Intra-bone Injection Research Articles

S106: LONG-TERM FOLLOW-UP OF BETA-THALASSEMIA PATIENTS TREATED WITH HEMATOPOIETIC STEM CELL GENE THERAPY

Background: Transfusion-dependent ß-thalassemia (TDT) is a disorder due to mutations in the gene encoding the ß-globin chain causing a reduced or absent production of haemoglobin A leading to severe anaemia and lifelong transfusion dependence. Gene therapy has now been accepted as a possible alternative cure to allogeneic bone marrow (BM) transplantation. Aims: We developed a gene therapy approach based on autologous mobilized hematopoietic stem cell transduced by lentiviral vector, expressing human ß-globin gene, administered by intrabone injection, following a myeloablative conditioning (NCT02453477). Methods and Results: Nine patients with severe TDT with different genotypes have been treated with a drug product with a median cell dose of 19.5x106 CD34+ cells/kg, a transduction efficiency from 38 to 77% and a median vector copy number/genome (VCN) in bulk CD34+ cells of 0.9 (range: 0.7–1.5). Overall, gene therapy was generally well-tolerated with no adverse events related to the investigational product. No severe infectious-related adverse events were reported, except for those related to neutropenia as expected after myeloablative conditioning. Polyclonal vector integrations profiles with no evidence of clonal dominance have been detected in all patients with the expected genomic distribution for lentiviral vectors. Clinical outcome showed a reduction of transfusion requirement both in frequency and volume in adult patients up to more than 50%. Among the pediatric patients, 4 out of 6 discontinued transfusions shortly after gene therapy and are transfusion-independent at the last follow-up (up to 75 months). A robust and persistent engraftment was observed in 7 out of 9 patients, with a marking of BM progenitors that, in engrafted patients, ranged between 25.3 and 79.8% and with a median VCN in CD34+ cells of 0.53 (range: 0.34–2.21). As a relevant target for transgene expression, BM erythroid cells were stably marked (VCN range 0.3 - 2.5). Summary and Conclusions: A longer follow-up will provide further results on long-term clinical efficacy and safety of this approach.

Paracrine Effects of Recombinant Human Adiponectin Promote Bone Regeneration.

Bone regeneration is a delicate physiological process. Non-union and delayed fracture healing remains a great challenge in clinical practice nowadays. Bone and fat hold a close relationship to remain balanced through hormones and cytokines. Adiponectin is a well-known protein to maintain the hemostasis, which may be an interesting target for fracture healing. Herein, we provided a facile and efficient method to obtain high-purity and high-yield recombinant human adiponectin (ADPN). The biocompatibility and the pharmaceutical behaviors were evaluated in Sprague–Dawley rats. The paracrine effects of adiponectin on bone fracture healing were investigated with a rat tibia fracture model via intrabone injection. Significantly accelerated bone healing was observed in the medulla injection group, indicating the paracrine effects of adiponectin could be potentially utilized for clinical treatments. The underlying mechanism was primarily assessed, and the expression of osteogenic markers, including bone morphogenic protein 2, alkaline phosphatase, and osteocalcin, along with adiponectin receptor 1 (AdipoR1), was markedly increased at the fracture site. The increased bone healing of ADPN treatment may result from both enhanced osteogenic proliferation as well as differentiation. Cell experiments confirmed that the expression of osteogenesis markers increased significantly in ADPN treatment groups, while it decreased when the expression of AdipoR1 was knocked down by siRNA. Our study provided a feasible and efficacious way for bone fracture treatment with local administration of ADPN, which could be rapidly translated into the clinics.

Effect of Rap-536 on MDS PDX-Murine Models with or without SF3B1 Mutation

Introduction: Anemia is the most frequent cytopenia in myelodysplastic syndromes (MDS) and patients will require red blood cell transfusion. Most low-risk MDS patients with anemia are currently treated with erythropoietin stimulating agents (ESA). This treatment gives 45-60% of erythroid response but responders will lose their erythroid response after a median time of 20-24 months (Park, Blood, 2008). Recently, luspatercept (RAP-536), a trap ligand of transforming growth factor-β superfamily, including activins and growth differentiation factors (GDFs), has shown positive effects on erythroid response in MDS patients especially in SF3B1 mutated patients (Platzbecker, Lancet Oncology, 2017). The objective of our work was to evaluate the effect of RAP-536 on human engraftment in PDX mice models of MDS. Methods: We performed patient derived xenograft (PDX) NOD SCID gamma (NSG) murine model by intrabone injections of mesenchymal stromal cells (MSC) and CD34+ hematopoietic stem progenitor cells (HSPC) from 3 MDS patients with SF3B1 mutations and 3 MDS patients without SF3B1 mutations but carrying other recurrent mutations (ASXL1, TET2, SRSF2, CBL, RUNX1, STAG2) seen in MDS (Table A). Three months after xenograft, we have realized a bone marrow aspiration for monitoring human engraftment using flow cytometry to check that there was a human engraftment. Then, the mice were treated for 2 months with 30mg/kg/week of RAP-536. At the end of the procedure, serum, blood and bone marrow were collected. Results: As GDF11 is a target of RAP-536, we have measured GDF11 level in serum in order to confirm the expected effects of RAP-536. We observed decreased levels of GDF11 in RAP-536 treated mice versus mock treated (p<0.0001). Concerning blood counts, we observed a higher level of hemoglobin (p=0.002) (Fig. B) and a higher level of white blood cells (p=0.04) (Fig. C) in RAP-536 treated mice, independently of the SF3B1 mutated status and no impact on platelet count (Fig. D). Human myeloid engraftment was determined by flow cytometry. If we consider human DAPI-/murine CD45-/ human CD45+ positive cells, the average percentage was 2.09% for mock treated mice and 2.4% for treated mice (no statistically significant difference between treated mice or not (p=0.09) and no impact of SF3B1 mutated status) (Fig. E). Interestingly, if we focused on human CD45+/CD33+ among the human CD45+ cells, we observed a higher engraftment 66.35% of positive cells versus 55.89% in treated mice vs mocked treated mice respectively (p=0.014), independently of SF3B1 mutated status (Fig. F). Molecular analyzes on CD45+ cells harvested from the PDX model are ongoing to confirm the human engraftment by detecting the recurrent gene mutations present in the initial MDS clones. Mutation burden level will be analyzed in the presence or absence of RAP-536. Conclusion: RAP-536 improves hemoglobin and WBC levels and seems to enhance the engraftment of human myeloid hematopoiesis in this PDX murine model of MDS. Molecular analyses are ongoing to clarify the relative proportions of normal and pathological human hematopoiesis before and after RAP-536. Figure Disclosures Park: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: RAP-536

Current status of umbilical cord blood transplantation in children

The first umbilical cord blood (UCB) transplantation was performed 30years ago. UCB transplantation (UCBT) is now widely used in children with malignant and non-malignant disorders who lack a matched family donor. UCBT affords a lower incidence of graft-versus-host disease compared to alternative stem cell sources, but also presents a slower immune recovery and a high risk of infections if serotherapy is not omitted or targeted within the conditioning regimen. The selection of UCB units with high cell content and good human leucocyte antigen match is essential to improve the outcome. Techniques, such as double UCBT, ex vivo stem cell expansion and intra-bone injection of UCB, have improved cord blood engraftment, but clinical benefit remains to be demonstrated. Cell therapies derived from UCB are under evaluation as potential novel strategies to reduce relapse and viral infections following transplantation. In recent years, improvements within haploidentical transplantation have reduced the overall use of UCBT as an alternative stem cell source; however, each may have its relative merits and disadvantages and tailored use of these alternative stem cell sources may be the optimal approach.

Intrabone transplant provides full stemness of cord blood stem cells with fast hematopoietic recovery and low GVHD rate: results from a prospective study

Umbilical Cord Blood (UCB) represents a valid option for patients with hematopoietic malignancies lacking an HLA matched donor. To overcome the limitation of the low stem cell dose of UCB, the intrabone (IB) route has been proposed. We report the results of a prospective study on a poor-prognosis cohort of 23 patients receiving intrabone single UCB transplant (Clinicaltrials.gov NCT00886522). Cumulative incidence of hematological recovery at day 90 was 82 ± 9% (ANC > 0.5 × 109/L) and 70 ± 10% (platelet > 50 × 109/L) and correlated with CD34 + cells in the graft. NRM was 20 ± 9%. No severe aGVHD and only one extensive cGVHD occurred, with fast immune reconstitution. To test the hypothesis that the direct IB injection could affect the expression of stem cells regulatory pathways, CD34 + cells from BM aspirates at day + 10, + 20, + 30, processed in hypoxic conditions mimicking the BM-microenvironment (7%pO2), were studied for the expression of c-Mpl, Notch1 and CXCR4. We found that the expression of c-Mpl in CD34 + cells at day + 10 significantly correlated with hematological recovery. In conclusion, IB-UCB transplant success is associated with low incidence of GVHD and high-speed platelet recovery; intrabone route may preserve full hematopoietic stemness by direct delivery of UCB stem cells into the hypoxic HSC niche.

Essential roles of the interaction between cancer cell-derived chemokine, CCL4, and intra-bone CCR5-expressing fibroblasts in breast cancer bone metastasis

From a murine breast cancer cell line, 4T1, we established a subclone, 4T1.3, which consistently metastasizes to bone upon its injection into the mammary fat pad. 4T1.3 clone exhibited similar proliferation rate and migration capacity as the parental clone. However, the intra-bone injection of 4T1.3 clone caused larger tumors than that of the parental cells, accompanied with increases in fibroblast, but not osteoclast or osteoblast numbers. 4T1.3 clone displayed an enhanced expression of a chemokine, CCL4, but not its specific receptor, CCR5. CCL4 shRNA-transfection of 4T1.3 clone had few effects on its in vitro properties, but reduced the tumorigenicity arising from the intra-bone injection. Moreover, intra-bone injection of 4T1.3 clone caused smaller tumors in mice deficient in CCR5 or those receiving CCR5 antagonist than in wild-type mice. The reduced tumor formation was associated with attenuated accumulation of CCR5-positive fibroblasts expressing connective tissue growth factor (CTGF)/CCN2. Tumor cell-derived CCL4 could induce fibroblasts to express CTGF/CCN2, which could support 4T1.3 clone proliferation under hypoxic culture conditions. Thus, the CCL4-CCR5 axis can contribute to breast cancer metastasis to bone by mediating the interaction between cancer cells and fibroblasts in bone cavity.

Specially modified stromal and immune microenvironment in injected bonemarrow following intrabone transplantation facilitates allogeneichematopoietic stem cell engraftment.

For allogeneic hematopoietic stem cell transplantation (HSCT), the first key step is the engraftment of hematopoietic stem cells (HSCs) across the major histocompatibility complex (MHC) barrier. Intrabone bone marrow transplantation (IBBMT) could replace more recipient stromal cells with donor cells and facilitate allogeneic organ transplantation compared with the conventional intravenous approach. However, it remains unknown whether and how IBBMT reconstructs the immune microenvironment for allogeneic HSCs. We explored where the BM microenvironment changes by determining BM stromal cell chimerism and measuring the change in CXCL-12 expression and regulatory Tcells in recipient BM. We found that most stromal cells were replaced by allogeneic cells in the injected BM, with higher expression of immune regulatory cytokines (interleukin-10) compared with the contralateral BM and the intravenous group BM. This difference was independent of injury caused by intrabone injection. Consistent with the microenvironment modification, the allogeneic the engraftment rate and reconstitution capacity of HSCs were enhanced in the injected BM compared with the contralateral BM and intravenous group BM. Surgical removal of the injected bone at 7days rather than 21days reduced the levels of allogeneic granulocytes and HSCs in the peripheral blood. In conclusion, IBBMT specially modifies stromal cells in the injected BM which provide immune protective cues that improve the engraftment of allogeneic HSCs in an early period.

Development of a Patient-Derived Xenograft Model of Human Myelodysplastic Syndromes

Background: Myelodysplastic syndromes (MDS) comprise a group of hematologic disorders characterized by ineffective hematopoiesis and cytopenias that may lead to acute myeloid leukemia (AML) or bone marrow failure. Although a few genetic mouse models have been generated, they cannot recapitulate the heterogeneity of the disease. The attempts for the development of a xenograft model have been challenging in the past, mainly due to poor engraftment of MDS hematopoietic cells. Recent scientific evidence pinpoint the importance of the niche in the establishment and progression of hematological malignancies and suggested a novel approach to establishing MDS xenotransplantation models. In this study we aim to develop a mouse xenotransplantation model of human MDS and to evaluate the role of mesenchymal stem cells (MSCs) in the engraftment process.Methods: MSC's from normal donors and MDS patient samples were generated and characterized using standard culture methods. NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS) mice were used as recipients. Mice were injected with 1 x 106 bone marrow mononuclear cells (MNC) alone or in combination with 5 x 105 MSC's. MSC's were from normal donors, from same patient as MNC's (autologous) or from other MDS patients (MDS allogeneic). All cells were injected intrafemorally. Mice were assessed for engraftment by bone marrow aspiration in the ipsilateral bone at 6 weeks, in the contralateral bone at 10 weeks and at necropsy at 12-22 weeks (depending on initial assessment). Long-term engrafted samples were further assessed for presence of stem cells by secondary transplantation into NSGS mice. 1 x 106 human CD45 selected hematopoietic cells were passaged again without or with 5 x 105 MSC's. Secondary recipients were assessed at 12 weeks for engrafment. MDS samples represented both high risk (refractory anemia with excess blasts, RAEB)) and low risk (refractory anemia (RA), refractory cytopenia with myelodysplasia (RCMD)) disease. The overall degree of engraftment was assessed by bone marrow aspiration and measurement of human CD45+ cells. Lymphoid, myeloid and erythroid engraftment was assessed.Results: Five out of five injected MDS samples showed persistent human cells when assessed at 6 weeks. For most samples, engraftment at 6 weeks was low (<2%) and was not consistently influences by the presence or absence of MSC. On week 10, only two out of five patients had increased engraftment and only one showed higher engraftment levels in the mice co-transplanted with MSCs. Both of these better engrafting samples were from patients with RAEB. Engrafting samples demonstrated both myeloid and erythroid engraftment. Molecular analysis to confirm engraftment of MDS clone will be presented. In total, 2 out of 5 patients showed long-term engraftment (> 12 weeks). One of these samples has been transferred to secondary animals. Secondary transplanted mice injected with selected hCD45+ cells with or without MSC's showed variable engraftment levels on week 10 after injections, with the majority of those not reaching long-term engraftment.Conclusions: Passive transferof MDS hematopoietic cells as assessed at 6 weeks after intrabone injection is highly consistent and not dependent on MSC when total MNC fraction is used. However, long-term engraftment of MDS cells in xenotransplanted mice is uncommon suggesting that true MDS stem cells do not consistently engraft. Further analysis with injection into different mouse strains with purified CD34+ cells is under way. DisclosuresDos Santos:Amgen: Employment.

Intrabone Injection of T-Cell Depleted Peripheral Blood Stem Cells from HLA-Haploidentical Donors to Reduce the Risk of Graft Rejection in Children

▪Background. Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is an effective treatment option for patients with malignant and non-malignant hematologic disorders lacking an HLA-compatible donor. Strategies for T-cell depletion (TCD) of the graft, such as positive selection of CD34+ cells, offer the potential to prevent acute and chronic graft-versus-host disease (GVHD). The risk of graft rejection associated with the extensive depletion of both T lymphocytes and accessory cells can be overcome by infusing a very high number (megadose) of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSC) (exceeding 10x106/kg recipient body weight) to overcome the HLA barrier (Aversa F. et al. Blood 1994). Moreover, the infusion of a megadose of CD34+ cells (higher than 20x106/kg and 12.4x106/kg, respectively) has been shown to result in faster immunological recovery and improved leukemia-free survival probability in children (Handgretinger R. et al. Bone Marrow Transplant 2001; Klingebiel T. et al. Blood 2010).Nevertheless, in the case of donors considered “poor mobilizers” (10-30% of cases), the threshold dose of CD34+ cells needed to ensure the inoculum of a megadose of stem cells might not be achieved. In the setting of cord blood (CB) transplantation, one of the strategies aimed at overcoming the problem of low cellularity is represented by the intrabone injection of CB stem cells, with good engraftment rates even in adult patients. We explored the same strategy in the context of T-cell depleted haplo-HSCT and low graft cellularity due to poor donor mobilization, ensuing in inadequate dose of CD34+cells available after positive selection TCD.Patients and methods. From September 2009 to April 2013, 11 pediatric patients affected by malignant or non-malignant hematological disorders (5 acute lymphoblastic leukemias, 1 acute myeloid leukemia, 1 myelodysplastic syndrome, 2 dyskeratosis congenita, 1 Fanconi anemia) received a T-cell depleted CD34+positively selected PBSC allograft from an HLA-haploidentical related donor.Due to the failure to achieve a target cell dose higher than 12x106 purified CD34+ cells/kg, part of the stem cell inoculum was infused as intrabone injection. The procedure was carried out at the patient bedside by multiple intrabone injections in the superior-posterior iliac crests under sedoanalgesia, as previously described (Frassoni F. et al. Lancet Oncol 2008). The median dose of CD34+ cells infused was 9x106/kg (range, 5-12) while the median number of CD3+ lymphocytes was 0.7x104/kg recipient body weight (range, 0.3-11). About one third of the stem cell inoculum, corresponding to a total volume of 20-40 ml, was given intrabone, while the remaining stem cell portion was infused intravenously.Results.No complication occurred during, or immediately after, the intrabone injection. Nine out of the 11 patients achieved a complete donor engraftment, while graft rejection occurred in 2 patients. The median time for neutrophil engraftment was 13.5 days (range, 12-20), while the median time for platelet recovery was 14 days (range, 13-24). One patient developed grade II acute GVHD and only 1 case of limited chronic GVHD was observed. No transplant-related deaths were observed.Conclusions. Our data suggest that, in the haplo-HSCT setting, the intrabone injection of positively selected CD34+ cells, can be safely used in cases of low graft cellularity due to poor donor mobilization, with the aim of minimizing the risk of graft rejection or poor engraftment. Our preliminary data need to be confirmed in larger series of patients and compared with those obtained with conventional intravenous administration of comparable dose of CD34+ cells. DisclosuresNo relevant conflicts of interest to declare.

Discoidin domain receptor 2 facilitates prostate cancer bone metastasis via regulating parathyroid hormone-related protein

Prostate cancer frequently metastasizes to the skeleton but the underlying mechanism remains largely undefined. Discoidin domain receptor 2 (DDR2) is a member of receptor tyrosine kinase (RTK) family and is activated by collagen binding. This study aimed to investigate the function and detailed mechanism of DDR2 in prostate cancer bone dissemination. Herein we found that DDR2 was strongly expressed in bone-metastatic prostate cancer cells and tissues compared to that in normal controls. Enhanced expression of constitutively activated DDR2 led to elevation in motility and invasiveness of prostate cancer cells, whereas knockdown of DDR2 through specific shRNA caused a dramatic repression. Knockdown of DDR2 in prostate cancer cells resulted in significant decrease in the proliferation, differentiation and function of osteoblast. Over-expression of DDR2 in prostate cancer cells resulted in notable acceleration of osteoclast differentiation and bone resorption, whereas knockdown of DDR2 exhibited the opposite effects. An intrabone injection bone metastasis animal model demonstrated that DDR2 promoted osteolytic metastasis in vivo. Molecular evidence demonstrated that DDR2 regulated the expression, secretion, and promoter activity of parathyroid hormone-related protein (PTHrP), via modulating Runx2 phosphorylation and transactivity. DDR2 was responsive to TGF-β and involved in TGF-β-mediated osteoclast activation and bone resorption. In addition, DDR2 facilitated prostate cancer cells adhere to type I collagen. This study reveals for the first time that DDR2 plays an essential role in prostate cancer bone metastasis. The mechanism disclosure may provide therapeutic targets for the treatment of prostate cancer.

Effects of MSC Coadministration and Route of Delivery on Cord Blood Hematopoietic Stem Cell Engraftment

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.

Unrelated Cord Blood Transplantation

Unrelated cord blood transplantation (UCBT) is associated with delayed hematopoietic recovery. Intrabone injection of cord blood cells (IB-UCBT) and double-UCBT (dUCBT) are designed to circumvent this problem. In a retrospective registry-based analysis, we compared outcomes of 87 IB-UCBT with 149 dUCBT recipients, after myeloablative conditioning regimen adjusting for the differences between the two groups. Median-infused total nucleated cells were 2.5×10/kg for IB-UCBT and 3.9×10/kg for dUCBT (P<0.001). At day +30, cumulative incidence (CI) of neutrophil recovery was 76% and 62% (P=0.014) with a median time to engraftment of 23 and 28 days (P=0.001), after IB-UCBT and dUCBT, respectively. At day +180, CI of platelets recovery was 74% after IB-UCBT, and 64%, after dUCBT (P=0.003). In multivariate analysis, IB-UCBT was associated with neutrophil and platelets recovery and lower acute graft versus host disease (II-IV) (P<0.01). At 2 years, CI of nonrelapse mortality and relapse incidence were 30% and 25% after IB-UCBT and 34% and 29% after dUCBT, and disease-free survival was 45% and 37%, respectively. However, after landmark analysis at 4.7 months from transplantation, in multivariate analysis, relapse incidence was reduced (P=0.03), and there was a trend for better disease-free survival after IB-UCBT (P=0.09). Both approaches expand the possibility of offering UCBT to patients with hematopoietic malignancies; IB-UCBT is associated with faster myeloid and platelet recovery and lower acute graft versus host disease and may reduce the total cost. However, studies on cost effectiveness are needed to compare both strategies.

Stem cell bioengineering strategies to widen the therapeutic applications of haematopoietic stem/progenitor cells from umbilical cord blood

Umbilical cord blood (UCB) transplantation has observed a significant increase in recent years, due to the unique features of UCB haematopoietic stem/progenitor cells (HSCs) for the treatment of blood-related disorders. However, the low cell numbers available per UCB unit significantly impairs the widespread use of this source for transplantation of adult patients, resulting in graft failure, delayed engraftment and delayed immune reconstitution. In order to overcome this issue, distinct approaches are now being considered in clinical trials, such as double-UCB transplantation, intrabone injection or ex vivo expansion. In this article the authors review the current state of the art, future trends and challenges on the ex vivo expansion of UCB HSCs, focusing on culture parameters affecting the yield and quality of the expanded HSC grafts: novel HSC selection schemes prior to cell culture, cytokine/growth factor cocktails, the impact of biochemical factors (e.g. O2 ) or the addition of supportive cells, e.g. mesenchymal stem/stromal cell (MSC)-based feeder layers) were addressed. Importantly, a critical challenge in cellular therapy is still the scalability, reproducibility and control of the expansion process, in order to meet the clinical requirements for therapeutic applications. Efficient design of bioreactor systems and operation modes are now the focus of many bioengineers, integrating the increasing 'know-how' on HSC biology and physiology, while complying with the GMP standards for the production of cellular products, i.e. through the use of commercially available, highly controlled, disposable technologies.

Alternative transplant donor sources

To review the data supporting the use of alternative donors for hematopoietic cell transplantation of patients with high-risk or advanced hematological malignancies. Advances in supportive therapy and technology have improved the safety and efficacy of alternative donors for hematopoietic cell transplantation. Molecular techniques have allowed for better human leukocyte antigen matching of unrelated adult donors. Novel strategies such as adoptive regulatory T cells or posttransplantation cyclophosphamide contributed to better outcomes after partially matched related donors. In umbilical cord blood transplantation, the ability to find adequately dosed single-unit grafts, the utilization of double-unit grafts, and novel methodologies such as ex-vivo expansion, intrabone injection, and priming to accelerate engraftment are promising. Available retrospective studies suggest despite the differences in hematopoietic recovery, risk of graft-versus-host disease, and relapse, long-term outcomes are similar between different alternative donor types. In the absence of a suitable matched related donor, most patients will be able to find an alternative donor to proceed to a potentially curative allogeneic transplantation. Emerging new technologies will further improve the safety and efficacy of alternative donor transplantation. Ongoing and future randomized studies will better define the relative efficacy of alternative donor types.

Optimization of an Intra-Bone Hematopoietic Stem Cell Delivery Technique in a Swine Model.

AbstractAbstract 2990 IntroductionTransplantation of allogeneic hematopoietic progenitor cells (HPC) is an effective treatment for a variety of hematological diseases. Intravenous (IV) injection is the routine method for HPC transplantation, based on the concept of “homing” to the bone marrow. To date, intrabone (IB) HPC transplantation has largely been investigated in the preclinical setting with only limited application in humans. Furthermore, an optimized method for the direct IB infusion of HPC in humans which maximizes cellular retention in the bone has not yet been developed. In this study, we aimed to optimize the IB transplantation procedure using a large animal model with the goal of retaining HPCs within the pelvic bone and to explore the feasibility of radionuclide labeling to assess the trafficking of HPCs using PET/CT imaging early after IB transplantation. MethodsHPC collection: a) Human HPCswere mobilized from healthy volunteers using G-CSF then were positively selected for CD34+ cells using immuno-magnetic beads (MiltenyiBiotec, MA) then were cryopreserved. b) Porcine bone marrow (BM) cells were aspirated (approximately 40 ml) from the iliac crest of swine then were filtered and mononuclear cells (MNCs) were isolated using Ficoll-Paque™ with density gradient separation. All animal procedures were conducted using domestic swine (Susscrofadomesticus) on NHLBI Animal Use Committee approved protocols. IB access in animals was initially achieved using the OnControl driver (Vidacare Corp. TX). To evaluate flow through the marrow and venous drainage, direct IB injection into the hemipelvis with iopamidol-370 contrast was performed under anesthesia with dynamic CT images acquired using a 320-detector row scanner (Aquilion One, Toshiba Medical, Japan). Human CD34+ and swine BM MNCs were labeled with Zirconium-89 (89Zr) then were assessed for viability, cell number, and the level of cellular radioactivity. Radiolabeled cells were then injected into pigs either IV or directly IB into the porcine pelvis at different infusion rates. Intramarrow(IM) pressures were measured continuously during IB injection using Millarcatheters and acquired simultaneously with intra-arterial pressure and electrocardiography on a PowerLab data acquisition system (ADInstruments, CO) and analyzed using LabChart 7. After injection of labeled cells, positron emission tomography (PET) images were acquired for up to 180 minutes with a clinical PET/CT system (Gemini TF, Philips Medical Systems, MA) to assess cellular distribution and homing. ResultsPeak IM pressures during bolus hand IB injection were high, substantially exceeding systemic systolic arterial pressures. In contrast, IM pressures during slow IB infusion were significantly lower, remaining well below diastolic arterial pressures. During manual sequential hand IB injection of 5 ml aliquots of contrast at two different sites in the ipsilateral iliac crest, dynamic CT images revealed leakage from the initial access site after the first injection as well as immediate drainage into the ipsilateral iliac vein. Following manual hand injection of 89Zr labeled human CD34+ cells (89Zr-hCD34+) given IV in swine via the external jugular vein, there was persistent PET activity noted in the lungs for up to 3 hrs. Bolus hand IB injection of 89Zr labeled swine BM MNCs or 89Zr-hCD34+ cells revealed PET activity in the iliac bone as well as activity in the lungs. Furthermore, PET activity following bolus hand IB injection was also noted in surrounding tissues outside the bone when more than a single ipsilateral injection site was used. In contrast, slow infusion of 89Zr labeled swine BMMNCs or 89Zr-hCD34+cells resulted in PET activity that was limited to the iliac bone, indicating retention of cells within the marrow space with no leakage of cells to the lungs. ConclusionRapid hand infusion of HPCs into the pelvic bone results in cellular leakage out of the marrow space into the lungs. In contrast, slow IB infusion of HPCs localizes cells to the bone marrow without leakage to the lungs. These data suggest maintaining low IM pressures may be critical to maximize cellular trapping in the marrow space following IB HPC transplantation in humans. Further study will be required to determine whether this optimized IB transplantation approach can be used to improve engraftment in recipients of transplants containing low HPC numbers. Disclosures:No relevant conflicts of interest to declare.

Reduced HMOX1 Expression in Myelomatous Bones Is Associated with Poor Survival and Its Induction Regulates Osteoclastogenesis and Osteoblastogenesis

Abstract 1815The bone marrow (BM) microenvironment plays critical role in the progression of multiple myeloma (MM) and identification of dysregulated microenvironmental pathways is essential for development of effective interventions for this disease. We and others have shown that bone disease is both a consequence of and a necessity for MM progression and that restoring bone turnover helps control MM. Prooxidant environment has been shown to promote tumorigenesis and bone disease, while circulating levels of antioxidants are reduced in MM patients (Sharma et al., 2009). Heme oxygenase 1 (HMOX1) is an intracellular inducible antioxidant that acts in response to oxidative stress and mediates anti-inflammatory and wound healing processes. The aim of the study was to identify dysregulated microenvironmental factors in myelomatous bone using mesenchymal stem cell (MSC) cytotherapy as an approach previously shown by us to induce bone formation and inhibit osteolysis and MM progression (Li et al., 2011; 2012). Intrabone injection of fetal MSCs into myelomatous bones of SCID-hu mice engrafted with a patient’s MM cells resulted in significant upregulation(≥2 folds) of 120 probe sets and downregulation of 44 probe sets 24 hours after MSC cytotherapy, assessed by global gene expression profile (GEP, 9 mice/group). HMOX1, one of top overexpressed genes by MSC cytotherapy (5.2 folds, p<0.009), was further found to be underexpressed in myelomatous versus non-myelomatous bones from SCID-hu mice using GEP (p<0.04), and immunohistochemistry. HMOX1 was also induced in myelomatous bone following cytotherapy with healthy donors MSCs (n=3, p<0.04) and was consistently upregulated by MSCs cytotherapy in bones engrafted with myeloma cells from 4 patients (p<0.0001) using qRT-PCR. GEP of clinical biopsies revealed lower HMOX1 expression in newly diagnosed MM patients (n=352) than healthy donors (n=20, p<0.001) and normal levels in biopsies from patients at variable time during remission (n=98, p<0.0001 vs. active MM). In our Total Therapy 2 (TT2) and 3 (TT3) patients, using optimal HMOX1 signal cut-point based on statistical significance value, lower HMOX1 expression in biopsies was associated with poor survival in each of the trials; the 5-years estimated overall survival was 62% vs. 85% in TT2 (p<0.0001) and 57% vs. 81% in TT3 (p<0.002) patients with low and high HMOX1 expression, respectively. Survival analysis based on HMOX1 expression in purified plasma cells revealed similar significant trend in TT2 (p<0.003) but not TT3 (p<0.056) patients. Induction of HMOX1 in osteoclast precursors through noncontact co-culture with MSC or with the HMOX1 inducer, hemin, resulted in downregulation of RANK and NFATC1 and inhibition of osteoclast formation demonstrated by reduced expression of osteoclast markers cathepsin K, vitronectin receptor and acid phosphates 5, and by fewer numbers of TRAP-expressing multinucleated osteoclasts (p<0.0001). In the human hFOB preosteoblastic cell line, HMOX1 expression was increased during osteoblast differentiation and treatment with hemin or MSC conditioned media further stimulated mineral deposition in these cells. These data highlight the involvement of HMOX1 in controlling osteoclastogenesis and osteoblastogenesis. We conclude that HMOX1 is suppressed in experimental and clinical myelomatous bone and that higher expression is associated with superior outcome. Approaches to induce HMOX1 expression may help control bone disease and MM progression. Disclosures:No relevant conflicts of interest to declare.

Advances in unrelated cord blood transplants in malignancies.

Cord blood is an unlimited source of hematopoietic stem cells (HSCs) for allogeneic HSC transplant. Since the first human cord blood transplant performed 20 years ago, cord blood banks have been established worldwide for collection and cryopreservation of cord blood for allogeneic HSC transplant. More than 500 000 cord blood units are now available for international exchange of cord blood units. A global network of cord blood banks and transplant centers has been established for a common inventory and study of clinical outcomes. Results of unrelated allogeneic cord blood transplants in malignant and nonmalignant diseases, in adults and children, show that, compared with HLA-matched unrelated bone marrow transplant, cord blood has several advantages including prompt availability of the transplant, decrease of graft versus host disease and better long-term immune recovery resulting in a similar long-term survival. Several studies have shown that the number of cells is the most important factor for engraftment while some degree of HLA mismatches is acceptable. Progresses in this field are expected to facilitate engraftment including ex vivo expansion of stem cells, intra-bone injection of cord blood cells and double cord blood transplants.

Engraftment defect of cytokine‐cultured adult human mobilized CD34+ cells is related to reduced adhesion to bone marrow niche elements

In vitro exposure of haematopoietic stem and progenitor cells (HSPC) to cytokines in expansion or gene therapy protocols reduces homing and engraftment in vivo. We have previously reported that this is related in part to altered tissue specificity of short-term homing, leading to loss of cells in non-haematopoietic tissues. Here we demonstrate that defective engraftment persists when cultured HSPC are transplanted by intrabone injection. Changes in engraftment function occur within 24h of cytokine exposure, and are evident when engraftment is analysed solely in the injected bone. A novel ex vivo model of the bone marrow was developed, in which the attachment of infused HSPC in rodent long bones is reduced following culture with cytokines. Finally, cultured HSPC demonstrated reduced adhesion to N-cadherin, osteopontin and vascular cell-adhesion molecule-1, ligands present in bone marrow niches. These changes in adhesive function occur rapidly, and are not related to downregulation of the relevant receptors. Our findings suggest that cytokine exposure of adult human HSPC results in altered adhesion within bone marrow niches, further leading to reduced engraftment potential in vivo.

Umbilical cord stem cell transplantation – two decades of clinical experience

Umbilical cord blood (UCB) is an abundant source of haematopoietic stem cells (HSC) for allogeneic HSC transplant. Since the first UCB transplant in 1988, many scientific data have been collected. Many umbilical cord blood banks have been established worldwide for the collection and cryopreservation as well as for the international exchange with more than 400,000 UC units available. There is probably a twofold figure of UCB units collected for the private UCB banks. More than 20,000 UCB units have been used for treating malignant and non-malignant diseases both in adults and children. Unrelated UCB HSCs became an alternative to bone-marrow derived HSCs based on their advantages, including a prompt availability, decrease of graftversus- host disease (GVHD) and better longterm immune recovery, resulting in a similar long-term survival. UCB stem cells can be transplanted to an allogeneic patient in spite of the high degree of HLA mismatches, which is highly important for the patients who cannot find a HLA-identical allogeneic donor. Since the low number of cells in the UCB units still represents the main cause of failure of engraftment, new strategies, such as double-unit UCB transplantation, intra-bone injection of UCB cells, simultaneous transplantation of accessory cells and the reduced intensity conditioning regimen, were developed to increase the success of UCB transplantation. Several clinical studies using UCB stem cells that have been previously expanded in vitro are underway. These procedures and the future development of regenerative medicine will further broaden possible indications for the use of UCB derived stem cells in adults.

Therapeutic effects of intrabone and systemic mesenchymal stem cell cytotherapy on myeloma bone disease and tumor growth

The cytotherapeutic potential of mesenchymal stem cells (MSCs) has been evaluated in various disorders including those involving inflammation, autoimmunity, bone regeneration, and cancer. Multiple myeloma (MM) is a systemic malignancy associated with induction of osteolytic lesions that often are not repaired even after prolonged remission. The aims of this study were to evaluate the effects of intrabone and systemic injections of MSCs on MM bone disease, tumor growth, and tumor regrowth in the severe combined immunodeficiency (SCID)-rab model and to shed light on the exact localization of systemically injected MSCs. Intrabone injection of MSCs, but not hematopoietic stem cells, into myelomatous bones prevented MM-induced bone disease, promoted bone formation, and inhibited MM growth. After remission was induced with melphalan treatment, intrabone-injected MSCs promoted bone formation and delayed myeloma cell regrowth in bone. Most intrabone or systemically injected MSCs were undetected 2 to 4 weeks after injection. The bone-building effects of MSCs were mediated through activation of endogenous osteoblasts and suppression of osteoclast activity. Although a single intravenous injection of MSCs had no effect on MM, sequential weekly intravenous injections of MSCs prevented MM-induced bone disease but had no effect on tumor burden. MSCs expressed high levels of anti-inflammatory (eg, HMOX1) and bone-remodeling (eg, Decorin, CYR61) mediators. In vitro, MSCs promoted osteoblast maturation and suppressed osteoclast formation, and these effects were partially prevented by blocking decorin. A subset of intravenously or intracardially injected MSCs trafficked to myelomatous bone in SCID-rab mice. Although the majority of intravenously injected MSCs were trapped in lungs, intracardially injected MSCs were mainly localized in draining mesenteric lymph nodes. This study shows that exogenous MSCs act as bystander cells to inhibit MM-induced bone disease and tumor growth and that systemically injected MSCs are attracted to bone by myeloma cells or conditions induced by MM and inhibit bone disease.