bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5 Umbilical Cord Blood Treatment to Improve Gross Motor Function in Individuals with Cerebral Palsy: Results from an Individual Participant Data Meta-Analysis
Authors: Finch-Edmondson Megan , Paton Madison , Webb Annabel , Ashrafi Mahmoud , Blatch-Williams Remy , Cox, Jr. Charles , Crompton Kylie , Griffin Alexandra , Kim MinYoung , Kosmach Steven , Kurtzberg Joanne , Nouri Masoumeh , Sun Jessica , Zarrabi Morteza , Novak Iona
Published: September 2023

Introduction

Umbilical cord blood (UCB) is a novel treatment for cerebral palsy (CP) with clinical trials indicating UCB is safe and can improve gross motor function. However, heterogeneity across research design, participant selection and treatment protocols has limited the ability to interpret overall treatment effect, identify best responders and dosing thresholds.

Objectives

We sought to conduct a systematic review and individual participant data meta-analysis (IPDMA) to 1) determine the effect of UCB treatment measured by Gross Motor Function Measure (GMFM) in individuals with CP, and 2) determine the effect of UCB cell dose on clinical improvement.

Methods

CENTRAL, Embase and Medline were systematically searched for clinical studies administering UCB to individuals with CP with or without a control group/comparator, collecting GMFM scores at baseline and 1, 3, 6 and/or 12 months. Individual de-identified participant data were obtained before being cleaned and coded. A one-stage IPDMA was conducted in R to obtain the pooled effect of UCB and the pooled cell dose effect via linear mixed models, including random effects terms to investigate between-study heterogeneity.

Results

Individual participant data from nine published and two unpublished studies (seven RCTs and four single-arm studies) were obtained, comprising 285 UCB (+/- erythropoietin, EPO)- treated participants and 171 controls for analysis. Median baseline age was 43 months (range 8-227). Most UCB infusions were allogeneic (80%), and 10/11 studies applied UCB intravenously. The median pre-thaw dose of intravenous-infused UCB was 46.7x10^6 total nucleated cells/kg (range 7.7-210.3).

The pooled effect at 6 months post-intravenous UCB was an average change from baseline GMFM-66 of 0.93 (95% CI: 0.12, 1.75; p=0.027) points greater among UCB-treated participants (+/- EPO) compared to controls. There was also a statistically significant positive pooled cell dose effect at 6 months post-intervention (p=0.005), indicating that higher cell doses were associated with greater GMFM-66 increases. There was negligible between-study heterogeneity in both the treatment effect and the cell dose effect at 6 months.

Discussion

UCB improves gross motor function, and higher dose is beneficial. Future exploration of additional timepoints in addition to co-variates including age at infusion, severity, and etiology is underway and may elucidate responder subgroups and reveal increased efficacy.
Abstract 9 Identification of Effective Changes to Workflow Triaging Criteria and Production Planning
Authors: Shields Sara , Walker Molly , Laster Candice , DeDino Faith , Wood Bridget , Bobik-Kurz Kathy , Thut Dawn , Vincent Kim
Published: September 2023

Introduction

The Cleveland Cord Blood Center (CCBC) analyzes fresh incoming cord blood units for the total nucleated cell (TNC) count. Units that meet the TNC criteria are processed for clinical banking and units not meeting the defined criteria are made available for research collaborations. CCBC sought to understand if adding additional pre-screening criteria would reduce post-manufacturing deferral rates and increase production of licensed units.

Objectives

To identify the main failure modes resulting in cord blood unit deferral for clinical banking post-manufacturing. To identify key factors resulting in unlicensed status of processed cord blood units. To implement changes to pre-screening strategies to reduce deferral rates and increase production of licensed units.

Methods

CCBC created four cross-departmental teams to evaluate the main reasons for deferrals post-manufacturing and to evaluate travel-risk history. Each committee had representation from collections, quality, and laboratory staff. Data was evaluated that was collected between January and June 2022.

Results

The committees identified that 19% of all deferrals post-manufacturing were related to low CD34 count and/or viability and that 17% of all processed units were unlicensed due to travel to Zika related areas. In March 2023, pre-screening criteria was updated to include a pre-processed CD34 count and viability and a pre-screening of the donating mother for travel history after consenting and prior to manufacturing. Units that fulfilled both the TNC and CD34 cut offs and did not have a high-risk travel history were processed for clinical banking. Data evaluated from March and April 2023 indicated that the deferral rates for low viable CD34 counts post-manufacturing went from 19% down to 0% and the percentage of high-risk travel units manufactured was reduced from 17% to 7%.

Discussion

Review of failure modes identified two main factors to guide improved triage efficiency. By implementing a pre-processing CD34 count, the larger TNC and CD34 cord blood units were identified for banking for clinical use. By using a pre-screening process at collection sites, high-risk travel donations were identified prior to processing. In the two-month timeframe evaluated, the overall deferral rates were reduced by roughly 7% and post-manufacturing licensure status increased from 83% to 93%.
Abstract 10 Experience with Enrollments and Releases Within a Medical Needs Program at a Large U.S. Private Cord Blood Bank
Authors: Fernekes Michelle , Brown Heather , Pandolfi Erica , Stroh Eliza , Isley Lauren
Published: September 2023

Introduction

Medical Needs / Directed Donor programs provide families with access to cord blood for potential future use. In situations where a diagnosis, potentially treatable with cord blood, exists in an immediate relative of the newborn, these programs often allow families to preserve newborn stem cells at little to no cost.

Objectives

The objective of this study was to examine indications for enrollment and unit releases in a medical needs program at a large U.S. private newborn stem cell bank.

Methods

Enrollment data over a 24-year period was analyzed. Data was categorized by indications for enrollment and indications for which units were released.

Results

There was a total of 7,034 enrollments within the specified timeframe. Most enrollments were for indications treatable by hematopoietic stem cell transplant given the program did not include experimental indications until 2019. Approximately one-third of enrollments (33.4%) for transplant indications were for various types of leukemia. The experimental indications most frequently represented in enrollments were autism (44.3%) and cerebral palsy (38.4%).

For all cord blood units stored under the medical needs program, the majority (70%) of releases were intended for use in transplant medicine. The most frequently represented transplant indication for releases was hemoglobinopathies including sickle cell anemia and beta thalassemia (44.6%). Approximately one-third (36.1%) of transplant releases were related to leukemia. Autism and cerebral palsy were the two most frequently represented experimental indications for units released at 39% and 36%, respectively.

Discussion

Clinical indications were mostly consistent between enrollments and releases through this cohort. This suggests the structure of the program is effective in identifying families who have a higher likelihood of utilizing their cord blood unit for a transplant or experimental infusion.

Cord blood banks should continue to re-evaluate the eligibility criteria for their medical needs programs in light of relevant updates in the potential utility of newborn stem cells including changes in transplant medicine indications and treatment procedures, as well as clinical trials and experimental protocols using related cord blood units. Future research related to pathways of referral to medical needs programs may inform education gaps among women’s health providers.
Abstract 13 Long-Term Stability of Dus Cord Blood Units After >29 Years of Cryopreservation: Follow-Up Data from the José Carreras Cord Blood Bank
Authors: Liedtke Stefanie , Többen Sabine , Gressmann Holger , Meyer Andrea , Gluckman Eliane , Kögler Gesine
Published: September 2023

Introduction

The José Carreras Cord Blood Bank (CBB) located in Düsseldorf stores 21215 active cryopreserved cord blood units (CBUs) and has released 1476 CBUs for allogeneic transplantation. The success of transplantations, but also the generation of cells for regenerative medicine for e.g. iPSC reprogramming, significantly depends on the quality of the cryopreserved CBUs after thaw.

Objectives

Therefore, typical parameters like cell count, recovery and viability are analysed from thawed CBUs yearly by a stability monitoring program according to the FACT standards. The longest cryopreservation period in liquid nitrogen tested to date is 338 months (>29 years) for unseparated units and 300 months (25 years) for volume-reduced units that are licensed by the Paul-Ehrlich Institute.

Methods

A retrospective analysis of unseparated CBUs processed from 1993-1997, CBUs processed from 1998-2005 by manual volume-reduction and CBUs processed since 2005 by automated volume-reduction applying the Sepax100 system was performed. In consideration of the distinct processing methods, storage time and existing follow-up data of released transplants, this approach should demonstrate the high CBU quality and proof the long-term engrafting capability of CBUs cryopreserved in liquid nitrogen.

Results

According to the CBB stability program means of TNC count (16.32±2.61 x 108), TNC recovery after thaw (106.8±13.91%, above 100% due to earlier applied cell-count method), TNC viability (88.91±5.01%), CD34+7AAD- viability (90.73±3.69%), CD45+7AAD- viability (69.09±5.38%) and CFC count (7.10±2.45 x 106) were determined after >29 years of cryopreservation for unseparated CBUs. In volume-reduced CBUs the mean TNC count (9.37±2.14 x 108), TNC recovery (97.78±3.70%), TNC viability (84.22±10.02%), CD34+7AAD- viability (88.11±5.16%), CD45+7AAD- viability (73.67±8.31%) and CFC count (1.37±0.59 x 106) were determined after 25 years of cryopreservation.

Discussion

In addition, these relevant parameters were retrospectively analyzed for released transplants in correlation to the storage time. The follow-up data of recipients from CBUs demonstrate an earlier engraftment of volume-reduced CBUs as compared to unseparated CBUs in accordance to an increased TNC count over time. With regard to applied processing methods, engraftment time and patient age (children and adults) this retrospective analysis confirms the high quality of old CBUs stored in the liquid phase at -196°C.
Abstract 16 Insights into Highly Engraftable Hematopoietic Cells from 27-Year Cryopreserved Umbilical Cord Blood
Authors: Broxmeyer Hal , Luchsinger Larry , Weinberg Rona , Jimenez Alexandra , Frenet Emeline Masson , Hof Wouter van't , Capitano Maegan , Hillyer Christopher , Kaplan Mark , Cooper Scott , Ropa James
Published: September 2023

Introduction

Cord blood banking has consistently outpaced the utilization of cord blood units (CBUs). Thus, the average duration of cryopreservation among banked CBUs will likely continue to increase. It remains unclear how long cryopreserved CBUs remain functional, and it is critical to determine whether duration of cryopreservation should be used as an exclusionary criterion during selection for clinical use or if alternative post-thaw metrics can identify potent cryopreserved CBUs regardless of age.

Objectives

Our goal was to determine whether long-term (27-year) cryopreserved CBUs retain viable and functional hematopoietic stem (HSCs) and progenitor cells (HPCs). We further sought to leverage differences in HSC/HPC function (measured by in vivo engraftment) to demonstrate the utility of using omics approaches to identify candidate genes for use as molecular potency markers.

Methods

We performed comprehensive ex vivo, in vivo, and molecular analyses on the numbers, viability, and function of three 27-year cryopreserved CBUs using 3-year cryopreserved and fresh CBUs for comparison. Assays included viability staining, immunophenotyping by flow cytometry, primary and secondary colony forming unit (CFU) assays, ex vivo expansion of immunophenotypic HSCs/HPCs/CFUs, limiting dilution transplantations into immune-deficient mice, secondary transplantations, and RNA-sequencing of sorted HSCs and multipotent progenitor cells.

Results

Compared to fresh and recently cryopreserved CBU controls, long-term cryopreserved CBUs yield statistically similar numbers of viable immunophenotypic HSCs, multipotent HPCs, and committed myeloid and lymphoid HPCs. They retain highly functional cells, demonstrating similar primary and secondary CFU numbers and expansion capacity compared to controls, as well as robust engraftment, SCID repopulating cell frequency, and secondary engraftment capacity in mouse models of transplantation. Transcriptomic modelling revealed 18 genes, including MALT1 and MAP2K1, and several gene programs, including lineage determination programs and oxidative stress responses, that are strongly enriched in high engrafting HSCs/HPCs.

Discussion

CBUs cryopreserved for up to 27 years retain highly functional HSCs/HPCs. Thus, duration of cryopreservation alone is not an ideal exclusionary criteria for selection of CBUs. Preserving older CBUs may help to maintain a large and diverse pool of donors for clinical selection. Further, transcriptomics can identify candidate genes associated with engraftment for elucidation of possible CBU potency markers regardless of the duration of cryopreservation.
Abstract 19 CD34+ Cell Isolation from Cord Blood: A Novel Option for Faster, More Consistent Outcomes
Authors: Fedenko Shannon , Sherouse Hallie , Messer Jonathan , Dailey Jordan , Namin Shabnam
Published: September 2023

Introduction

Immunomagnetic isolation of cells has been long established but still suffers from drawbacks when used for hemopoietic stem cells (HSCs) in cord blood. [Kekarainen et al., 2006] Limitations with HSC isolation from cord blood include steep learning curves, time consuming manual labor, and inconsistent outcomes. [de Wynter et al., 1999; Perdomo-Arciniegas and Vernot, 2011] FerroBioTM has developed a semi-automated system for CD34+ cell isolation from unmodified cord blood (Fig 1A) as a means to provide fast, effective, and consistent isolations.

Objectives

The purpose of this work is to explore process improvements for the FerroBioTM system. Additionally, this work aims to characterize the resulting cell isolates.

Methods

The cell isolation protocol was evaluated by flow cytometry for cell counts, viability, and purity (total n=195). Protocol modification included incubation time at various steps, magnetic bead dosing, temperatures, and volumes. The ‘percent of cells captured’ was calculated as the difference between starting CD34 and non-captured CD34 cells. Cryopreservation tolerance was evaluated by freeze-thawing cells in a dimethyl sulfoxide-based media and measuring viability via hemocytometry (n=3). Sterility testing on cell isolates was performed via BacT/ALERT iNST & iAST (n=2).

Results

Incubation time did not have a significant effect on the efficiency of bead-cell binding (Fig 1B). As the FerroBioTM protocol progressed, final cell purity and viability increased and became more consistent (Fig 1C, D). Additionally, over the same phases of process development, final cell recovery increased 4-fold, from 6.5%±5.9% to 28.1%±9.7%. Post-cryopreservation viability was 78.8%±3.3%. Sterility tests were negative for anaerobic and aerobic bacteria.

Discussion

By establishing that bead-cell incubation duration did not significantly influence outcomes, the incubation time was considerably reduced, bringing the total processing time to ~3hrs. That said, if an overnight incubation is preferable for an operator’s workflow, the data suggests the process could be flexible to meet such needs. Iterations of the process have also resulted in significant improvements to final cell recovery while maintaining high purity and viability. Additional work is being done to further improve recovery. The initial investigation of sterility and cryopreservation was encouraging, and assays for cell functionality are underway.

Figure 1. (A) The FerroBioTM hardware, shown with a cartridge in which cell isolation occurs. (B) The proportion of cells that successfully bound magnetic beads as a function of bead-cell incubation time. Final cell (C) purity and (D) viability, shown at different phases of chronological process development. Statistics: GraphPad Prism 9, 1-way ANOVA followed by a Tukey’s HSD. * p < 0.05, **** p < 0.0001. Each data point corresponds to one CBU.
Abstract 21 Machine Learning Approach for Automatic Enumeration of Cord Blood Stem Cells by Flow Cytometry
Authors: Trépanier Patrick , Simard Carl , Fournier Diane
Published: September 2023

Introduction

Stem cell laboratories measure the number of viable CD34+ and CD45+ cells in their products by flow cytometry following a standard protocol (ISHAGE). Although the ISHAGE protocol is well documented, its correct application can be challenging. Automated gating algorithms have been created to address this issue, but they have limitations and can only be used in certain conditions. Recent developments in artificial intelligence (AI) and machine learning (ML) have made these approaches more accessible and practical to use. For flow cytometry data, the Cytobank platform now offers the Automatic Gating Algorithm (AGA), an AI-ML-based feature allowing users to easily implement their own gating strategies.

Objectives

The performance of the AGA based on the ISHAGE protocol gating was evaluated using cord blood post-thaw flow cytometry data.

Methods

In this study, the AGA was trained using 29 flow cytometry cord blood data files analyzed according to the ISHAGE gating strategy. Using the model, an inference analysis was conducted on two series of 12 data samples acquired from two independent operators. The enumeration of viable CD34+ cells was manually performed and compared to the AI analyses.

Results

The mean differences and standard deviation in vCD34+ counts between the manual and the automatic gating are 6.0% ± 2.7 for Operator #1 and 7.7% ± 2.1 for Operator #2. The correlation coefficients between vCD34+ results are 0.9981 for Operator #1 vs AI, and 0.9699 for Operator #2 vs AI. These results show that the AI inference provides results comparable to human manual gating, and are within the accepted difference of 10% between duplicates as defined by ISHAGE convention.

Discussion

This study provides a testing ground for using AI-ML-based automatic gating and supports its use in stem cell laboratories with acceptable performance and accuracy. The application of AI and ML for vCD34+ enumeration can potentially lead to the development of more widely applicable automatic gating algorithms. This could improve standardization by providing a common tool for analyzing flow cytometry data.
Abstract 22 Anomalous HLA-A Typing Found in Patient Using PacBio Sequencing Technology
Authors: Peavy Lydia , Norvell Brian , Liu Haipeng , Steege Eline , Williams Jon
Published: September 2023

Introduction

Labcorp’s DNA Identification division offers HLA typing at various levels of resolution using commercially available kits as well as lab developed assays. A patient sample was received by the lab requiring HLA typing for a disease association screen, and was found to have anomalous HLA-A results.

Objectives

To investigate a patient typing with results initially showing multiple variants to any known reference sequence.

Methods

This sample was sequenced using a lab developed whole gene assay that utilizes Pacific Biosciences’s® (PacBio) SMRT® NGS sequencing technology on a Sequel® I with LAA2 contig generation. Due to ambiguous results, the sample was repeated for confirmation using the same test, but a more sensitive data pipeline was used. Subreads coming off of the sequencer were converted to high fidelity or Closed Circular Consensus (CCS) reads using the PBAA tool from PacBio®. The sample was also processed with the GenDx NGSgo®-MX11-3 HLA typing kit with Illumina® sequencing, and the results were sent to GenDx for analysis.

Results

Initial testing yielded a high resolution fourth field result for HLA Class I, though the result remained ambiguous. Repeat sequencing resulted three HLA-A alleles with a similar number of CCS reads. The sample was typed as A*01:01:01:01, A*02:01:01:01, and A*02:New which contained a large exonic deletion compared to the A*02:01:01:01 reference. GenDX output confirmed previous findings, indicating a clear first allele of A*01:01:01:01, and reads for the second allele split between A*02:01:01:01 and A*02:New.

Discussion

Using Pacbio® sequencing technology with CCS analysis, and confirmation with GenDx technology, it was determined that this individual possesses 3 HLA-A alleles. When aligned with 02:01:01:01, both Pacbio® and GenDx assays determined the A*02:New allele to have a 34bp deletion in exon 2 (position 196-229). As a result of the frame shift caused by this deletion, a premature stop codon was found 43bp downstream from the anomaly, potentially rendering this allele null. Possible explanations for the presence of three HLA-A alleles in one patient include partial allele loss during an autoimmune event, a mutation within a chromosomal duplication of the region, or the patient has undergone a hematopoietic stem cell transplant.
Abstract 23 HLA-Homozygous iPSC from Cord Blood for the Generation of Cardiomyocytes for Allogeneic Cell Therapy to the Heart
Authors: Becker Fabienne , Liedtke Stefanie , Greber Boris , Kriedemann Nils , Martin Ulrich , Zweigerdt Robert , Koegler Gesine
Published: September 2023

Introduction

Based on a previous grant on HLA-homozygous (HLAh) iPS-GMP (and ATMP conform) generation from allogeneic licensed cord blood (CB) units, the Düsseldorf CBB could provide the most frequent HLA-types for reprogramming (Liedtke et al. 2020). These HLAh iPSC provide the basis for the generation of cardiomyocytes from iPSC. Patients with acute heart failure or cardiomyopathies have limited options of treatment. iPSC technology allows for de novo generation of functional heart muscle in vitro, enabling the implementation of new therapy concepts for heart regeneration.

Objectives

The GMP-grade iPSC-lines are the backbone for the granted EU project (HEAL (101056712) on iPSC-cardiomyocyte aggregate manufacturing for allogeneic cell therapy. Besides GMP-compliant up-scaled cell production of cardiomyocytes, the project is designed to overcome many of the scientific, safety and regulatory hurdles necessary to initiate a first-in-man clinical HLAh-based heart regeneration study.

Methods

After CD34+ selection/short expansion of CD34, cells were reprogrammed by GMP-conform plasmids and stored in a master cell bank with most frequent HLA-haplotypes. All iPSC lines were tested extensively for genetic stability, mycoplasma, virus markers, endotoxins and microorganisms. The differentiation into cardiomyocytes was performed in correspondence to the Wnt modulation, i.e. first activating Wnt pathway and then inhibiting the pathway with the addition of small molecules (Halloin et al. 2019). For the up-scaled production, a 3D-cardiomyocyte aggregate culture in large stirred tank bioreactors (STBR) was applied. Cryopreservation using controlled rate freezing was adapted to tissue freezing.

Results

Selection of the most frequent homozygous haplotypes facilitated the production of HLAh iPSC lines for differentiation into cardiomyocytes. Efficient progress had been made for the improvement of STBR platform for sufficient production of cardiomyocytes, enabling the differentiation with a purity of up to 95%, measured via cardiac troponin T (cTnT).

Discussion

Although high yields of cardiomyocytes in a GMP-compliant process is getting more achievable by using STBRs, the safety and regulatory hurdles are still a major problem. For safety testing, toxicology, tumorigenicity assays will be performed in mice and in pig models. This will enable the prediction of arrhythmia-related risks. For a safe distribution of the finally released cell product optimization of cryopreservation of cardiomyocytes aggregates will be performed.

Disclaimer: Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Health and Digital Executive Agency (HADEA). Neither the European Union nor the granting authority can be held responsible for them.
Abstract 24 Development of an In Vitro B-Cell Differentiation Model for the Characterization of the Origin and Causation of ETV6::RUNX1+ Preleukemic Cells
Authors: Bhave Rigveda , Kameri Ersen , Körschgen Lutz , Jepsen Vera , Hecker Katharina , Pandyra Aleksandra , Borkhardt Arndt , Fischer Ute , Koegler Gesine
Published: September 2023

Introduction

Pregnant women are exposed to various carcinogenic substances that can subsequently affect their progeny. Recurrent preleukemic chromosomal translocations characteristic for childhood acute lymphoblastic leukemia (ALL) frequently emerge before birth in utero. The most common translocation t (12;21) occurs in 25% of B-cell precursor ALL and results in the formation of the chimeric oncogenic transcription factor ETV6::RUNX1.

Objectives

We aim to analyze factors promoting the generation of preleukemic ETV6::RUNX1 translocations, investigate the biological origin of these preleukemic cells, and develop an in vitro model of early B-cell differentiation to analyze the mechanisms underlying the persistence of ETV6::RUNX1+ preleukemic cells and leukemia evolution in children.

Methods

Genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) (Schafer et. al., 2018) was applied to identify ETV6::RUNX1+ cord blood units (CBU). Screening results were validated by Sanger sequencing and quantified by real-time PCR (ipsogen ETV6-RUNX1 Kit, Qiagen). ETV6::RUNX1+ CBUs were thawed, sorted for CD34+ and CD19+ cells, and analyzed by qPCR. CD34+ were differentiated towards B-cell lineage on MSC feeder lines according to a modified protocol (Ichii et. al., 2008).

Results

We screened a cohort of 1405 cord bloods collected in 2004 and discovered that 103 (7.3%) carried the ETV6::RUNX1 fusion gene. The preleukemic translocation was detected in CD34+ stem and early B progenitor cells. For the establishment of an in vitro B-cell differentiation model, bone marrow (BM-MSCs) and CB (USSCs, CB-MSCs) stromal feeder cells were compared and the effect of different media (SCGM, DMEM/5 % FCS, MSCBM/10 % FCS) in the presence of stem cell factor (SCF), Flt-3, IL3, +/- IL7 was assessed. BM-MSCs demonstrated the highest ability to support B-lymphopoieses towards CD19+ cell differentiation in MSCBM media (~30%), primarily due to the release of IL-7 by the feeder layer. Currently, factors that may promote generation of preleukemic translocations in utero e.g. nitrosamines contained in food products, are being tested in this model.

Discussion

We identified the HSC/early B progenitor state as the origin of ETV6::RUNX1 translocations in CB. The establishment of an in vitro B-cell differentiation system will allow to test the impact of environmental carcinogens on the generation of ETV6::RUNX1+ pre-leukemic cells and their leukemic evolution.
Abstract 26 The Role of Oxygen in Cord Blood Hematopoietic Stem and Progenitor Cell Expansion and Engraftment
Authors: Ropa James , Gutch Sarah , Beasley Lindsay , Hof Wouter Van't , Kaplan Mark , Capitano Maegan
Published: September 2023

Introduction

Hematopoietic stem (HSC) and progenitor cells (HPCs) are exposed to differing oxygen tensions ranging from <1% to 21% as they reside in/move through different tissues or are harvested for clinical utility. Functional changes in HSCs/HPCs are induced by acute changes in oxygen tension (e.g., a change in percent of cells in cycle).

Objectives

We sought to determine if variable oxygen levels affect expansion and/or functional properties of cord blood (CB) HSCs/HPCs ex vivo and in vivo.

Methods

Human CB CD34+ cells were grown in expansion culture +/-UM171, an agonist of HSC self-renewal that expands transplantable CB HSCs, in five oxygen tensions: 1%O2, 3%O2, 5%O2, 14%O2, and 21%O2. HSCs/HPCs were enumerated by flow cytometry. Functional HPCs were enumerated by plating in semi solid media for colony forming unit assays (CFU). Cell cycle and reactive oxygen species (ROS) were measured by flow cytometry. Ability of expanded cells to engraft was determined by transplantation in non-lethally irradiated NSG mice.

Results

Immunophenotypic HPCs and functional HPC CFUs expanded significantly more after 7 days of growth in higher oxygen tensions (5%O2-21%O2) compared to lower (1%O2-3%O2), while immunophenotypic HSCs expanded best at 5% O2. HSCs/HPCs grown in low oxygen tensions had significantly lower ROS levels, significantly higher percentage of cells in G0, and were slightly but reproducibly smaller/less granular than those grown in high oxygen levels. HSC/HPC numbers were reduced in high oxygen tensions 1-2 days after plating but were better maintained in low, suggesting cells undergo a culture shock/stress after plating that is mitigated by reduced oxygen. In the presence of UM171, HSCs expanded significantly better at higher oxygen levels, but HPCs are better maintained in 5%O2. Ex vivo CD34+ expansions maintained under physiological O2 levels (1-14%O2) demonstrated significantly better/faster neutrophil recovery following transplantation compared to cells expanded at 21%O2 or input.

Discussion

HSCs/HPCs proliferate rapidly in high oxygen but have fewer quiescent cells, higher ROS, and are larger and more granular which are all characteristics associated with exhaustion. While high oxygen allows for faster growth, low tensions may mitigate cell stress and allow for prolonged growth (i.e., HSC/HPC expansion) while maintaining functional properties.
Abstract 29 Umbilical Cord-derived Mesenchymal Stromal Cells Suppress Microglia Activation Induced by Demyelination
Authors: Xu Li , Min Hyunjung , Parrott Roberta , Kurtzberg Joanne , Filiano Anthony
Published: September 2023

Introduction

White matter diseases are often accompanied by microglial activation and neuroinflammation. Mesenchymal stromal cells (MSCs) manufactured from umbilical cord tissue possess immunomodulatory properties, making them promising candidates for cell therapy in white matter disease. MSCs can interact with resident macrophages and modify the course of inflammation. However, the influence MSCs have on microglia, the resident macrophages of the central nervous system (CNS), and its implications for white matter disease remain uncertain.

Objectives

From the blood, MSCs traffic to lung and interact with resident macrophages to alter the trajectory of inflammation. We hypothesized that MSCs injected into the cerebrospinal fluid (CSF) would similarly suppress the activation of microglia. Our goals in this study were to test the efficacy MSCs have on microglia activation and develop a relevant potency assay.

Methods

In this study, we employed a range of assays, including an in vivo acute spinal cord demyelination model, organotypic brain slice cultures, and microglia activation assays with primary microglia and an immortalized cell line. Using these assays, we assessed the impact cord tissue derived MSCs have on microglial activation.

Results

In spinal cords and brain slices, lysophosphatidylcholine produced robust demyelination and microglia activation that was suppressed by MSCs. To determine if MSCs could directly impact microglia, we isolated primary microglia and activated them in culture with lipopolysachride (LPS), a toll-like receptor agonist. We screen 6 cytokines induced by LPS and determined that MSCs suppress the release of tumor necrosis factor (TNF) from LPS-activated microglia. We confirmed these results in vivo as MSCs blocked TNF production by spinal cord microglia after acute demyelination in the spinal cord. Additionally, we confirmed the ability of MSCs to suppress TNF in a simple assay with a microglia cell line (IMG).

Discussion

In this study, we demonstrated that cord tissue MSCs altered the immune response, suppressed microglia activation, and the release of TNF, a cytokine known to contribute to white matter damage. Among these assays, IMG could be standardized as an effective potency assay to determine the efficacy of MSC for the use in treatment of white matter disease.
Transcriptional Targets of TWIST1 in Human Mesenchymal Stem/Stromal Cells Mechanistically Link Stem/Progenitor and Paracrine Functions
Authors: Haga Christopher L , Booker Cori N , Carvalho Ana , Boregowda Siddaraju V , Phinney Donald G
Published: September 2023

Despite extensive clinical testing, mesenchymal stem/stromal cell (MSC)-based therapies continue to underperform with respect to efficacy, which reflects the paucity of biomarkers that predict potency prior to patient administration. Previously, we reported that TWIST1 predicts inter-donor differences in MSC quality attributes that confer potency. To define the full spectrum of TWIST1 activity in MSCs, the present work employed integrated omics-based profiling to identify a high-confidence set of TWIST1 targets, which mapped to cellular processes related to ECM structure/organization, skeletal and circulatory system development, interferon gamma signaling, and inflammation. These targets are implicated in contributing to both stem/progenitor and paracrine activities of MSCs indicating these processes are linked mechanistically in a TWIST1-dependent manner. Targets implicated in extracellular matrix dynamics further implicate TWIST1 in modulating cellular responses to niche remodeling. Novel TWIST1-regulated genes identified herein may be prioritized for future mechanistic and functional studies.
Hydraulic traits and photosynthesis are coordinated with trunk sapwood capacitance in tropical tree species
Authors: Wei Yang , Chen Ya-Jun , Siddiq Zafar , Zhang Jiao-Lin , Zhang Shu-Bin , Jansen Steven , Cao Kun-Fang
Published: September 2023

Water stored in trunk sapwood is vital for the canopy to maintain its physiological function under high transpiration demands. Little is known regarding the anatomical properties that contribute to the hydraulic capacitance of tree trunks and whether trunk capacitance is correlated with the hydraulic and gas exchange traits of canopy branches. We examined sapwood capacitance, xylem anatomical characteristics of tree trunks, embolism resistance, the minimal xylem water potential of canopy branches, leaf photosynthesis and stomatal conductance in 22 species from a tropical seasonal rainforest and savanna. The results showed that the mean trunk sapwood capacitance did not differ between the two biomes. Capacitance was closely related to the fiber lumen fraction and fiber wall reinforcement and not to the axial and ray parenchyma fractions. Additionally, it was positively correlated with the theoretical hydraulic conductivity of a trunk and the specific hydraulic conductivity of branches, and showed a trade-off with branch embolism resistance. Species with a high trunk sapwood capacitance maintained less negative canopy water potentials in the dry season, but higher leaf photosynthetic rates and stomatal conductance in the wet season. This study provides a functional link among trunk sapwood capacitance, xylem anatomy, canopy hydraulics and photosynthesis in tropical trees.
No secondary impact of ivermectin mass drug administration for onchocerciasis elimination on the prevalence of scabies in northwestern Ethiopia
Authors: Yirgu Robel , Middleton Jo , Fekadu Abebaw , Davey Gail , Bremner Stephen , Jones Christopher Iain , Cassell Jackie A
Published: September 2023

Background

Mass drug administration (MDA) is among the five major strategies that are currently in use to control, eliminate or eradicate Neglected Tropical Diseases (NTDs). Optimising MDA to control multiple NTDs maximises impact. The objective of this study is to estimate the secondary impact of ivermectin MDA for onchocerciasis on the prevalence of scabies.

Methods

This quasi-experimental study was conducted in Ayu Guagusa district, northwestern Ethiopia. Scabies prevalence was estimated in surveys before the MDA, at 6 and 12 months afterwards. The sample size was 1437 people from a panel of 381 randomly selected study households. Multistage sampling was employed in randomly selecting six kebeles (the lowest administrative unit) with respective gotes (small villages) and households. All members of the selected households were invited to participate in the study and participants who were available in all three surveys formed a cohort.

Results

Scabies prevalence was similar prior to the MDA (13.4%, 95% CI 11.7 to 15.2%) and 6 months after (11.7%, 95% CI 10.1 to 13.2%) but was substantially greater at 12 months (22.1%, 95% CI 20.1 to 24.1%). The 6-month incidence and disappearance rates were 10.8% (95% CI 8.8 to 13.2%) and 82.6% (95% CI 75.0 to 88.6%), respectively.

Conclusions

Ivermectin MDA for onchocerciasis was not observed to have a secondary impact on the prevalence of scabies over the follow-up period of 12 months.
A tale of 141 municipalities: the spatial distribution of dengue in Mato Grosso, Brazil
Authors: Fernandes Keli Aparecida Paludo , de Almeida Filho Ariel Rocha , Moura Alves Taynná Vacaro , Bernardo Christine Steiner São , Montibeller Maria Jara , Mondini Adriano , Bronzoni Roberta Vieira de Morais
Published: September 2023

Background

In recent years, the state of Mato Grosso has presented one of the highest dengue incidence rates in Brazil. The meeting of the Amazon, Cerrado and Pantanal biomes results in a large variation of rainfall and temperature across different regions of the state. In addition, Mato Grosso has been undergoing intense urban growth since the 1970s, mainly due to the colonization of the Mid-North and North regions. We analyzed factors involved in dengue incidence in Mato Grosso from 2008 to 2019.

Methods

The Moran Global Index was used to assess spatial autocorrelation of dengue incidence using explanatory variables such as temperature, precipitation, deforestation, population density and municipal development index. Areas at risk of dengue were grouped by the Local Moran Indicator.

Results

We noticed that areas at risk of dengue expanded from the Mid-North region to the North; the same pattern occurred from the Southeast to the Northeast; the South region remained at low-risk levels. The increase in incidence was influenced by precipitation, deforestation and the municipal development index.

Conclusions

The identification of risk areas for dengue in space and time enables public health authorities to focus their control and prevention efforts, reducing infestation and the potential impact of dengue in the human population.
Cocoa Apoplastome Contains Defense Proteins Against Pathogens
Authors: de Oliveira Ivina Babosa , Moura Igor Moutinho , Santana Juliano Oliveira , Gramacho Karina Peres , dos Santos Alves Saline , Ferreira Monaliza Macêdo , Santos Ariana Silva , de Novais Diogo Pereira Silva , Pirovani Carlos Priminho
Published: September 2023

The apoplast performs important functions in the plant, such as defense against stress, and compounds present form the apoplastic washing fluid (AWF). The fungus Moniliophthora perniciosa, the causal agent of witches’ broom disease (WBD) in Theobroma cacao, initially colonizes the apoplast in its biotrophic phase. In this period, the fungus can remain for approximately 60 days, until it changes to its second phase, causing tissue death and consequently large loss in the production of beans. To better understand the importance of the apoplast in the T. cacao– M. perniciosa interaction, we performed the first apoplastic proteomic mapping of two contrasting genotypes for WBD resistance (CCN51-resistant and Catongo-susceptible). Based on two-dimensional gel analysis, we identified 36 proteins in CCN-51 and 15 in Catongo. We highlight PR-proteins, such as peroxidases, β-1,3-glucanases, and chitinases. A possible candidate for a resistance marker of the CCN-51 genotype, osmotin, was identified. The antioxidative metabolism of the superoxide dismutase (SOD) enzyme showed a significant increase ( P < 0.05) in the AWF of the two genotypes under field conditions (FD). T. cacao AWF inhibited the germination of M. perniciosa basidiospores (>80%), in addition to causing morphological changes. Our results shed more light on the nature of the plant's defense performed by the apoplast in the T. cacao–M. perniciosa interaction in the initial (biotrophic) phase of fungal infection and therefore make it possible to expand WBD control strategies based on the identification of potential targets for resistance markers and advance scientific knowledge of the disease.
Heat Treatments at Varying Ambient Temperatures and Durations Differentially Affect Plant Defense to Blumeria hordei in a Resistant and a Susceptible Hordeum vulgare Line
Authors: Fodor József , Nagy Judit Kolozsváriné , Király Lóránt , Mészáros Klára , Bányai Judit , Cséplő Mónika Károlyiné , Schwarczinger Ildikó , Künstler András
Published: September 2023

Our previous research showed that a powdery mildew resistant barley line (MvHV07-17) maintains its resistance to Blumeria hordei (Bh) even if plants are exposed to a long-term high temperature of 35°C for 120 h before Bh inoculation, whereas such high temperature pretreatment further increases susceptibility to infection in the susceptible barley line MvHV118-17. In the present study, we extended this approach using short-term high-temperature water treatment (49°C for 30 s) to determine how it affects powdery mildew resistance in these barley lines. We found that this short-term heat shock (HS) impaired plant defense responses, as reflected by development of Bh colonies and visible necrotic spots on leaves of MvHV07-17, which does not develop visible symptoms upon Bh inoculation under optimal growth conditions. In contrast, both HS and long-term heat stress enhanced susceptibility to Bh in MvHV118-17 plants. These results were supported by the measurement of Bh biomass using a qPCR method. Furthermore, microscopic examinations showed that HS elevated the rate of successful Bh penetration events and the spread of cell death in the surrounding mesophyll area and allowed for colony formation and sporulation in resistant barley, whereas early and effective plant defense responses, such as papilla formation and single-cell epidermal hypersensitive response, were significantly reduced. Furthermore, we found that the accumulation of hydrogen peroxide in both resistant and susceptible barley was correlated with susceptibility induced by HS and long-term heat-stress. This study may contribute to a better understanding of plant defense responses to Bh in barley exposed to heat.

[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
A ‘Candidatus Liberibacter solanacearum’ Haplotype B-Specific Family of Candidate Bacterial Effectors
Authors: Levy Julien G. , Oh Junepyo , Mendoza Herrera Azucena , Parida Adwaita , Lao Loi , Starkey Jesse , Yuan Peiguo , Kan Chia-Cheng , Tamborindeguy Cecilia
Published: September 2023

‘ Candidatus Liberibacter solanacearum’ (Lso) is a phloem-limited pathogen associated with devastating diseases in members of the Solanaceae and Apiaceae and vectored by several psyllid species. Different Lso haplotypes have been identified, and LsoA and LsoB are responsible for diseases in Solanaceae crops. Our efforts are aimed at identifying pathogenicity factors used by this bacterium to thrive in different hosts. Bacterial secreted proteins can play a role in host colonization or the manipulation of the host immune responses; these proteins are called effectors. In this study, we identified six LsoB-specific proteins with a conserved secretion motif as well as a conserved N-terminal domain in the mature protein. These proteins had different expression and secretion patterns but a similar subcellular localization in Nicotiana benthamiana leaves, suggesting that they play different roles regardless of their conserved secretion motif. One of these proteins, CKC_04425, was expressed at high levels in the insect vector and the host plant, indicating that it could play a role in both the plant and insect hosts, whereas the others were mainly expressed in the plant. One protein, CKC_05701, was able to efficiently suppress programmed cell death and reactive oxygen species production, suggesting that it may have a virulence role in LsoB-specific pathogenesis.
CXCL8 induces M2 macrophage polarization and inhibits CD8+ T cell infiltration to generate an immunosuppressive microenvironment in colorectal cancer
Authors: Shao Ying , Lan Yan , Chai Xinyue , Gao Shuhua , Zheng Jinxiu , Huang Rui , Shi Yu , Xiang Yi , Guo Hongmei , Xi Yanfeng , Yang Lijun , Yang Tao
Published: September 2023

The poor prognosis of immunotherapy in patients with colorectal cancer (CRC) necessitates a comprehensive understanding of the immunosuppressive mechanisms within tumor microenvironment (TME). Undoubtedly, the anti‐tumor immune cells play an indispensable role in immune tolerance. Therefore, it is imperative to investigate novel immune‐related factors that have the capacity to enhance anti‐tumor immunity. Here, we employed bioinformatic analysis using R and Cytoscape to identify the hub gene chemokine (C‐X‐C motif) ligand 8 (CXCL8), which is overexpressed in CRC, in the malignant progression of CRC. However, its specific role of CXCL8 in CRC immunity remains to be elucidated. For this purpose, we evaluated how tumor‐derived CXCL8 promotes M2 macrophage infiltration by in vivo and in vitro, which can be triggered by IL‐1β within TME. Mechanistically, CXCL8‐induced polarization of M2 macrophages depends on the activation of the STAT3 signaling. Finally, immunohistochemistry and multiplexed immunohistochemistry analysis identified that CXCL8 not only enhances PD‐L1+ M2 macrophage infiltration but also attenuates the recruitment of PD‐1+CD8+ T cells in murine CRC models. Together, these findings emphasize the critical role for CXCL8 in promoting M2 macrophage polarization and inhibiting CD8+ T cell infiltration, thereby links CXCL8 to the emergency of immunosuppressive microenvironment facilitating tumor evasion. Overall, these findings may provide novel strategy for CRC immunotherapy.