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.

Expression of CD95 on mature leukocytes of MRL/lpr mice after transplantation of genetically modified bone marrow stem cells

Bone marrow transplantation (BMT) is commonly used for the treatment of severe haematological and immunological diseases. For instance, the autoimmune lymphoproliferative syndrome (ALPS) caused by a complete expression defect of CD95 (Fas, APO-1) can be cured by allogeneic BMT. However, since this therapy may not generate satisfactory results when only partially compatible donors are available, we were interested in the development of a potential alternative treatment by using lentiviral gene transfer of a normal copy of CD95 cDNA in hematopoietic stem cells. Here, we show that this approach applied to MRL/lpr mice results in the expression of functional CD95 receptors on the surface of lymphocytes, monocytes, and granulocytes. This suggests that correction of CD95 deficiency can be achieved by gene therapy.

Intraarterial administration of bone marrow mononuclear cells in patients with critical limb ischemia: a randomized-start, placebo-controlled pilot trial (PROVASA)

Critical limb ischemia due to peripheral arterial occlusive disease is associated with a severely increased morbidity and mortality. There is no effective pharmacological therapy available. Injection of autologous bone marrow-derived mononuclear cells (BM-MNC) is a promising therapeutic option in patients with critical limb ischemia, but double-blind, randomized trials are lacking.

Intracoronary injection of bone marrow-derived mononuclear cells early or late after acute myocardial infarction: effects on global left ventricular function

BACKGROUND Intracoronary administration of autologous bone marrow-derived mononuclear cells (BM-MNC) may improve remodeling of the left ventricle (LV) after acute myocardial infarction. The optimal time point of administration of BM-MNC is still uncertain and has rarely been addressed prospectively in randomized clinical trials. METHODS AND RESULTS In a multicenter study, we randomized 200 patients with large, successfully reperfused ST-segment elevation myocardial infarction in a 1:1:1 pattern into an open-labeled control and 2 BM-MNC treatment groups. In the BM-MNC groups, cells were administered either early (i.e., 5 to 7 days) or late (i.e., 3 to 4 weeks) after acute myocardial infarction. Cardiac magnetic resonance imaging was performed at baseline and after 4 months. The primary end point was the change from baseline to 4 months in global LV ejection fraction between the 2 treatment groups and the control group. The absolute change in LV ejection fraction from baseline to 4 months was -0.4±8.8% (mean±SD; P=0.74 versus baseline) in the control group, 1.8±8.4% (P=0.12 versus baseline) in the early group, and 0.8±7.6% (P=0.45 versus baseline) in the late group. The treatment effect of BM-MNC as estimated by ANCOVA was 1.25 (95% confidence interval, -1.83 to 4.32; P=0.42) for the early therapy group and 0.55 (95% confidence interval, -2.61 to 3.71; P=0.73) for the late therapy group. CONCLUSIONS Among patients with ST-segment elevation myocardial infarction and LV dysfunction after successful reperfusion, intracoronary infusion of BM-MNC at either 5 to 7 days or 3 to 4 weeks after acute myocardial infarction did not improve LV function at 4-month follow-up.

Platelet released growth factors boost expansion of bone marrow derived CD34(+) and CD133(+) endothelial progenitor cells for autologous grafting

Stem cell based autologous grafting has recently gained mayor interest in various surgical fields for the treatment of extensive tissue defects. CD34(+) and CD133(+) cells that can be isolated from the pool of bone marrow mononuclear cells (BMC) are capable of differentiating into mature endothelial cells in vivo. These endothelial progenitor cells (EPC) are believed to represent a major portion of the angiogenic regenerative cells that are released from bone marrow when tissue injury has occurred. In recent years tissue engineers increasingly looked at the process of vessel neoformation because of its major importance for successful cell grafting to replace damaged tissue. Up to now one of the greatest problems preventing a clinical application is the large scale of expansion that is required for such purpose. We established a method to effectively enhance the expansion of CD34(+) and CD133(+) cells by the use of platelet-released growth factors (PRGF) as a media supplement. PRGF were prepared from thrombocyte concentrates and used as a media supplement to iscove's modified dulbecco's media (IMDM). EPC were immunomagnetically separated from human bone morrow monocyte cells and cultured in IMDM + 10% fetal calf serum (FCS), IMDM + 5%, FCS + 5% PRGF and IMDM + 10% PRGF. We clearly demonstrate a statistically significant higher and faster cell proliferation rate at 7, 14, 21, and 28 days of culture when both PRGF and FCS were added to the medium as opposed to 10% FCS or 10% PRGF alone. The addition of 10% PRGF to IMDM in the absence of FCS leads to a growth arrest from day 14 on. In histochemical, immunocytochemical, and gene-expression analysis we showed that angiogenic and precursor markers of CD34(+) and CD133(+) cells are maintained during long-term culture. In summary, we established a protocol to boost the expansion of CD34(+) and CD133(+) cells. Thereby we provide a technical step towards the clinical application of autologous stem cell transplantation.

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.

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.

Bone marrow-derived cells in palatal wound healing

OBJECTIVE: Myofibroblasts are responsible for contraction and scarring after cleft palate repair. This leads to growth disturbances in the upper jaw. We hypothesized that cells from the bone marrow are recruited to palatal wounds and differentiate into myofibroblasts. METHODS: We transplanted bone marrow from green fluorescent protein (GFP)-transgenic rats into lethally irradiated wild-type rats. After recovery, experimental wounds were made in the palatal mucoperiosteum, and harvested 2 weeks later. GFP-expressing cells were identified using immunostaining. Myofibroblasts, activated fibroblasts, endothelial cells, and myeloid cells were quantified with specific markers. RESULTS: After transplantation, 89 ± 8.9% of mononuclear cells in the blood expressed the GFP and about 50% of adherent cells in the bone marrow. Tissue obtained during initial wounding contained only minor numbers of GFP-positive cells, like adjacent control tissue. Following wound healing, 8.1 ± 5.1% of all cells in the wound area were positive, and 5.0 ± 4.0% of the myofibroblasts, which was significantly higher than in adjacent tissue. Similar percentages were found for activated fibroblasts and endothelial cells, but for myeloid cells it was considerably higher (22 ± 9%). CONCLUSIONS: Bone marrow-derived cells contribute to palatal wound healing, but are not the main source of myofibroblasts. In small wounds, the local precursor cells are probably sufficient to replenish the defect.

Haematopoietic stem cell transplantation in Switzerland. Report from the Swiss Transplant Working Group Blood and Marrow Transplantation (STABMT) Registry 1997-2003

In 1997, the Swiss Transplant Working Group Blood and Marrow Transplantation (STABMT) initiated a mandatory national registry for all haematopoietic stem cell transplants (HSCT) in Switzerland. As of 2003, information was collected of 2010 patients with a first HSCT (577 allogeneic (29%) and 1433 autologous (71%) HSCT) and 616 additional re-transplants. This included 1167 male and 843 female patients with a median age of 42.4 years (range 0.2-76.6 years). Main indications were leukaemias (592; 29%) lymphoproliferative disorders (1,061; 53%), solid tumours (295; 15%) and non-malignant disorders (62; 3%). At the time of analysis 1,263 patients were alive (63%), 747 had died (37%). Probability of survival, transplant related mortality or relapse at 5 years was 52%, 21%, 36% for allogeneic and 54%, 5%, 60% for autologous HSCT. Outcome depended on indication, donor type, stem cell source and age of patient. HSCT is an established therapy in Switzerland. These data describe current practice and outcome.

Liver repopulation with bone marrow derived cells improves the metabolic disorder in the Gunn rat

BACKGROUND: Reversible ischaemia/reperfusion (I/R) liver injury has been used to induce engraftment and hepatic parenchymal differentiation of exogenous beta2-microglubulin(-)/Thy1(+) bone marrow derived cells. AIM: To test the ability of this method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, to correct the metabolic disorder in a rat model of congenital hyperbilirubinaemia. METHODS AND RESULTS: Analysis by confocal laser microscopy of fluorescence labelled cells and by immunohistochemistry for beta2-microglubulin, 72 hours after intraportal delivery, showed engraftment of infused cells in liver parenchyma of rats with I/R, but not in control animals with non-injured liver. Transplantation of bone marrow derived cells obtained from GFP-transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP positive hepatocytes in I/R liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP negative bone marrow derived cells into GFP positive transgenic rats resulted in the appearance of GFP negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow derived cells into hyperbilirubinaemic Gunn rats with deficient bilirubin conjugation after I/R damage resulted in 30% decrease in serum bilirubin, the appearance of bilirubin conjugates in bile, and the expression of normal UDP-glucuronyltransferase enzyme evaluated by polymerase chain reaction. CONCLUSIONS: I/R injury induced hepatic parenchymal engraftment and differentiation into hepatocyte-like cells of bone marrow derived cells. Transplantation of bone marrow derived cells from non-affected animals resulted in the partial correction of hyperbilirubinaemia in the Gunn rat.

Generation and functional characterization of ovine bone marrow-derived macrophages.

A method for the culturing and propagation of ovine bone marrow-derived macrophages (BMM) in vitro is described. Bone marrow cells from sterna of freshly slaughtered sheep were cultured in hydrophobic (teflon foil) bags in the presence of high serum concentrations (20% autologous serum and 20% fetal calf serum). During an 18 day culture period in the absence of added conditioned medium, and without medium change, a strong enrichment of mononuclear phagocytes was achieved. Whereas the number of macrophages increased four to fivefold during this time, granulocytes, lymphoid cells, stem cells and undifferentiated progenitor cells were reduced to less than 3% of their numbers at Day 0. This resulted in BMM populations of 94 +/- 3% purity. These cells had morphological and histochemical characteristics of differentiated macrophages, and they performed functions similar to those of non-activated, unprimed human monocyte-derived macrophages. Thus, they avidly ingested erythrocytes coated with IgG of heterologous or homologous origin. They expressed a modest level of procoagulant activity, but upon triggering with lipopolysaccharide (LPS), a marked increase in cell-associated procoagulant activity was observed. LPS triggering promoted the secretion of interleukin-1, as evidenced by measurement of murine thymocyte costimulatory activity, and transforming growth factor-beta. Using the mouse L929 cell cytotoxicity assay as an indication of tumor necrosis factor (TNF) activity, no TNF activity was detected in the same supernatants, a result possibly due to species restriction. BMM generated low levels of O2- upon triggering with phorbol 12-myristate 13-acetate (PMA). On the other hand, no O2- production was observed upon stimulation with zymosan opsonized with ovine or human serum. Using luminol-enhanced chemiluminescence (CL) as a more sensitive indicator of an oxidative burst, both PMA or zymosan were able to trigger CL, but the response was subject to partial inhibition by sodium azide, an inhibitor of myeloperoxidase. This points to non-macrophage cells contributing also to the CL response, and is consistent with the view that unprimed BMM elicit a low oxidative burst upon triggering with strong inducers of a burst. Our functional characterization now allows us to apply priming and activation protocols and to relate their effect to functional alterations.

Culture of ovine bone marrow-derived macrophages and evidence for serum factors distinct from M-CSF contributing to their propagation in vitro.

An in vitro system allowing the culture of ovine bone marrow-derived macrophages (BMMs) is described. Bone marrow (BM) cells from the sternum of 4- to 9-month-old sheep were cultured in liquid suspension in hydrophobic bags with medium containing 20% autologous serum and 20% fetal calf serum (FCS). Cells with macrophage characteristics were positively selected and increased four- to five-fold between day (d) 0 and d18. Granulocytes and cells of lymphoid appearance including progenitor cells were negatively selected and were diminished 50-fold during this 18-d culture. The addition of macrophage colony-stimulating factor (M-CSF)-containing supernatants to liquid cultures did not significantly improve the yield of BMM in 18-d cultures. In contrast, cell survival at d6 and macrophage cell yield at d18 depended on the concentration and source of serum in the culture medium. FCS and 1:1 mixtures of FCS and autologous serum were superior to autologous serum alone. Analysis of growth requirements of ovine BMMs suggested that they are under more complex growth control than their murine counterparts. In an [3H]thymidine incorporation assay with BM cells collected at different times of culture, d3 or d4 BM cells responded to human recombinant M-CSF, human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), bovine GM-CSF, murine M-CSF or murine M-CSF-containing supernatants, and bovine interleukin 1 beta (IL-1 beta) in decreasing order of magnitude. Likewise, pure murine BMM populations harvested at d6 responded to homologous GM-CSF, IL-3, and human or murine M-CSF. FCS did not stimulate the proliferation of murine BMMs (d6) and of ovine BM cells (d3 or d4). In contrast, ovine BM cells harvested at d12 responded to FCS by proliferation in a dose-dependent manner but failed to proliferate in the presence of human or murine M-CSF or M-CSF-containing supernatants of mouse and sheep fibroblasts containing mouse macrophage growth-promoting activity. Likewise, various cytokine-containing supernatants and recombinant cytokines (murine IL-3, murine and human GM-CSF, murine and bovine IL-1 beta) did not promote proliferation of ovine d12 BM cells to an extent greater than that achieved with 15% FCS alone. Thus, ovine BMM proliferation is under the control of at least two factors acting in sequence, M-CSF and an unidentified factor contained in FCS. The ovine BMM culture system may provide a model for the analysis of myelomonocytopoiesis in vitro.

Varicella zoster virus reactivation after autologous SCT is a frequent event and associated with favorable outcome in myeloma patients.

The occurrence of varicella zoster virus (VZV) reactivation is increased after allogeneic transplantation, whereas limited data are available for herpes zoster (HZ) after autologous SCT (ASCT). We determined the incidence and the prognostic significance of HZ and its correlation with VZV serology in 191 consecutive myeloma patients undergoing high-dose melphalan chemotherapy with ASCT. We found that VZV reactivation occurred in 57 (30%) patients, in 8.5% during induction and in 21.5% after ASCT peaking at 8 months after ASCT. Disease burden due to HZ was assessed as high or rather high in 70% of the patients. By immune fluorescence and Serion Elisa VZV IgG assessment, 90.8% of all patients had specific anti-VZV antibodies at ASCT. Lower specific antibody titers at transplantation were observed in patients with HZ after ASCT than in those without reactivation (P=0.009). Finally, OS was better in myeloma patients with HZ after ASCT compared with patients without HZ (P=0.007). Our data indicate that VZV reactivation after ASCT is a frequent event carrying a significant disease burden and it is associated with improved survival. Low levels of specific VZV antibodies at ASCT suggest increased vulnerability for VZV reactivation.Bone Marrow Transplantation advance online publication, 19 January 2015; doi:10.1038/bmt.2014.290.

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.

Outcome of aplastic anaemia in children. A study by the severe aplastic anaemia and paediatric disease working parties of the European group blood and bone marrow transplant.

This study analysed the outcome of 563 Aplastic Anaemia (AA) children aged 0-12 years reported to the Severe Aplastic Anaemia Working Party database of the European Society for Blood and Marrow Transplantation, according to treatment received. Overall survival (OS) after upfront human leucocyte antigen-matched family donor (MFD) haematopoietic stem cell transplantation (HSCT) or immunosuppressive treatment (IST) was 91% vs. 87% (P 0·18). Event-free survival (EFS) after upfront MFD HSCT or IST was 87% vs. 33% (P 0·001). Ninety-one of 167 patients (55%) failed front-line IST and underwent rescue HSCT. The OS of this rescue group was 83% compared with 91% for upfront MFD HSCT patients and 97% for those who did not fail IST up-front (P 0·017). Rejection was 2% for MFD HSCT and HSCT post-IST failure (P 0·73). Acute graft-versus-host disease (GVHD) grade II-IV was 8% in MFD graft vs. 25% for HSCT post-IST failure (P < 0·0001). Chronic GVHD was 6% in MFD HSCT vs. 20% in HSCT post-IST failure (P < 0·0001). MFD HSCT is an excellent therapy for children with AA. IST has a high failure rate, but remains a reasonable first-line choice if MFD HSCT is not available because high OS enables access to HSCT, which is a very good rescue option.