Umbilical Cord Blood Units Research Articles

HLA diversity in the Argentinian Umbilical Cord Blood Bank: frequencies according to donor\u2019s reported ancestry and geographical distribution

Umbilical cord blood (UCB) is a suitable source for hematopoietic stem cell transplantation. The study of HLA genes by next generation sequencing is commonly used in transplants. Donor/patient HLA matching is often higher within groups of common ancestry, however “Hispanic” is a broad category that fails to represent Argentina’s complex genetic admixture. Our aim is to describe HLA diversity of banked UCB units collected across the country taking into consideration donor’s reported ancestral origins as well as geographic distribution. Our results showed an evenly distribution of units mainly for 2 groups: of European and of Native American descent, each associated to a defined geographic location pattern (Central vs. North regions). We observed differences in allele frequency distributions for some alleles previously described in Amerindian populations: for Class I (A*68:17, A*02:11:01G, A*02:22:01G, B*39:05:01, B*35:21, B*40:04, B*15:04:01G, B*35:04:01, B*51:13:01) and Class II (DRB1*04:11:01, DRB1*04:07:01G/03, DRB1*08:02:01, DRB1*08:07, DRB1*09:01:02G, DRB1*14:02:01, DRB1*16:02:01G). Our database expands the current knowledge of HLA diversity in Argentinian population. Although further studies are necessary to fully comprehend HLA heterogeneity, this report should prove useful to increase the possibility of finding compatible donors for successful allogeneic transplant and to improve recruitment strategies for UCB donors across the country.

Complete Restoration of Motor Function in Acute Cerebral Stroke Treated with Allogeneic Human Umbilical Cord Blood Monocytes: Preliminary Results of a phase I Clinical Trial

Stem cell therapy has been explored for the treatment of cerebral stroke. Several types of stem cells have been investigated to ensure the safety and efficacy in clinical trials. Cryopreserved umbilical cord blood (UCB) mononuclear cells (MNCs) obtained from healthy donors have a more stabilized quality, thereby ensuring a successful therapy. A phase I study was conducted on patients aged 45–80 years who sustained acute ischemic stroke. An UCB unit was obtained from a public cord blood bank based on ABO/Rh blood type, HLA matching score (6/6), and cell dose (total MNC count of 0.5–5 × 107 cells/kg). In addition, to facilitate blood brain barrier penetration of UCB, 4 doses of 100 mL mannitol was administered intravenously after 30 min after UCB transplantation and every 4 h thereafter. The primary outcomes were the number of disease (GVHD) within 100 days after transfusion. The secondary outcomes were changes in the National Institutes of Health Stroke Scale (NIHSS), Barthel index, and Berg Balance Scale scores. A 46-year-old male patient with identical ABO/Rh blood type, HLA matching score of 6/6, and MNC count of 2.63 × 108 cells/kg was enrolled. The patient did not present with serious AEs or GVHD during the 12-month study period. The patient’s NIHSS score decreased from 9 to 1. Moreover, the Berg Balance Scale score increased from 0 to 48 and the Barthel index score from 0 to 90. This preliminary study showed that an adult patient with hemiplegia due to ischemic stroke completely recovered within 12 months after receiving allogeneic UCB therapy.

Isolation and <i> Ex Vivo</i> Expansion of Human Hematopoietic Stem Cells Derived from Umbilical Cord Blood

Umbilical cord blood (UCB) is a current major source of hematopoietic stem cells (HSCs) for cell transplantation therapy. Cell transplantation with HSCs derived from UCB is advantageous over transplantation with HSCs from adult tissues. However, the low number of HSC derived from a single unit of UCB limits its application. Thus, ex vivo expansion is a good option to create more UCB HSCs for clinical application. The strategies for HSC expansion in vitro focus on mimicking the composition and structure of HSC natural niche by enhancing self-renewal and inhibiting lineage differentiation of HSCs. In the past decade, the mechanisms of the interaction between HSC and the natural niche have been deeply investigated. This great progress in basic research has led to advancements in UCB HSC ex vivo expansion. In addition, the biological characteristics of the originally isolated UCB HSCs correlate with outcome of subsequent ex vivo expansion. In this paper, we summarize the late progress achieved in isolation and ex vivo expansion of UCB HSCs. Importantly, we attempt to provide an impact and practicable procedure to expand UCB HSC in vitro from isolation of original HSCs to identification of expanded HSCs.

Evaluation of factors affecting total nucleated cells in umbilical cord blood collected for the Calabria cord blood bank.

Total nucleated cell (TNC) count is the most important biological feature to consider in assessing the quality of umbilical cord blood (UCB) for haematopoietic stem cell (HSC) transplantation. Certain obstetric factors have been reported to increase TNC count in UCB units collected for transplantation. The aim of our study was to analyze how various maternal, neonatal and obstetric factors affected TNC count in the UCBs we collected for our cord blood bank in southern Italy. We performed a retrospective analysis of 634 medical records of UCBs collected by Calabria Cord Blood Bank (CCBB), between January 1, 2010 and December 31, 2016. We analyzed various maternal, neonatal and obstetric variables factors and related this factor with the characteristic of TNC. We found that the average number of TNCs was significantly greater in vaginal delivery than in caesarean delivery. We also found that TNCs were higher in the 40th week of pregnancy and when Apgar 1' scores were ≤ 9. The effect of a newborn's gender was less evident on TNC count. Knowledge of factors predictive of a higher TNC count would help cord blood banks more efficiently identify donors likely to yield high-quality UCBs for transplantation.

Optimization of the criteria for cord blood unit selection for transplantation in recipients of the different age groups: current view and prospects in Ukraine

Umbilical cord blood has been widely used to treat both malignant and non-malignant hematological diseases for over 30 years. During this time, more than 40,000 successful hematopoietic stem cell (HSC) transplantations of umbilical cord blood have been performed. However, today in Ukraine there is no public umbilical cord blood bank established for unrelated HSC transplantation to patients with oncohematological disorders (both children and adults). In this regard, the HSC units must be purchased abroad or the patients are sent to foreign clinics for high-cost treatment. The organization of a public umbilical cord blood bank in Ukraine would help in a short time to meet the needs of patients with oncohematological disorders for donor HSCs for unrelated transplantation and save significant funds for the treatment of patients abroad. According to the experience of the world's leading oncohematological centers, when it is impossible to find either a related or haploidentical donor or in all available registries – a unrelated transplant, the search continues in the registers of public cord blood banks and an umbilical cord blood unit that matches the criteria is usually found. The optimal choice of umbilical cord blood unit is crucial to maximize the likelihood of successful transplant engraftment and recipient survival after the transplantation, so the criteria for cord blood unit selection for unrelated transplantation are a bit broader than those used when matching donor-recipient pairs. The review presents the main criteria for cord blood unit selection according to the assessment of its quality, cell dose, HLA matching for unrelated transplantation to recipients of different age groups in accordance with international guidelines developed by the National Marrow Donor Program (NMDP), USA Center for International Blood and Brain Transplantation Research (CIBMTR), in collaboration with the NMDP Council Advisory Group, as well as in accordance with the American Society for Transplantation and Cellular Therapy (ASTCT) and the Seventh Edition of the NetCord-FACT International Standards for Cord Blood Collection, Banking, and Release for Administration.

Potentiation of cord blood cell therapy with erythropoietin for children with CP: a 2\u2009\xd7\u20092 factorial randomized placebo-controlled trial

BackgroundConcomitant administration of allogeneic umbilical cord blood (UCB) infusion and erythropoietin (EPO) showed therapeutic efficacy in children with cerebral palsy (CP). However, no clinical studies have investigated the effects of UCB and EPO combination therapy using a 2 × 2 four-arm factorial blinded design with four arms. This randomized placebo-controlled trial aimed to identify the synergistic and individual efficacies of UCB cell and EPO for the treatment of CP.MethodsChildren diagnosed with CP were randomly segregated into four groups: (A) UCB+EPO, (B) UCB+placebo EPO, (C) placebo UCB+EPO, and (D) placebo UCB+placebo EPO. Based on the UCB unit selection criteria of matching for ≥ 4/6 of human leukocyte antigen (HLA)-A, -B, and DRB1 and total nucleated cell (TNC) number of ≥ 3 × 107/kg, allogeneic UCB was intravenously infused and 500 IU/kg human recombinant EPO was administered six times. Functional measurements, brain imaging studies, and electroencephalography were performed from baseline until 12 months post-treatment. Furthermore, adverse events were closely monitored.ResultsEighty-eight of 92 children enrolled (3.05 ± 1.22 years) completed the study. Change in gross motor performance measure (GMPM) was greater in group A than in group D at 1 month (△2.30 vs. △0.71, P = 0.025) and 12 months (△6.85 vs. △2.34, P = 0.018) post-treatment. GMPM change ratios were calculated to adjust motor function at the baseline. Group A showed a larger improvement in the GMPM change ratio at 1 month and 12 months post-treatment than group D. At 12 months post-treatment, the GMPM change ratios were in the order of groups A, B, C, and D. These results indicate synergistic effect of UCB and EPO combination better than each single therapy. In diffusion tensor imaging, the change ratio of fractional anisotropy at spinothalamic radiation was higher in group A than group D in subgroup of age ≥ 3 years. Additionally, higher TNC and more HLA-matched UCB units led to better gross motor outcomes in group A. Adverse events remained unchanged upon UCB or EPO administration.ConclusionsThese results indicate that the efficacy of allogeneic UCB cell could be potentiated by EPO for neurological recovery in children with CP without harmful effects.Trial registrationClinicalTrials.gov, NCT01991145, registered 25 November 2013.

Ex Vivo Expansion of Adult Hematopoietic Stem and Progenitor Cells with Valproic Acid.

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic stem cell transplantation but lack a matched donor. However, the limited number of HSCs within each UCB unit remains a major challenge for their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of human HSCs in ex vivo cultures initiated with CD34+ cells isolated from UCBs can overcome this limitation. The method described here utilizes a deacetylase inhibitor, valproic acid (VPA), to rapidly expand to a high degree the numbers of functional HSCs and committed progenitors (HPCs). The expanded HSCs are capable of establishing both short-term and long-term multilineage hematopoietic reconstitution. This highly reproducible and simple protocol can be also applied to expansion of both HSCs and HPCs from different sources including the bone marrow and peripheral blood.

Prognostic Value of Refined Disease Risk Index (rDRI) and Conditioning Regimen in Adult Patients Receiving Unrelated Cord Blood Transplantation (UCBT) for Haematological Malignancies - 15-Year Follow-up of Multicenter Study in Singapore

Introduction: Umbilical cord blood (UCB) is an established stem cell source for transplantation in patients with hematological diseases who lack suitable related or unrelated donors. Refined Disease Risk Index (rDRI) is a prognostic tool used in hematopoietic cell transplant (HCT), solely based on disease type and status. Its value in patients undergoing UCB transplants (UCBT) with different conditioning regime intensity is unclear. We sought to evaluate the predictive value of rDRI in patients undergoing UCBT. Methods: We performed a retrospective analysis of 107 adult patients with various hematological malignancies who underwent 4-6/6 HLA-matched UCBT using single unit UCB (n=14) or double unit UCB (n=93) between Aug 2006 and Nov 2019 at 2 academic tertiary hospitals in Singapore. Patients received pre-transplant conditioning with either myeloablative conditioning (MAC, n=63) regimen consisting of Fludarabine (Flu) 75mg/m2, Cyclophosphamide (Cy) 120mg/kg and total body irradiation (TBI) 12- 13.2Gy , intensified reduced intensity conditioning (i-RIC, N=22) regimen consisting of Flu 150 mg/m2, Cy 50mg/kg, thiotepa 10mg/kg and TBI 4 Gy, or nonmyeloablative conditioning (NM, n=22) regimen, consisting of Flu 200 mg/m2, Cy 50mg/kg and TBI 2 Gy. The median total nucleated cell dose infused was 5.07 x 107/kg (range, 2.61 to 11.5) Results: Median follow-up was 84 months (range, 6 to 167 months).. Kaplan-Meier estimates of overall (OS) and event-free (EFS) survival at 10 years were 40 % (95% CI, 31-50) and 37% (95% CI, 28-47) respectively. At 2 years, cumulative incidence (CI) of relapse-related mortality (RRM) and non-relapse mortality (NRM) were 27% (95% CI, 18-35) and 35 % (95% CI, 25-45), respectively. In multivariate analysis (MVA), rDRI showed significant correlation with OS (HR 2.41; 95% CI 1.41-4.11; p=0.001), EFS (HR 2.54; 95% CI 1.51-4.28; p=0.0004) and RRM (HR 2.71; 95% 1.36-5.41; p=0.005). Conditioning regimen intensity was found to have significant impact on NRM (p=0.047) and RRM (p=0.002) in MVA. When patients were further stratified into 4 risk groups incorporating both rDRI and conditioning regimen intensity, significant differences in OS (p=0.002) and EFS (p=0.001) were seen. 5-year OS (Figure 1) in patients receiving MAC/i-RIC and NM conditioning with low-intermediate risk rDRI were 52% and 36% respectively, compared with 24% and 13% in patients with high-very high risk rDRI (p=0.002). The corresponding ES were 50%, 29%, 20%, and 13%, respectively (Figure2). Conclusions: Our results confirm that rDRI has good predictive value for relapse and survival in UCBT recipients. Conditioning regime intensity was also found to have a significant impact on patient outcomes when evaluated together with rDRI. Prognostic scoring systems in HSCT are revised constantly with further studies needed to potentially incorporate the intensity of the conditioning regime into the rDRI. Outcomes in rDRI high risk patients remain poor and further studies are needed to define the best strategy that can induce sustained engraftment without increasing risk of relapse and toxicity death. Disclosures No relevant conflicts of interest to declare.

Umbilical Cord Blood Transplants: Current Status and Evolving Therapies.

Hematopoietic cell transplants using stem cells from umbilical cord blood are used worldwide for the treatment of malignant and non-malignant disorders. Transplant procedures from this stem cell source have shown promising outcomes in successfully treating various hematologic, immunologic, malignant, and inherited metabolic disorders. Rapid availability of these stem cells is an important advantage over other unrelated donor transplants, especially in situations where waiting can adversely affect the prognosis. The umbilical cord blood is rich in CD34+ stem cells, though with a limited cell dose and usually takes longer to engraft. Limitations around this have been addressed by in vivo and ex vivo expansion techniques as well as enhanced engraftment kinetics. Development of adoptive immunotherapy using other components of umbilical cord blood such as regulatory T cells, virus-specific T cells, and natural killer cells has further transformed the field and enhanced the utility of umbilical cord blood unit.

Identifying Barriers to Umbilical Cord Blood Banking in Jordan: A Cross-Sectional Survey of Obstetricians.

Background: The expansion of umbilical cord blood (UCB) banking necessitates a greater understanding among obstetricians in order to responsibly inform parents about UCB collection and storage. Gaps in knowledge can compromise public UCB banking efforts and result in missed opportunities and public misguidance about UCB banking. Materials and Methods: A cross-sectional survey was disseminated among obstetricians in Amman, Jordan. The questionnaire aimed to evaluate obstetricians’ knowledge of and attitude toward UCB storage and applications, as well as current practice patterns. Results: Ninety-six obstetricians responded (55% response rate), most of whom were Jordanian (71%), female (83%), resident physicians (59%), and working in either private (43%) or public (42%) hospitals, with an average of 6.5 years in practice. Only 26% had personal experience in UCB collection, and 20% had received education on UCB collection. Nearly 75% said their hospitals lacked standard operating procedures, guidelines, or infectious disease screening for UCB units. Overall knowledge about UCB was moderate, and the internet was the most common information source (54%). Overall attitudes were positive, especially in desire to expand personal knowledge about UCB, integrate information into medical residency curricula, and establish a public UCB bank in Jordan. However, many believed that ethical (61%) and religious (56%) controversies surround UCB donation. Conclusion: This study identifies deficiencies in quality control and experience in UCB collection in Jordan, as well as areas of inadequate knowledge and ethical controversies among obstetricians. These issues contribute to public misinformation and limit public UCB donation programs, and requires improved medical education on this topic

Tailored Cytokine Optimization for ex vivo Culture Platforms Targeting the Expansion of Human Hematopoietic Stem/Progenitor Cells.

Umbilical cord blood (UCB) has been established as an alternative source for hematopoietic stem/progenitor cells (HSPC) for cell and gene therapies. Limited cell yields of UCB units have been tackled with the development of cytokine-based ex vivo expansion platforms. To improve the effectiveness of these platforms, namely targeting clinical approval, in this study, we optimized the cytokine cocktails in two clinically relevant expansion platforms for HSPC, a liquid suspension culture system (CS_HSPC) and a co-culture system with bone marrow derived mesenchymal stromal cells (BM MSC) (CS_HSPC/MSC). Using a methodology based on experimental design, three different cytokines [stem cell factor (SCF), fms-like tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin (TPO)] were studied in both systems during a 7-day culture under serum-free conditions. Proliferation and colony-forming unit assays, as well as immunophenotypic analysis were performed. Five experimental outputs [fold increase (FI) of total nucleated cells (FI TNC), FI of CD34+ cells, FI of erythroid burst-forming unit (BFU-E), FI of colony-forming unit granulocyte-monocyte (CFU-GM), and FI of multilineage colony-forming unit (CFU-Mix)] were followed as target outputs of the optimization model. The novel optimized cocktails determined herein comprised concentrations of 64, 61, and 80 ng/mL (CS_HSPC) and 90, 82, and 77 ng/mL (CS_HSPC/MSC) for SCF, Flt-3L, and TPO, respectively. After cytokine optimization, CS_HSPC and CS_HSPC/MSC were directly compared as platforms. CS_HSPC/MSC outperformed the feeder-free system in 6 of 8 tested experimental measures, displaying superior capability toward increasing the number of hematopoietic cells while maintaining the expression of HSPC markers (i.e., CD34+ and CD34+CD90+) and multilineage differentiation potential. A tailored approach toward optimization has made it possible to individually maximize cytokine contribution in both studied platforms. Consequently, cocktail optimization has successfully led to an increase in the expansion platform performance, while allowing a rational side-by-side comparison among different platforms and enhancing our knowledge on the impact of cytokine supplementation on the HSPC expansion process.

TNFSF15 facilitates human umbilical cord blood haematopoietic stem cell expansion by activating Notch signal pathway.

The lack of efficient ex vivo expansion methods restricts clinical use of haematopoietic stem cells (HSC) for the treatment of haematological malignancies and degenerative diseases. Umbilical cord blood (UCB) serves as an alternative haematopoietic stem cell source. However, currently what limits the use of UCB‐derived HSC is the very low numbers of haematopoietic stem and progenitor cells available for transplantation in a single umbilical cord blood unit. Here, we report that TNFSF15, a member of the tumour necrosis factor superfamily, promotes the expansion of human umbilical cord blood (UCB)‐derived HSC. TNFSF15‐treated UCB‐HSC is capable of bone marrow engraftment as demonstrated with NOD/SCID or NOD/Shi‐SCID/IL2Rgnull (NOG) mice in both primary and secondary transplantation. The frequency of repopulating cells occurring in the injected tibiae is markedly higher than that in vehicle‐treated group. Additionally, signal proteins of the Notch pathway are highly up‐regulated in TNFSF15‐treated UCB‐HSC. These findings indicate that TNFSF15 is useful for in vitro expansion of UCB‐HSC for clinical applications. Furthermore, TNFSF15 may be a hopeful selection for further UCB‐HSC application or study.

Reduced-intensity single-unit unrelated cord blood transplant with optional immune boost for nonmalignant disorders

AbstractChildren with many inherited nonmalignant disorders can be cured or their condition alleviated by hematopoietic stem cell transplantation (HSCT). Umbilical cord blood (UCB) units are a rapidly available stem cell source and offer great flexibility in HLA matching, allowing nearly uniform access to HSCT. Although reduced-intensity conditioning (RIC) regimens promise decreased treatment-related morbidity and mortality, graft failure and infections have limited their use in chemotherapy-naive patients. We prospectively evaluated a novel RIC regimen of alemtuzumab, hydroxyurea, fludarabine, melphalan, and thiotepa with a single-unit UCB graft in 44 consecutive patients with inborn errors of metabolism, immunity, or hematopoiesis. In addition, 5% of the UCB graft was re-cryopreserved and reserved for cord donor leukocyte infusion (cDLI) posttransplant. All patients engrafted at a median of 15 days posttransplant, and chimerism was >90% donor in the majority of patients at 1-year posttransplant with only 1 secondary graft failure. The incidence of grade II to IV graft-versus-host disease (GVHD) was 27% (95% confidence interval [CI], 17-43) with no extensive chronic GVHD. Overall survival was 95% (95% CI, 83-99) and 85% (95% CI, 64-93) at 1 and 5 years posttransplant, respectively. No significant end-organ toxicities were observed. The use of cDLI did not affect GVHD and showed signals of efficacy for infection control or donor chimerism. This RIC transplant regimen using single-unit UCB graft resulted in outstanding survival and remarkably low rates of graft failure. Implementation of the protocol not requiring pharmacokinetic monitoring would be feasible and applicable worldwide for children with inherited disorders of metabolism, immunity, or hematopoiesis. This trial was registered at www.clinicaltrials.gov as #NCT01962415.

Cord blood transplants supported by unrelated donor CD34+ progenitor cells.

Alternative donor transplantation with the haplo-cord platform allows the use of a lower-dose single umbilical cord blood unit (CBU) by co-infusion of third-party CD34+-selected cells from a haploidentical relative, which provides early transient engraftment while awaiting durable CBU engraftment. In our experience, ~15% of patients lack a suitable haploidentical donor. Here we report 26 patients who underwent haplo-cord transplant using CD34+-selected partially matched unrelated donor grafts. Twenty-four were conditioned with fludarabine/melphalan +/- low-dose TBI (n = 16). Twenty-five received ATG and all received posttransplant tacrolimus and mycophenolate mofetil. Median time to neutrophil and platelet recovery was 11 and 18 days. CBU engraftment, with CD33 and CD3 >5% cord chimerism in the myeloid/lymphoid compartment by day +60, occurred in 20 of 24 patients (83%). Incidence of grade 2-4 acute graft-versus-host disease (GVHD) was 27% at day +100, and chronic GVHD was 4% at 1 year. Overall survival at 1 year was 54%. For patients in need of an alternative transplant who lack a haploidentical donor, haplo-cord transplantation using CD34+-selected partially matched unrelated donor grafts results in rapid engraftment with no increased rate of cord graft failure or GVHD.

A Review of Evaluating Hematopoietic Stem Cells Derived from Umbilical Cord Blood's Expansion and Homing.

Transplantation of hematopoietic stem cells (HSCs) derived from umbilical cord blood (UCB) has been taken into account as a therapeutic approach in patients with hematologic malignancies. Unfortunately, there are limitations concerning HSC transplantation (HSCT), including (a) low contents of UCB-HSCs in a single unit of UCB and (b) defects in UCB-HSC homing to their niche. Therefore, delays are observed in hematopoietic and immunologic recovery and homing. Among numerous strategies proposed, ex vivo expansion of UCB-HSCs to enhance UCB-HSC dose without any differentiation into mature cells is known as an efficient procedure that is able to alter clinical treatments through adjusting transplantation-related results and making them available. Accordingly, culture type, cytokine combinations, O2 level, co-culture with mesenchymal stromal cells (MSCs), as well as gene manipulation of UCB-HSCs can have effects on their expansion and growth. Besides, defects in homing can be resolved by exposing UCB-HSCs to compounds aimed at improving homing. Fucosylation of HSCs before expansion, CXCR4-SDF-1 axis partnership and homing gene involvement are among strategies that all depend on efficiency, reasonable costs, and confirmation of clinical trials. In general, the present study reviewed factors improving the expansion and homing of UCB-HSCs aimed at advancing hematopoietic recovery and expansion in clinical applications and future directions.

Cellular Enumeration of Tregs and CD3+ Cells in Cryopreserved Umbilical Cord Blood Units

Background Allogeneic haematopoietic cell transplantation (HCT) is a curative therapy for severe haematological disorders, including acute leukaemia. However, it carries significant morbidity and mortality due to infection, graft-versus-host disease and relapse. The cellular content of HCT grafts, in particular the ratio between regulatory T cells (Treg) and conventional T cells, is an important factor influencing the severity of these complications. Whilst previous studies have been performed in fresh adult peripheral blood mobilised grafts, it has not been applied in umbilical cord blood (CB) HCTs. CB enumerations are more challenging as they must be performed from small cryopreserved segments stored alongside the CB units used for HCT. Study and design methods Fresh CB units and thawed segments were analysed for their Treg and T cell content using both flow cytometry (the benchmark technique) and an epigenetic, DNA-based methodology. The two methods were compared for their agreement, consistency and susceptibility to error when assessing Treg and CD3+ cell numbers in both fresh and cryopreserved samples. Results Epigenetic enumeration gave consistent results in both fresh and frozen samples, providing Treg/CD3 estimates that were similar. Assessment of Tregs and CD3+ cells by flow cytometry and epigenetic assessments in fresh samples showed that these two methods were correlated. Conversely, significant cell death was observed in the thawed segments which affected Treg and CD3 cell estimates by flow cytometry. Conclusion Epigenetic assessments offer significant advantages over flow cytometry for analysing cryopreserved CB. Unlike with flow cytometry assessments of thawed segments, similar cell numbers were observed in fresh and frozen samples, with material requirements not being limiting and being unaffected by high cell death. With this method, multiple epigenetic assessments can be performed from extracted DNA, to provide statistical confidence and confirm observed results. Finally, the method raises the possibility of retrospective studies of historical samples where only DNA is available.

Three obstetric factors should be considered in umbilical cord blood donor selection

Background/Aim. The umbilical cord blood (UCB) volume and hematopoietic stem cells count are used as indicators for hematopoietic potential of UBC units. These indicators are affected by a collection method and obstetric factors. It was established that birth weight and placental weight affect the volume and hematopoietic stem cells count in UCB units. The influence of other obstetric factors is less clear. The aim of this study was to investigate the impact of obstetric factors on hematopoietic potential of UCB units. Methods. The study involved 103 consecutive UCB units collected during 2013. Relationship of UCB volume, total nucleated cells, CD34+ cells and Colony Forming Unit- Granulocyte Monocyte count with maternal and neonatal characteristics was retrospectively analyzed. Results. It was shown that birth weight, placental weight and umbilical cord length ? 31 cm significantly increased the volume of collected samples, total nucleated cells, CD34+ cells and Colony Forming Unit-Granulocyte Monocyte count. Gestational age between 38?40 weeks increased significantly all umbilical factors (volume, total nucleated cells, CD34+ cells, and Colony Forming Unit-Granulocyte Monocyte count). Gender did not have an influence on quality of UCB units except on total nucleated cells and CD34+ cells count. Other obstetric factors did not affect significantly the quality of UCB units. Conclusion. Our study confirmed that birth weight, placenta weight, length of the umbilical cord and gestational age independently influenced the UCB unit volume, and absolute count of nuclear cells and hematopoietic stem cells. Due to a positive correlation between birth weight and placental weight, only birth weight, umbilical cord length and gestational age should be standard parameters in procedure of donor selection.

Scalable Manufacturing of Human Hematopoietic Stem/Progenitor Cells Exploiting a Co-culture Platform with Mesenchymal Stromal Cells.

In the context of hematopoietic cell transplantation, hematopoietic stem/progenitor cells (HSPC) from the umbilical cord blood (UCB) present several advantages compared to adult sources including higher proliferative capacity, abundant availability and ease of collection, non-risk and painless harvesting procedure, and lower risk of graft-versus-host disease. However, the therapeutic utility of UCB HSPC has been limited to pediatric patients due to the low cell frequency per unit of UCB. The development of efficient and cost-effective strategies to generate large numbers of functional UCB HSPC ex vivo would boost all current and future medical uses of these cells. Herein, we describe a scalable serum-free co-culture system for the expansion of UCB-derived CD34+-enriched cells using microcarrier-immobilized human bone marrow-derived mesenchymal stromal cells as feeder cells.

Umbilical Cord Blood Units Cryopreserved in the Public Cord Blood Bank: A Breakthrough in iPSC Haplobanking?

The use of induced pluripotent stem cells (iPSCs) is an emerging therapeutic option for precision medicine. Cord blood (CB) cells with lower immunogenicity, fewer genomic changes, and persistent epigenetic memory might be ideal candidates for iPSC production. Based on the human leukocyte antigen (HLA) distribution of cord blood units (CBUs) in the public CB bank, we estimated the coverage of the Korean population with HLA-homozygous iPSCs to repurpose cryopreserved CBUs. We analyzed a total of 27,904 Korean CBUs donated to the public CB bank. Low-to-intermediate resolution typing was performed for HLA-A, -B, and -DRB1 alleles, and individuals possessing homozygous HLA haplotypes were identified by direct counting. Moreover, the matching probabilities for zero-mismatch transplantation were calculated for 27,904 CBUs and 50,000,000 potential Korean patients. Among the preserved CBUs, 15 HLA-A, 40 HLA-B, and 13 HLA-DRB1 alleles as well as 48 homozygous HLA-A-B-DRB1 haplotypes were identified at serological equivalents (2 digits). The 48 identified homozygous haplotypes cumulatively matched 78.18% of the 27,904 Korean CB donors as zero HLA-mismatch iPSC sources. Among the combinations of 1,699 haplotypes with frequencies greater than 0.001%, assuming a population of 50 million, those 48 haplotypes can provide a match for 78.37% of potential Korean recipients. A practicable number of HLA-A, -B, and -DRB1 homozygous iPSC lines derived from CBUs may be an efficient option in allogeneic iPSC therapy because this type of haplobanking may provide cell lines with optimal HLA matching for up to three-quarters of the Korean population.

Regimen-dependent synergism and antagonism of treprostinil and vildagliptin in hematopoietic cell transplantation

The cell dose in umbilical cord blood units is a major determinant for the outcome of hematopoietic cell transplantation. Prostaglandin analogs and dipeptidylpeptidase-4 (DPP4/CD26)-inhibitors enhance the ability of hematopoietic stem cells (HSCs) to reconstitute hematopoiesis. Here we explored the synergism between treprostinil, a stable prostaglandin agonist, and the DPP4/CD26-inhibitor vildagliptin. The combination of treprostinil and forskolin caused a modest but statistically significant increase in the surface levels of DPP4/CD26 on hematopoietic stem and progenitor cells (HSPCs) derived from murine bone and human cord blood. Their migration towards stromal cell-derived factor-1 (SDF-1/CXCL12) was enhanced, if they were pretreated with treprostinil and forskolin, and further augmented by vildagliptin. Administration of vildagliptin rescued 25% of lethally irradiated recipient mice injected with a limiting number of untreated HSPCs, but 90 to 100% of recipients injected with HSPCs preincubated with treprostinil and forskolin. The efficacy of vildagliptin surpassed that of treprostinil (60% rescue). Surprisingly, concomitant administration of vildagliptin and treprostinil resulted in poor survival of recipients indicating mutual antagonism, which was recapitulated when homing of and colony formation by HSPCs were assessed. These observations of regimen-dependent synergism and antagonism of treprostinil and vildagliptin are of translational relevance for the design of clinical trials.Key messagesPretreatment with treprostinil increases surface levels of DPP4/CD26 in HSPCs.Vildagliptin enhances in vitro migration of pretreated HSPCs.Vildagliptin enhances in vivo homing and engraftment of pretreated HSPCs.Unexpected mutual antagonism in vivo by concomitant administration of vildagliptin and treprostinil.