Human Bone Marrow Transplantation Research Articles

Expression of the miR-221/222 cluster can safeguards transplanted HSC from loss of lymphoid-myeloid multipotency

A microRNA cluster, miR‑221/222, is expressed in quiescent and activated HSC of mice. The expressions of fos and jun, hence AP-1, and a collection of other immediate early genes (IEG), are up-regulated in miR-221/222-deficient HSC. Social stress and HSC transplantation induces comparable up-regulation of IEGs. Stress, as well as miR-221/222-deficiency leads to decreases in the numbers of quiescent HSC and increases in the number of activated hematopoietic (MPP2-4) progenitors, as well as numbers of progenitors of emergency granulopoiesis. Serial transplantations of miR-221/222-deficient HSC generated HSC in the recipient bone marrow, prematurely lose their lymphoid repopulation capacities – normally a sign of aged HSC. Consequently, enforced expression of miR-221/222 should preserve and enhance lymphoid potency of HSC. These results suggest that the expression of miR-221/222 genes themselves and target genes of miR-221/222 in HSC should improve HSC performance in bone marrow niches and their lymphoid development in the transplanted host. Since the miR-221 and -222 genes are also expressed in human HSC, it is likely, that these new targets should be applicable to the improvement of human bone marrow transplantation.

Allogeneic peripheral blood haematopoietic stem cell transplantation for the treatment of dogs with high-grade B-cell lymphoma.

Autologous peripheral blood haematopoietic stem cell transplantation (HCT) cures 33%-40% of dogs with high-grade B-cell lymphoma. We hypothesized, based on human allogeneic bone marrow transplantation literature, that transplanting dogs using canine donor leukocyte-matched CD34+ cells would lead to fewer relapses and increased cure rates. We retrospectively reviewed medical records of dogs diagnosed with high-grade B-cell lymphoma who received an identical allogeneic HCT. A total of 15 dogs transplanted at four facilities were identified. Five of fifteen dogs relapsed before transplant. The mean number of donor CD34+ cells/kg harvested and infused into recipient dogs was 8.0 × 106 /kg (range: 2.08 × 106 /kg-2.9 × 107 /kg). The median disease-free interval and overall survival of all dogs was 1095 days (range: 9-2920 days) and 1115 days (range: 9-2920 days), respectively. Two of five dogs, not in remission at transplant, died in the hospital. The median disease-free interval and overall survival of the remaining three dogs was 25 days (range: 15-250 days) and 1100 days (range: 66-1902 days), respectively. The median disease-free interval and overall survival of the 10 dogs who had not relapsed was 1235 days (range: 19-2920 days) and 1235 days (range: 19-2920 days), respectively. One dog died soon after discharge of presumed gastric-dilatation-volvulus. Eight of nine remaining dogs lived >4yrs post-alloHCT, leading to a cure rate of 89%. Acute graft versus host disease was seen in three dogs. These results suggest that allogeneic HCT can cure ~50% more dogs than those treated with autologous HCT.

3156 – BONE MARROW TRANSPLANTATION COMPROMISES THE REGENERATIVE CAPACITY OF THE NICHE

In bone marrow transplantation (BMT), the recipient environment is conditioned by myeloablative treatment. In many patients, this is associated with long-term osteoporotic changes. To assess the contributions of recipient conditioning and aging of the niche, we compared multipotent stromal and progenitor cells (MSPCs) from young (young: 3 mo old), middle-aged mice (aged: 13 mo old) with mice 10 months after BMT (BMT; total age of 13 mo). Our experiments show reduced numbers of (CD45/Ter119)- CD31- CD166low/med SCA1+ MSPCs with a strongly reduced CFU-F frequency, suggesting a functional compromise of MSPC in BMT. On the cellular level, BMT MSPCs show an increased ROS production compared to aged and young mice. In addition, BMT MSPC show disorganized F-actin stress fibers with increased activation of the small GTPase CDC42 compared to both young and aged MSPCs. In experiments with patient-derived human BMT MSPC, we found similar deregulation of F-Actin, compared to MSPC from young and aged healthy donors (HD). To prevent damaging functional and cytoskeletal changes after conditioning, we treated a small cohort of control and BMT mice with CASIN, an inhibitor of CDC42, in the first week after transplantation. This resulted in a 1.2-fold higher engraftment without affecting early regeneration. Interestingly, CASIN-treated male BMT mice also show elevated trabecular separation and thickness with a slightly higher bone volume compared to treated controls. Our results show that early CASIN-treatment after BMT increases osteogenesis and reduces osteoporotic changes commonly noted in human patients after BMT. Our findings could help in devising strategies to improve long-term restoration of tissues after transplantation, particularly in aging graft recipients. In bone marrow transplantation (BMT), the recipient environment is conditioned by myeloablative treatment. In many patients, this is associated with long-term osteoporotic changes. To assess the contributions of recipient conditioning and aging of the niche, we compared multipotent stromal and progenitor cells (MSPCs) from young (young: 3 mo old), middle-aged mice (aged: 13 mo old) with mice 10 months after BMT (BMT; total age of 13 mo). Our experiments show reduced numbers of (CD45/Ter119)- CD31- CD166low/med SCA1+ MSPCs with a strongly reduced CFU-F frequency, suggesting a functional compromise of MSPC in BMT. On the cellular level, BMT MSPCs show an increased ROS production compared to aged and young mice. In addition, BMT MSPC show disorganized F-actin stress fibers with increased activation of the small GTPase CDC42 compared to both young and aged MSPCs. In experiments with patient-derived human BMT MSPC, we found similar deregulation of F-Actin, compared to MSPC from young and aged healthy donors (HD). To prevent damaging functional and cytoskeletal changes after conditioning, we treated a small cohort of control and BMT mice with CASIN, an inhibitor of CDC42, in the first week after transplantation. This resulted in a 1.2-fold higher engraftment without affecting early regeneration. Interestingly, CASIN-treated male BMT mice also show elevated trabecular separation and thickness with a slightly higher bone volume compared to treated controls. Our results show that early CASIN-treatment after BMT increases osteogenesis and reduces osteoporotic changes commonly noted in human patients after BMT. Our findings could help in devising strategies to improve long-term restoration of tissues after transplantation, particularly in aging graft recipients.

Chronic active Epstein\u2013Barr virus-associated secondary hemophagocytic lymphohistiocytosis in pregnancy: a case report

BackgroundSecondary hemophagocytic lymphohistiocytosis (sHLH) is a rare and fatal disease characterized by uncontrolled immune cell activation that can lead to a cytokine storm. Unfortunately, this condition can occur even during pregnancy, threatening both maternal and fetal lives.Case presentationA 23-year-old nulliparous woman at 26 weeks of gestation presented with continuous fever, coughing, and sore throat. Upon arrival at our hospital, her temperature was >38°C and laboratory findings indicated cytopenia (neutrophil count, 779/μL; hemoglobin level, 10.2 g/dL; platelet count, 29,000/μL), elevated ferritin level (1,308 ng/mL), and elevated soluble interleukin-2 receptor level (11,200 U/mL). Computed tomography showed marked splenomegaly. Bone marrow examination revealed hemophagocytosis, and blood examination showed a plasma Epstein–Barr virus (EBV) DNA level of 8.9 × 105 copies/μg. The monoclonal proliferation of EBV-infected T cells was confirmed by Southern blotting, and the patient was diagnosed with chronic active EBV-associated sHLH and T-cell lymphoproliferative disease. Immediately after admission, the patient’s condition suddenly deteriorated. She developed shock and disseminated intravascular coagulation, requiring endotracheal intubation along with methylprednisolone pulse and etoposide therapy. Although the patient recovered, she delivered a stillborn baby. After delivery, she was treated with reduced-dose dexamethasone, etoposide, ifosfamide, and carboplatin (DeVIC) and steroid (dexamethasone), methotrexate, ifosfamide, L-asparaginase, and etoposide (SMILE) chemotherapies. Five months after diagnosis, she received human leukocyte antigen-haploidentical allogeneic bone marrow transplantation from her sister. She remains in remission for 5 months from the time of transplantation to the present.ConclusionssHLH, which may cause maternal and fetal death, should be carefully considered in critically ill pregnant women, particularly those presenting with continuous fever and cytopenia.

The E3 Ligase Itch Is a Regulator of Bone Marrow Mesenchymal Stem Cells and the Hematopoietic Stem Cell Niche

Hematopoietic stem cells (HSCs) reside in a complex cellular microenvironment within the bone marrow. Bone marrow mesenchymal stromal/stem cells (BM-MSCs) have emerged as an important cellular component of this hematopoietic stem cell niche. BM-MSCs have been implicated in physiological hematopoiesis as well as in hematological malignancies. The intracellular regulatory network that governs BM-MSC homeostasis and their function within the HSC niche remains poorly understood. Posttranslational modification of proteins through ubiquitylation has been proven an integrative component in the regulation of quiescence, proliferation, self-renewal and differentiation of stem cells. We have previously demonstrated that the E3 ubiquitin ligase Itch is a negative regulator of HSC homeostasis and function (Rathinam et al., Nat.Immunol., 2011). Notably, patients suffering from a rare ITCH deficiency syndrome display morphologic abnormalities with dysmorphic features, suggesting a role for this E3 ligase within the skeletal tissue and mesenchymal lineage cells (Lohr et al., Am.J.Hum.Genet., 2010).In the present study, we identify Itch as a critical regulator of BM-MSCs. Itch-/- mice exhibit an at least two-fold reduction of MSCs (immunophenotypically identified as CD45-TER119-CD31-PDGFRα+Sca-1+CD90+) in the BM. Consistently, Colony Forming Unit-Fibroblasts (CFU-F) assays revealed a remarkable reduction of MSCs in the BM (p=0.0392). Moreover, in-vitro culture studies identified a hyperproliferative phenotype of Itch mutant MSCs. However, further studies revealed that Itch deficiency leads to reduced differentiation of MSCs into osteoblasts, both in-vivo and in-vitro, and therefore resulting in osteopenic bone phenotype. Reciprocal and Serial Bone marrow transplantation studies suggested an alteration of the niche for HSCs in Itch deficient BM. Interestingly, Itch-/- recipients exhibited a five-fold increase in their donor (wild-type) hematopoietic stem and progenitor cell compartment. Molecular and biochemical studies documented an increase of signals mediated by Notch in Itch deficient MSCs and co-localization of Itch and Notch-1 intracellular domain. Of note these findings gain importance, as Notch signaling has been previously implicated in the regulation of MSC differentiation into osteoblasts, as well as in the regulation of the HSC niche.In summary, our studies identified Itch as a positive regulator of BM-MSC homeostasis. Our results suggest that the Itch-Notch axis within the HSC niche may serve as a potential pharmaceutical target, especially in the setting of bone marrow transplantation in humans. Treatment of the transplant recipient with an Itch inhibitor may avoid graft failure or rescue poor hematopoietic recovery. DisclosuresSchünemann:German Academic Exchange Service: Other: scholarship; Boehringer Ingelheim Fonds: Other: travel grant.

A Functional Single Nucleotide Polymorphism in the Aγ Globin Gene Promoter Affects Globin Chain Synthesis

Beta Thalassemia and related hemoglobinopathies (such as sickle cell disease), are usually characterized by high levels of HbF since this compensates for the lack of, or abnormal HbA that is expressed in vivo. Further insight on how the hemoglobin switch is controlled and regulated can become crucial in the treatment of hemoglobin disorders. The present treatment options currently include routine blood transfusions with the occasional complications attributed to iron overload in major organs. Human stem cell therapy and bone marrow transplantation are some other options that are very risky and not without complications. Previous studies have shown that HbF levels differ in adults and that at least three loci independent of the β globin locus are responsible including XMN1, the HBS1L-MYB intergenic interval, and BCL11A. In another study conducted on an extended Maltese family, revealed that KLF1 also controls HbF levels in vivo. This study also suggested that other genes in conjunction with KLF1, play a part in activating the HbF switch upon birth. To date, the complete mechanism of how globin gene switching works is still elusive. A total of 355 DNA samples were tested. The majority of the DNA samples were obtained from an existing collection of normal to borderline HbA2 and HbF cases, while other samples were collected along the course of the study. Data from anonymous and randomized cord blood DNA samples were also used and obtained from the Laboratory of Molecular Genetics, University of Malta. These were tested for the rs368698783 polymorphism under study and were used for reference purposes. The SNP rs368698783 was amplified by PCR and checked by agarose gel electrophoresis using a 1.5% agarose gel. Restriction enzyme fragment length polymorphism analysis was then performed using Mnl1 restriction enzyme. Agarose gel electrophoresis was performed again in order to separate the Mnl1- incubated products according to size and thus determine the frequency of the mutation identified. Some samples were chosen at random and were genotyped by DNA sequencing. Amongst the total study population (~355 individuals), the frequency of the mutant allele was 0.14 and close to 0.25 for the random newborn cord blood DNA analyzed in a previous study. The total study population was divided into 125 healthy adults and 193 into borderline HbF/HbA2 The mean fetal hemoglobin and HbA2 for the normal sub-population were 0.39± 0.034 and 2.68± 0.02 respectively. The mean levels in the borderline population were slightly higher with a mean of 0.6± 0.04 for fetal hemoglobin and 3.7±0.04 for HbA2 The effects of rs368698783 A allele on HbF levels examined in the total population and furthermore in the two sub populations of (i) normal healthy adults and (ii) adults with borderline levels of HbF/HbA2 showed that the mutant A allele of HBG1-rs368698783 exhibited a significantly elevated level of HbF (p < 0.05) compared with the wildtype G allele in each of the cohorts from Malta. Intriguingly no association was determined between this point mutation and HbA2 levels. The data is suggestive that the variant HBG1-rs368698783 could act as a modifier associated with elevated levels of HbF in human adults. The SNP is located within the LYAR binding motif. The LYAR is a transcriptive factor which is said to silence the expression of fetal hemoglobin. thus, the G to A polymorphism reduces the DNA binding affinity increasing HbF levels invivo. Figure Disclosures No relevant conflicts of interest to declare.

3036 – BONE MARROW TRANSPLANTATION COMPROMISES THE REGENERATIVE CAPACITY OF THE BONE MARROW NICHE

Successful tissue regeneration depends on the regenerative potential of the graft and its integration in recipients tissue. In bone marrow transplantation (BMT), age-related factors negatively impact on BMT. To dissect the separate contributions of recipient conditioning and aging for the success of BMT, we compared in vitro behavior and transcriptomes of multipotent stromal cells (MSCs) from young (Y: 3 months old), middle aged mice (A: 13 months old) with mice 10 months after BMT (BMT; total age of 13 months). Our experiments show that although HSC numbers are similar in A, and BMT mice, repopulating activity is significantly reduced in BMT HSC. This is accompanied by a reduction of (CD45/Ter119) - CD31 - CD166 - SCA1 + MSCs in the BM with a strongly reduced CFU-F frequency, indicating a functional compromise of BMT MSC. On the cellular level, BMT MSC show a reduced number of mitochondria with increased ROS production compared to A mice. In addition, BMT MSC show disorganized F-actin stress fibers compared to both Y and A MSC. To understand the mechanisms underlying the compromised function of BMT MSC, we analyzed the transcriptome of uncultured primary MSCs from the BM of Y, A, and BMT mice. These analyses show that in comparison to Y and A MSC, BMT MSC downregulate genes involved in intracellular nutrient transport and mitochondrial clearance, but upregulate metabolic processes, lysosomal genes and calcium-dependent non-canonical Wnt signaling. In experiments to assess similar changes in human BMT MSC, we found that MSC from patients undergoing BMT (2 and 4 weeks after BMT) show a severe dysregulation of F-Actin organization, compared to MSC from young and aged healthy donors (HD). Our results show deregulation of HSCs and MSCs in murine or human recipients undergoing BMT. Our results further suggest underlying defects in mitochondrial clearance and metabolic activation with deregulated F-actin organization. Our findings help to dissect and understand mechanisms of age-related factors hampering BMT and could help in devising strategies to improve long-term restoration of tissues after transplantation in aging graft recipients.

Poly dispersed acid-functionalized single walled carbon nanotubes target activated T and B cells to suppress acute and chronic GVHD in mouse model

Graft versus host disease (GVHD) results from hyper-activation of transplanted lymphocytes against the host antigens. Bone marrow transplantation in humans as well as some cases of blood transfusion and organ transplantation are associated with a strong GVH reaction resulting in GVHD that in many cases may be fatal. We had previously shown that poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) specifically target activated T and B lymphocytes and kill them. In the present study, efficacy of AF-SWCNTs to suppress the GVH reaction was tested in the mouse model. Acute GVHD was induced in mice by administering intravenously 30 or 60 million spleen cells from a parental strain (C57bl/6 mouse, MHC haplotype H-2b) to host (C57bl/6 x Balb/c) F1 mice (MHC haplotype H-2b/d)and waiting for 8–10 days. Chronic GVHD was similarly induced by administration of 30 million parent spleen cells to F1 mice and waiting for a period of 60 days. Our results demonstrate a marked decline in splenomegaly and recovery of spleen T (both CD4 and CD8) and B cells in GVHD mice treated with AF-SWCNTs. AF-SWCNTs treatment also limited T and B cell proliferation by restricting S-phage of cell cycle. Generation of anti-host cytotoxic T cells (CTLs) was also markedly suppressed by AF-SWCNT treatment of acute GVHD mice, and a significant reduction in the generation of anti-host antibodies could also be demonstrated. Taken together, our results suggest that the AF-SWCNTs can be considered as a potential therapeutic agent for treating GVHD.

Hax1 Is Indispensable for Neutrophil Development in Zebrafish

Inherited deficiency of HCLS1-associated protein X1 (HAX1) in human leads to the development of severe congenital neutropenia (CN), which is characterized by impaired neutrophil development. Patients with HAX1 deficiency are prone to life-threatening infections beginning in their first months of life unless treated by recombinant human granulocyte colony-stimulating factor (rhG-CSF) or bone marrow transplantation. But approximately 10% of these patients do not respond to the rhG-CSF therapy. Therefore, there is an urgent need for new tailored therapeutic strategies for the treatment of this disease. However, there is a lack of an animal model that allows high-throughput compound screening for the HAX1 associated neutropenia. In this study, we sought to determine the role of hax1 in zebrafish hematopoiesis. We used two approaches to interfere with the zebrafish hax1. First, we injected antisense morpholino that efficiently blocked the translation of hax1 mRNA. As a second approach, we used the CRISPR-Cas9 technique to introduce mutations in the zebrafish hax1 gene. Both approaches were used in the in wild-type embryos and transgenic (mpo:gfp) reporter line, where the neutrophils are labeled with GFP. We found that hax1 knockdown reduced the number of neutrophils, without affecting the development of HSPCs and monocytes/macrophages. Compared with their wild-type counterparts, hax1 morphants exhibited reduced expression levels of hcls1 and cebpa. Whereas the expression level of cebpb, a marker of emergency granulopoiesis, was upregulated. Next, we examined whether hax1 knockdown impairs the cellular viability because CN patients who harbor HAX1 mutations exhibit increased apoptosis of myeloid progenitors. Although zebrafish hax1 morphants showed increased cell death throughout the embryo, apoptosis was not triggered in the hematopoietic site. Therefore, our results suggested that reduced neutrophil numbers in zebrafish hax1 morphants is due to decreased production of neutrophils rather than increased cell death. To determine whether our zebrafish model is suitable for discovering human drugs, we treated zebrafish hax1 morphants with human G-CSF and rescued the quantitative reduction of neutrophils. Overall, we have successfully established a novel in vivo model for studying the HAX-1 role in the granulopoiesis, which might open new avenues for developing therapeutic strategies for CN. Disclosures No relevant conflicts of interest to declare.

Transmission of Toxoplasma gondii Infection Due to Bone Marrow Transplantation: Validation by an Experimental Model.

Toxoplasmosis is an opportunistic infectious disease and may present a fatal outcome for human bone marrow transplant (BMT) recipients, due to the rapid disease course in immunosuppressed individuals. Several reports about occurrence of toxoplasmosis after BMT have been published in the literature, but this disease has been associated mainly due to reactivation of latent infection rather than primary infection. Even though there are reports of acute toxoplasmosis in recipients who were seronegative for T. gondii, suggesting transmission of infection after BMT, the source of infection in those cases has not been clearly demonstrated, whether it is due to the transplantation procedure by itself or from environmental source. Thus, the present study aimed to observe if it could be possible to demonstrate the parasite‘s ability to infect bone marrow (BM) cells and cause toxoplasmosis, when using an experimental model. Our results showed that 11% of hematopoietic and 7.1% of nonhematopoietic lineages may become infected when using in vitro experiments. Also, in vivo experiments demonstrated that, when C57BL/6 mice were infected with RH-RFP or ME-49-GFP T. gondii strains, the BM cells may be infected at different time points of infection. The parasites were detected by both fluorescent microscopy and qPCR. Also, when those BM samples were collected and used for BMT, the transplanted animals presented high rates of mortality and 87.5% of them became seropositive for T. gondii. Taken together, our results clearly demonstrated that it is possible to acquire primary T. gondii infection from the donor cells after BMT. Therefore, we are emphasizing that, before transplantation, serological screening for T. gondii infection from both donors and recipients, in addition to DNA search for this parasite from donor bone marrow cells, are necessary procedures to avoid the risk of T. gondii infection for immunocompromised patients.

Developmental origin, functional maintenance and genetic rescue of osteoclasts

Osteoclasts are multinucleated giant cells that resorb bone, ensuring development and continuous remodelling of the skeleton and the bone marrow haematopoietic niche. Defective osteoclast activity leads to osteopetrosis and bone marrow failure1–9, whereas excess activity can contribute to bone loss and osteoporosis10. Osteopetrosis can be partially treated by bone marrow transplantation in humans and mice11–18, consistent with a haematopoietic origin of osteoclasts13,16,19 and studies that suggest that they develop by fusion of monocytic precursors derived from haematopoietic stem cells in the presence of CSF1 and RANK ligand1,20. However, the developmental origin and lifespan of osteoclasts, and the mechanisms that ensure maintenance of osteoclast function throughout life in vivo remain largely unexplored. Here we report that osteoclasts that colonize fetal ossification centres originate from embryonic erythro-myeloid progenitors21,22. These erythro-myeloid progenitor-derived osteoclasts are required for normal bone development and tooth eruption. Yet, timely transfusion of haematopoietic-stem-cell-derived monocytic cells in newborn mice is sufficient to rescue bone development in early-onset autosomal recessive osteopetrosis. We also found that the postnatal maintenance of osteoclasts, bone mass and the bone marrow cavity involve iterative fusion of circulating blood monocytic cells with long-lived osteoclast syncytia. As a consequence, parabiosis or transfusion of monocytic cells results in long-term gene transfer in osteoclasts in the absence of haematopoietic-stem-cell chimerism, and can rescue an adult-onset osteopetrotic phenotype caused by cathepsin K deficiency23,24. In sum, our results identify the developmental origin of osteoclasts and a mechanism that controls their maintenance in bones after birth. These data suggest strategies to rescue osteoclast deficiency in osteopetrosis and to modulate osteoclast activity in vivo. Multinucleated osteoclasts required for normal bone development and tooth eruption in the mouse originate from embryonic erythro-myeloid progenitors and are maintained after birth by fusion with circulating monocytes.

Developmental origin, functional maintenance and genetic rescue of osteoclasts.

Osteoclasts are multinucleated giant cells that resorb bone, ensuring development and continuous remodelling of the skeleton and the bone marrow haematopoietic niche. Defective osteoclast activity leads to osteopetrosis and bone marrow failure1-9, whereas excess activity can contribute to bone loss and osteoporosis10. Osteopetrosis can be partially treated by bone marrow transplantation in humans and mice11-18, consistent with a haematopoietic origin of osteoclasts13,16,19 and studies thatsuggest that they develop by fusion of monocytic precursors derived from haematopoietic stem cells in the presence of CSF1 and RANK ligand1,20. However, the developmental origin and lifespan of osteoclasts, and the mechanisms that ensure maintenance of osteoclast function throughout life in vivo remain largely unexplored. Here we report that osteoclasts that colonize fetal ossification centres originate from embryonic erythro-myeloid progenitors21,22. These erythro-myeloid progenitor-derived osteoclasts are required for normal bone development and tooth eruption. Yet, timely transfusion of haematopoietic-stem-cell-derived monocytic cells in newborn mice is sufficient to rescue bone development in early-onset autosomal recessive osteopetrosis. We also found that the postnatal maintenance of osteoclasts, bone mass and the bone marrow cavity involve iterative fusion of circulating blood monocytic cells with long-lived osteoclast syncytia. As a consequence, parabiosis or transfusion of monocytic cells results in long-term gene transfer in osteoclasts in the absence of haematopoietic-stem-cell chimerism, and can rescue an adult-onset osteopetrotic phenotype caused by cathepsin K deficiency23,24. In sum, our results identify the developmental origin of osteoclasts and a mechanism that controls their maintenance in bones after birth. These data suggest strategies to rescue osteoclast deficiency in osteopetrosis and to modulate osteoclast activity in vivo.

PC-1 Genetic and pharmaceutical inactivation of hypoxia-inducible factor 1α reveals a molecular switch from graft vs host diseases to graft vs leukemia effect

PC-1 Genetic and pharmaceutical inactivation of hypoxia-inducible factor 1α reveals a molecular switch from graft vs host diseases to graft vs leukemia effect

Summary of the Third International Workshop on Clinical Tolerance.

The Third International Workshop on Clinical Tolerance was held in Stanford, California, September 8-9, 2017. This is a summary of Workshop presentations of clinical trials designed to withdraw or minimize immunosuppressive (IS) drugs in kidney and liver transplant patients without subsequent evidence of rejection. All clinical protocols had in common the use of donor or recipient cell therapy combined with organ transplantation. Tolerance to HLA matched and mismatched living donor kidney transplants with complete withdrawal of IS drugs without subsequent rejection for up to 14 years of observation was achieved in more than 50 patients enrolled in trials in four medical centers after the establishment of transient or persistent chimerism. Complete IS drug withdrawal without chimerism was reported in a prospective trial of liver transplantation combined with injection of regulatory T cells. IS drug minimization without rejection was reported in recipients of living donor kidney transplants enrolled in the One Study consortium after injection of recipient regulatory T cells, or injection of donor regulatory monocytes or dendritic cells. In conclusion, considerable progress has been made in achieving IS drug withdrawal after cell therapy in recipients of organ transplants.

PGE2 pulsing of murine bone marrow cells reduces migration of daughter monocytes/macrophages in vitro and in vivo

Monocytes/macrophages differentiating from bone marrow (BM) cells pulsed for 2 hours at 37°C with a stabilized derivative of prostaglandin E2, 16,16-dimethyl PGE2 (dmPGE2), migrated less efficiently toward a chemoattractant than monocytes/macrophages differentiated from BM cells pulsed with vehicle. To confirm that the effect on BM cells was long lasting and to replicate human BM transplantation, chimeric mice were established with donor BM cells pulsed for 2 hours with dmPGE2 before injection into marrow-ablated congenic recipient mice. After 12 weeks, when high levels (90%) of engraftment were obtained, regenerated BM-derived monocytes/macrophages differentiating in vitro or in vivo migrated inefficiently toward the chemokines colony-stimulating factor-1 (CSF-1) and chemokine (C-C motif) ligand 2 (CCL2) or thioglycollate, respectively. Our results reveal long-lasting changes to progenitor cells of monocytes/macrophages by a 2-hour dmPGE2 pulse that, in turn, limits the migration of their daughter cells to chemoattractants and inflammatory mediators.

Biological Age and Circulating Progenitor Cell Levels as Predictors Heart Disease Events

Identifying patients with stable coronary artery disease (CAD) at high risk for future heart attacks or death remains a challenge for all cardiologists. The study entitled Telomere Shortening, Regenerative Capacity, and Cardiovascular Outcomes in this issue has shed new light in the field.1 Hammadah et al1 focused on the relationship between circulating CD34+ cell levels, telomere length, and cardiovascular outcomes in subjects with stable CAD. Their study yielded 2 novel findings: (1) 10% shorter telomeres correlated with 5% lower levels of circulating CD34+ cells and (2) both short leukocyte telomere length (LTL) and low CD34+ cell levels independently predict adverse cardiovascular disease (CVD) outcomes (death, myocardial infarction [MI], coronary revascularization, or cerebrovascular events). Measurements of LTL and circulating CD34+ cells may facilitate risk stratification of patients with stable CAD distinct from traditional CVD risk factors. Prior studies reported associations between shortened LTL and CVD,2 and postulated that decreased regenerative capacity may be a primary cause of CVD events. The study by Hammadah et al1 is the first and largest study to test the hypothesis that LTL may be linked to the levels of CD34+ cells as a surrogate for regenerative capacity, and correlate these measures with CVD events. Article, see p 1130 Telomeres prevent chromosome ends from being recognized as a DNA break and activating DNA damage response pathways …

Plasma-derived proteomic biomarkers in human leukocyte antigen-haploidentical or human leukocyte antigen-matched bone marrow transplantation using post-transplantation cyclophosphamide.

Recent studies have suggested that plasma-derived proteins may be potential biomarkers relevant for graft-versus-host disease and/or non-relapse mortality occurring after allogeneic blood or marrow transplantation. However, none of these putative biomarkers have been assessed in patients treated either with human leukocyte antigen-haploidentical blood or marrow transplantation or with post-transplantation cyclophosphamide, which has been repeatedly associated with low rates of severe acute graft-versus-host disease, chronic graft-versus-host disease, and non-relapse mortality. We explored whether seven of these plasma-derived proteins, as measured by enzyme-linked immunosorbent assays, were predictive of clinical outcomes in post-transplantation cyclophosphamide-treated patients using plasma samples collected at serial predetermined timepoints from patients treated on prospective clinical studies of human leukocyte antigen-haploidentical (n=58; clinicaltrials.gov Identifier: 00796562) or human leukocyte antigen-matched-related or -unrelated (n=100; clinicaltrials.gov Identifiers: 00134017 and 00809276) T-cell-replete bone marrow transplantation. Day 30 levels of interleukin-2 receptor α, tumor necrosis factor receptor 1, serum STimulation-2 (IL1RL1 gene product), and regenerating islet-derived 3-α all had high areas under the curve of 0.74–0.97 for predicting non-relapse mortality occurrence by 3 months post-transplant in both the human leukocyte antigen-matched and human leukocyte antigen-haploidentical cohorts. In both cohorts, all four of these proteins were also predictive of subsequent non-relapse mortality occurring by 6, 9, or 12 months post-transplant and were significantly associated with non-relapse mortality in univariable analyses. Furthermore, day 30 elevations of interleukin-2 receptor α were associated with grade II–IV and III–IV acute graft-versus-host disease occurring after day 30 in both cohorts. These data confirm that plasma-derived proteins previously assessed in other transplantation platforms appear to retain prognostic and predictive utility in patients treated with post-transplantation cyclophosphamide.

Identity, proliferation capacity, genomic stability and novel senescence markers of mesenchymal stem cells isolated from low volume of human bone marrow.

Human bone marrow mesenchymal stem cells (hBM-MSCs) hold promise for treating incurable diseases and repairing of damaged tissues. However, hBM-MSCs face the disadvantages of painful invasive isolation and limited cell numbers. In this study we assessed characteristics of MSCs isolated from residual human bone marrow transplantation material and expanded to clinically relevant numbers at passages 3-4 and 6-7. Results indicated that early passage hBM-MSCs are genomically stable and retain identity and high proliferation capacity. Despite the chromosomal stability, the cells became senescent at late passages, paralleling the slower proliferation, altered morphology and immunophenotype. By qRT-PCR array profiling, we revealed 13 genes and 33 miRNAs significantly differentially expressed in late passage cells, among which 8 genes and 30 miRNAs emerged as potential novel biomarkers of hBM-MSC aging. Functional analysis of genes with altered expression showed strong association with biological processes causing cellular senescence. Altogether, this study revives hBM as convenient source for cellular therapy. Potential novel markers provide new details for better understanding the hBM-MSC senescence mechanisms, contributing to basic science, facilitating the development of cellular therapy quality control, and providing new clues for human disease processes since senescence phenotype of the hematological patient hBM-MSCs only very recently has been revealed.

Allogeneic Bone Marrow Transplant in Pediatric Patients in Kuwait

To describe outcomes of human leukocyte antigen-matched sibling bone marrow transplantation (BMT) in 12 patients: 9 with ?- thalassemia major (TM) and 1 each with severe aplastic anemia, myelodysplastic syndrome and congenital dyserythropoietic anemia. The median age at transplantation was 9 years and the median follow-up was 24 months. Among the TM patients, 6 were in Pesaro risk class II and 3 in class III. The median neutrophil recovery and platelet recovery were 19 and 25 days, respectively. None of the patients had primary graft failure. At the last follow-up, 11 out of 12 patients were alive, free of disease and living normal quality of life. Most of the patients (75%) had acute GVHD grade I-II, in the skin. None of the patients had grade IV regimen-related toxicity. Six patients developed Gram-negative while two had Gram-positive bacteremia, one had probable lung aspergillosis and four developed febrile neutropenia without identifiable microbiological growth during the aplastic phase. Cytomegalovirus reactivation occurred in five patients. We lost one patient who developed severe acute graft-versus-host disease (GVHD) affecting the skin, gut and liver, who later progressed to severe chronic GVHD and severe interstitial pneumonitis. In conclusion, allogenic BMT is feasible and safe in pediatric patients with hematological disorders in Kuwait.

Very Small Embryonic-Like Stem Cells: A Potential Developmental Link Between Germinal Lineage and Hematopoiesis in Humans.

It has been suggested that hematopoietic stem/progenitor cells (HSPCs) could become specified from a population of migrating primordial germ cells (PGCs), precursors of gametes, during embryogenesis. Some recent experimental data demonstrated that the cell population that is usually considered to be PGCs, moving toward the gonadal ridges of an embryo, contains a subset of cells coexpressing several germ cell and hematopoietic markers and possessing hematopoietic activity. Experimental data showed that bone morphogenetic protein 4 (BMP4) generates PGCs from mouse bone marrow-derived pluripotent stem cells. Interestingly, functional reproductive hormone receptors have been identified in HSPCs, thus indicating their potential role in reproductive function. Several reports have demonstrated fertility restoration and germ cell generation after bone marrow transplantation in both animal models and humans. A potential link between HSPCs and germinal lineage might be represented by very small embryonic-like stem cells (VSELs), which have been found in adult human bone marrow, peripheral blood, and umbilical cord blood, express a specific pattern of pluripotency, germinal lineage, and hematopoiesis, and are proposed to persist in adult tissues and organs from the embryonic period of life. Stem cell populations, similar to VSELs, expressing several genes related to pluripotency and germinal lineage, especially to PGCs, have been discovered in adult human reproductive organs, ovaries and testicles, and were related to primitive germ cell-like cell development in vitro, thus supporting the idea of VSELs as a potential link between germinal lineage and hematopoiesis.