Umbilical Cord Blood Units Research Articles

Umbilical cord blood mesenchymal stem cells application in hematopoietic stem cells expansion on nanofiber three-dimensional scaffold.

Umbilical cord blood (UCB) hematopoietic stem cells (HSCs) transplantation (HSCTs) is considered as a therapeutic strategy for malignant and nonmalignant hematologic disorders. Nevertheless, the low number of HSCs obtained from each unit of UCB can be a major challenge for using these cells in adults. In addition, UCB is a rich source of mesenchymal stem cells (MSCs) creating hopes for nonaggressive and painless treatment in tissue engineering compared with bone marrow MSCs. This study was designed to evaluate the effects of UCB-MSCs application in UCB-HSCs expansion on the nanoscaffold that mimics the cell's natural niche. To achieve this goal, after flow cytometry confirmation of isolated HSCs from UCB, they were expanded on three-dimensional (3D) poly-l-lactic acid (PLLA) scaffolds fabricated by electrospinning and two-dimensional (2D)-culture systems, such as (1) HSCs-MSCs culturing on the scaffold, (2) HSCs culturing on the scaffold, (3) HSCs-MSCs culturing on 2D, and (4) HSCs culturing on 2D. After 7 days, real-time polymerase chain reaction (PCR) was performed to evaluate the CXCR4 gene expression in the mentioned groups. Moreover, for the next validation, the number of total HSCs, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay, scanning electron microscopy imaging, and colony-forming unit assay were evaluated as well. The results of the study indicated that UCB-MSCs interaction with HSCs in 3D-culture systems led to the highest expansion of UCB-HSCs on day 7. Flow cytometry results showed the highest purity of HSCs cocultured with MSCs. Real-time PCR showed a significant increase in gene expression of CXCR4 in the mentioned group. The highest viability and clonogenicity were detected in the mentioned group too. Considered together, our results suggest that UCB-HSCs and MSCs coculturing on PLLA scaffold could provide a proper microenvironment that efficiently promotes UCB-HSCs expansion and UCB-MSCs can also be considered as a promising candidate for UCB-HSCTs.

Use of Unlicensed Unrelated Umbilical Cord Blood Expands Access to Underserved Patients: Report of 2466 Transplants in a Racially/Ethnically Diverse Population

Background Umbilical Cord Blood (UCB) transplant (UCBT) is a curative procedure for patients with hematologic malignancies and genetic disorders, and expands transplant options for a racially/ethnically diverse population, who are often unable to find a fully matched unrelated donor. In 2011, the Food and Drug Administration (FDA) required that all unrelated UCBT use either licensed UCB or unlicensed UCB via an Investigational New Drug (IND). Methods To allow continued access to unlicensed UCB units, the National Marrow Donor Program® (NMDP) manages a prospective distribution protocol for unlicensed UCB units, under an IND, 10-Cord Blood Access. This is an interim report of outcomes of 2466 US patients receiving unlicensed UCB units from 2011-2016. Of note, only 6% of available UCB units were licensed by December 2016. Results 1509 adults and 957 children (564 malignant disease and 393 non-malignant disease) received single or double UCBT between October 2011 and December 2016. Median age was 31 years ( Conclusions Use of unlicensed UCB units is safe and effective, and provides an important graft source for a diverse population.

Safety and Efficacy of Donor Leukocyte Infusion for Single Unit Umbilical Cord Blood Transplantation to Accelerate Immune Reconstitution Prior to Thymopoiesis

Success of reduced intensity umbilical cord blood transplantation (UCBT) in chemotherapy naive patients is limited by an increased risk of mixed chimerism, infection, and delayed immune reconstitution (IR). In a prospective clinical trial, we utilized risk-adapted alemtuzumab dosing, tested the safety of donor leukocyte infusion (cDLI) from a fraction of the thawed graft and evaluated it's efficacy in impacting IR, viral infection, and/or donor chimerism. Patients with inborn errors of metabolism, immunity, or hematopoiesis underwent a single unit UCBT following a reduced-intensity conditioning regimen of alemtuzumab, hydroxyurea, fludarabine, melphalan, and thiotepa (ClinicalTrials.gov: NCT00744692). Up to 5% of the graft was re-cryopreserved for potential cDLI and administered in cases of declining donor Tcell chimerism, viral infection, or delayed IR. Alemtuzumab levels were obtained on the day of UCBT. Chimerism and IR were measured at standard time points. 33 patients had cDLI stored and 18 patients received cDLI at a median of 65 days post-UCBT. Indications included mixed whole blood or CD3 chimerism, viral infection, or delayed IR. All patients engrafted with neutrophils at a median of 15 days post-UCBT, however, one patient experienced secondary graft failure at day +38, despite cDLI 28 days post-UCBT. Improvement in chimerism was seen in 5 of 9 patients, viral load and/or symptoms of infection in 5 of 9 patients and absolute CD3 count in 5 of 5 patients. 15 patients developed acute grade I-III GVHD at a median of 69 days post-UCBT. In those who received cDLI, 8 developed grade I-II skin GVHD at a median of 19 days post-cDLI. 2 patients with a history of GVHD received subsequent cDLI without GVHD flare. No patients who received cDLI developed grade III or IV acute or extensive chronic GVHD, while 4 patients who did not receive cDLI developed grade III GVHD affecting skin and/or the gastrointestinal tract. There was no difference in the cumulative incidence of acute GVHD between those who received cDLI and those who did not (44% vs. 33%; p=0.81). The 3-year overall and event-free survival of all patients was 94% and 91% respectively, with treatment-related mortality (TRM) of 5% 1 year post-UCBT. We report outcomes on the largest prospective trial utilizing cord blood grafts for nonmalignant disease. cDLI is safe in patients undergoing single-unit UCBT as all engrafted. Greater than half of the patients had improvement in the primary indication for its use, without increased GVHD risk. Greatest benefit was seen in patients with delayed IR and TRM at 1 year was exceptionally low. In sum, cDLI may be a simple and effective way to address mixed chimerism, infection, and accelerate IR during the vulnerable time prior to the onset of thymopoiesis. Further characterization of the merits of cDLI should be explored in a larger clinical trial.

Adaptive NK cell reconstitution is associated with better clinical outcomes.

Human cytomegalovirus (CMV) reactivation is a common occurrence early after transplant and is associated with heterogeneous NK cell subset expansion. These adaptive NK cell expansions are highly variable between recipients, with respect to magnitude and relative frequencies of adaptive NK cell subsets. To gain insight into the factors that influence adaptive NK cell expansion from a CMV naive graft source, we performed a high-resolution NK cell and CD8+ T cell phenotypic analysis of 215 patients with hematological malignancies that were transplanted with 2 partially HLA matched CMV negative umbilical cord blood units. We found that adaptive NK cells were significantly higher in recipients who received nonmyeloablative conditioning (NMAC) relative to myeloablative conditioning (MAC), and high CMV neutralizing antibody titers correlated with the degree of adaptive NK cell expansion. The frequencies of adaptive NK cell subsets (defined by NKG2C, FcεRγ, EAT-2, and SYK expression) that reconstitute from donor hematopoietic progenitor cells largely matched the frequencies observed in the NK cell compartment of the recipient prior to conditioning, suggesting that host - as well as viral reactivation factors - may determine the phenotypic diversification after transplant. Additionally, multivariable analyses show that higher adaptive NK cell expansion associated with better disease-free survival. Our findings provide important insights into adaptive NK cell reconstitution after transplant and support a role for adaptive NK cells in promoting better clinical outcomes. The NIH and the National Marrow Donor Program.

Umbilical cord blood quality and quantity: Collection up to transplantation.

Umbilical cord blood (UCB) is an attractive source of hematopoietic stem cells for transplantation in some blood disorders. One of the major factors that influence on transplantation fate is cord blood (CB) cell count, in addition to human leukocyte antigen similarity and CD34+ cell number. Here, we review the factors that could effect on quality and quantity of CBUs. Relevant English-language literatures were searched and retrieved from PubMed using the terms: CB, quality, collection, and transplantation. The numbers of total nucleated cells (TNCs) and CD34+ cells are good indicators of CB quality because they have been associated with engraftment; thereby, whatever the TNCs in a CB unit (CBU) are higher, more likely they led to successful engraftment. Many factors influence the quantity and quality of UCB units that collect after delivery. Some parameters are not in our hands, such as maternal and infant factors, and hence, we cannot change these. However, some other factors are in our authority, such as mode of collection, type and amount of anticoagulant, and time and temperature during collection to postthaw CBUs and freeze-and-thaw procedures. By optimizing the CB collection, we can improve the quantity and quality of UCB for storage and increase the likelihood of its use for transplantation.

Positive Correlation between Very Small Embryonic Stem Cell, Hematopoietic Stem Cell, and Endothelial Progenitor Cell in Umbilical Cord Blood Unit

BACKGROUND: Umbilical Cord Blood (UCB) has been widely use as regenerative medicine due to the content of undifferentiated cell which have capability to do self-renewal and differentiation into various type of cell called stem cells. Recent studies show that UCB contains not only hematopoietic stem cell (HSC) but also others stem cell and progenitor cell such as endothelial progenitor cell (EPC) and very small embryonic-like stem cell (VSEL). It is beliefs that HSC and EPC shared the same progenitor. In this study, correlation between the cell number of HSC, EPC and VSEL is analyzed in umbilical cord blood as the source of stem cell for clinical application.METHODS: The cell number of HSC, EPC and VSEL is counted from cryopreserved UCB collected from 22 women delivered via cesarean section which already stored for more than 2 years in this study. Sample were incubated with antibodies such as cluster of differentiation (CD)34-phycoerythrin (PE)/CD45-fluorescein isothiocyanate (FITC), CD133/1 (anti-CD (AC)133)-antigen-presenting cell (APC) and, CD184 (C-X-C chemokine receptor (CXCR)4)-PE.Vio770 to detect the present of HSC, EPC and VSEL in UCB. Sample were analyze using flowcytometer BD FACS Canto II.RESULTS: The cell population of HSC and late-EPC is 0.009% and 0.01% of total cell in UCB. VSEL only represented 0.001% from total cell in UCB, showing the lowest number of cell population in UCB. The correlation between the cell number of HSC and EPC is r=0,483*, p=0.023) and between HSC and VSEL is r=0.510*, p=0.015.CONCLUSION: In this study, both EPC and VSEL have a significant positive correlation with HSC.KEYWORDS: stem cell, umbilical cord blood, endothelial progenitor, flowcytometry

Nucleated red blood cells as a novel indicator of CD34+ cell content in umbilical cord blood

Umbilical cord blood (UCB) has become an important source of transplantable CD34+ hematopoietic progenitor cells. Cord blood banks (CBBs) can increase their efficiency by minimizing the processing of UCB units with low CD34+ content, which have a lower likelihood of transplant utilization. We sought to identify a readily available preprocessing metric that would correlate with CD34+ cell counts, without the cost of additional analysis. Data were compiled for 131 UCB units processed at the regional CBB. Preprocessing hematologic metrics, including complete blood count and differential, were compared to postprocessing CD34+ cell quantities. The data were divided into six groups of varying preprocessing metrics, then compared for significant differences in postprocessing CD34+ cell quantities to develop a screening guidance. UCB units with nucleated RBC (nRBC) content of 15% or greater were found to have a significant increase in CD34+ cell percentage (p < 0.00001) and total CD34+ cell content (p < 0.0001). Units with preprocessing total nucleated cell count (TNC) of ≥ 1.50 × 109 with nRBC content of 15% or greater, and for TNC ≥ of 2.00 × 109 with nRBC content less than15%, had a significant increase in CD34+ content (p < 0.05 and p < 0.001, respectively). Applied as a screening guideline, these units had an increase in mean CD34+ content from 6.24 × 106 to 9.27 × 106 . Units originally in the bottom and top quartiles of CD34+ content constitute 5% and 53% of processed units meeting these TNC/nRBC criteria, respectively. These screening criteria utilizing nRBC provides a guideline that public CBBs may use to increase their efficiency by minimizing the processing of UCB units with lower CD34+ cell content.

Phase I/II Study of Stem-Cell Transplantation Using a Single Cord Blood Unit Expanded Ex Vivo With Nicotinamide

PurposeIncreasing the number of hematopoietic stem and progenitor cells within an umbilical cord blood (UCB) graft shortens the time to hematopoietic recovery after UCB transplantation. In this study, we assessed the safety and efficacy of a UCB graft that was expanded ex vivo in the presence of nicotinamide and transplanted after myeloablative conditioning as a stand-alone hematopoietic stem-cell graft.MethodsThirty-six patients with hematologic malignancies underwent transplantation at 11 sites.ResultsThe cumulative incidence of neutrophil engraftment at day 42 was 94%. Two patients experienced secondary graft failure attributable to viral infections. Hematopoietic recovery was compared with that observed in recipients of standard UCB transplantation as reported to the Center for International Blood and Marrow Transplant Research (n = 146). The median time to neutrophil recovery was 11.5 days (95% CI, 9 to 14 days) for recipients of nicotinamide-expanded UCB and 21 days (95% CI, 20 to 23 days) for the comparator (P < .001). The median time to platelet recovery was 34 days (95% CI, 32 to 42 days) and 46 days (95% CI, 42 to 50 days) for the expanded and the comparator cohorts, respectively (P < .001). The cumulative incidence of grade 2 to 4 acute graft-versus-host disease (GVHD) at day 100 was 44%, and grade 3 and 4 acute GVHD at day 100 was 11%. The cumulative incidence at 2 years of all chronic GVHD was 40%, and moderate/severe chronic GVHD was 10%. The 2-year cumulative incidences of nonrelapse mortality and relapse were 24% and 33%, respectively. The 2-year probabilities of overall and disease-free survival were 51% and 43%, respectively.ConclusionUCB expanded ex vivo with nicotinamide shortens median neutrophil recovery by 9.5 days (95% CI, 7 to 12 days) and median platelet recovery by 12 days (95% CI, 3 to 16.5 days). This trial establishes feasibility, safety, and efficacy of an ex vivo expanded UCB unit as a stand-alone graft.

Intrabone Delivery of CD34+ Cells Using an Optimized Delivery System Does Not Enhance Engraftment in a Rhesus Macaque Model of Hematopoietic Stem Cell Transplantation

Introduction: Umbilical cord blood (UCB) grafts are the only option for a significant minority of patients who require hematopoietic stem cell transplantation (HSCT) but lack a suitable related or unrelated donor. While UCB can serve as a suitable ‘off the shelf’ graft for many patients, units with the best HLA match often contain low and sometimes insufficient numbers of CD34+ cells for use in transplantation, particularly for adult patients. Furthermore, UCB grafts contain lower CD34+ cells number compared to BM or PBSC grafts, which leads to longer engraftment times and a higher risk of graft failure. BM and PBSC grafts are usually injected intravenously, homing to the bone marrow after several hours in the circulation. During this time, CD34+ cells are lost in the lungs, liver and spleen with typically < 20% making it to the bone marrow.1 Investigators have sought to overcome the limitation of low cell dose in UCB grafts and loss of CD34+ cells in the circulation by injecting CD34+ cells directly into the bone marrow space. However, we have recently shown that conventional intrabone delivery methods used in investigational trials to transplant UCB, result in low-level retention of hematopoietic progenitor cells in the intrabone space. Recently, we developed an optimized intrabone (OIB) transplant method using computer controlled low pressure and low volume injection (controlled infusion rate <0.2ml/min, total volume <5ml) under CT guidance. Utilizing porcine and rhesus macaque models, we have shown that OIB delivery of CD34+ cells improves intrabone retention, preventing circulation of CD34+ cells through the lungs, which is observed with conventional non-optimized intrabone methods.2 Here we conducted experiments using an autologous myeloablative transplant model in rhesus macaques comparing engraftment of gene-marked CD34+ cells transplanted intravenously, with cells transplanted using OIB delivery.Methods: Rhesus macaques received GCSF and plerixafor mobilization prior to apheresis. Products were CD34+ selected using Miltenyi beads. CD34+ cells were split equally for transduction with lentiviral vectors encoding the reporters GFP and YFP. After myeloablative conditioning with 10Gy total body irradiation, half the graft was injected directly intra-bone using the OIB method, with the other half of the autograft simultaneously being injected intravenously via slow iv push. Peripheral blood samples were measured daily by flow cytometry to assess the proportion of engrafting cells deriving from each source. To address whether OIB transplantation could allow engraftment of low CD34+ cell numbers, as found in UCB units, the cell doses transplanted in one rhesus macaque recipient were reduced to only 0.5 x 106CD34+ cells/kg.Results: CD34+ cells injected intrabone utilizing the OIB method engrafted in all 3 animals. However, flow cytometric analysis gating on GFP vs YFP positive neutrophils showed CD34+ cells injected utilizing OIB delivery did not engraft quicker than IV transplanted cells. Sequential monitoring of neutrophils over 30 days showed the contribution to hematopoiesis of OIB delivered cells was significantly lower than the cells injected IV (Table 1.)Conclusions: We developed a novel intrabone delivery system that optimizes the retention of CD34+ cells into the bone marrow space. Although autologous CD34+ cells injected using this OIB transplant method were capable of engrafting in rhesus macaques that had undergone myeloablative conditioning, they did not engraft faster and contributed less to hematopoiesis than CD34+ cells simultaneously transplanted using conventional IV infusion. These data raise questions over whether intrabone delivery, even when using techniques to optimize intrabone retention, has utility in improving CD34+ cell engraftment.

Role of RasGRP3 in EPO/EPOR Signaling and Transmigration of Human Hematopoietic CD34+ Cells

Introduction: Umbilical cord blood (UCB) is a salient source of primitive hematopoietic stem progenitor cells (HSPCs) for bone marrow (BM) reconstitution in patients with hematologic and non-hematologic malignancies. However, a relatively low number of HSPCs in UCB units and poor BM homing efficiency greatly hinders the clinical application of UCB CD34+ cells for transplantation. To overcome these hurdles, we developed two independent strategies that increase CD34+ cell numbers and improve BM homing efficiency of UCB HSPCs. First, we expanded UCB HSPCs by culturing them in decellularized Wharton's jelly matrix (DWJM), a biometric scaffold mimicking the 3-dimenstional (3D) microenvironment of BM. Second, we enhanced the in vitro transmigration and in vivo BM homing efficiency of UCB CD34+ cells by blocking EPO/EPOR signaling. Both approaches enhance UCB CD34+ cell migration toward stromal cell-derived factor 1 (SDF1). In this study, we employed RNA-Seq and RT-PCR approaches to analyze UCB HSPCs treated with EPO and co-cultured with DWJM, aiming to identify molecules that regulate UCB HSPC transmigration via EPO/EPOR signaling.Methods: CD34+ cells from highly enriched UCB units (>90% purity) were treated with EPO for 24 hours and separately co-cultured with DWJM for 1 week. UCB CD34+ cells were collected and subjected to RNA-Seq and real-time PCR (RT-PCR) analyses. In vitro transmigration toward SDF-1 was assessed by transwell assay. To assess the involvement of RasGRP3 in UCB CD34+ cell mobility, cells were treated with 100 nM ingenol-3-angelate (I3A), a diacylglycerol (DAG) analog that specifically targets RAS Guanyl Releasing-Protein 3 (RasGRP3), for 16 hours followed by transwell assay. Anti-EPOR antibody-treated or EPO-treated cells were used as controls. In addition, RasGRP3 gene expression was examined in CD34+ cells from peripheral blood (PB) and BM samples collected from the same donor, and compared to RasGRP3 expression in UCB CD34+ cells. Unpaired, 2-tailed t-test was used to analyze results.Results: RasGRP3 was identified by RNA-Seq from the two independent approaches, EPO treatment and DWJM co-culture. EPO downregulated and DWJM upregulated RasGRP3 gene expression in UCB CD34+ cells. RasGRP3 expression was confirmed by qPCR. UCB CD34+ cells that migrated to the bottom chamber of the transwell assays, a population that has a higher mobility, showed an elevated RasGRP3 gene expression and a decreased EPOR cell surface expression. Activation of RasGRP3 by DAG analog I3A induced a significant increase in RasGRP3 gene expression (control: I3A treatment = 1: 202 ± 58, p=0.00012) that was associated with an enhanced transmigration capability (control: I3A = 41%+/-5: 54%+/- 3, p=0.032). Knocking-down of RasGRP3 in K562 cells, a known EPOR expressing cell line, impaired the transmigration capability of K562. CD34+ cells in peripheral blood (PB) showed a higher level of RasGRP3 gene expression compared to CD34+ cells in BM samples from the same healthy donors. RasGRP3 expression in PB CD34+ cells was significantly higher than BM and UCB CD34+ cells (qPCR signals relative to BM, BM: PB: UCB = 1: 431±65: 21±8, p=0.0012, 0.0023, and <0.0001 for BM vs. PB, BM vs. UCB and PB vs. UCB, respectively).Conclusions: By employing transwell assays, flow cytometry and molecular analyses, we demonstrate for the first time that RasGRP3, a protein responsible for GDP/GTP exchange of Ras, regulates the transmigration ability of human CD34+ cells. In addition, our findings connect RasGRP3 expression to the EPOR-mediated signaling pathway in CD34+ cells. A significantly higher level of RasGRP3 expression in PB CD34+ cells than its counterparts in BM might provide an explanation for why PB HSPCs show relatively faster BM engraftment than BM HSPCs during transplantation. Ongoing follow-up studies will elucidate the molecular mechanism(s) underlying EPOR signaling, which holds clinical potential to improve the BM homing deficiency of UCB CD34+ cells via modulating EPOR and RasGRP3 expression (Figure 1). [Display omitted] DisclosuresLiesveld:Onconova: Other: DSMB; Abbvie: Honoraria. Aljitawi:Medpace: Consultancy; The University of Rochester Medical Center: Patents & Royalties: Pending patent related to decellularized Wharton's jelly matrix.

CD34+ Derived Microparticles Regulate Apoptosis in Normal and Acute Myeloid Leukemia Cells

Introduction Microparticles (MPs) are small vesicles 100nm-1μm derived from the apoptotic or stimulated cells. The mechanism of their production is distinctive from exosomes or apoptotic bodies. MPs have been detected in the blood in many pathological conditions associated mainly with endothelial injury, thrombosis and inflammation. Our previous study showed that MPs originated also from CD34+ cells of umbilical cord blood, which is an alternative source for hemopoeitic stem cell transplantation. MPs considered as markers of cell activation, as well as apoptosis. Apoptosis is a complex interaction network regulated either through death receptor like FAS or through the internal pathway of Bcl2, Bax. The two pathways activate the executor of cell death program, caspases. The aim of this study is to elucidate the role of MPs in apoptosis of hemopoietic cells.Methods Umbilical cord blood units (UCB) were collected after informed consent. The units that were used in this study, were rejected as not appropriate for transplantation due to low volume. The HL60 promyelocytic leukemia cell line was cultured in RPMI (Life Technologies) supplemented with 10% fetal calf serum (FCS) and 1% penicillin-strepromycin. CD34+ MPs were isolated from the plasma of UCBs after centrifugation and magnetic bead MACS purification (Miltenyi Biotec). The number of CD34+ MPs was estimated by flow cytometry using CD34-PE and Annexin V-FITC Abs. Mononuclear cells (MNC) were collected after density gradient centrifugation on lymphoprep (Fresenius) and were cultured for 3 and 6 days in the presence of CD34+ MPs. Viability assays were performed using 7-AAD in flow cytometry. In another set of experiments different numbers of CD34+ MPs were used in MNC cultures. RNA was extracted from MNC using Qiagen RNA extraction kit and reverse transcribed into cDNA using Superscript II reverse transcriptase (Invitrogen) with random primers (Promega). RT-PCR was performed using Platinum Pfx polymerase (Invitrogen). The primers for FAS, BCL2, BAX, caspase 3, survivin and GAPDH genes were used. The PCR products were analysed in an agarose gel electrophoresis.Results Cell viability increased in UCB derived MNC (UCB-MNC) incubated with CD34+ MPs (800 /ml) after 3 day vs. 6 days of culture. The UCB-MNC viability was higher using 800/ml CD34+ MPs vs. 400/ml. In contrast, CD34+ MPs (800 /ml vs. 400/ml) did not affect the viability in one day MNC culture. Purified CD34+ MPs were applied to UCB-MNC cultures and by RT-PCR was shown increased expression of BCL2 gene as well as FAS and caspase-3 genes. The promyelocytc cell line HL60 has been used in order to analyze the effect of CD34+ MPs in leukemic cells. The expression of Bcl2 was decreased in HL60 cells co-incubated with CD34+ MPs. This result shows an opposite effect of CD34+ MPs in the apoptotic gene Bcl2 for the HL60 cells indicating that there are different mechanisms of MP function in various cell types.Conclusions In this study we have identified and monitored the time- and dose-dependent effect of CD34-derived microparticles in the viability of UCB mononuclear cells. Additionally, CD34+ MPs function is accosiated with the high expression of the pro- and anti-apoptotic Bcl2 and apoptotic FAS. In contrast CD34+ MPs decreases the expression of Bcl2 in the promyelocytic leukemia cell line HL60. Therefore the stem cell derived microparticles might serve as a potential regulator of apoptosis in normal and malignant hematopoietic cells. DisclosuresNo relevant conflicts of interest to declare.

Haploidentical Stem Cell Transplantation with Post-Transplant Cyclophosphamide As Graft-Versus-Host Disease Prophylaxis in Pediatric Hematologic Malignancies

BackgroundHematopoietic stem cell transplantation (HSCT) is used in pediatric patients with acute leukemia, after a relapse to bone marrow, or in first remission in case of high risk disease. If necessary, 75% of the cases do not have a compatible related donor and it is not always possible to have a compatible unrelated donor or an umbilical cord blood unit. Therefore, using haploidentical alternative donors of hematopoietic stem cells (HSC) is becoming more frequent.ObjectiveThe purpose of this is to report the results of haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide (PTCy) in a group of pediatric patients, which lacks a related HLA compatible donor, at the National Pediatrics Institute in México City, México, in the period between January 2012 thru December 2016.MethodsWe retrospectively reviewed 27 patients´ files <18 years old, who had been diagnosed with acute lymphoblastic leukemia (ALL, eighteen patients), or acute myeloid leukemia (AML, nine patients), and underwent on haploidentical transplantation with post-transplant cyclophosphamide (PTCy). The group of patients consisted of eighteen men and nine women. Age ranged from one to eighteen years (median age eight years). Sixteen patients were in their first hematologic remission, eight in their second, two in their third and one in their fourth hematologic remission, respectively. All donors were ⩾ 4 HLA loci mismatched parents (mother, n = 17; father, n = 8), or siblings, n = 2. Conditioning was based on a nonmyeloablative regimen comprised of fludarabine 50 mg/m2, days -6 to -2, cyclophosphamide 14.5 mg/kg, days -6 and -5, and low-dose total body irradiation 200 cGy at day -1. Graft versus host disease prophylaxis comprised Cy 50 mg/kg intravenous on day 3 and 4 after transplantation, followed by tacrolimus 0.06 mg/kg and mycophenolate mofetil 15 mg/kg, each beginning on day 5. According to the availability of bone marrow harvesting kit, the HSC source was peripheral blood in twenty patients and bone marrow in seven. The median number of CD34+ cells infused was 5.41 × 106/kg (range, 1.27-22.6).ResultsFourteen patients (51.6%) presented complete and sustained chimerism. Overall survival is presented in Figure 1. Graft failure occurred (48.1%) more frequently in the group of patients in whom bone marrow was used as a source of HSC (p=0.26). Nine patients with complete chimera, whose HSC source turned out to be PB, presented acute-GVHD III-IV (p = 0.06). Four of the patients whom presented full engraftment died, three of them due to infectious processes (cytomegalovirus pneumonia, AH1N1 pneumonia, abdominal sepsis secondary to intestinal perforation and E. coli sepsis), before +100-day post-transplantation. All these patients presented acute-GVHD III-IV. The fourth patient who died, also the cause was infectious (pulmonary sepsis secondary to Morganella morganii), thirteen months after the transplant and without history of GVHD. Of the thirteen patients who presented primary graft failure, seven of them are alive and without evidence of tumoral activityDiscussionHaploidentical transplantation with PTCy is a feasible therapeutic option in pediatric patients with malignant hematological diseases who require a HSCT and do not have a matched sibling or unrelated donor available.Conflict-of-interest disclosure: The authors declare they have nothing to disclose.BibliographyKlein OR, Buddenbaum J, Tucker N, et al. Nonmyeloablative Haploidentical Bone Marrow Transplantation with Post-Transplant Cyclophosphamide for Pediatric and Young Adult Patients with High-Risk Hematologic Malignancies. Biol Blood Marrow Transplant. 2017; 23(2): 325-332.González-Llano O, González-López EE, Ramírez-Cázares AC, et al. Haploidentical peripheral blood stem cell transplantation with posttransplant cyclophosphamide in children and adolescents with hematological malignancies. Pediatr Blood Cancer. 2016; 63: 2033-2037.Robinson TM, O'Donnell PV, Fuchs EJ, Luznik L. Haploidentical bone marrow and stem cell transplantation: experience with post-transplantation cyclophosphamide. Semin Hematol. 2016; 53(2): 90-97.Fuchs EJ, Huang XJ, Miller JS. HLA-haploidentical stem cell transplantation for hematologic malignancies. Biol Blood Marrow Transplant. Biol Blood Marrow Transplant. 2010; 16 (1 Suppl): S57-S63. DisclosuresNo relevant conflicts of interest to declare.

KIR Expression on inVitro-Derived Natural Killer Cells Does Not Regulate Killing of Allogeneic Targets

Natural killer (NK) cells play an essential role in early innate killing of virally-infected and tumor targets. NK cell-mediated activity is regulated by a repertoire of activating and inhibitory receptors that recognize ligands on diseased, stressed, or tumor targets. Killer cell immunoglobulin-like receptors (KIRs) are a family of polymorphic receptors that can be inhibitory or activating based on their intracellular signaling motifs. Expression of certain KIR haplotypes plays a key role in survival and relapse prevention for patients with acute myelogenous leukemia (AML) who receive allogeneic hematopoietic cell transplantation. Therefore, KIR haplotypes are an important consideration in selecting allogeneic donors for patients with AML. However, it is unclear if KIRs play a role in adoptive transfer of NK cells that are becoming more routinely utilized to treat refractory AML and other malignancies. To better address this question we used umbilical cord blood to isolate both CD34+ hematopoietic stem cells and CD45+CD56+ NK cells (UCB-56-NK) from the same umbilical cord blood unit. The CD34+ hematopoietic stem cells were then differentiated in vitro into CD56+ NK cells (UCB-34-NK). Despite originating from the same donor and sharing the same genetic background, as well as comparable expression of Fas ligand, TRAIL, NKp46, NKp44, NKG2A, and NKG2D, the UCB-34-NK cells have characteristically low KIR expression, whereas the UCB-56-NK cells have high KIR expression. This phenotype was further confirmed by mass cytometric (CyTOF) analysis of UCB-56-NK cells and UCB-34-NKcells with a panel of 36 phenotypic and functional NK cell markers. This unique system allows us to study the role of KIR expression independent of any other variations in donor or cell characteristics. The cytotoxicity and NK cell activation of UCB-34-NK cells and UCB-56-NK cells are compared to control NK cells isolated from peripheral blood (PB-NK cells) with standard in vitro cytotoxicity assays against neuroblastoma lines with varying HLA genotypes and a control K562 leukemic targets. Our data demonstrates that there is no statistical difference in NK cytotoxicity and activation of UCB-34-NK cells and UCB-56-NK cells across a spectrum of target cell HLA types, despite the differences in KIR expression. For example, at effector to target (E:T) ratios of 1:5 and 1:20 against neuroblastoma line IMR32, UCB-34-NK cells (KIR-low) demonstrated 68.5% and 84.1% maximal Caspase 3/7 activation, compared to 81.3% and 89.6% by UCB-56-NK cells (KIR-high). Additionally, we have used human induced pluripotent stem cells to derive NK cells (iPSC-NK cells) that vary in KIR expression levels. These CD45+CD56+ iPSC-NKs are differentiated from the same well-established iPSC line in the laboratory and therefore again share the same genetic background, and they have similar NK cell surface receptor expression of Fas ligand, TRAIL, NKp46, NKp44, and NKG2D, but differ in levels of KIR expression. Again in vitro cytotoxicity against hematopoietic tumor targets such as K562 and MOLM13 do not demonstrate a significant difference in killing, despite these KIR differences. For example, in targeting erythroleukemia K562 cells, iPSC-NK cells with high levels of KIR expression at E:T ratios of 1:2.5, 1:5, and 1: 10 have Caspase 3/7 activation of 21.1%, 28.2%, and 41.0%, compared to 20.1%, 22.0%, and 31.2% by iPSC-NK cells with low KIR expression. Together, these studies demonstrate that in vitro-derived NK cells do not require KIR expression to become licensed for anti-tumor activity and these cells are able to kill tumor targets whether or not they express KIRs. These studies better enable use of these allogeneic NK cell populations for off-the-shelf NK cell-based therapies without the need to optimize for KIR profiles for patients of differing HLA haplotypes. DisclosuresMalmberg:Fate Therapeutics Inc.: Consultancy, Research Funding. Kaufman:Fate Therapeutics: Consultancy, Research Funding.

Co-Transplantation of Ucbmn with UC-MSC Enhances Engraftment of Ucbmn in Iron Overload NOD/SCID Mice

Background: Umbilical cord blood (UCB)-derived hematopoietic stem cells (HSC) are ideal for allogeneic HSCT when a compatible donor is unavailable. However, the insufficient number of HSCs per UCB unit delays engraftment and may lead to graft failure and/or mortality. Besides, transfusion-induced iron overload has been reported to be involved in the delayed engraftment of HSC. Previously mesenchymal stem cells (MSC) have been implicated in playing a significant role in HSC engraftment. This study aimed to determine the effect of co-transplantation of umbilical cord mesenchymal stem cells (UC-MSC) on engraftment and hematopoietic recovery.Methods: The iron overload NOD/SCID mice were induced by dose-escalation injection of iron dextran. And the sublethally irradiated iron overload NOD/SCID mice were divided into four groups: (1) intravenous injection of normal saline (control group, n=8), (2) intravenous injection of umbilical cord mesenchymal MN) alone (n=10), (3) intravenous injection of UCBMN and UC-MSC (n=10), and (4) intrafemoral injection of UCBMN and UC-MSC (n=12). Six weeks after injection of UC, the bone marrow (BM) was harvested and analyzed. The semi-quantitative analysis of cytokines including SDF-1a, OPN, and VEGF-α were examined by immunohistochemistry.Results: As a result, iron deposits in bone liver, BM and spleen. In the BM of control group, no human CD45+ cells were found. Via the intra-bone marrow (iBM) injection, the percentage of human CD45+ cells was 9.86% ±1.86% (n=10) in the presence and 3.18% ±0.98% (n=6) in the absence of MSC, respectively, via the tail vein, the percentage of human CD45+ cells was 3.5%±0.94%. In additional, the levels of SDF-1a, OPN, and VEGF-a were all significantly higher in The mice that received MSC showed significantly enhanced engraftment in all three experiments (P=0.000).Conclusion: Co-transplantation of UC-MSC is effective for improving engraftment in iron overload NOD/SCID mice. The point underlying the positive outcome may relate to cytokines including SDF-1a, OPN, and VEGF-a secreted by UC-MSC, which has been indicated to play a crucial part in homing and engraftment. DisclosuresNo relevant conflicts of interest to declare.

Donor Leukocyte Infusion for Single Unit Umbilical Cord Blood Transplantation: 5% of Thawed Single Cord Blood Unit Refrozen on Day of Transplant Is Safe with Signals of Efficacy in Improving Donor Derived Immunity

Introduction:Success of umbilical cord blood transplantation (UCBT) is limited by an increased risk of graft rejection, mixed chimerism, infection, and delayed immune reconstitution. Additionally, strategies such as donor lymphocyte infusion (DLI) to improve declining donor chimerism or augment immune reconstitution are extremely limited in UCBT. We hypothesized that removing and storing a portion of the thawed cord blood graft for future use as a cord DLI (cDLI) could be used to improve mixed chimerism or augment immunity without adverse impact on engraftment or graft versus host disease (GVHD).Methods:Patients with inborn errors of metabolism, immunity, or hematopoiesis underwent a single unit UCBT following a reduced-intensity conditioning regimen of alemtuzumab, hydroxyurea, fludarabine, melphalan, and thiotepa (ClinicalTrials.gov: NCT00744692) with a thaw and dilute (no wash) methodology. Up to 5% of the cord blood graft was removed from the thawed graft and re-cryopreserved for potential cDLI. The cDLI was infused at the discretion of the treating physician in discussion with the study PI (P.S.) in cases of mixed or declining donor chimerism, viral infection, or delayed immune reconstitution.Results:A total of 33 patients had cells cryopreserved prior to UCBT for potential cDLI. The characteristics of these patients are shown in Table 1. A total of 18 patients received cDLI at a median of 65 days (range 14-124) post-UCBT. The median cell dose of the cDLI was 8.46e+05 CD3/kg and 1.53e+04 CD34/kg. The indication for cDLI was mixed whole blood or CD3 chimerism (n=9), viral infection (n=9), and delayed immune reconstitution (n=5); 5 patients had more than one indication for cDLI, most commonly infection with delayed immune reconstitution (n=4).All patients engrafted with neutrophils at a median of 15 days post-transplant (range: 10-31 days); however, one patient experienced secondary graft failure at day 38 post-transplant despite cDLI at day 28 post-transplant. Improvement in chimerism was seen in 5 of 9 patients, viral load and/or clinical symptoms of viral infection in 5 of 9 patients (including gastroenteritis due to rotavirus (n=1) or adenovirus (n=1)), and absolute CD3 count in 5 of 5 patients (Figure).A total of 15 patients (45%) developed acute grade I-III GVHD at a median of 69 days (range: 13-95 days) post-UCBT. In the group of patients who received cDLI, 8 developed grade I-II skin GVHD at a median of 19 days (range 4-81 days) post-cDLI. In addition, 2 patients with a history of GVHD received subsequent cDLI without GVHD flare post-cDLI and were analyzed for progression post-cDLI. No patients who received cDLI developed grade III or IV acute GVHD or extensive chronic GVHD, while 4 patients who did not receive cDLI developed grade III GVHD affecting skin and/or the gastrointestinal tract. There was no difference in the cumulative incidence of acute GVHD between those who received cDLI and those who did not (44% and 33%, respectively; p=0.81; Figure). The 3-year overall and event-free survival of the entire group was 94% and 91% respectively (Figure), with treatment-related mortality of 6% at 1 year post-transplant.Conclusions:The use of cDLI is safe in patients undergoing single-unit UCBT. Despite the removal of a small portion of the cord blood graft for cDLI, all patients engrafted with neutrophils and no increased morbidity was seen. In addition, although the sample size is small, over half of the patients who received cDLI had improvement in the primary indication (chimerism, viral infection, or immune reconstitution) for cDLI, with the most benefit seen in patients receiving cDLI for delayed immune reconstitution, as evidenced by both clinical improvement in infectious symptoms and increases in absolute CD3 count. This suggests that cDLI may be an effective way to address mixed chimerism, infection, and particularly, delayed immune reconstitution and should be explored in a larger clinical trial. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Factors influencing the success of cord blood collection: a tertiary perinatal medicine center’s experience

Background/aim: This study aimed to evaluate the effects of certain maternal, fetal, and umbilical cord blood unit factors on storage and/or discard incidence of collected cord blood units from perinatal medicine patients. Materials and methods: A total of 273 cord blood units collected between January 2011 and December 2016 in the Division of Perinatology of Hacettepe University Hospital were evaluated retrospectively in this study. Results: Of the collected cord blood units, 53.8% (147/273) were stored. Infant birth weight, cord blood unit volume, total nucleated cell count, and CD34+ cell count were statistically significantly different between the eligible and discarded cord blood unit groups (P < 0.001 for all). No cord blood units were discarded owing to contamination-related issues. The mean gestational age for pregnant women whose umbilical cord blood was stored was 36.6 ± 1.0 weeks. Conclusion: Infant birth weight, cord blood unit volume, total nucleated cell count, and CD34+ cell count were significantly different between the eligible and discarded cord blood unit groups. The low rate of specimen storage was most likely because of the unique characteristics of perinatal medicine patients. Physicians should choose appropriate donors for cord blood collection to increase the rate of cord blood utilization.

Ex vivo human HSC expansion requires coordination of cellular reprogramming with mitochondrial remodeling and p53 activation

AbstractThe limited number of hematopoietic stem cells (HSCs) in umbilical cord blood (UCB) units restricts their use for stem cell transplantation. Ex vivo treatment of UCB-CD34+ cells with valproic acid (VPA) increases the number of transplantable HSCs. In this study, we demonstrate that HSC expansion is not merely a result of proliferation of the existing stem cells but, rather, a result of a rapid reprogramming of CD34+CD90− cells into CD34+CD90+ cells, which is accompanied by limited numbers of cell divisions. Beyond this phenotypic switch, the treated cells acquire and retain a transcriptomic and mitochondrial profile, reminiscent of primary HSCs. Single and bulk RNA-seq revealed a signature highly enriched for transcripts characteristic of primary HSCs. The acquisition of this HSC signature is linked to mitochondrial remodeling accompanied by a reduced activity and enhanced glycolytic potential. These events act in concert with a modest upregulation of p53 activity to limit the levels of reactive oxygen species (ROS). Inhibition of either glycolysis or p53 activity impairs HSC expansion. This study indicates that a complex interplay of events is required for effective ex vivo expansion of UCB-HSCs.

Outcome of combination of HLA-haploidentical hematopoietic SCT with an unrelated cord blood unit for 127 patients with acquired severe aplastic anemia

目的评价单倍型造血干细胞移植（haplo-HSCT）联合第三方脐血干细胞移植治疗重型再生障碍性贫血（SAA）的疗效及安全性。方法对2011年9月至2017年4月间接受haplo-HSCT联合第三方脐血干细胞移植的127例SAA患者进行回顾性研究。结果全部127例SAA患者中，男74例，女53例，中位年龄23.5（3~54）岁，其中极重型再生障碍性贫血65例。诊断至移植中位时间2（0.5~180）个月。单倍型造血干细胞来源为骨髓+外周血。脐血均选用单份，HLA配型≥4/6相合。127例患者均接受改良Bu/Cy+ATG/ALG预处理方案（白消安+环磷酰胺+抗胸腺细胞球蛋白/抗淋巴细胞球蛋白）。以环孢素A、霉酚酸酯联合短程甲氨蝶呤预防GVHD。回输单倍型供者单个核细胞10.87（3.61~24.00）×108/kg，CD34+细胞3.49（1.02~8.89）×106/kg；回输脐血单个核细胞2.22（1.10~7.30）×107/kg，CD34+细胞0.56（0.16~2.27）×105/kg。127例患者中5例发生早期死亡。在可评估的122例患者中，1例发生原发植入失败，其余121例患者成功植入（均为单倍型造血干细胞植入）。中性粒细胞、血小板植入时间分别为11（9~28）d、15（9~330）d，5例患者发生血小板植入不良。移植过程中74例（58.27%）发生感染。存活患者中位随访20.5（4.0~60.0）个月，Ⅱ~Ⅳ度急性GVHD发生率为24.79%（30/121），中/重度慢性GVHD发生率为14.15%（15/106），预期4年总生存率为（78.5±4.3）%，无失败生存率为（77.4±4.3）%。结论haplo-HSCT联合第三方脐血干细胞移植治疗SAA疗效确切且安全性较好，在无全相合供者情况下可作为有价值的治疗选择。

The number of CD34+CD133+ hematopoietic stem cells residing in umbilical cord blood (UCB) units is not correlated with the numbers of total nucleated cells and CD34+ cells: a possible new indicator for quality evaluation of UCB units.

Umbilical cord blood transplantation (UCBT) is often associated with delayed neutrophil and platelet recovery. Engraftment failure is another major obstacle. Several factors influence these serious complications, including the numbers of total nucleated cells (TNCs) and CD34+ cells which have been used as reliable factors for selecting UCB units for transplantation. However, whether both factors are reliable indices of the hematopoietic stem cell (HSC) activity of UCB units remains unknown. To evaluate the quality of UCB units, we quantified the actual number of transplantable CD34+CD133+ HSCs (tHSCs) residing in UCB units. The number of tHSCs was not correlated with the numbers of TNCs or CD34+ cells. These results strongly suggest that neither factor reflects the numbers of tHSCs residing in UCB units. To validate the significance of the number of tHSCs, further analysis is required to determine whether the number of tHSCs residing in UCB units is useful as a new indicator for the quality assessment of UCB units.

Maternal and neonatal predictive variables for quality of cord blood as a source of stem cells

Objective: This study aimed to identify the maternal and neonatal predictive variables for quality of cord blood as a source of stem cells.Methods: A descriptive design was followed in this study. A purposive sample of 143 pairs; pregnant women assigned for elective cesarean section and their newborns, was enrolled in current study. The subjects were recruited from the Operating Theater of Mansoura University Hospital, Egypt. One hundred forty three umbilical cord blood units were collected in utero from cesarean deliveries. The collected units were assessed for its blood volume and total nucleated cells content. An assessment sheet of 12 items was used as a tool of data collection.Results: The greater blood volume and higher nucleated cells obtained from the primigravida, between 37 to 40 gestation weeks, and those not exposed to cigarette smoke during pregnancy (163.1 ± 24.8 ml and 10.2 ± 2.8 × 106 cells/ml, 169.3 ± 22.6 ml and 11.2 ± 1.9 × 106 cells/ml, 169.8 ± 21.4 ml and 10.0 ± 2.9 × 106 cells/ml respectively). The birth weight ≥ 3,600 g, the first born baby, and the heavier placenta (≥ 550 g) produced significantly greater blood volume and cellular content (161.5 ± 20.6 ml and 9.8 ± 2.4 × 106 cells/ml, 164.6 ± 19.4 ml and 9.6 ±2.7 cells/ml, 164.8 ± 19.6 ml and 10.2 ± 2.6 × 106 cells/ml respectively).Conclusions: Primigravida, delivery between 37 to 40 gestation weeks, and no exposure to cigarette smoke during pregnancy were the maternal variables that were associated with larger blood volume and higher cellular content. Even though, first born baby, birth weight ≥ 3,600 g, and placenta weight ≥ 550 g were the neonatal variables.