Allogeneic haematopoietic cell transplantation (HCT) is a curative therapy for severe haematological disorders. However, it carries significant risk of morbidity and mortality. To improve patient ...outcomes, better graft selection strategies are needed, incorporating HLA matching with clinically important graft characteristics. Studies have shown that the cellular content of HCT grafts, specifically higher ratios of T regulatory (Tregs)/T cells, are important factors influencing outcomes when using adult peripheral blood mobilised grafts. So far, no equivalent study exists in umbilical cord blood (CB) transplantation due to the limitations of cryopreserved CB samples.
To establish the most robust and efficient way to measure the Treg content of previously cryopreserved CB units, we compared the enumeration of Treg and CD3+ cells using flow cytometry and an epigenetic, DNA-based methodology. The two methods were assessed for their agreement, consistency and susceptibility to error when enumerating Treg and CD3+ cell numbers in both fresh and cryopreserved CB samples.
Epigenetic enumeration gave consistent and comparable results in both fresh and frozen CB samples. By contrast, assessment of Tregs and CD3+ cells by flow cytometry was only possible in fresh samples due to significant cell death following cryopreservation and thawing.
Epigenetic assessment offers significant advantages over flow cytometry for analysing cryopreserved CB; similar cell numbers were observed both in fresh and frozen samples. Furthermore, multiple epigenetic assessments can be performed from DNA extracted from small cryopreserved CB segments; often the only CB sample available for clinical studies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background
The therapeutic benefit of convalescent plasma (CP) therapy to treat COVID‐19 may derive from neutralizing antibodies (nAbs) to SARS‐CoV‐2. To investigate the effects of antigenic ...variation on neutralization potency of CP, we compared nAb titers against prototype and recently emerging strains of SARS‐CoV‐2, including Delta and Omicron, in CP donors previously infected with SARS‐CoV‐2 before and after immunization.
Methods and Materials
Samples were assayed from previously SARS‐CoV‐2 infected donors before (n = 17) and after one (n = 43) or two (n = 71) doses of Astra‐Zeneca or Pfizer vaccinations. Ab titers against Wuhan/wild type (WT), Alpha, Beta, and Delta SARS‐CoV‐2 strains were determined by live virus microneutralization assay while titers to Omicron used a focus reduction neutralization test. Anti‐spike antibody was assayed by Elecsys anti‐SARS‐CoV‐2 quantitative spike assay (Roche).
Results
Unvaccinated donors showed a geometric mean titer (GMT) of 148 against WT, 80 against Alpha but mostly failed to neutralize Beta, Delta, and Omicron strains. Contrastingly, high GMTs were observed in vaccinated donors against all SARS‐CoV‐2 strains after one vaccine dose (WT:703; Alpha:692; Beta:187; Delta:215; Omicron:434). By ROC analysis, reactivity in the Roche quantitative Elecsys spike assay of 20,000 U/mL was highly predictive of donations with nAb titers of ≥1:640 against Delta (90% sensitivity; 97% specificity) and ≥1:320 against Omicron (89% sensitivity; 81% specificity).
Discussion
Vaccination of previously infected CP donors induced high levels of broadly neutralizing antibodies against circulating antigenic variants of SARS‐CoV‐2. High titer donations could be reliably identified by automated quantitative anti‐spike antibody assay, enabling large‐scale preselection of high‐titer convalescent plasma.
Human-induced pluripotent stem cells (hiPSCs) are a potentially invaluable resource for regenerative medicine, including the in vitro manufacture of blood products. HiPSC-derived red blood cells are ...an attractive therapeutic option in hematology, yet exhibit unexplained proliferation and enucleation defects that presently preclude such applications. We hypothesised that substantial differential regulation of gene expression during erythroid development accounts for these important differences between hiPSC-derived cells and those from adult or cord-blood progenitors. We thus cultured erythroblasts from each source for transcriptomic analysis to investigate differential gene expression underlying these functional defects.
Our high resolution transcriptional view of definitive erythropoiesis captures the regulation of genes relevant to cell-cycle control and confers statistical power to deploy novel bioinformatics methods. Whilst the dynamics of erythroid program elaboration from adult and cord blood progenitors were very similar, the emerging erythroid transcriptome in hiPSCs revealed radically different program elaboration compared to adult and cord blood cells. We explored the function of differentially expressed genes in hiPSC-specific clusters defined by our novel tunable clustering algorithms (SMART and Bi-CoPaM). HiPSCs show reduced expression of c-KIT and key erythroid transcription factors SOX6, MYB and BCL11A, strong HBZ-induction, and aberrant expression of genes involved in protein degradation, lysosomal clearance and cell-cycle regulation.
Together, these data suggest that hiPSC-derived cells may be specified to a primitive erythroid fate, and implies that definitive specification may more accurately reflect adult development. We have therefore identified, for the first time, distinct gene expression dynamics during erythroblast differentiation from hiPSCs which may cause reduced proliferation and enucleation of hiPSC-derived erythroid cells. The data suggest several mechanistic defects which may partially explain the observed aberrant erythroid differentiation from hiPSCs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Allogeneic haematopoietic cell transplantation (HCT) is a curative therapy for severe haematological disorders, including acute leukaemia. However, it carries significant morbidity and mortality due ...to infection, graft-versus-host disease and relapse. The cellular content of HCT grafts, in particular the ratio between regulatory T cells (Treg) and conventional T cells, is an important factor influencing the severity of these complications. Whilst previous studies have been performed in fresh adult peripheral blood mobilised grafts, it has not been applied in umbilical cord blood (CB) HCTs. CB enumerations are more challenging as they must be performed from small cryopreserved segments stored alongside the CB units used for HCT.
Fresh CB units and thawed segments were analysed for their Treg and T cell content using both flow cytometry (the benchmark technique) and an epigenetic, DNA-based methodology. The two methods were compared for their agreement, consistency and susceptibility to error when assessing Treg and CD3+ cell numbers in both fresh and cryopreserved samples.
Epigenetic enumeration gave consistent results in both fresh and frozen samples, providing Treg/CD3 estimates that were similar. Assessment of Tregs and CD3+ cells by flow cytometry and epigenetic assessments in fresh samples showed that these two methods were correlated. Conversely, significant cell death was observed in the thawed segments which affected Treg and CD3 cell estimates by flow cytometry.
Epigenetic assessments offer significant advantages over flow cytometry for analysing cryopreserved CB. Unlike with flow cytometry assessments of thawed segments, similar cell numbers were observed in fresh and frozen samples, with material requirements not being limiting and being unaffected by high cell death. With this method, multiple epigenetic assessments can be performed from extracted DNA, to provide statistical confidence and confirm observed results. Finally, the method raises the possibility of retrospective studies of historical samples where only DNA is available.
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