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

Transient expression of calretinin in the trout habenulo-interpeduncular system during development

Calcium-binding proteins control calcium homeostasis during neural development. The expression of one of these proteins, calretinin (CR), was monitored by immunohistochemistry in the developing habenulo-interpeduncular system of the rainbow trout, a conserved region of the brain along vertebrate phylogeny that undergoes a neurochemical reorganization in late development. No CR-immunoreactivity was observed in the habenulo-interpeduncular system during the embryonic development. CRimmunolabeling appeared in newly hatched fry, and during the fry development the number of CR-immunostained elements increased progressively. During the juvenile stages (from 30 days post-hatching onwards) a gradual decrease in the number of CRimmunostained cells occurred, until its complete disappearance in adults. These variations in CR expression may represent variable calcium-buffering needs during different developmental stages

Regeneration of hyaline cartilage promoted by xenogeneic mesenchymal stromal cells embedded within elastin-like recombinamer-based bioactive hydrogels

Producción CientíficaOver the last decades, novel therapeutic tools for osteochondral regeneration have arisen from the combination of mesenchymal stromal cells (MSCs) and highly specialized smart biomaterials, such as hydrogel-forming elastin-like recombinamers (ELRs), which could serve as cell-carriers. Herein, we evaluate the delivery of xenogeneic human MSCs (hMSCs) within an injectable ELR-based hydrogel carrier for osteochondral regeneration in rabbits. First, a critical-size osteochondral defect was created in the femora of the animals and subsequently filled with the ELR-based hydrogel alone or with embedded hMSCs. Regeneration outcomes were evaluated after three months by gross assessment, magnetic resonance imaging and computed tomography, showing complete filling of the defect and the de novo formation of hyaline-like cartilage and subchondral bone in the hMSC-treated knees. Furthermore, histological sectioning and staining of every sample confirmed regeneration of the full cartilage thickness and early subchondral bone repair, which was more similar to the native cartilage in the case of the cell-loaded ELR-based hydrogel. Overall histological differences between the two groups were assessed semi-quantitatively using the Wakitani scale and found to be statistically significant (p < 0.05). Immunofluorescence against a human mitochondrial antibody three months post-implantation showed that the hMSCs were integrated into the de novo formed tissue, thus suggesting their ability to overcome the interspecies barrier. Hence, we conclude that the use of xenogeneic MSCs embedded in an ELR-based hydrogel leads to the successful regeneration of hyaline cartilage in osteochondral lesions.Ministerio de Economía, Industria y Competitividad (Project (MAT2016-78903-R, MAT2016-9 79435-R, MAT2013-42473-R, MAT2013-41723-R and MAT2012-38043)Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA244U13, VA313U14 and GRS/516/A/10

Dasatinib as a Bone-Modifying Agent: Anabolic and Anti-Resorptive Effects

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: Bone loss, in malignant or non-malignant diseases, is caused by increased osteoclast resorption and/or reduced osteoblast bone formation, and is commonly associated with skeletal complications. Thus, there is a need to identify new agents capable of influencing bone remodeling. We aimed to further pre-clinically evaluate the effects of dasatinib (BMS-354825), a multitargeted tyrosine kinase inhibitor, on osteoblast and osteoclast differentiation and function. [Methods]: For studies on osteoblasts, primary human bone marrow mensenchymal stem cells (hMSCs) together with the hMSC-TERT and the MG-63 cell lines were employed. Osteoclasts were generated from peripheral blood mononuclear cells (PBMC) of healthy volunteers. Skeletally-immature CD1 mice were used in the in vivo model. [Results]: Dasatinib inhibited the platelet derived growth factor receptor-β (PDGFR-β), c-Src and c-Kit phosphorylation in hMSC-TERT and MG-63 cell lines, which was associated with decreased cell proliferation and activation of canonical Wnt signaling. Treatment of MSCs from healthy donors, but also from multiple myeloma patients with low doses of dasatinib (2-5 nM), promoted its osteogenic differentiation and matrix mineralization. The bone anabolic effect of dasatinib was also observed in vivo by targeting endogenous osteoprogenitors, as assessed by elevated serum levels of bone formation markers, and increased trabecular microarchitecture and number of osteoblast-like cells. By in vitro exposure of hemopoietic progenitors to a similar range of dasatinib concentrations (1-2 nM), novel biological sequelae relative to inhibition of osteoclast formation and resorptive function were identified, including F-actin ring disruption, reduced levels of c-Fos and of nuclear factor of activated T cells 1 (NFATc1) in the nucleus, together with lowered cathepsin K, αVβ3 integrin and CCR1 expression. [Conclusions]: Low dasatinib concentrations show convergent bone anabolic and reduced bone resorption effects, which suggests its potential use for the treatment of bone diseases such as osteoporosis, osteolytic bone metastasis and myeloma bone disease. © 2012 Garcia-Gomez et al.This work was supported by grants from the Spanish Ministry of Science and Innovation – ISCIII (PI081825); Mutua Madrileña Medical Research Foundation (AP27262008); Centro en Red of Regenerative Medicine and Cellular Therapy from Castilla y León, Consejería de Sanidad JCyL – ISCIII; the Cooperative Research Thematic Network in Cancer (RTICC; RD06/0020/0006 and RD03/0020/0041); and Spanish FIS (PS09/01897). AG-G and CS are supported by the Centro en Red of Regenerative Medicine and Cellular Therapy from Castilla y León Project.Peer Reviewe

Parvalbumin immunoreactivity during the development of the cerebellum of the rainbow trout

The distribution of parvalbumin immunoreactivity in the developing cerebellum ofthe rainbow trout was studied by using a specific monoclonal antibody and the avidin-biotin peroxidase method. Parvalbumin immunoreactivity was absent during the embryonic development of the cerebellum. The first immunoreactive elements, identified by their localization and posterior morphological evolution as immature Purkinje cells, appeared at 6 days posthatching in the presumptive corpus cerebelli and lobus vestibulolateralis. The labeling extended throughout the cerebellum following a caudorostral gradient, and in 21 days alevins, parvalbumin immunoreactive Purkinje cells were also observed in the valvula cerebelli. The appearance of PV-immunostaining in the Purkinje cells was not simultaneous; the labeling was observed initially in the cell body, extending gradually to the dendritic branches and finally to the axon. From one year onwards, parvalbumin immunoreactive terminal puncta from the Purkinje cell axons were observed surrounding the cell bodies of eurydendroid cells, that were parvalbumin immunonegative in all developmental stages studied. The spatio-temporal pattern of parvalbumin immunoreactivity in the rainbow trout cerebellum is different to previous observations in the cerebellum of amniotes

Neurocalcin immunoreactivity in the rato accessory olfactory bulb

The distribution and morphology of neurocalcin-immunopositive neurons have been studied in the rat accessory olfactory bulb. Different subsets of neurons displaying neurocalcin-immunoreactivity were found in the glomerular layer, the external plexiform layer and the internal plexiform layer. The most abundant staining was detected in the glomerular layer where neurocalcin-immunoreactive periglomerular cells and external tufted cells were observed in the lateral glomeruli, whereas the central region of this layer was practically devoid of immunopositive neurons. In the external plexiform layer, medial tufted cells and Van Gehuchten cells displayed neurocalcin-immunoreactivity. In the internal plexiform layer, interneurons classified as horizontal cells and vertical cells of Cajal were neurocalcin-immunostained. The staining pattern for neurocalcin in the accessory olfactory bulb showed similarities with the immunostaining described in this brain region for another EF-hand calcium binding protein, calbindin D-28k. However, after double immunohistochemical labeling, colocalization of both proteins in the same neuron was not observed, reflecting a biochemical heterogeneity within morphologically homogeneous neuronal groups

Nitric oxide synthase activity in the olfactory bulb of anuran and urodele amphibians

Nitric oxide synthase activity was studied by means of NADPH-diaphorase activity and nitric oxide synthase immunoreactivity in the main and accessory olfactory bulbs of the frog Rana perezi and the newt Triturus marmoratus. In both species, NADPH-diaphorase staining was observed in all olfactory fibers. Vomeronasal fibers were NADPH-diaphorase labeled in Triturus but they were NADPH-diaphorase negative in Rana. Nitric oxide synthase immunoreactivity was not observed in the primary afferents in any case.Granule cells were NADPH-diaphorase positive and nitric oxide synthase immunopositive in the main and accessory olfactory bulb of Rana, and in the main olfactory bulb of Triturus. The homogeneous NADPH-diaphorase staining of olfactory fibers is similar to what has been reported in teleosts, and it contrasts with the spatial segregation of NADPH-diaphorase positive and negative olfactory projections in rodents. These results confirm the interspecies variability of the NADPH-diaphorase/nitric oxide synthase distribution in the olfactory system of vertebrates

Calbindin D-28k immunoreactivity in the rat accessory olfactory bulb

The distribution pattern and the morphology of calbindin D-28k-immunoreactive neurons were studied in the accessory olfactory bulb of the rat using a monoclonal antibody and the avidin-biotin-immunoperoxidase method. Positive neurons were observed in all layers but the vomeronasal nerve layer. Scarce monodendritic periglomerular neurons were calbindin D-28k immunoreactive. Different morphological types of short-axon cells were calbindin D-28k immunostained, with different degrees of intensity, in the boundary between the internal and external plexiform layer. In addition, deep short-axon cells located in the granule cell layer were calbindin D-28k-immunopositive. By contrast, previous studies described all cells in the rat accessory olfactory bulb as calbindin D-28kimmunonegative.The staining pattern in the rat accessory olfactory bulb showed both similarities and differences with the distribution pattern of the same calcium-binding protein in the main olfactory bulb

Calretinin immunoreactivity in the developing olfactory system of the rainbow trout

The distribution of calretinin immunoreactivity in the developing olfactory system of the rainbow trout was studied by using an indirect immunocytochemical method. Calretinin immunoreactivity was firstly detected at 150 day-degrees in the olfactory placode, where labeled primordial cells were observed. At 250 day-degrees, precursor cells of the olfactory receptor neurons located in the olfactory pit were calretininimmunoreactive. At 300 day-degrees, recognizable olfactory receptor neurons displayed calretinin immunoreactivity in the olfactory epithelium, and calretinin-immunopositive olfactory axons reached the presumptive olfactory bulb. After hatching (400 day-degrees) and during the subsequent development and maturation of the olfactory system, the number of calretinin-immunopositive olfactory receptor cells increased and distributed homogeneously throughout the olfactory epithelium. Accordingly, new positive olfactory fibers arrived to the olfactory bulb arborizing in olfactory glomeruli distributed in nine different terminal fields. Six days after hatching, calretinin-immunopositive interneurons within the olfactory bulb were also observed. The size and number of calretinin immunoreactive interneurons increased from this stage to adulthood. The adult pattern demonstrated both similarities and differences with the distribution of calretinin immunoreactivity previously described in the olfactory system of mammals

Isolation and characterization of mesenchymal stromal cells from human degenerated nucleus pulposus: Comparison with bone marrow mesenchymal stromal cells from the same subjects

Study Design.: To identify mesenchymal stromal cells (MSC) from degenerate human nucleus pulposus (NP) and compare them with bone marrow (BM) MSC. Objective.: To test whether MSC obtained from NP and BM from the same subjects share similar biologic characteristics. Summary of Background Data.: Recent studies have proposed biologic strategies for the treatment of intervertebral disc degeneration, including cell therapy. Bone marrow (BM) MSC could be an attractive approach to restore disc function, and there is evidence that NP may contain MSC-like cells. Methods.: Tissue samples were obtained from degenerate lumbar NP and from iliac crest of the same 16 patients with degenerative disc diseases, undergoing discectomy and fusion procedures. MSC isolated from both sources were compared regarding their expansion time, immunophenotype, differentiation ability, and molecular analysis. Results.: In all cases, MSC from NP were isolated and expanded. They fulfil nearly all morphological, inmunophenotypical, and differentiation criteria described by the International Society of Cell Therapy for MSC, with the exception that NP-MSC are not able to differentiate into adipocytes. Slight differences were observed with BM-MSC from the same subjects. Conclusion.: The NP contains mesenchymal stem cells. These cells were quite similar to mesenchymal stem cellsfrom BM, with the exception of their adipogenic differentiation ability. These findings suggest that we may treat intervertebral disc degeneration by cell therapy (MSC from BM) and by stimulating endogenous MSC from NP.Peer Reviewe