Engraftment of autologous bone marrow cells into the injured cranial cruciate ligament in dogs

Current research indicates that exogenous stem cells may accelerate reparative processes in joint disease but, no previous studies have evaluated whether bone marrow cells (BMCs) target the injured cranial cruciate ligament (CCL) in dogs. The objective of this study was to investigate engraftment of BMCs following intra-articular injection in dogs with spontaneous CCL injury. Autologous PKH26-labelled BMCs were injected into the stifle joint of eight client-owned dogs with CCL rupture. The effects of PKH26 staining on cell viability and PKH26 fluorescence intensity were analysed in vitro using a MTT assay and flow cytometry. Labelled BMCs in injured CCL tissue were identified using fluorescence microscopy of biopsies harvested 3 and 13 days after intra-articular BMC injection. The intensity of PKH26 fluorescence declines with cell division but was still detectable after 16 days. Labelling with PKH26 had no detectable effect on cell viability or proliferation. Only rare PKH26-positive cells were present in biopsies of the injured CCL in 3/7 dogs and in synovial fluid in 1/7 dogs. No differences in transforming growth factor-beta1, and interleukin-6 before and after BMC treatment were found and no clinical complications were noted during a 1 year follow-up period. In conclusion, BMCs were shown to engraft to the injured CCL in dogs when injected into the articular cavity. Intra-articular application of PKH26-labelled cultured mesenchymal stem cells is likely to result in higher numbers of engrafted cells that can be tracked using this method in a clinical setting.

Saliva suppresses osteoclastogenesis in murine bone marrow cultures.

Saliva can reach mineralized surfaces in the oral cavity; however, the relationship between saliva and bone resorption is unclear. Herein, we examined whether saliva affects the process of osteoclastogenesis in vitro. We used murine bone marrow cultures to study osteoclast formation. The addition of fresh sterile saliva eliminated the formation of multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). In line with the histochemical staining, saliva substantially reduced gene expression of cathepsin K, calcitonin receptor, and TRAP. Addition of saliva led to considerably decreased gene expression of receptor activator of nuclear factor kappa-B (RANK) and, to a lesser extent, that of c-fms. The respective master regulators of osteoclastogenesis (c-fos and NFATc1) and the downstream cell fusion genes (DC-STAMP and Atp6v0d2) showed decreased expression after the addition of saliva. Among the costimulatory molecules for osteoclastogenesis, only OSCAR showed decreased expression. In contrast, CD40, CD80, and CD86-all costimulatory molecules of phagocytic cells-were increasingly expressed with saliva. The phagocytic capacity of the cells was confirmed by latex bead ingestion. Based on these in vitro results, it can be concluded that saliva suppresses osteoclastogenesis and leads to the development of a phagocytic cell phenotype.

Cytotoxic CD8+ T cells stimulate hematopoietic progenitors by promoting cytokine release from bone marrow mesenchymal stromal cells.

Cytotoxic CD8(+) T cells (CTLs) play a major role in host defense against intracellular pathogens, but a complete clearance of pathogens and return to homeostasis requires the regulated interplay of the innate and acquired immune systems. Here, we show that interferon γ (IFNγ) secreted by effector CTLs stimulates hematopoiesis at the level of early multipotent hematopoietic progenitor cells and induces myeloid differentiation. IFNγ did not primarily affect hematopoietic stem or progenitor cells directly. Instead, it promoted the release of hematopoietic cytokines, including interleukin 6 from bone marrow mesenchymal stromal cells (MSCs) in the hematopoietic stem cell niche, which in turn reduced the expression of the transcription factors Runx-1 and Cebpα in early hematopoietic progenitor cells and increased myeloid differentiation. Therefore, our study indicates that, during an acute viral infection, CTLs indirectly modulate early multipotent hematopoietic progenitors via MSCs in order to trigger the temporary activation of emergency myelopoiesis and promote clearance of the infection.

Should the standard dimethyl sulfoxide concentration be reduced? Results of a European Group for Blood and Marrow Transplantation prospective noninterventional study on usage and side effects of dimethyl sulfoxide.

BACKGROUND Dimethyl sulfoxide (DMSO) is essential for the preservation of liquid nitrogen-frozen stem cells, but is associated with toxicity in the transplant recipient. STUDY DESIGN AND METHODS In this prospective noninterventional study, we describe the use of DMSO in 64 European Blood and Marrow Transplant Group centers undertaking autologous transplantation on patients with myeloma and lymphoma and analyze side effects after return of DMSO-preserved stem cells. RESULTS While the majority of centers continue to use 10% DMSO, a significant proportion either use lower concentrations, mostly 5 or 7.5%, or wash cells before infusion (some for selected patients only). In contrast, the median dose of DMSO given (20 mL) was much less than the upper limit set by the same institutions (70 mL). In an accompanying statistical analysis of side effects noted after return of DMSO-preserved stem cells, we show that patients in the highest quartile receiving DMSO (mL and mL/kg body weight) had significantly more side effects attributed to DMSO, although this effect was not observed if DMSO was calculated as mL/min. Dividing the myeloma and lymphoma patients each into two equal groups by age we were able to confirm this result in all but young myeloma patients in whom an inversion of the odds ratio was seen, possibly related to the higher dose of melphalan received by young myeloma patients. CONCLUSION We suggest better standardization of preservation method with reduced DMSO concentration and attention to the dose of DMSO received by patients could help reduce the toxicity and morbidity of the transplant procedure.

Bone marrow-derived mesenchymal stromal cells improve vascular regeneration and reduce leukocyte-endothelium activation in critical ischemic murine skin in a dose-dependent manner

BACKGROUND AIMS Stem cells participate in vascular regeneration following critical ischemia. However, their angiogenic and remodeling properties, as well as their role in ischemia-related endothelial leukocyte activation, need to be further elucidated. Herein, we investigated the effect of bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a critically ischemic murine skin flap model. METHODS Groups received either 1 × 10(5), 5 × 10(5), or 1 × 10(6) BM-MSCs or cell-free conditioned medium (CM). Controls received sodium chloride. Intravital fluorescence microscopy was performed for morphological and quantitative assessment of micro-hemodynamic parameters over 12 days. RESULTS Tortuosity and diameter of conduit-arterioles were pronounced in the MSC groups (P < 0.01), whereas vasodilation was shifted to the end arteriolar level in the CM group (P < 0.01). These effects were accompanied by angiopoietin-2 expression. Functional capillary density and red blood cell velocity were enhanced in all treatment groups (P < 0.01). Although a significant reduction of rolling and sticking leukocytes was observed in the MSC groups with a reduction of diameter in postcapillary venules (P < 0.01), animals receiving CM exhibited a leukocyte-endothelium interaction similar to controls. This correlated with leukocyte common antigen expression in tissue sections (P < 0.01) and p38 mitogen-activated protein kinase expression from tissue samples. Cytokine analysis from BM-MSC culture medium revealed a 50% reduction of pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-12, tumor necrosis factor-α, interferon-γ) and chemokines (keratinocyte chemoattractant, granulocyte colony-stimulating factor) under hypoxic conditions. DISCUSSION We demonstrated positive effects of BM-MSCs on vascular regeneration and modulation of endothelial leukocyte adhesion in critical ischemic skin. The improvements after MSC application were dose-dependent and superior to the use of CM alone.

Improved relapse-free survival after autologous stem cell transplantation does not translate into better quality of life in chronic lymphocytic leukemia: lessons from the randomized European Society for Blood and Marrow Transplantation-Intergroup study

In chronic lymphocytic leukemia (CLL) medical progress is driven by clinical studies with relapse-free survival (RFS) as the primary endpoint. The randomized EBMT-Intergroup trial compared high-dose therapy and autologous stem cell transplantation (ASCT) to observation and demonstrated a substantial improvement of RFS without showing improved overall survival for the transplant arm. Here we report quality of life (QoL) information of the first 3 years following randomization from that study. The main objective was to assess the impact of treatment on QoL over time. Two secondary analyses were performed to further investigate the impact of ASCT and relapse on QoL. In the primary analysis, we demonstrate an adverse impact of ASCT on QoL which was largest at 4 months and continued throughout the first year after randomization. Further, we demonstrated a sustained adverse impact of relapse on QoL which worsened over time. Despite better disease control by ASCT the side effects thus turned the net effect towards inferior QoL in the first year and comparable QoL in the following 2 years after randomization. This study emphasizes the importance of information concerning QoL impacts when patients are counseled about treatments aimed at improving RFS in the absence of a survival benefit.

Retinal differentiation of human bone marrow-derived stem cells by co-culture with retinal pigment epithelium in vitro.

The goal of this study was to assess the in vitro differentiation capacity of human bone marrow-derived stem cells (hBMSCs) along retinal lineages. Mononuclear cells (MNC) were isolated from bone marrow (BM) and mobilized peripheral blood (mPB) using Ficoll-Paque density gradient centrifugation, and were sorted by magnetic-activated cell sorting (MACS) for specific stem cell subsets (CD34(+)CD38(+)/CD34(+)CD38(-)). These cells were then co-cultured on human retinal pigment epithelial cells (hRPE) for 7 days. The expression of stem cell, neural and retina-specific markers was examined by immunostaining, and the gene expression profiles were assessed after FACS separation of the co-cultured hBMSCs by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, in vitro functionality of the differentiated cells was analyzed by quantifying phagocytosis of CY5-labeled photoreceptor outer segments (POS). After 7 days of co-culture, hBMSCs adopted an elongated epithelial-like morphology and expressed RPE-specific markers, such as RPE65 and bestrophin. In addition, these differentiated cells were able to phagocytose OS, one of the main characteristics of native RPE cells. Our data demonstrated that human CD34(+)CD38(-) hBMSC may differentiate towards an RPE-like cell type in vitro and could become a new type of autologous donor cell for regenerative therapy in retinal degenerative diseases.

The generation of neutrophils in the bone marrow is controlled by autophagy.

Autophagy has been demonstrated to have an essential function in several cellular hematopoietic differentiation processes, for example, the differentiation of reticulocytes. To investigate the role of autophagy in neutrophil granulopoiesis, we studied neutrophils lacking autophagy-related (Atg) 5, a gene encoding a protein essential for autophagosome formation. Using Cre-recombinase mediated gene deletion, Atg5-deficient neutrophils showed no evidence of abnormalities in morphology, granule protein content, apoptosis regulation, migration, or effector functions. In such mice, however, we observed an increased proliferation rate in the neutrophil precursor cells of the bone marrow as well as an accelerated process of neutrophil differentiation, resulting in an accumulation of mature neutrophils in the bone marrow, blood, spleen, and lymph nodes. To directly study the role of autophagy in neutrophils, we employed an in vitro model of differentiating neutrophils that allowed modulating the levels of ATG5 expression, or, alternatively, intervening pharmacologically with autophagy-regulating drugs. We could show that autophagic activity correlated inversely with the rate of neutrophil differentiation. Moreover, pharmacological inhibition of p38 MAPK or mTORC1 induced autophagy in neutrophilic precursor cells and blocked their differentiation, suggesting that autophagy is negatively controlled by the p38 MAPK-mTORC1 signaling pathway. On the other hand, we obtained no evidence for an involvement of the PI3K-AKT or ERK1/2 signaling pathways in the regulation of neutrophil differentiation. Taken together, these findings show that, in contrast to erythropoiesis, autophagy is not essential for neutrophil granulopoiesis, having instead a negative impact on the generation of neutrophils. Thus, autophagy and differentiation exhibit a reciprocal regulation by the p38-mTORC1 axis.

Conditioned medium from fresh and demineralized bone enhances osteoclastogenesis in murine bone marrow cultures

OBJECTIVES Osteoclasts rapidly form on the surface of bone chips at augmentation sites. The underlying molecular mechanism, however, is unclear. Soluble factors released from bone chips in vitro have a robust impact on mesenchymal cell differentiation. Whether these soluble factors change the differentiation of hematopoietic cells into osteoclasts remains unknown. METHODS Osteoclastogenesis, the formation of tartrate-resistant acid phosphatase-positive multinucleated cells, was studied with murine bone marrow cultures exposed to RANKL and M-CSF, and conditioned medium from fresh (BCM) and demineralized bone matrix (DCM). Histochemical staining, gene and protein expression, as well as viability assays were performed. RESULTS This study shows that BCM had no impact on osteoclastogenesis. However, when BCM was heated to 85°C (BCMh), the number of tartrate-resistant acid phosphatase-positive multinucleated cells that developed in the presence of RANKL and M-CSF approximately doubled. In line with the histochemical observations, there was a trend that BCMh increased expression of osteoclast marker genes, in particular the transcription factor c-fos. The expression of c-fos was significantly reduced by the TGF-β receptor I antagonist SB431542. DCM significantly stimulated osteoclastogenesis, independent of thermal processing. CONCLUSIONS These data demonstrate that activated BCM by heat and DBM are able to stimulate osteoclastogenesis in vitro. These in vitro results support the notion that the resorption of autografts may be supported by as yet less defined paracrine mechanisms.

Osteogenic differentiation of bone marrow stromal cells is hindered by the presence of intervertebral disc cells.

BACKGROUND Clinical observations indicate that the presence of nucleus pulposus (NP) tissue during spinal fusion hinders the rate of disc ossification. While the underlying mechanism remains unknown, this observation could be due to incomplete removal of NP cells (NPCs) that secrete factors preventing disc calcification, such as bone morphogenetic protein (BMP) antagonists including noggin and members of the DAN (differential screening selected gene aberrative in neuroblastoma) family. METHODS Monolayer human bone marrow-derived mesenchymal stem cells (MSCs) were cocultured withNPCs and annulus fibrosus cells (AFCs) embedded in alginate for 21 days. At the end of coculture, MSCs were stained for mineral deposition by alizarin red, and relative expression of bone-related genes [Runt-related transcription factor 2, (RUNX2), Osteopontin (OPN), and Alkaline phosphatase (ALP)] and ALP activity were analyzed. Relative expression of three BMP antagonists, chordin (CHRD), gremlin (GREM1), and noggin (NOG), was determined in primary human NPCs and AFCs. These cells were also stained for Gremlin and Noggin by immunocytochemistry. RESULTS Alizarin red staining showed that MSC osteogenesis in monolayer cultures was inhibited by coculture with NPCs or AFCs. ALP activity and RT-PCR analyses confirmed these results and demonstrated inhibition of osteogenesis of MSC in the presence of disc cells. NOG was significantly up-regulated in MSCs after coculture. Relative gene expression of intervertebral disc (IVD) cells showed higher expression of GREM1 in NPCs than in AFCs. CONCLUSIONS We show that primary IVD cells inhibit osteogenesis of MSCs. BMP inhibitors NOG, GREM1 and CHRD were expressed in IVD cells. GREM1 appears to be differentially expressed in NPCs and AFCs. Our results have implications for the design and development of treatments for non-union in spinal fusion.

Fatal hyperammonemia and carbamoyl phosphate synthetase 1 (CPS1) deficiency following high-dose chemotherapy and autologous hematopoietic stem cell transplantation

Fatal hyperammonemia secondary to chemotherapy for hematological malignancies or following bone marrow transplantation has been described in few patients so far. In these, the pathogenesis of hyperammonemia remained unclear and was suggested to be multifactorial. We observed severe hyperammonemia (maximum 475 μmol/L) in a 2-year-old male patient, who underwent high-dose chemotherapy with carboplatin, etoposide and melphalan, and autologous hematopoietic stem cell transplantation for a neuroblastoma stage IV. Despite intensive care treatment, hyperammonemia persisted and the patient died due to cerebral edema. The biochemical profile with elevations of ammonia and glutamine (maximum 1757 μmol/L) suggested urea cycle dysfunction. In liver homogenates, enzymatic activity and protein expression of the urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) were virtually absent. However, no mutation was found in CPS1 cDNA from liver and CPS1 mRNA expression was only slightly decreased. We therefore hypothesized that the acute onset of hyperammonemia was due to an acquired, chemotherapy-induced (posttranscriptional) CPS1 deficiency. This was further supported by in vitro experiments in HepG2 cells treated with carboplatin and etoposide showing a dose-dependent decrease in CPS1 protein expression. Due to severe hyperlactatemia, we analysed oxidative phosphorylation complexes in liver tissue and found reduced activities of complexes I and V, which suggested a more general mitochondrial dysfunction. This study adds to the understanding of chemotherapy-induced hyperammonemia as drug-induced CPS1 deficiency is suggested. Moreover, we highlight the need for urgent diagnostic and therapeutic strategies addressing a possible secondary urea cycle failure in future patients with hyperammonemia during chemotherapy and stem cell transplantation.

Three-dimensional culture and characterization of mononuclear cells from human bone marrow

BACKGROUND AIMS The diverse phenotypic changes and clinical and economic disadvantages associated with the monolayer expansion of bone marrow-derived mesenchymal stromal cells (MSCs) have focused attention on the development of one-step intraoperative cells therapies and homing strategies. The mononuclear cell fraction of bone marrow, inclusive of discrete stem cell populations, is not well characterized, and we currently lack suitable cell culture systems in which to culture and investigate the behavior of these cells. METHODS Human bone marrow-derived mononuclear cells were cultured within fibrin for 2 weeks with or without fibroblast growth factor-2 supplementation. DNA content and cell viability of enzymatically retrieved cells were determined at days 7 and 14. Cell surface marker profiling and cell cycle analysis were performed by means of multi-color flow cytometry and a 5-ethynyl-2'-deoxyuridine incorporation assay, respectively. RESULTS Total mononuclear cell fractions, isolated from whole human bone marrow, was successfully cultured in fibrin gels for up to 14 days under static conditions. Discrete niche cell populations including MSCs, pericytes and hematopoietic stem cells were maintained in relative quiescence for 7 days in proportions similar to that in freshly isolated cells. Colony-forming unit efficiency of enzymatically retrieved MSCs was significantly higher at day 14 compared to day 0; and in accordance with previously published works, it was fibroblast growth factor-2-dependant. CONCLUSIONS Fibrin gels provide a simple, novel system in which to culture and study the complete fraction of bone marrow-derived mononuclear cells and may support the development of improved bone marrow cell-based therapies.