Source Of Hematopoietic Stem Cells Research Articles

Comparison of T Cell Subsets and Cytokines between Steady-State and G-CSF-Primed Bone Marrow and Its Association with Graft-Versus-Host-Disease

BackgroundThe widespread use and success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) can be affected by graft-versus-host-disease (GVHD), an immunological syndrome that involves tissue damage mediated by donor lymphocytes. Both, acute and chronic GVHD, result from an imbalance between the effector and regulatory arms of the immune system. On the other hand, worldwide, the main source of stem cells is peripheral blood (PBSC). However, its use has been associated with higher incidence of GVHD when compared with bone marrow. Although the fast engraftment using PBSC as a source of stem cells compares to that seen with G-CSF-primed bone marrow (G-BM), the latter, also associates with a decreased incidence of GVHD. At our institution, patients transplanted with G-BM, have a lower incidence of GVHD when compared to those transplanted with PBSC and steady state bone marrow (SS-BM). Some microenvironment characteristics of these sources of hematopoietic stem cells (HSCS) remain unknown, including differences between Tregs, Th17 cells, and cytokines, and its association with GVHD.AimThe aim of this study was to analyze lymphocyte subsets and associated cytokines in donor G-BM and SS-BM, comparing patients who developed GVHD and those who did not.Material and MethodsA prospective analysis was performed in 48 donors SS-BM and G-BM collected from March 2002 to April 2017. Twenty two samples collected since March 2002 were thawed; since 2014, samples (20 mL) were immediately processed and analyzed. A 24 hour resting with RPMI, and posterior activation with PMA (50 ng/ml) for 48 hours was performed. Cells were harvested and cytokines were evaluated by flow cytometry (CBA assay).From each sample, one million mononuclear cells were permeabilized, fixed, and stained with CD4-FITC, IL17A-PE, IFN -γ APC, and IL-4 PECy7, for their posterior phenotipication by flow cytometry. The samples were obtained in a BD LSR Fortessa cytometry, and analyzed with the Flow-Jo software. Comparison between variables was made by means of Student's t test using SPSS v.21.ResultsWe found increased Tregs (1.46% ± 0.69 vs 1.01% ± 0.49, p=0.01) and Th2 (1.55% ± 0.36 vs 1.28% ± 0.39, p=0.02) subsets in G-BM compared to SS-BM. Nonetheless, although differences in cytokine production were observed, we did not find any statistical significance. Acute GVHD was diagnosed in 9% (n=3) and 20% (n=3) of patients receiving G-BM and SS-BM, respectively. Chronic GVHD was seen in 33% of patients in both groups (receiving SS-BM or G-BM). Thirty one patients did not present either GVHD. In all the cohort, indistinctively from the HSCs used, acute GVHD was characterized by a predominant pro-inflammatory profile, with increased TNFα (59.28 ± 56.6 vs 14.21 ± 36.2 pg/mL, p=0.011), and a higher percentage of Th1 (22.55% ± 4.8 vs 20.71% ± 4.8, p=0.04) and Th17 (0.87% ± 0.33 vs 0.57% ± 0.24, p=0.009) cells. Furthermore, a deficient suppressor response, with decreased IL-10 production (1.17 ± 1.26 vs 33.55 ± 49.3 pg/mL, p<0.0001) as well as lower Tregs percentage (0.76% ± 0.12 vs 1.40% ± 0.68, p<0.0001) was found. Likewise, chronic GVHD showed the same profile, but with more striking differences in pro-inflammatory cytokine production. The T cell subset profile in chronic GVHD also showed an imbalance between pro-inflammatory and suppressor subsets percentage (figure 1 ).ConclusionsThis is the first study exploring the differences in T cells and cytokine profile between G-BM and SS-BM. Our results showed that G-BM has an enhanced suppressive phenotype compared to SS-BM. Also, patients who developed GVHD were characterized by an imbalance in pro-inflammatory and suppressor responses, with enhanced Th17 cells, as well as increased TNF-α, IL-6 and IL-17A production in contrast to a dampened Tregs frequency and IL-10 production which can explain the low frequency of GVHD in our transplanted patients using G-BM. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Validation of a Predictive Model for Survival in Patients with Acute Myeloid Leukemia and Myelodysplastic Syndrome Receiving Hematopoietic Stem Cell Transplantation

▪IntroductionAllogeneic hematopoietic stem-cell transplantation (AHSCT) is an effective treatment for patients with high-risk or relapsed acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Several factors are known to influence survival post-transplant. Our group developed a model to predict survival in a small number of patients with AML/MDS using 3 disease (cytogenetics and disease status at transplant) and patient-related [Hematopoietic stem cell transplant comorbidity index (HCT-CI)] variables (Bachegowda et al.Blood.2017) . This model effectively stratified AML/MDS patients into 3 risk groups with very different survival. Here we aim to validate this model in a large cohort of AML/MDS patients receiving AHSCT with various donor types.MethodsA total of 934 patients with AML/MDS consecutively treated at the University of Texas MD Anderson Cancer Center were included in this analysis. Donors were HLA matched related (n=377, 40%), matched unrelated (n=416, 45%) mismatched unrelated (n=68, 7%) and T-cell replete haploidentical transplants (n=73, 8%). The median age was 53 years (range 18-65 years). Cytogenetics data at diagnosis was available in 920 patients and was favorable, intermediate and adverse-risk in 340 (37%), 517 (56%) and 63 (7%), respectively. Five hundred and forty patients (58%) were in first or second complete remission (CR1/2). The hematopoietic stem cell transplant comorbidity index (HCT-CI) was available in 914 patients and the median score was 2 (range 0-17). The sources of hematopoietic stem cells were peripheral blood for 586 patients (63%) and bone marrow for 348 patients (37%). Seven hundred and eighty patients (84%) received myeloablative-conditioning chemotherapy.ResultsPatients were stratified into 3 risk groups according to the previously published model: 1) low-risk group (patients in CR1/2 with intermediate/favorable risk cytogenetics, irrespective of HCT-CI (HCT-CI did not impact survival in this group) (n=351), 2) intermediate-risk group (patients beyond CR1/2 and/or with adverse-risk cytogenetics and HCT-CI score ≤4) (n=412), and 3) high-risk group (patients with at least one negative prognostic factor - beyond CR1/2 and/or with adverse-risk cytogenetics and a HCTCI score >4) (n=155).Median follow up duration for 436 survivors was 48 months. For the whole group, the 5-year progression-free survival (PFS), treatment-related mortality (TRM) and relapse rate were 39%, 21% and 39%, respectively. TRM at 5 years for low-risk group was 17%, for the intermediate-risk group was 18% (HR [95% CI] 1.12 [0.79-1.59]; P=0.52), and for the high-risk group was 40% (3.24 [2.26-4.64]; P<0.0001), respectively, whereas cumulative incidence of relapse at 5 years was 24% for low-risk, 52% for intermediate-risk (2.86 [2.20-3.71]; P<0.0001) and 38% for the high-risk group (1.88 [1.34-2.63]; P<0.0001), respectively. In multivariable model, transplantation in >CR1/2 (2.84 [2.38-3.40]; P<0.0001), adverse cytogenetic risk (1.54 [1.29-1.83]; P<0.0001), males (1.23 [1.03-1.46]; P=0.022) were significantly associated with worse PFS while higher age-adjusted HCT-CI scores (1.20 [0.99-1.47]; P=0.06) approached significance. Other factors including conditioning intensity (1.02 [0.82- 1.29]; P=0.84, age (1.01 [1.00-1.01]; P=0.17) and donor type (haploidentical (1.2 [0.83-1.72]; P=0.33), matched unrelated (1.06 [0.88-1.28]; P=0.55) and mismatched unrelated donor (1.07 [0.76-1.50]; P=0.70) as compared with matched related donor) did not significantly influence PFS. Using our previously described predictive model, the patients separated in 3 risk groups with significantly different median PFS, which was not reached for the low-risk group, 9.2 months for the intermediate-risk group (2.4 [1.97-2.99]; P<0.0001), and 6.0 months for the high-risk group (3.3 [2.57-4.21]; P<0.0001), respectively. (Figures 1A and 1B)In conclusion, we were able to stratify allogeneic stem cell transplant patients in 3 risk groups based on a combination of disease (cytogenetics and disease status at transplant) and patient-related factors (HCT-CI) with significantly different survival. HCT-CI appears to have a much higher discriminatory impact in patients with higher risk disease (not in CR1/CR2 and/or adverse risk cytogenetics) compared with those in the lower risk group (CR1/CR2 and intermediate/favorable risk cytogenetics). [Display omitted] DisclosuresOran:AROG: Research Funding; Celgene: Research Funding; Astex: Research Funding. Khouri:Novartis: Research Funding.

Impact of Splenomegaly on Outcomes of Fully Matched Sibling Bone Marrow Transplant Among Children with Low Risk Thalassemia

Objective:Severe thalassemia syndromes (ST) are the most common non-infectious life-threatening disease in South Asia. ST is highly curable by BMT but rejection may occur in 5 to 30% of cases. In a limited resource setting appropriate case selection for BMT and aggressive down staging pre-transplant are crucial in order to achieve optimal outcomes. Though hepatomegaly > 2 cm has clearly been proven to be associated with poor transplant outcomes whereas the role of splenomegaly as an independent risk factor for rejection in low risk thalassemia has not been clarified.Methods:We analyzed a total of 87 consecutive first bone marrow transplants carried out between August 2013 and Dec 2016 after conditioning with busulfan (14 mg/kg oral, not adjusted to serum levels), cyclophosphamide (200 mg/kg) and anti-thymocyte globulin (Genzyme 4 mg/kg or Fresenius 16 mg/kg on days -12 to -10). 74 patients received cyclosporine and methotrexate, and 13 patients received cyclosporine and mycophenolate as GVHD/rejection prophylaxis. G-CSF-primed bone marrow (5 μg/kg/dose twice daily for 3 to 5 days prior to harvest) was the source of hematopoietic stem cells in all cases. All patients were considered low risk based on liver size < 2 cm from costal margin and age less than 15 years (median 6.9 years, range 1.1 to 14.5). All sibling couples were HLA-compatible.Results:Of the 87 low risk transplants 12 children rejected (13.8%) of which 5 were primary and 7 were secondary graft failures. Out of the total 87 patients 11 patients had significant splenomegaly at transplant (spleen > 3 cm below costal margin and/or z score > 3 spleen size measured by ultrasound adjusted for height). Four of the rejections (1 primary and 3 secondary) occurred in children with significant splenomegaly which is statistically significant (log rank p=0.02) (figure 1). Splenomegaly also seems to increase treatment-related mortality albeit not statistically significant (log rank p=0.17). Out of the 7 children who had treatment related mortality 2 had splenomegaly. One of them died as a consequence of primary graft failure and the other due to a large intracranial bleed. We could not detect any difference in other variables potentially associated with rejection risk such as gender mismatch, serum ferritin, ABO mismatch, mean cell dose (median 6.75; range 2.6 to 29.44 x 108 nucleated cells /kg) and immunosuppressive therapy between splenomegaly and no splenomegaly groups. In the latter group 41% of children received hydroxyurea pre-BMT compared to 81% in the splenomegaly group (P= 0.01) which in fact should have helped to reduce rejection rates in the splenomegaly group. By multivariate analysis significant splenomegaly (p= 0.0049) seems to be more relevant than gender mismatch in terms of rejection risk (p = 0.0379), while cell dose (p=0.9546) was not a significant factor.Conclusion:Splenomegaly may increase rejection risk in low risk thalassemia transplants transplanted with ATG-BuCy. Strategies to reduce spleen size prior to transplant should be considered. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CD3 Lymphocyte Graft Content and Not Hematopoietic Stem Cell Source Is Associated to Higher Chronic GVHD after Haploidentical Transplantation with Post-Transplant Cyclophosphamide

Background: Haploidentical hematopoietic cell transplantation (HCT) using post-transplant cyclophosphamide (PT-Cy) as graft-versus-host disease (GVHD) prophylaxis has shown promising results. A possible influence on outcome according to graft type [peripheral blood stem cells (PBSC) vs bone marrow (BM), Asad Bashey JCO 2017 ] has been recently shown, however there are still no data regarding graft composition and donor type influence on survival outcomes. We conducted a retrospective study on haploidentical HCT using PT-Cy according to graft source, donor characteristics and graft cell composition.Methods: 234 (>18 years old) patients who underwent HCT between 2010 and 2016 from 5 European hospitals were included. Patients, disease and transplant characteristics were collected from centers' registries and medical records. Donor characteristics (age, type, gender) and graft cell composition (CD34+, CD3+, CD4+, CD8+, CD56+, CD19+) data were also analyzed. Graft cell counts were considered as continuous variables. The primary end-point was overall survival (OS). Secondary endpoints included cumulative incidence of acute and chronic GVHD, non-relapse mortality (NRM), relapse/progression (RI/PD), progression-free survival (PFS) and GVHD and Relapse-Free Survival (GRFS). Median follow up for surviving patients was 18 months. Only variables significant in univariate analysis were included in the multivariate models with the graft cell counts.Results: Study cohort characteristics are shown in Table 1. Day +30 and +90 neutrophil and platelet recovery were 92%, 95% and 61% and 81% respectively with no differences between PBSC and BM. Acute GVHD grade II-IV incidence at day +100 was 34% (BM 26% vs PBSC 41%, p=0.002) while grade III-IV was 11% (BM 6% vs 17%, p=0.003). Chronic GVHD incidence at 18 months was 19% (BM 6% vs PBSC 33%, p<0.001) and extensive chronic GVHD incidence was 10% (BM 1% vs PBSC 20%, p<0.001). At 18 months, OS, PFS and GRFS were 46%, 60% and 36% respectively. Cumulative incidence of NRM and RI/PD were 15% and 35% with no differences between BM and PBSC.On multivariate analysis, OS was favorably affected by CR at transplant (HR 0.47, p=0.002) and negatively by age>50yrs (HR 1.95, p=0.012), Comorbidity Score >2 (HR 1.83, p=0.004), and higher CD8+ graft cell content (HR 1.15, p=0.034). PFS was positively affected by CR at transplant (HR 0.53, p=0.002) and negatively by Disease Risk Index > Intermediate-High (HR 1.66, p=0.030) and Comorbidity Score >2 (HR 1.45, p=0.046). Similarly, GRFS was positively influenced by CR at transplant (HR 0.60, p=0.006) and negatively by Comorbidity Score >2 (HR 1.45, p=0.032). As expected RI/PD was negatively affected by Disease Risk Index > Intermediate-High (HR 1.91, p=0.030), but CR at transplant (HR 0.59, p=0.033) and higher CD4 graft cell count showed a protective effect (HR 0.83, p=0.007). NRM was favorably influenced only by higher CD3 graft cell count (HR 0.08, p=<0.001) while age>50yrs (HR 1.98, p=0.033), Comorbidity Score >2 (HR 1.97, p=0.049), higher CD8 graft count (HR 1.13, p=0.005) and higher CD4 graft count (HR 1.34, p=0.010) had a detrimental effect. Interestingly, when analyzing GVHD incidence in the multivariate model, we observed that graft cell composition variables abrogated the graft source effect (PBSC vs BM). In particular, no prognostic factors influenced acute GVHD grade II-IV while all grade and extensive chronic GVHD incidence were strongly increased when a higher CD3 graft cell composition was used (HR 3.49, p=0.008 for all grade chronic GVHD and HR 5.54, p=0.017 for extensive chronic GVHD). A cut off value of 0.9 x 10^8/kg CD3 graft cell count was able to effectively split both all grade chronic GVHD (33% vs 6%, p<0.001) and extensive chronic GVHD (19% vs 2%, p<0.001) curves (Figure 1). Donor characteristics did not influence any outcomes.Conclusion: PT-Cy mediated T cell repletion abrogates the effects of donor characteristics on survival outcomes as opposed to ATG-based haploidentical platform (Yu Wang, Blood 2014). The higher incidence of chronic GVHD is probably explained by a higher number of graft CD3+ cells (>0.9 x 10^8/kg) and not merely by graft type (BM vs PBSC). These results could help in adapting GVHD prophylaxis for those high risk patients who infused high CD3 content grafts. These observations should be validated on prospective multicentric studies. [Display omitted] DisclosuresCiceri:GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Corradini:Gilead: Honoraria; Takeda: Honoraria; Amgen: Honoraria; Celgene: Honoraria; Janssen: Honoraria; Roche: Honoraria; Sanofi: Honoraria; Novartis: Honoraria.

A Bioreactor Simulating Pulse-Pressure Mediated Circumferential Stretch Stimulates Hematopoietic Stem Cell Formation

During fetal development, heartbeat precedes blood circulation and definitive hematopoietic stem cell (HSC) formation in the aorta-gonad-mesonephros (AGM) region. However, how the function and growth of heart and blood vessels are linked to the development of blood is largely unknown. Blood flow-mediated shear stress on the endothelial lining of the AGM stimulates the endothelial emergence of HSCs. A zebrafish mutant for cadherin 5, which has normal blood formation despite an early circulation arrest, enabled us to analyze the timing and intertwined roles of heartbeat in blood formation. Here we show that heartbeat and stretching of the blood vessel stimulate the HSC formation. We used three-dimensional (3D) digital Doppler ultrasound, microangiography, and time-lapse confocal microscopy to demonstrate that a beating heart, in cdh5-silenced zebrafish embryos, produces pulsation in blood vessels despite the lack of cardiac output and active blood circulation. Our time-lapse confocal imaging of cd41:eGFP+ HSCs emerging from flk1:mCherry+ endothelium from the cdh5-silenced embryos, followed by machine learning analysis of pulsating blood vessels, further establishing that pulse-pressure on the arterial endothelial lining of AGM regulates the endothelial emergence of HSCs. To understand the relationship between pulse pressure, biomechanical stretching of endothelial lining, and HSC formation, we recreated pulsating blood vessel-like conditions in a bioreactor by applying cyclic strain on mouse E11.5 AGM-derived hemogenic endothelial cells. Cyclic strain-mediated Trpv4 activation in hemogenic endothelial cells both stimulated HSC development and rescued hematopoiesis in the silent heart (tnnt2)-silenced embryos, which lacked blood flow and heartbeat. Therefore, heartbeat-mediated biomechanical stretching of hemogenic endothelial cells in the AGM stimulates trpv4 channels during the endothelial-to-HSC transition. Our findings advance our fundamental understanding of the developmental cues and 3D microenvironmental mechanisms regulating HSC formation. Our model could establish hemogenic endothelial cells as a potential source of bone marrow-independent HSCs. DisclosuresNo relevant conflicts of interest to declare.

The Combination of GROβ and AMD3100 Leads to Rapid and Robust Mobilization of Hematopoietic Stem Cells in Nonhuman Primates

▪Introduction: Mobilized peripheral blood grafts are currently the predominant source of hematopoietic stem and progenitor cells (HSPC) for both autologous and allogeneic transplantation. The most common clinical hematopoietic stem cell mobilization protocol is five days of Filgrastim (G-CSF). This regimen requires daily injections, has been associated with bone pain and often results in unpredictably low yields. A rapid mobilization method that ideally only required a single treatment and had robust and predictable kinetics would be a significant improvement over the current standard of care. In mice, a unique CXCR2 agonist, GROβ, induces rapid mobilization of stem and progenitor cells 15 minutes after a single injection. When co-administered with plerixafor (AMD3100), an inhibitor of CXCR4, a synergistic increase in mobilization results, with a graft enriched in highly engraftable hematopoietic stem cells (Hoggatt et al., Blood 2016 128:368). Here, we present data demonstrating that combination treatment with GROβ and AMD3100 synergistically mobilizes CD34+ cells and colony forming units (CFU) in nonhuman primates (NHP).Methods: Cynomolgus macaques were injected with G-CSF (10 µg/kg/day, s.c. for four days), AMD3100 (1 mg/kg, s.c. once) or GROβ (various doses and routes) alone and in combination with AMD3100. Blood was collected at various times post treatment and analyzed by multicolor flow cytometry to quantitate HSPC numbers. Additional aliquots of mobilized blood were plated in methylcellulose and CFU were enumerated seven days later.Results: We tested the effect of GROβ alone or in combination with AMD3100 in Cynomolgus monkeys. As published previously, administration of G-CSF, AMD3100 or G-CSF+AMD3100 induced significant mobilization of hematopoietic stem and progenitor cells. In comparison, a single injection each of GROβ+AMD3100 yielded 4-fold more CD34+ cells compared to four days of G-CSF (p < 0.01). While the combination of G-CSF+AMD3100 mobilized significantly more CD34+ cells compared to GROβ+AMD3100 (p < 0.0001), the numbers of CD34+ CD90+ CD45RA- cells, representing the most primitive of hematopoietic stem and progenitor cells, were equivalent between treatment groups. Similarly, GROβ+AMD3100 treatment elicited 7-fold more CFU compared to animals treated with G-CSF (p < 0.01), 3.5-fold more CFU compared to AMD3100 (p < 0.01) and equivalent CFU compared to G-CSF+AMD3100. Importantly, the frequency of CFU among the CD34+ cells was significantly greater with GROβ+AMD3100 (1 in 4) compared to G-CSF (1 in 10) or G-CSF+AMD3100 (1 in 25). In contrast to the increased CD34+ numbers and CFU, white blood cell (WBC) and neutrophil counts were significantly lower with GROβ+AMD3100 compared to AMD3100 alone or G-CSF+AMD3100.Conclusions: We describe a rapid mobilization method that within four hours of a single treatment results in robust hematopoietic mobilization in nonhuman primates. The GROβ+AMD3100 regimen enriches for primitive CD34+ CD90+ CD45RA- stem and progenitor cells with increased colony forming capacity compared to CD34+ cells mobilized with G-CSF+AMD3100, suggesting a significant graft quality difference with the new regimen. The ability to rapidly mobilize primitive HSC with increased CFU frequency may allow effective single day mobilization of HPSCs for patients requiring a HSCT and be especially useful for HSC based gene therapy and gene editing protocols where HSPC grafts enriched for primitive HSC may reduce manufacturing costs and ensure durability of the gene modified HSC. The ability to achieve increased HSPC numbers without dramatically increasing WBC and neutrophils may be especially advantageous in patient populations where G-CSF mobilization is not well tolerated such as Sickle Cell Disease, or where G-CSF leads to increased disease activity such as in autologous transplant for Multiple Sclerosis. A single treatment that results in robust mobilization that is equivalent to G-CSF may allow for a one-day collection method of donor stem cells, and these findings in nonhuman primates support clinical exploration. DisclosuresFalahee:Magenta Therapeutics: Employment, Equity Ownership. Goncalves:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Hoggatt:Magenta Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Morrow:Magenta Therapeutics: Employment, Equity Ownership. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Bone Marrow Transplant Product Quality Has Decreased Over Time— a Retrospective Examination of NMDP Collected Bone Marrow Products from 1994-2016

▪BackgroundBone marrow (BM) is an essential hematopoietic stem cell (HSC) source for many allogeneic transplants (HCT) and is the preferred cell source for specific disease indications in adults (e.g., Aplastic Anemia), the majority of pediatric transplants, and when the benefits of a decreased risk of chronic graft versus host disease (GVHD) outweigh other considerations. The advent of G-CSF mobilized peripheral blood stem cells (PBSC), with logistic benefits over the collection of BM, has resulted in a significant decrease in the utilization of BM. Since the advent of PBSC, BM has declined from 100% in the early 1990s to 15% in 2015, of unrelated donor products. This decreased utilization of BM has a number of potential consequences from a quality perspective. Many centers perform a limited number of BM collections per year and thus, the ability to ensure ongoing competency of staff in this technical procedure can be challenging. Proper assessment of BM harvest metrics is difficult in an individual collection facility when limited procedures are performed. Previous data from a single center noted that the total number of nucleated cells (TNC) received in BM products for individual patients decreased over the years. The aim of this study was to examine the trends in BM harvest quality in a large cohort of NMDP donors to see if this observation can be generalized.MethodsThe primary outcome of this study was examination of the total nucleated cells collected per milliliter (TNC/mL) of BM as an estimate of product quality. The population studied was BM donations from domestic unrelated donors reported to the NMDP/CIBMTR from 1994-2016 (analyzed over 5 time periods). The TNC/mL for each harvest was calculated based on the total number of nucleated cells (TNC) collected in harvest and the volume (mL) of final product including additives. Additional covariates, including time period of harvest, the number of harvests performed per year at the center, donor age, donor body mass index (BMI), donor weight, donor race/ethnicity, and donor gender were examined.ResultsExamination of >15,000 BM donations revealed a significant decline in the concentration of total nucleated cells from 21.8 TNC x 106/mL in the earliest era to 18.7 TNC x106/mL in the 2012-2016 era (p<0.001). (Table 1). The number of overall collections performed per center in the 2012-2016 era was similar to other eras (Table 2). The number of collections performed by a collection center did not appear to explain the decrease in BM quality as median TNC/mL decreased over time intervals regardless of collection centre size (Figure 1). Interestingly, the total product volume harvest increased over the eras (p<0.001), while the duration of the harvest decreased (p<0.001). Donor characteristics were examined across eras. In recent years, donors are significantly more likely to be male (p<0.001) and young (p<0.001). For example, in the 2012-2016 era, 54% of the donors were under the age of 30 as compared to the 1994-1996 and 1997-2001 eras where only 25% of the donors were under the age of 30. Similarly, 65% of the donors were male in the 2012-2016 era, compared to 53% in the 1994-1996 era.ConclusionIn conclusion, we showed a significant decrease in the TNC/mL in BM products over time. This did not appear to be related to the number of collections per center, suggesting that cell number collected is not directly related to the number of procedures performed but rather other factors in the collection process. Interestingly, the significant decrease in TNC concentration in BM observed in this study persisted despite the increased use of optimal donors. Further analysis will be required to elucidate the exact cause of decreased concentration of TNC collected in products. Factors such as technique, rate of collection, materials /equipment used, training, and collector experience may have profound influences on the quality of BM collected. For example, the number of personnel performing harvests, number of harvests performed per collector, or turn-over of personnel at each collection center maybe contributing factors. These further studies will help to better understand the entire process and help improve the quality of the products, an issue crucial to ensuring bone marrow remains a quality cell source for transplant. [Display omitted] DisclosuresPulsipher:Jazz Pharmaceuticals: Other: advisory board, education; CSL Behring: Other: advisory board Feb 2017; Adaptive: Other: Advisory board 6/17; Chimerix: Other: Advisory board Dec 2015; Novartis Pharmaceuticals Corporation: Consultancy, Other: Phase II steering committee.

The Hematopoietic Reconstitution through Intra-Bone-Marrow Injection Using Murine Fetal Liver Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) were considered an effective treatment option for many blood diseases and autoimmune diseases. HSCs can currently be isolated from bone marrow and umbilical cord blood. Due to the shortage of these resources, an alternative source of HSCs preparation were sorted out in this study and the ability of hematopoietic reconstruction were examined using such HSCs obtained from fetal liver.Fetal liver hematopoietic stem cells (FL-HSCs) were isolated from embryos of transgenic mice with an erythoid-specific GFP expression that was driven by a human β-globin gene promoter (HS23-GFP mice). Thus, GFP expression could be used as tracer of marker genes of red blood cells derived from the donor FL-HSCs visually. Single-cell suspension of E13.5 HS23-GFP murine liver was used for Ficoll density gradient separation, and the FL-HSC enriched portion was collected for intra-bone-marrow injection (IBMI) into 6-8 week-old recipient mice post 137Cs γ irradiation treatment. Three doses were tested for injection, i.e. 1×105, 1×106 and 1×107 cells, respectively.Hematopoietic reconstruction ability was observed every week after injection using blood smear and routine blood panel such as RBC, HGB and HCT. Bone marrow smear and pathological sections of liver and spleen of recipient mice were analyzed 8 weeks post FL-HSC transplantation. GFP expressed erythrocyte was captured in the peripheral blood starting one week after HL-HSC transplantation. The ability of hematopoietic reconstruction continued to rise until week 7, and became stable thereafter. Among the three treatment groups, the highest cell number group (1×107 ) had the best hematopoietic restoration, according to the RBC, HGB and HCT data. No abnormality such as lymphocytic infiltration in pathological sections of liver and spleen of recipients were noted. Bone marrow smear of recipient mice showed no difference when comparing to that of the wide type mice. This demonstrated HSC obtained from fetal liver, are capable of reconstructing hematopoietic function after transplantation using IBMI. This could provide an alternative source of HSC for future study of hematopoietic function and for future therapeutic treatment for blood diseases. DisclosuresNo relevant conflicts of interest to declare.

Bioactive SL-13R Peptide Expands Human Umbilical Cord Blood Hematopoietic Stem Cells and Maintains Long-Term Reconstitution Ability

Hematopoietic stem cell (HSC) transplantation is a curative treatment of hematological disorders that has been utilized for several decades. Although umbilical cord blood (UCB) is a promising source of HSCs, the presence of an insufficient number of HSCs in these preparations limits their use, prompting need for ex vivo HSC amplification. To establish a more efficient method to expand UCB HSCs, we developed the SL-13R peptide and cultured UCB CD34+ cells with peptide in serum-free medium containing a cytokine cocktail. Following 9 days of culture with peptide, the number of total cells, CD34+CD38- cells and hematopoietic colonies significantly increased relative to control cells grown without peptide. Transplantation of these cells into immunodeficient NOD/Shi-scid/IL-2Rγ knockout mice confirmed that they possess long-term reconstitution ability. To understand how the peptide promotes HSC expansion, we identified PLEC and ERLIN2 proteins as peptide interactors and undertook loss-of-function analysis to determine whether either was required for peptide function in our culture system. PLEC knockdown UCB CD34+ cells cultured with peptide showed a decreased number of hematopoietic colonies relative to peptide-treated, non-knockdown controls. By contrast, ERLIN2 knockdown had little effect in the presence of peptide. This work suggests that PLEC functions in HSC expansion promoted by SL-13R. In summary, we have identified a novel peptide promoting expansion of UCB CD34+ cells with long-term reconstitution ability. Its use may facilitate clinical use of UCB HSCs. DisclosuresSugiyama:Science Lustre, Ltd: Equity Ownership.

Extracellular vesicles from human iPSCs enhance reconstitution capacity of cord blood-derived hematopoietic stem and progenitor cells

Cord blood (CB) represents a source of hematopoietic stem and progenitor cells (CB-HSPCs) for bone marrow (BM) reconstitution, but clinical CB application is limited in adult patients due to the insufficient number of CB-HSCPCs and the lack of effective ex vivo approaches to increase CB-HSPC functionality. Since human-induced pluripotent stem cells (hiPSCs) have been indicated as donor cells for bioactive extracellular vesicles (EVs) modulating properties of other cells, we are the first to employ hiPSC-derived EVs (hiPSC-EVs) to enhance the hematopoietic potential of CB-derived CD45dimLin-CD34+ cell fraction enriched in CB-HSPCs. We demonstrated that hiPSC-EVs improved functional properties of CB-HSPCs critical for their hematopoietic capacity including metabolic, hematopoietic and clonogenic potential as well as survival, chemotactic response to stromal cell-derived factor 1 and adhesion to the model components of hematopoietic niche in vitro. Moreover, hiPSC-EVs enhanced homing and engraftment of CB-HSPCs in vivo. This phenomenon might be related to activation of signaling pathways in CB-HSPCs following hiPSC-EV treatment, as shown on both gene expression and the protein kinases activity levels. In conclusion, hiPSC-EVs might be used as ex vivo modulators of CB-HSPCs capacity to enhance their functional properties and augment future practical applications of CB-derived cells in BM reconstitution.

Cord-Blood-Derived Professional Antigen-Presenting Cells: Functions and Applications in Current and Prospective Cell Therapies.

Human umbilical cord blood (UCB) represents a valuable source of hematopoietic stem cells, particularly for patients lacking a matching donor. UCB provides practical advantages, including a lower risk of graft-versus-host-disease and permissive human leukocyte antigen mismatching. These advantageous properties have so far been applied for stem cell, mesenchymal stromal cell, and chimeric antigen receptor T cell therapies. However, UCB-derived professional antigen-presenting cells are increasingly being utilized in the context of immune tolerance and regenerative therapy. Here, we review the cell-specific characteristics as well as recent advancements in UCB-based cell therapies focusing on dendritic cells, monocytes, B lymphocytes, innate lymphoid cells, and macrophages.

ТРАНСПЛАНТАЦИЯ ПУПОВИННОЙ КРОВИ У ДЕТЕЙ: ОБЗОР ЛИТЕРАТУРЫ И ОПЫТ ОДНОГО ЦЕНТРА

Umbilical cord blood (CB) has been used successfully as hematopoietic stem cells (HSCs) in adults and adults with malignant and non-malignant diseases in which there are indications for HSC transplantation (HSCT), and when an HLA-compatible related or unrelated donor is not available. CB, like any source of HSC, has certain advantages and disadvantages, which also determine the features of its use in transplantation. The article presents the experience of using CB as a source of HSC in pediatric patients at the Russian Children's Clinical Hospital (RCCH). Materials and methods of research: in a retrospective single-center continuous non-randomized uncontrolled open study includes 16 patients with various malignant diseases and 16 patients with non-malignant diseases, who underwent CB transplantation in the bone marrow transplantation department of the Russian Children's Clinical Hospital from 1997 to 2012. The peak of CB transplant activity occurred in the period from 2006 to 2010. The age of the patients at the time of the transplantation ranged from 6 months to 14 years. In 10 patients, a CB transplant from HLA-identical related donors was used, in 22 – from unrelated HLA-identical and HLA-incompatible for 2 or more antigens obtained from domestic CB banks. In addition to CB, six children were given bone marrow or peripheral blood stem cells from HLA-identical siblings as a transplant. The majority of patients (n=20) received one CB sample, 10 patients – 2 samples and two – 3. Results: implantation of the transplant was recorded in 27 children (84%), which 17 (53%) were alive, 10 (31%) died at different timesof post-transplant period. In 5 (16%) patients, implantation did not occur, of which one (3%) died, the cause of mortality in pediatric patients was a relapse of the underlying diseases. Acute graftversus-host reaction (GVHD) after CB transplantation was assessed in 27 (84%) patients of this study, among them 17 (63%) were diagnosed with acute GVHD stages I-II, 3 (11%) – stage III–IV (life-threatening form). Chronic GVHD was assessed in 18 (56%) patients included in the study, with 5 (28%) diagnosed with a limited form and 3 (17%) with an extensive form. Overall survival (OS) for the entire group of patients (n=32) was 58,3±8,8%, 5-year dormant survival rate (DSR) – 53±8.8%. OS in non-malignant diseases was higher (68%) than for malignant diseases (56%), but no statistically significant differences in OS were obtained in the two groups (p&gt;0,05), as well as when comparing OS and DSR when using one or several samples of CB. Conclusion: CB remains available and acceptable source of stem cells for HSCT. At our center, the survival rates of patients after CB transplantation are comparable to those of international registries. The improvement of the method and the results of haploidentical SCT in recent years has led to a decrease in interest and transplantation activity (including in our center) using CB as a source of HSC. However, the unexplored antileukemic potential of CB stem cells, as well as a number of potential resources for improving and optimizing the use of this HSC source, may increase the chances of future CB transplantation.

Women’s attitude towards umbilical cord blood banking in Poland

Introduction: Umbilical cord blood (UCB) is considered as a valuable potential source of hematopoietic stem and progenitor cells. A process of collecting and storing UCB in the immediate period after the birth is called UCB banking.

Factors Predicting the Presence of Maternal Cells in Cord Blood and Associated Changes in Immune Cell Composition.

BackgroundCord blood (CB) samples are increasingly used as a source of hematopoietic stem cells in transplantation settings. Maternal cells have been detected in CB samples and their presence is associated with a better graft outcome. However, we still do not know what influences the presence of maternal microchimerism (MMc) in CB samples and whether their presence influences CB hematopoietic cell composition.Patients and MethodsHere we test whether genetic, biological, anthropometric and/or obstetrical parameters influence the frequency and/or quantity of maternal Mc in CB samples from 55 healthy primigravid women. Mc was evaluated by targeting non-shared, non-inherited Human Leukocyte Antigen (HLA)-specific real-time quantitative PCR in whole blood and four cell subsets (T, B lymphocytes, granulocytes and/or hematopoietic progenitor cells). Furthermore CB samples were analyzed for their cell composition by flow cytometry and categorized according to their microchimeric status.ResultsMMc was present in 55% of CB samples in at least one cell subset or whole blood, with levels reaching up to 0.3% of hematopoietic progenitor cells. Two factors were predictive of the presence of MMc in CB samples: high concentrations of maternal serological Pregnancy-Associated-Protein-A at first trimester of pregnancy (p=0.018) and feto-maternal HLA-A and/or –DR compatibility (p=0.009 and p=0.01 respectively). Finally, CB samples positive for MMc were significantly enriched in CD56+ cells compared to CB negative for MMc.ConclusionsWe have identified two factors, measurable at early pregnancy, predicting the presence of maternal cells in CB samples at delivery. We have shown that MMc in CB samples could have an influence on the hematopoietic composition of fetal cells. CD56 is the phenotypic marker of natural killer cells (NK) and NK cells are known to be the main effector for graft versus leukemia reactions early after hematopoietic stem cell transplantation. These results emphasize the importance of MMc investigation for CB banking strategies.

Eltrombopag for thrombocytopenia in 24 children after hematopoietic stem cell transplantation

Objective: To evaluate the efficacy and safety of eltrombopag for children with thrombocytopenia after hematopoietic stem cell transplantation (HSCT). Methods: Clinical data of 24 patients with thrombocytopenia after HSCT,who were treated with eltrombopag in the Department of Pediatrics, Sun Yat-sen Memorial Hospital, Sun Yat-sen University from August 1, 2018 to April 1, 2019 were analyzed retrospectively. The response rate and adverse reactions of eltrombopag were evaluated. Patients were divided into groups by source of hematopoietic stem cells (umbilical cord blood group and peripheral stem cell group) and type of disease (malignant and non-malignant disease group) and the clinical outcomes between groups were compared. Rank Sum test was used for comparisons between groups. Results: Among 24 cases, 15 were males and 9 females, the age of starting eltrombopag was 7.7 (2.6-13.7) years, the time of eltrombopag treatment after HSCT was 27.5 (8.0-125.0) days, the time from treatment to complete response (CR) was 23.5 (6.0-83.0) days, with the treatment course 36.5 (8.0-90.0) days. The total dose of eltrombopag was 1 400(200-5 900) mg. Complete response rate was 92% (22/24),without eltrombopag related adverse reactions. Comparing with peripheral stem cell group (n=8), the course and total dose of eltrombopag in umbilical cord blood group (n=16) were significantly reduced(24.5 (8.0-81.0) vs. 65.5 (35.0-90.0) d, Z=-3.004, P=0.002; 900.0 (200.0-3 850.0) vs. 2 862.5 (1 175.0-5 900.0) mg, Z=-2.604, P=0.007), but no significant differences were found in the time from treatment to complete response, platelet count after 2 weeks of eltrombopag withdrawal or platelet count at the end point of follow-up (all P>0.05). Comparing malignant patients (n=12) and non-malignant patients (n=12), no significant differences were found in the time from treatment to complete response, course, total dose, platelet count after 2 weeks of eltrombopag withdrawal, and platelet count at the end point of follow-up in non-malignant patients (all P>0.05). Conclusion: Eltrombopag is safe and maybe effective for thrombocytopenia after HSCT, especially for umbilical cord blood transplantation.

Women\u2019s attitude towards umbilical cord blood banking in Poland

Umbilical cord blood (UCB) is considered as a valuable potential source of hematopoietic stem and progenitor cells. A process of collecting and storing UCB in the immediate period after the birth is called UCB banking. The study was conducted in order to determine women’s knowledge, awareness, preferences and attitude towards UCB banking in Poland, considering the sociodemographic and obstetric factors. A cross-sectional, self-administered, online questionnaire-based study including mostly multiple choice questions concerning attitude and awareness regarding UCB banking was conducted entirely online among Facebook female users in Poland. A total of 1077 participants correctly completed the survey. Most participants (n = 911, 84.6%) were aware of the possibility of UCB banking. Social media were considered as the main source of information (47.5%). However, the participants mostly indicated the doctor as their preferred source of reliable information (86.8%). The majority of women (61.8%) assessed their level of knowledge of UCB banking as still insufficient. Among the participants who supported UCB banking (70%), the following reasons were considered as the most vital: potential possibility of helping their child (93.9%) and helping other relatives (64.4%). More than half of the respondents (66.9%), who have not stored and are not willing to store their children’s UCB, indicated the high cost of UCB banking as the main reason of this decision. The knowledge and awareness of UCB storage and banking possibilities amongst women in Poland could be improved. The professional medical personnel should be a source of reliable information.

Characterization of hematopoietic stem cells from the canine yolk sac.

The characterization of hematopoietic stem cells (HSC) from the canine yolk sac (cYS) can contribute to future gene therapies because it is possible to obtain information about the beginning of the development of the circulatory system through the characterization. The cYS is a likely source of HSC, which is a source of blood cell development in mammals. Studies in this field have been conducted for decades; however, interest in cellular therapy is currently at its peak with greater visibility, and these cells are a promising therapeutic tool for the treatment of diseases related to animals and humans. The aim of this study was to isolate and characterize HSC from the cYS embryos at 30 to 45 days of gestational age. Our results showed that the cYS was macroscopically located in the ventral region with a central portion and extremities. The cells in culture presented a circular morphology and cell clusters. The average cell viability was 22.55% dead cells out of 6.5 × 104 total cells. The cells were also able to form colonies on methylcellulose. Flow cytometry analysis revealed the expression of CD34, CD117, and CD45. Our results suggest that the cYS can be used as a source of hematopoietic cells, and this study is very important to understand the mechanism and development of the hematopoietic system in dogs.

Analysis on the Composition of Leukapheresis Product – A Comparison Between MCS+® + and Spectra Optia® Apheresis Equipment

INTRODUCTION: Peripheral blood stem cell has become the preferred source for hematopoietic stem cells in both autologous and allogenic transplants. CD34+ cells represent a small proportion of the leukapheresis product content. The present study aims to analyze the cellular composition of leukapheresis product collected by intermittent-flow Mobile Collection System®+ (MCS+) and continuous-flow (Spectra Optia®) apheresis equipment. METHODS: In this retrospective study, 97 leukapheresis procedures for 85 individuals mobilized only with granulocyte colony-stimulating factor were analyzed. The product samples were analyzed for CD34+ cells, red blood cells (RBCs), white blood cells, and platelet contents. The difference in overall product composition was compared between the equipment through Mann–Whitney U-test. RESULTS: Both the equipment had similar CD34 and mononuclear cells (MNC) harvested in the product. However, Spectra Optia had statistically significantly lower product volume than MCS+ (215 mL vs. 260 mL, P = 0.04). Similarly non-CD34 composition such as RBC content per apheresis was six-fold higher (27 mL vs. 4 mL) and platelet contamination (1601 vs. 1275 × 109/L) was relatively higher with MCS+ over Spectra Optia. No relationship was observed between the CD34 concentration in the product and RBC and platelet contamination between both the equipment. CONCLUSION: Both equipment collect adequate CD34 and MNC cells; however, Spectra Optia is preferred due to product quality in terms of less product volume with minimal RBC and platelet contamination.

Isolation and &amp;lt;i&amp;gt; Ex Vivo&amp;lt;/i&amp;gt; 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.

Differentiated Cells Derived from Hematopoietic Stem Cells and Their Applications in Translational Medicine.

Hematopoietic stem cells (HSCs) and their development are one of the most widely studied model systems in mammals. In adults, HSCs are predominantly found in the bone marrow, from where they maintain homeostasis. Besides bone marrow and mobilized peripheral blood, cord blood is also being used as an alternate allogenic source of transplantable HSCs. HSCs from both autologous and allogenic sources are being applied for the treatment of various conditions like blood cancers, anemia, etc. HSCs can further differentiate to mature blood cells. Differentiation process of HSCs is being extensively studied so as to obtain a large number of pure populations of various differentiated cells in vitro so that they can be taken up for clinical trials. The ability to generate sufficient quantity of clinical-grade specialized blood cells in vitro would take the field of hematology a step ahead in translational medicine.