Bone marrow mesenchymal stem cells for improving hematopoietic function: An in vitro and in vivo model. Part 2: Effect on bone marrow microenvironment

9 páginas, 4 figuras, 1 tabla.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.The aim of the present study was to determine how mesenchymal stem cells (MSC) could improve bone marrow (BM) stroma function after damage, both in vitro and in vivo. Human MSC from 20 healthy donors were isolated and expanded. Mobilized selected CD34+ progenitor cells were obtained from 20 HSCT donors. For in vitro study, long-term bone marrow cultures (LTBMC) were performed using a etoposide damaged stromal model to test MSC effect in stromal confluence, capability of MSC to lodge in stromal layer as well as some molecules (SDF1, osteopontin,) involved in hematopoietic niche maintenance were analyzed. For the in vivo model, 64 NOD/SCID recipients were transplanted with CD34+ cells administered either by intravenous (IV) or intrabone (IB) route, with or without BM derived MSC. MSC lodgement within the BM niche was assessed by FISH analysis and the expression of SDF1 and osteopontin by immunohistochemistry. In vivo study showed that when the stromal damage was severe, TP-MSC could lodge in the etoposide-treated BM stroma, as shown by FISH analysis. Osteopontin and SDF1 were differently expressed in damaged stroma and their expression restored after TP-MSC addition. Human in vivo MSC lodgement was observed within BM niche by FISH, but MSC only were detected and not in the contralateral femurs. Human MSC were located around blood vessels in the subendoestal region of femurs and expressed SDF1 and osteopontin. In summary, our data show that MSC can restore BM stromal function and also engraft when a higher stromal damage was done. Interestingly, MSC were detected locally where they were administered but not in the contralateral femur. © 2011 Carrancio et al.This study was supported in part by a grant from Consejeria de Educación de Castilla y León (ref: HUS003A10-2), Gerencia Regional de Salud de Castilla y León (ref: GRS/222/A/08) and Fondo de Investigaciones Sanitarias (ISCIII) (ref: PS09/01530), Ministerio de Sanidad, Spain. S.C. was supported by Junta de Castilla y Leon (FPI Grant EDU/1878/2006). B.B. was supported by Fondo de Investigaciones Sanitarias (FIS) from the Instituto de Salud Carlos III (ref. CD06/00042).Peer Reviewe

Effects of MSC coadministration and route of delivery on cord blood hematopoietic stem cell engraftment

Licencia Creative Commons Reconocimiento-No comercial.-- et al.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 immunofluorescence. 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.This study was supported in part by a grant from Gerencia Regional de Salud de Castilla y León (ref. GRS/222/A/08) and by a grant from Consejería de Educación de la Junta de Castilla y León (ref. HUS003A10-2). S.C. was supported by Junta de Castilla y Leon (FPI grant EDU/1878/2006).Peer Reviewe

Human Bone Marrow Stromal Cells Differentiate Into Corneal Tissue and Prevent Ocular Graft-Versus-Host Disease in Mice

Clinical trials have assessed the use of human bone marrow stromal cells (hBMSCs) for the treatment of immune-related disorders such as graft-versus-host disease (GVHD). In the current study, we show that GFP+-transduced hBMSCs generated from bone marrow migrate and differentiate into corneal tissue after subconjunctival injection in mice. Interestingly, these hBMSCs display morphological features of epithelial, stromal, and endothelial cells and appear at different layers and with different morphologies depending on their position within the epithelium. Furthermore, these cells display ultrastructural properties, such as bundles of intermediate filaments, interdigitations, and desmosomes with GFP- cells, which confirms their differentiation into corneal tissues. GFP+-transduced hBMSCs were injected at different time points into the right eye of lethally irradiated mice undergoing bone marrow transplantation, which developed ocular GVHD (oGVHD). Remarkably, hBMSCs massively migrate to corneal tissues after subconjunctival injection. Both macroscopic and histopathological examination showed minimal or no evidence of GVHD in the right eye, while the left eye, where no hBMSCs were injected, displayed features of GVHD. Thus, in the current study, we confirm that hBMSCs may induce their therapeutic effect at least in part by differentiation and regeneration of damaged tissues in the host. Our results provide experimental evidence that hBMSCs represent a potential cellular therapy to attenuate oGVHD

Bone Marrow Mesenchymal Stem Cells for Improving Hematopoietic Function: An In Vitro and In Vivo Model. Part 2: Effect on Bone Marrow Microenvironment

The aim of the present study was to determine how mesenchymal stem cells (MSC) could improve bone marrow (BM) stroma function after damage, both in vitro and in vivo. Human MSC from 20 healthy donors were isolated and expanded. Mobilized selected CD34+ progenitor cells were obtained from 20 HSCT donors. For in vitro study, long-term bone marrow cultures (LTBMC) were performed using a etoposide damaged stromal model to test MSC effect in stromal confluence, capability of MSC to lodge in stromal layer as well as some molecules (SDF1, osteopontin,) involved in hematopoietic niche maintenance were analyzed. For the in vivo model, 64 NOD/SCID recipients were transplanted with CD34+ cells administered either by intravenous (IV) or intrabone (IB) route, with or without BM derived MSC. MSC lodgement within the BM niche was assessed by FISH analysis and the expression of SDF1 and osteopontin by immunohistochemistry. In vivo study showed that when the stromal damage was severe, TP-MSC could lodge in the etoposide-treated BM stroma, as shown by FISH analysis. Osteopontin and SDF1 were differently expressed in damaged stroma and their expression restored after TP-MSC addition. Human in vivo MSC lodgement was observed within BM niche by FISH, but MSC only were detected and not in the contralateral femurs. Human MSC were located around blood vessels in the subendoestal region of femurs and expressed SDF1 and osteopontin. In summary, our data show that MSC can restore BM stromal function and also engraft when a higher stromal damage was done. Interestingly, MSC were detected locally where they were administered but not in the contralateral femur

Papel de las células mesenquimales expandidas in vitro para mejorar el injerto en el contexto del trasplante hematopoyético

[EN] The hematopoietic stem cell transplantation (HSCT) is a therapeutic procedure designed to reconstitute the hematopoietic tissue of the patient, usually presents a bone marrow failure or have a neoplastic disease. Since the late nineteenth century, Brown-Sequard raised the possibility of reconstituting the production of blood cells in cases of anemia with the administration of spleen of animals, raw or cooked. It was not until the 50's when it got to reconstitute hematopoiesis in mice subjected to a lethal dose of irradiation, by intravenous administration of hematopoietic cells from another mouse, confirming the real possibility of organ transplant.[ES] El trasplante de progenitores hematopoyéticos (TPH) es un procedimiento terapéutico destinado a reconstituir el tejido hematopoyético del paciente, que generalmente presenta una insuficiencia medular o sufre una enfermedad neoplásica. Ya a finales del S.XIX, Brown-Sequard planteó la posibilidad de reconstituir la producción de células sanguíneas en casos de anemia con la administración de bazo de animales, crudo o cocido. Pero no fue hasta los años 50 cuando se consiguió reconstituir la hematopoyesis en ratones sometidos a una dosis letal de irradiación, mediante la administración intravenosa de células hematopoyéticas de otro ratón, confirmando así la posibilidad real de un trasplante hematopoyético

Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification

[Objective]: Optimization of the mesenchymal stem cells (MSC) isolation and expansion method. [Materials and Methods]: Mononuclear cells (MNC) from bone marrow aspirates were obtained by both density gradient centrifugation (standard method) and gravity sedimentation. Cells were cultured in standard conditions (10% fetal calf serum and normal oxygen tension [21% O2]) and expansion results compared to those obtained with the same culture conditions to which platelet lysate (PL) preparations were added; in addition, the 21% O2 concentration was compared to a lower (5%) concentration (hypoxia) until the fourth cell passage. Time of expansion, number of cells obtained, morphology, cell surface markers, and differentiation potential were evaluated. [Results]: MSC obtained by any of the different culture conditions expressed comparable immunophenotype and were able to differentiate into osteoblasts, adipocytes, and chondrocytes. When the number of MSC obtained at fourth passage was analyzed, the highest cell numbers were obtained with gravity sedimentation isolation and PL-supplemented culture and the expansion time was the shortest when cells were cultured under hypoxic conditions. [Conclusion]: MSC isolation by MNC gravity sedimentation together with culture medium supplementation with 5% of PL in a hypoxic atmosphere (5% O2) significantly improved MSC yield and reduced expansion time compared to the standard accepted protocols. © 2008 ISEH - Society for Hematology and Stem Cells.S. Carrancio was supported by a grant from Junta de Castilla y León (FPI Grant EDU/1878/2006). V Barbado is supported by a research grant from Federación de Cajas de Ahorros de Castilla y León, Spain.Peer Reviewe

The novel combination of sirolimus and bortezomib prevents graft-versus-host disease but maintains the graft-versus-leukemia effect after allogeneic transplantation

This is an open-access paper.[Background]: We have previously shown that bortezomib induces a depletion of alloreactive T cells and allows the expansion of T cells with suppressive properties. In the current study, we analyzed the potential synergistic effect of bortezomib in conjunction with sirolimus in order to reducegraft- versus-host disease without hampering graft-versus-leukemia effect in the allogeneic transplant setting. [Design and Methods]: We evaluated the effect of sirolimus, bortezomib or the combination of both in the proliferation and activation of in vitro stimulated T lymphocytes. Pathways involved in this synergy were also analyzed using Western blot assays. Finally, BALB/c mice receiving C57BL/6 allogeneic donor bone marrow with splenocytes were used to measure in vivo the effect of this novel combination on the risk of graft-versus-host disease. [Results]: The combination of both drugs synergistically inhibited both activation and proliferation of stimulated T cells. Also, the production of Th1 cytokines (IFN γ, IL-2 and TNF) was significantly inhibited. This effect was due, at least in part, to the inhibition of Erk and Akt phosphorylation. In vivo, the combination reduced the risk of graft-versus-host disease without hampering graft-versus-leukemia effect, as shown in mice receiving graft-versus-host disease prophylaxis with sirolimus plus bortezomib being infused with tumor WEHI cells plus C57BL/6 donor BM and splenocytes. [Conclusions]: The current study reveals a synergistic effect of the combination sirolimus and bortezomib to prevent graft-versus-host disease while maintaining the graft-versus-leukemia effect.Supported by a grant Rio Hortega (code: CM10/00161) from the Instituto de Salud Carlos III and grant PI080047 from FIS.Peer Reviewe

Optimisation of mesenchymal stromal cells karyotyping analysis: Implications for clinical use

[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. © 2012 The Authors. Transfusion Medicine © 2012 British Blood Transfusion Society.This project had been partially supported by a grant from Ministerio de Ciencia e Innovación (reference code PLE2009-0094).Peer Reviewe

Impaired expression of DICER, DROSHA, SBDS and some microRNAs in mesenchymal stromal cells from myelodysplastic syndrome patients

This is an open-access paper.[Background]: Recent findings suggest that a specific deletion of Dicer1 in mesenchymal stromal cells-derived osteoprogenitors triggers several features of myelodysplastic syndrome in a murine model. Our aim was to analyze DICER1 and DROSHA gene and protein expression in mesenchymal stromal cells (the osteoblastic progenitors) obtained from bone marrow of myelodysplastic syndrome patients, in addition to microRNA expression profile and other target genes such as SBDS, a DICER1-related gene that promotes bone marrow dysfunction and myelodysplasia when repressed in a murine model. [Design and Methods]: Mesenchymal stromal cells from 33 bone marrow samples were evaluated. DICER, DROSHA and SBDS gene expression levels were assessed by real-time PCR and protein expression by Western blot. MicroRNA expresion profile was analyzed by commercial low-density arrays and some of these results were confirmed by individual real-time PCR. [Results]: Mesenchymal stromal cells from myelodysplastic syndrome patients showed lower DICER1 (0.65±0.08 vs. 1.91±0.57; P=0.011) and DROSHA (0.62±0.06 vs. 1.38±0.29; P=0.009) gene expression levels, two relevant endonucleases associated to microRNA biogenesis, in comparison to normal myelodysplastic syndrome. These findings were confirmed at protein levels by Western blot. Strikingly, no differences were observed between paired mononuclear cells from myelodysplastic syndrome and controls. In addition, mesenchymal stromal cells from myelodysplastic syndrome patients showed significant lower SBDS (0.63±0.06 vs. 1.15±0.28; P=0.021) gene expression levels than mesenchymal stromal cells from healthy controls. Furthermore, mesenchymal stromal cells from myelodysplastic syndrome patients showed an underlying microRNA repression compared to healthy controls. Real-time PCR approach confirmed that mir-155, miR-181a and miR- 222 were down-expressed in mesenchymal stromal cells from myelodysplastic syndrome patients. [Conclusions]: This is the first description of an impaired microRNA biogenesis in human mesenchymal stromal cells from myelodysplastic syndrome patients, where DICER1 and DROSHA gene and protein downregulation correlated to a gene and microRNA abnormal expression profile, validating the animal model results previously described.Grants from Consejeria de Educacion de la Junta de Castilla y León (HUS03A09) and Instituto de Salud Carlos III (FIS09/01530).Peer reviewe

Comparison of RNA-seq and microarray platforms for splice event detection using a cross-platform algorithm

Abstract Background RNA-seq is a reference technology for determining alternative splicing at genome-wide level. Exon arrays remain widely used for the analysis of gene expression, but show poor validation rate with regard to splicing events. Commercial arrays that include probes within exon junctions have been developed in order to overcome this problem. We compare the performance of RNA-seq (Illumina HiSeq) and junction arrays (Affymetrix Human Transcriptome array) for the analysis of transcript splicing events. Three different breast cancer cell lines were treated with CX-4945, a drug that severely affects splicing. To enable a direct comparison of the two platforms, we adapted EventPointer, an algorithm that detects and labels alternative splicing events using junction arrays, to work also on RNA-seq data. Common results and discrepancies between the technologies were validated and/or resolved by over 200 PCR experiments. Results As might be expected, RNA-seq appears superior in cases where the technologies disagree and is able to discover novel splicing events beyond the limitations of physical probe-sets. We observe a high degree of coherence between the two technologies, however, with correlation of EventPointer results over 0.90. Through decimation, the detection power of the junction arrays is equivalent to RNA-seq with up to 60 million reads. Conclusions Our results suggest, therefore, that exon-junction arrays are a viable alternative to RNA-seq for detection of alternative splicing events when focusing on well-described transcriptional regions