yield of metaphases in mesenchymal stromal cells (MSC) in vitro cultures and to study the karyotype of MSC expanded in good manufacturing practice (GMP) conditions for clinical use. BACKGROUND: MSC are being increasingly used in clinical trials for a number of diseases. Biosafety demonstration in all cases is mandatory. Unfortunately, current standard karyotyping methods fail to obtain enough number of evaluable metaphases. METHODS AND MATERIALS: In the present work, to optimise the yield of metaphases in MSC expanded in vitro, we have tested several conditions by modifying colcemid concentration (we have tested 0.01, 0.05 and 0.1 µg mL(-1) ) and exposure time (during 5, 15 and 24 h). We further applied these optimised conditions to 61 MSC expansions in GMP conditions for clinical use. RESULTS: Our results show that the highest number of metaphases was obtained when MSC were incubated with 0.05 µg mL(-1) of colcemid overnight (15 h), compared to the remaining experimental conditions. In most cases (59/61 cases) enough number of metaphases was obtained. And what is more relevant, only in one case a karyotypic abnormality was found (trisomy of chromosome 10), and cells were subsequently discarded for clinical use. CONCLUSION: We describe here an optimal method to obtain enough number of metaphases for karyotype analysis of in vitro expanded MSCs, what is essential for their clinical use in cell therapy programmes.
