Human Hematopoietic Stem Cell Transplantation Research Articles

Distributions of MICA and MICB Alleles Typed by Amplicon-Based Next-Generation Sequencing in South Koreans.

Major histocompatibility complex class I chain-related genes A and B (MICA and MICB) play a role as ligands in activating the NKG2D receptor expressed in natural killer cells, γδ T-cells and αβ CD8 T-cells and have been defined in human diseases and haematopoietic stem cell transplantation (HSCT). MICA and MICB alleles were genotyped at the three-field level by amplicon-based next-generation sequencing (NGS) using a MiSeqDx system and compared with the results from previous studies in healthy South Korean donors. Exons 2-5 of MICA and exons 2-4 of MICB were amplified using a multiplex polymerase chain reaction (PCR). Sequence reads of ≥ 51 depth counts were consistently obtained for each sample exon, and target exons were determined to match reference sequences contained in the IPD-IMGT/HLA database. MICA and MICB alleles were tested using exon combinations. The program was designed to recognise specific sequences and discriminate between the MICA*008:01:01/*027 alleles. A total of 22 alleles were found in MICA and MICB. We observed 1 HLA-C ~ HLA-B ~ MICA ~ MICB ~ HLA-DRB1 haplotype with significant linkage disequilibrium between alleles at all neighbouring HLA loci. These results are consistent with previous microarray results. Genotyping of MICA and MICB was possible using 11-loci HLA genes. We updated the distribution of MICA and MICB based on three-field allele and haplotype frequencies containing linkage disequilibrium in South Koreans using amplicon-based NGS. These data suggest that high-resolution MICA and MICB typing data obtained using NGS may aid in performing HSCT and disease association studies.

The Effects of Busulfan on Xenogeneic Transplantation of Human Peripheral Blood Mononuclear Cells in Recipient Mice

Background. Humanized mouse models with engraftment of human peripheral blood mononuclear cells (PBMCs) or hematopoietic stem cells (HSCs) are effective tools for the study of human immunity. Busulfan has been used as a substitute for irradiation in human hematopoietic stem cell (HSC) transplantation models, but it has not been tested in human peripheral blood mononuclear cell (PBMC) transplantation models.Materials and Methods. This study evaluated PBMC engraftment using cytometry and enzyme-linked immunosorbent assay (ELISA) in female NOD.CB17/Prkdcscid/JKrb/ IL2 receptor γ−/− (NIG) mice treated with busulfan.Results. In this model, the percentage of human CD3+ T cell engraftment in the blood was 28.2%, with dominant infiltration of CD8+ cells in the spleen 3 weeks post PBMC transplantation. Production of human cytokines, including IL-12p70, IL-4, IL-5, IFN-γ, IL-6, IL-8, IL-22, TNF-α, and IL-10, was determined in mice treated with busulfan.Conclusions. Our findings demonstrate that busulfan treatment is a beneficial alternative for simple and efficient PBMC engraftment in a rodent model, possibly helping to evaluate human immunity in preclinical studies.

Characterization of Hematopoietic Stem Cell Diversity Using Spatiotemporal Quantitative Phase Imaging

Cells undergo continual transformations, never retaining their original state. Hematopoietic stem cells (HSCs), at the apex of the hematopoietic hierarchy, are not exempt from this dynamic process. In recent years, the advent of single-cell analysis techniques such as scRNAseq has progressively unveiled the heterogeneity within cell populations previously presumed to be homogeneous. However, these techniques are limited to evaluating the cellular state at a single temporal point based on snapshot analysis, thus failing to account for temporal changes and dynamic behavior of cells. Therefore, the true diversity inherent to the ever-changing HSCs remains uncertain. In this study, we comprehensively elucidate HSC diversity, including temporal changes, by integrating our recently established ex vivo HSC expansion techniques (Wilkinson et al., Nature, Becker et al., Cell Stem Cell), single-cell quantitative phase imaging (QPI), and post-transplantation dynamics analysis. For live imaging, HSCs were harvested from hepatic leukemia factor ( Hlf)-tdTomato reporter mice, which specifically marks long-term HSCs. Index sorted single Hlf-tdTomato + HSC were expanded with a single-HSC culture system and monitored by QPI fluorescence time-lapse imaging. Surprisingly, even though the cells were derived from the same HSC fraction, significant variations in mitotic rate, motility, morphological changes, and Hlf expression levels were observed at the single-cell level. Stemness did not decrease stepwise over time, but always exhibited continuous changes. Thus, analysis including the temporal dimension illuminated the infinitely branched nature of HSC diversity. Interestingly, Hlf expression level correlated with the dynamic behavior of HSCs in the expansion culture system. Hlf-high HSCs exhibited a slow division rate, small, rounded morphology and low motility, while Hlf-low HSCs actively divided and differentiated, displayed larger morphological size with low sphericity, and exhibited high motility. Furthermore, spatiotemporal dynamics analysis of Hlf-high/low HSCs after transplantation, employing the highly sensitive in vivo imaging system Akaluc bioluminescence imaging (AkaBLI), revealed that Hlf-high HSCs initiate systemic hematopoiesis slowly, while Hlf-low HSCs expanded quickly, characterized by short-term explosive hematopoiesis in the spleen. As expected, only Hlf-high HSCs had long-term bone marrow reconstitution ability. Thus, based on the expression level of Hlf, it is possible to predict the dynamic behavior of the cells in the expansion medium and in the post-transplant body. Next, we investigated whether Hlf expression level could be predicted based on the kinetic features of cells during HSC ex vivo expansion. Interestingly, multidimensional analysis based on kinetic features extracted from time-lapse imaging showed multiple clusters, each linked to Hlf expression levels. In human HSCs, the dynamic diversity of HSCs was also observed in our recently established ex vivo expansion system (Sakurai et al., Nature). Notably, the kinetic features of human HSCs differed from those of mouse HSCs. Transplantation of human HSCs exhibiting specific morphological characteristics showed long-term reconstitution ability. Thus, classification of HSCs based on kinetic features facilitated enrichment of functional HSCs. This study demonstrates the importance of characterizing cells, including the temporal dimension, and illustrates the existence of infinite dynamic individuality in mouse and human HSCs.

175. Clindamycin versus linezolid for Group A Streptococcal bloodstream infections?: A comparative effectiveness study of adjunctive antitoxin therapy at 137 US Hospitals

Abstract Background Adjunctive clindamycin has survival benefit in invasive Group A streptococcus (GAS) infections. Like clindamycin, linezolid also leads to decreased toxin and virulence factor production. However, rising clindamycin resistance among GAS isolates and inadequate clinical data on linezolid both offer pause on which to choose. We examined the impact of adjunctive clindamycin vs. linezolid on survival among patients with GAS bloodstream infections (BSI) in the presence and absence of clindamycin resistant (clinda-R) isolates Methods Clinical characteristics, antimicrobial susceptibility testing (AST), and antibiotic therapy were examined for unique adult inpatient encounters with GAS BSIs in the PINC-AI Database. Patients treated with a β-lactam for ≥3 days ±3 days of culture who received clindamycin ±3 days of culture were overlap weighted on a propensity-score to those who received linezolid using basine patient and hospital factors. The primary outcome was odds ratio (OR) of in-hospital mortality associated with clindamycin (vs linezolid). The secondary outcome was length of stay (LOS) among survivors. Subgroups analyses were conducted excluding clindamycin receipients with clinda-R isolates (subgroup 1) and also missing clindamycin AST(including D-test; subgroup 2).Figure 1.Study Flow chart Selection of patients with Group A streptococcal blood stream infections. The database was queried for inpatients (aged ≥18 years) with blood culture displaying growth of Group A streptococcus, filtered on the basis of receiving β-lactam antibiotics within 3 days either side of culture sampling for a minimum duration of 3 days and received either adjunctive clindamycin or linezolid treatment within 3 days either side of culture sampling.Table 1:Baseline characteristics of all patients with Group A Streptococcal blood stream infections treated with β-lactam antibiotics and adjunctive clindamycin or linezolid (N=660)Abbreviations: IVIG: intravenous immunoglobulin, ICU: intensive care unit, IQR: interquartile range, SOFA: Sequential Organ Failure Assessment * This score was calculated by use of ICD-10-clinical modification codes and adapted from the methods used by Quan and colleagues ^ Calculated by use of an electronic health record-based adaption of the original Sequential Organ Failure Assessment ^^ Deteremined by clindamycin antimicrobial susceptibility and D-test results. Defined as resistant or intermediate clindamycin susceptibility results and/or a positive D-test result. † Defined as presence of ICD-10 codes coding for any of the following conditions: Human immunodeficiency virus, cancer, solid organ or hematopoietic stem cell transplantation, receipt of systemic corticosteroids, chemotherapy or other immunosuppressive therapy and immunodeficiency. ‡ Receipt of norepinephrine, epinephrine, phenylephrine, and dopamine administered within a 24-h period either side of culture sampling. Results Of 3019 β-lactam–treated inpatients with GAS BSI, 500 (17%) received clindamycin and 160 (5%) received linezolid. The prevalence of clinda-R isolates was 19%;1 isolate was linezolid resistant and excluded (Figure 1). Overlap weighting resulted in well balanced groups (Figure 2). In the overlap weighted cohort, mortality risk was similar between recipients of clindamycin (10%, [50/500]) vs linezolid (9%, [14/160]; OR 1.38 [95% CI: 0.76-2.49]). Among survivors median[interquartile range] LOS was similar between the two groups (8[9] vs. 9[8] days, p=0.45). Removing those with clinda-R isolates (N=84) and also missing AST (N=166) yielded similar results (Figure 3).Figure 2.Standardized mean differences for covariates included the propensity score calculation Standardized mean differences for covariates included in the propensity score generation averaged across exposure categories in the unweighted cohort (blue triangles) and overlap weighted cohort weight (red circles). After overlap weighting, the mean standard difference at each variable assessed was zero . Abbreviations: ICU: intensive care unit, IVIG: intravenous immunoglobulin, NSTI: necrotizing soft tissue infectionFigure 3.Odds Ratio of in-hospital mortality in patients with invasive group A streptococcal blood stream infection treated with adjunctive clindamycin versus adjunctive linezolid The ORs (95% CIs) of in-hospital mortality (including discharge to hospice) in the primary analysis and subgroup analysis with patients withclindamycin resistant isolates (subgroup 1) and those with clindamycin resistant isolates and missing clindamycin susceptibility results (subgroup 2) removed from clindamycin group. Abbreviations: CI: confidence interval Conclusion Among β-lactam-treated patients with GAS BSI, linezolid and clindamycin displayed comparable effectiveness as adjunctive antitoxin agents. Similar intrinsic effectiveness (i.e., in patients with only susceptible isolates) supports linezolid as an alternative even in low clinda-R settings. Disclosures Ahmed Babiker, MBBS, Roche: Advisor/Consultant Morgan Walker, MD, Cytovale: Advisor/Consultant

Racial Disparity in Hematopoietic Stem Cell Transplant Clinical Trial Recruitment

Introduction: Race significantly influences phenotypic and genetic diversity in hematological diseases, consequently affecting disease prognosis, Human Leukocyte Antigen-matching and Hematopoietic Stem Cell Transplant (HSCT) outcomes. To ensure the development of novel transplant strategies effective for patients from diverse backgrounds, all racial groups must have equal opportunities to take part in HSCT clinical trials. Methods: Using ClinicalTrials.gov, a web-based resource that registers all studies meeting the definition of a clinical trial according to the International Committee on Medical Journal Editors, we identified all phase 1 to phase 4 HSCT clinical trials registered from 2006 to 2022. We manually abstracted data on the racial distribution of enrolled participants, sex distribution, transplant type, trial phase, location, and year of trial reporting. We calculated observed ratios of racial participation and compared them with the estimated probability of finding adequate donors from different racial subgroups reported by the National Marrow Donor Program to define the disproportionate enrolment factor. We then conducted subgroup racial distribution based on the trial phase, location, and type of transplant. We further evaluated trends in racial disparity within clinical trial enrolment over the years. Results: We identified 398 clinical trials related to HSCT between 2006 and 2022, of which 72% were allogenic and 28% were autologous HSCT trials. Most of these were phase 2 clinical trials (64.3%) and the majority (78.1%) were conducted in the United States. The racial distribution of enrolled participants was publicly available in 235 (59%) of these clinical trials, including 57.7% allogeneic and 62.5% autologous HSCT. The race distribution of enrolled subjects was reported in 80% of Phase 1 and 79% of Phase 3, whereas only 48% of Phase 1/2 and 44% of Phase 2/3 clinical trials reported racial distribution. The race distribution among 25,075 study participants in 235 clinical trials was: 80.4% Whites (20,175), 7.4% Blacks (1858), 5.3% Asians (1336), 1.6% other races (396), and 5.3% (1310) subjects with unreported race. The same race distribution trend was observed across all subgroups of clinical trials based on phase, location, and transplant type (Table 1). The disproportionate enrolment factor was 0.27 for Black participants, and 0.11 for Asian participants, compared to 1.02 for Whites. From 2006 to 2022, yearly trends for the absolute number of clinical trial enrollees showed a 2.5% and 1.5% increase in Black and Asian subject representation; however, minorities still accounted for only 20.3% of total representation in 2022 (Figure 1). Conclusions: Our results emphasize the significant racial underrepresentation of minority populations with a very low disproportionate enrolment factor in HSCT clinical trials despite the increased probability of finding a suitable donor resulting from novel HSCT technologies in recent years. Addressing these disparities requires urgent action and a multi-faceted approach. Efforts such as targeted outreach, cultural competence training and community engagement to focus on eliminating barriers are urgently needed to increase the enrolment of the minority population to achieve racial equity in HSCT.

How to optimize outcome of patients undergoing HLA-matched related haematopoietic stem cell transplantation in acquired and inherited bone marrow failure syndromes.

Up-front allogeneic haematopoietic stem cell transplantation (allo-HSCT) after a reduced intensity conditioning regimen is the standard treatment in children with acquired severe aplastic anaemia (aSAA) and inherited bone marrow failure syndromes (iBMFs) in the presence of a healthy matched related donor (MRD). The paper by Alsultan et al. report the safety and efficacy of MRD HSCT conditioned with low-dose cyclophosphamide, fludarabine and thymoglobulin in both aSAA and non-Fanconi iBMFs, strengthening the concept of the pivotal role of immunosuppressive approach in allo-HSCT for specific subgroups of non-malignant diseases requiring a reduced risk of toxicities, offering the opportunity to discuss the essential points for achieving patients' long-term survival after MRD HSCT in BMF. Commentary on: Alsultan et al. Human leucocyte antigen-matched related haematopoietic stem cell transplantation using low-dose cyclophosphamide, fludarabine and thymoglobulin in children with severe aplastic anaemia. Br J Haematol 2023;203:255-263.

Human leucocyte antigen-matched related haematopoietic stem cell transplantation using low-dose cyclophosphamide, fludarabine and thymoglobulin in children with severe aplastic anaemia.

When human leucocyte antigen-matched related donors are available, haematopoietic stem cell transplantation (HSCT) in children with severe aplastic anaemia (SAA) represents the standard of care. Cyclophosphamide (Cy) 200 mg/kg and anti-thymocyte globulin (ATG) are frequently administered, but to-date, no standard conditioning regimen exists. In this study, we investigated the efficacy of a unified HSCT conditioning protocol consisting of low-dose Cy 80 mg/kg, fludarabine and ATG. Data were reviewed from children aged ≤14 years with either acquired SAA or non-Fanconi anaemia inherited bone marrow failure syndrome (IBMFS) between 2011 and 2022 at various Saudi institutions. Graft-versus-host disease (GVHD) prophylaxis included mycophenolate mofetil and calcineurin inhibitors. HSCT was performed in 32 children (17 females and 15 males). Nine patients had deleterious mutations (two ERCC6L2, two ANKRD26, two TINF2, one LZTFL1, one RTEL1 and one DNAJC21). Four patients had short telomeres. All 32 patients engrafted successfully. At 3 years post-transplant, the event-free survival was 93% and overall survival was 95%. Two patients experienced secondary graft failure or myelodysplastic syndrome. A low probability of GVHD was observed (one acute GVHD II and one mild chronic GVHD). These data highlight how HSCT using low-dose Cy as part of a fludarabine-based regimen is safe and effective in SAA/non-Fanconi anaemia IBMFS.

Nuclear Tubulin Enhances CXCR4 Transcription and Promotes Chemotaxis Through TCF12 Transcription Factor in human Hematopoietic Stem Cells.

Tubulins are cytoskeleton components in all eukaryotic cells and play crucial roles in various cellular activities by polymerizing into dynamic microtubules. A subpopulation of tubulin has been shown to localize in the nucleus, however, the function of nuclear tubulin remains largely unexplored. Here we report that microtubule depolymerization specifically upregulates surface CXCR4 expression in human hematopoietic stem cells (HSCs). Mechanistically, microtubule depolymerization results in accumulation of tubulin subunits in the nucleus, leading to elevated CXCR4 transcription and increased chemotaxis of human HSCs. Treatment with microtubule stabilizer Epothilone B strongly suppresses the phenotypes induced by microtubule depolymerizing agents in human HSCs. Furthermore, chromatin immunoprecipitation assay reveals an increased binding of nuclear tubulin and TCF12 transcription factor at the CXCR4 promoter region. Depletion of TCF12 significantly suppresses microtubule depolymerization mediated upregulation of CXCR4 surface expression. These results demonstrate a previously unknown function of nuclear tubulin in regulating gene transcription through TCF12. New strategy targeting nuclear tubulin-TCF12-CXCR4 axis may be applicable to enhance HSC transplantation.

The Impact of Graft CD3+ T-Cell Dose on the Outcome of T-Cell Replete Human Leukocyte Antigen-Mismatched Allogeneic Hematopoietic Peripheral Blood Stem Cells Transplantation.

Data on whether the graft CD3-positive (CD3+) T-cell dose in T-cell-replete human leukocyte antigen (HLA)-mismatched allogeneic hematopoietic peripheral blood stem cells transplantation (PBSCT) influences post-transplant outcomes are controversial. Using King Hussein Cancer Center (KHCC) Blood and Marrow Transplantation (BMT) Registry database, 52 adult subjects, receiving the first T-cell-replete HLA-mismatched allogeneic hematopoietic PBSCT for acute leukemias or myelodysplastic syndrome, were identified, from January 2017 to December 2020. The cutoff value of graft CD3+ T-cell dose was identified using the receiver operating characteristic (ROC) formula and Youden's analysis. Subjects were divided into two cohorts: cohort 1 with low CD3+ T-cell dose (n = 34) and cohort 2 with high CD3+ T-cell dose (n = 18). Correlative analyses were performed between CD3+ T-cell dose and the risk of graft-versus-host disease (GvHD), relapse, relapse-free survival (RFS), and overall survival (OS). P-values were two-sided and considered significant when P < 0.05. Subject covariates were displayed. Subject's characteristics were comparable, except for higher nucleated cells and more female donors in the high CD3+ T-cell cohort. The 100-day cumulative incidence of acute GvHD (aGvHD) was 45±7% and 3-year cumulative incidence of chronic GvHD (cGvHD) was 28±6.7%. There was no statistically significant difference between the two cohorts in aGvHD (50% vs. 39%, P = 0.4) or cGvHD (29% vs. 22%, P = 0.7). The 2-year cumulative incidence of relapse (CIR) was 67.5±16.3% for low compared with 14.3±6.8% for high CD3+ T-cell cohort (P = 0.018). Fifteen subjects relapsed and 24 have died, 13 due to disease relapse. There was an improvement in 2-year RFS (94% vs. 83%; P = 0.0022) and 2-year OS (91% vs. 89%; P = 0.025) in low CD3+ T-cell cohort compared with high CD3+ T-cell cohort. Graft CD3+ T-cell dose is the only significant risk factor for relapse (P = 002), and OS (P = 0.030) in univariate analysis which was maintained in multivariate for relapse (P = 0.003), but not for OS (P = 0.050). Our data suggest that high graft CD3+ T-cell dose is associated with lower risk of relapse, and might improve long-term survival, but has no influence on the risk of developing aGvHD or cGvHD.

Experimental Models of Mouse and Human Hematopoietic Stem Cell Transplantation.

Experimental hematopoietic stem cell transplantation (HSCT) is an invaluable tool in determining the function and characteristics of hematopoietic stem cells (HSC) from experimental mouse and human donor groups. These groups could include, but are not limited to, genetically altered populations (gene knockout/knockin models), ex vivo manipulated cell populations, or in vivo modulated cell populations. The basic fundamentals of this process involve taking cells from a mouse/human donor source and putting them into another mouse (recipient) after preconditioning of the recipient with either total body irradiation (TBI) for mouse donor cells or into sublethally irradiated immune-deficient mice for human donor cells. Then, at pre-determined time pointspost-transplant, sampling a small amount of peripheral blood (PB) and at the termination of the evalaution, bone marrow (BM) to determine donor contribution and function by phenotypic analysis. Exploiting the congenic mouse strains of C57BL/6 (CD45.1- CD45.2+), BoyJ (CD45.1+ CD45.2-), and their F1-crossed hybrid C57BL/6×BoyJ (CD45.1+ CD45.2+), we are able to quantify donor, competitor, and recipient mouse cell contributions to the engraftment state. Human donor cell engraftment (e.g., from the cord blood [CB], mobilized PB, or BM) is assessed by human cell phenotyping in sublethally irradiated immune-deficient mouse recipients (e.g., NOD scid gamma mice that are deficient in B cells, T cells, and natural killer cells and have defective dendritic cells and macrophages). Engraftment of cells from primary mouse recipients into secondary mice allows for an estimation of the self-renewal capacity of the original donor HSC. This chapter outlines concepts, methods, and techniques for mouse and human cell models of HSCT and for assessment of donor cells collected and processed in hypoxia versus ambient air.

Challenges in Cell Fate Acquisition to Scid-Repopulating Activity from Hemogenic Endothelium of hiPSCs Derived from AML Patients Using Forced Transcription Factor Expression.

The generation of human hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) represents a major goal in regenerative medicine and is believed would follow principles of early development. HSCs arise from a type of endothelial cell called a “hemogenic endothelium” (HE), and human HSCs are experimentally detected by transplantation into SCID or other immune-deficient mouse recipients, termed SCID-Repopulating Cells (SRC). Recently, SRCs were detected by forced expression of seven transcription factors (TF) (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, and SPI1) in hPSC-derived HE, suggesting these factors are deficient in hPSC differentiation to HEs required to generate HSCs. Here we derived PECAM-1-, Flk-1-, and VE-cadherin-positive endothelial cells that also lack CD45 expression (PFVCD45−) which are solely responsible for hematopoietic output from iPSC lines reprogrammed from AML patients. Using HEs derived from AML patient iPSCs devoid of somatic leukemic aberrations, we sought to generate putative SRCs by the forced expression of 7TFs to model autologous HSC transplantation. The expression of 7TFs in hPSC-derived HE cells from an enhanced hematopoietic progenitor capacity was present in vitro, but failed to acquire SRC activity in vivo. Our findings emphasize the benefits of forced TF expression, along with the continued challenges in developing HSCs for autologous-based therapies from hPSC sources.

A retrospective comparative study of haplotype hematopoietic stem cell transplantation and human leukocyte antigen-matched sibling donor hematopoietic stem cell transplantation in the treatment of acute B-lymphocyte leukemia

目的探讨单倍型造血干细胞移植（haplo-HSCT）治疗移植前微小残留病（Pre-MRD）阳性急性B淋巴细胞白血病（B-ALL）是否较全相合同胞造血干细胞移植（MSDT）具有生存优势，以及该作用是否受Pre-MRD的影响。方法对2009年6月至2018年6月在北京大学血液病研究所接受异基因造血干细胞移植（allo-HSCT）的998例移植前处于完全缓解（CR）的B-ALL患者进行回顾性分析，其中haplo-HSCT组788例、MSDT组210例，移植前用多参数流式细胞术（MFC）检测MRD水平。结果①全部998例B-ALL患者中，997例获得持续的完全供者嵌合状态，移植后100 d中性粒细胞、血小板植入率分别为99.9％（997/998）、95.3％（951/998），Ⅱ～Ⅳ度急性移植物抗宿主病（GVHD）发生率为26.6％（95％CI 23.8％～29.4％），3年慢性GVHD累积发生率为49.1％（95％CI 45.7％～52.4％），移植后3年白血病累积复发率（CIR）为17.3％（95％CI 15.0％～19.7％），非复发死亡率（NRM）为13.8％（95％ CI 11.6％～16.0％），移植后3年无白血病生存（LFS）率、总生存（OS）率分别为69.1％（95％CI 66.1％～72.1％）、73.0％（95％CI 70.2％～75.8％）。②Pre-MRD阳性组（282例）患者移植后3年CIR高于Pre-MRD阴性组（716例）［31.6％（95％CI 25.8％～37.5％）对14.3％（95％CI 11.4％～17.2％），P<0.001］。③在Pre-MRD阳性组中，haplo-HSCT患者（219例）移植后3年CIR低于MSDT患者（63例）［27.2％（95％CI 21.0％～33.4％）对47.0％（95％CI 33.8％～60.2％），P＝0.002］。④全部998例患者按照Pre-MRD结果分为阴性组（716例）、<0.01％组（46例）、0.01％～<0.1％组（117组）、0.1％～<1％组（87例）、≥1％组（32例）；5组患者中，<0.01％组haplo-HSCT患者（40例）移植后3年CIR低于MSDT患者（6例）［10.0％（95％CI 0.4％～19.6％）对32.3％（95％CI 0％～69.9％），P＝0.017］，0.01％～<0.1％组haplo-HSCT患者（81例）移植后3年CIR也低于MSDT患者（36例）［20.4％（95％CI 10.4％～30.4％）对47.0％（95％CI 29.2％～64.8％），P＝0.004］；其他三组中，haplo-HSCT和MSDT患者移植后3年CIR差异无统计学意义。⑤在Pre-MRD<0.1％组（163例）中，haplo-HSCT患者（121例）移植后3年CIR低于MSDT患者（42例）［16.0％（95％CI 9.4％～22.7％）对40.5％（95％ CI 25.2％～55.8％），P<0.001］，3年LFS率、OS率均高于MSDT组［78.2％（95％CI 70.6％～85.8％）对47.6％（95％CI 32.2％～63.0％），P<0.001；80.5％（95％CI 73.1％～87.9％）对54.6％（95％CI 39.2％～70.0％），P<0.001］，两组3年NRM差异无统计学意义［5.8％（95％CI 1.6％～10.0％）对11.9％（95％CI 2.0％～21.8％），P＝0.188］。多因素分析显示，haplo-HSCT是Pre-MRD<0.1％组移植后低CIR（HR＝0.248，95％CI 0.131～0.472，P<0.001）、高LFS率（HR＝0.275，95％CI 0.157～0.483，P<0.001）和高OS率（HR＝0.286，95％CI 0.159～0.513，P<0.001）的独立影响因素。结论haplo-HSCT治疗Pre-MRD<0.1％的B-ALL患者较MSDT具有生存优势。

Integrated Analysis Toolset for Defining and Tracking Alloreactive T-cell Clones After Human Solid Organ and Hematopoietic Stem Cell Transplantation.

We have developed a suite of tools for integrated analysis of T-Cell-Receptor Sequencing data to define and track alloreactive T-cells in human transplant studies. This has enabled discovery of sequences and patterns of T-cell enrichment and deletion associated with clinical outcomes such as transplant rejection and tolerance. The codebase includes user-friendly default analyses with customizable parameters which greatly accelerate computational workflows and provide robust statistics comparing post-transplant specimens to pre-transplant baseline. It also includes helper functions for robust characterization of T-cell-repertoire diversity, sample-to-sample divergence, resolution of sample-of-origin ambiguity in pooled assays, and functions to output all sequences defined as alloreactive.

Development of a novel humanized mouse model for improved evaluation of in vivo anti-cancer effects of anti-PD-1 antibody.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer in the clinic. Further discovery of novel drugs or therapeutic protocols that enhance efficacy requires reliable animal models that recapitulate human immune responses to ICI treatment in vivo. In this study, we utilized an immunodeficient NOG mouse substrain deficient for mouse FcγR genes, NOG-FcγR−/− mice, to evaluate the anti-cancer effects of nivolumab, an anti-programmed cell death-1 (PD-1) antibody. After reconstitution of human immune systems by human hematopoietic stem cell transplantation (huNOG-FcγR−/− mice), four different programmed death-ligand 1 (PD-L1)-positive human cancer cell lines were tested. Among them, the growth of three cell lines was strongly suppressed by nivolumab in huNOG-FcγR−/− mice, but not in conventional huNOG mice. Accordingly, immunohistochemistry demonstrated the enhanced infiltration of human T cells into tumor parenchyma in only nivolumab-treated huNOG-FcγR−/− mice. Consistently, the number of human T cells was increased in the spleen in huNOG-FcγR−/− mice by nivolumab but not in huNOG mice. Furthermore, human PD-L1 expression was strongly induced in the spleen of huNOG-FcγR−/− mice. Collectively, our results suggest that the anti-cancer effects of anti-PD-1 antibodies can be detected more clearly in NOG-FcγR−/− mice than in NOG mice.

An Enhanced Zebrafish Xenograft Model to Study the Behavior of Human Hematopoietic Stem Cells and Patient Derived Leukemia In Vivo

Human hematopoietic stem cells (HSCs) are characterized by their unique ability to self-renew and differentiate into multiple cell types. Transplantation of human HSCs into immunocompromised mice is the gold standard for evaluating the in vivo repopulation capacity of these cells and has been adapted to evaluate the leukemic potential of leukemia-initiating cells (LICs) in acute leukemia. In spite of the popularity of this approach, certain cellular subsets do not survive post xenotransplantation as they are dependent on human factors that may be missing in the murine microenvironment. We pioneered human leukemia xenografts in the zebrafish model (Corkery et al., 2011; Bentley et al., 2015) as a parallel strategy, taking advantage of the immuno-permissiveness and transparency of zebrafish larvae to provide unprecedented continuous observation of leukemia evolution. However, as in mice, the addition of human cytokines and growth factors may further improve engraftment and optimize the utility of the zebrafish as a preclinical transplant model for medium to high-throughput therapeutic screening by creating a microenvironment more comparable to that found in patients.Using transgenic technology, we created novel zebrafish lines that express human stem cell factor (SCF/KITLG), granulocyte-macrophage colony stimulating factor (GM-CSF/CSF2), and stromal cell-derived factor 1 alpha (SDF1α/CXCL12). All of these factors are essential for the survival and expansion of HSCs, progenitors and LICs. CMK, GRWi (Down Syndrome- acute myeloid leukemia (DS-AML)) and Jurkat (T-acute lymphoblastic leukemia) cell lines were injected into the yolk sac of these multi-cytokine zebrafish embryos. These cells engrafted and exhibited increased cell proliferation and physiological migration compared to control casper embryo transplants. Moreover, only in the multi-cytokine fish did these cells home to hematopoietic niches, including the caudal hematopoietic tissue (CHT) (equivalent to the fetal liver) at 7 days-post injection (dpi) and the kidney marrow at 11 dpi.The yolk sac is not the physiological site for hematopoietic cell growth and thus we injected patient-derived AML cells into the circulation of the multi-cytokine fish. We observed similar trends in proliferation and migration as seen with leukemia cell lines. Historically, human HSCs do not survive beyond 24h in zebrafish xenografts (Pruvot et al. 2011). Given our success with patient-derived leukemia samples, we wanted to see if our multi-cytokine fish would provide a more favorable environment for human HSC survival. Indeed, primary human umbilical cord blood-derived HSCs were injected into the circulation of zebrafish larvae and survived past 3 dpi in the multi-cytokine fish but not in casper control larvae.Transgenic zebrafish expressing human cytokines provide an enhanced model for studying human leukemia and stem cell biology in a visually tractable system that more closely recapitulates the human microenvironment. The inherent capacity of the zebrafish for rapid and efficient chemical screening will be exploited in this model for preclinical testing of anti-leukemic therapies and to evaluate HSC enhancing agents in vivo . DisclosuresBerman:AGADA Therapeutics: Research Funding.

Radiation-induced bystander effects impair transplanted human hematopoietic stem cells via oxidative DNA damage

AbstractTotal body irradiation (TBI) is commonly used in host conditioning regimens for human hematopoietic stem cell (HSC) transplantation to treat various hematological disorders. Exposure to TBI not only induces acute myelosuppression and immunosuppression, but also injures the various components of the HSC niche in recipients. Our previous study demonstrated that radiation-induced bystander effects (RIBE) of irradiated recipients decreased the long-term repopulating ability of transplanted mouse HSCs. However, RIBE on transplanted human HSCs have not been studied. Here, we report that RIBE impaired the long-term hematopoietic reconstitution of human HSCs as well as the colony-forming ability of human hematopoietic progenitor cells (HPCs). Our further analyses revealed that the RIBE-affected human hematopoietic cells showed enhanced DNA damage responses, cell-cycle arrest, and p53-dependent apoptosis, mainly because of oxidative stress. Moreover, multiple antioxidants could mitigate these bystander effects, though at different efficacies in vitro and in vivo. Taken together, these findings suggest that RIBE impair human HSCs and HPCs by oxidative DNA damage. This study provides definitive evidence for RIBE on transplanted human HSCs and further justifies the necessity of conducting clinical trials to evaluate different antioxidants to improve the efficacy of HSC transplantation for the patients with hematological or nonhematological disorders.

Standard operating Protocol (SOP) of Haematopoietic Stem Cells Enumeration

Human hematopoietic stem cells (HSCs) are obtained either from the bone marrow (BM), cord blood (CB) or peripheral blood (PB). Transplantation of HSCs occurs following various conditions like high doses of chemotherapy, diseases like; leukaemia, lymphoma, congenital metabolic defects, immunedeficiency illnesses and myeloblastic syndromes.

Limited Mitochondrial Activity Coupled With Strong Expression of CD34, CD90 and EPCR Determines the Functional Fitness of ex vivo Expanded Human Hematopoietic Stem Cells

Ex vivo expansion strategies of human hematopoietic stem cell (HSC) grafts with suboptimal stem cell dose have emerged as promising strategies for improving outcomes of HSC transplantation in patients with hematological malignancies. While exposure of HSCs to ex vivo cultures expands the number of phenotypically identifiable HSCs, it frequently alters the transcriptomic and metabolic profiles, therefore, compromising their long-term (LT) hematopoietic reconstitution capacity. Within the heterogeneous pool of expanded HSCs, the precise phenotypic, transcriptomic and metabolic profile and thus, the identity of HSCs that confer LT repopulation potential remains poorly described. Utilizing valproic acid (VPA) in ex vivo cultures of umbilical cord blood (UCB)-CD34+ cells, we demonstrate that expanded HSCs phenotypically marked by expression of the stem cell markers CD34, CD90 and EPCR (CD201) are highly enriched for LT-HSCs. Furthermore, we report that low mitochondrial membrane potential, and, hence, mitochondrial activity distinguishes LT-HSCs within the expanded pool of phenotypically defined HSCs. Remarkably, such reduced mitochondrial activity is restricted to cells with the highest expression levels of CD34, CD90 and EPCR phenotypic markers. Together, our findings reveal that high expression of CD34, CD90 and EPCR in conjunction with low mitochondrial activity is critical for identification of functional LT-HSCs generated within ex vivo expansion cultures.

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.

A study of human leukocyte antigen-haploidentical hematopoietic stem cells transplantation combined with allogenic mesenchymal stem cell infusion for treatment of severe aplastic anemia in pediatric and adolescent patients.

The clinical applications of human leukocyte antigen (HLA) haploidentical hematopoietic stem cells transplantation (haplo‐HSCT) have offered most of the young severe aplastic anemia (SAA) patients an opportunity to accept curative therapy at the early stage of bone marrow lesions. However, the outcome of juvenile SAA patients received haplo‐HSCT remain to be improved due to high incidence of graft failure and graft vs host disease (GVHD). Mesenchymal stem cells (MSCs) have been characterized by their hematopoiesis‐supporting and immunomodulatory properties. In the current study, we designed a combination of haplo‐HSCT with allogenic MSC for treatment of SAA in pediatric and adolescent patients and evaluated its effects. Juvenile patients (<18 years) with SAA (n = 103) were given HLA‐haploidentical HSC combined with allogenic MSC after a conditioning regimen consisting of busulfan, cyclophosphamide, fludarabine, and antithymocyte globulin and an intensive GVHD prophylaxis, including cyclosporine, short‐term methotrexate, mycophenolate mofetil, and basiliximab. Neutrophil engraftment was achieved in 102 of 103 patients in a median time of 14.3 days (range 9‐25 days). The median time of platelet engraftment was 25.42 days (range 8‐93 days). The cumulative incidence of II‐IV acute GVHD at day +100 was 26.32% ± 0.19% and III‐IV acute GVHD was 6.79% ± 0.06% at day +100, respectively. The cumulative incidence of chronic GVHD was 25.56% ± 0.26%. The overall survival was 87.15% ± 3.3% at a median follow‐up of 40 (1.3‐98) months. Our data suggest that cotransplantation of HLA‐haploidentical HSC and allogenic mesenchymal stem cell may provide an effective and safe treatment for children and adolescents with SAA who lack matched donors.