Validation of a rapid potency assay for cord blood stem cells using phospho flow cytometry: The IL-3-pSTAT5 assay.

INTRODUCTION: Public cord blood banks (CBBs) are required to measure cord blood units (CBUs) potency before their release, allowing for the identification of units that may be unsuitable for haematopoietic transplantation. We have developed a rapid flow cytometry assay based on the measurement of STAT-5 phosphorylation of CD34+ stem cells in response to IL-3 stimulation. METHOD: To adapt the assay from a research setting to its implementation within our CBB regulated operations, we proceded with a full method validation and a correlation comparison of the IL-3-pSTAT5 assay results with the colony-forming unit assay (CFU) results. A total of 60 CBUs cryopreserved in vials were analysed by flow cytometry to determine the sensitivity, specificity, intra-assay precision, robustness, reproducibility, and inter-laboratory agreement of the assay. The CFU assay was also done on the same samples for comparison purposes. RESULTS: The assay threshold was established at 50% CD34+CD45+pSTAT5+, which provides a 100% sensitivity and a 98.3% specificity. An average intra-assay CV of 7.3% was determined. All results met our qualitative results acceptance criteria regarding the inter-user and inter-laboratory agreements, IL-3 stimulation time, post-thaw incubation delay and staining time. The IL-3-pSTAT5 assay results correlated well with the total CFU determined using the CFU assay (r2  = 0.82, n = 56). CONCLUSION: This study shows that our rapid flow cytometry assay can be successfully validated and that the potency data obtained display good sensitivity, specificity and robustness. These results demonstrate the feasibility of implementing this assay within CBB operations, as a validated potency assay.

Rapid potency assessment of autologous peripheral blood stem cells by intracellular flow cytometry: the PBSC-IL-3-pSTAT5 assay.

BACKGROUND AIMS: The current gold standard for stem cell product potency assessment, the colony-forming unit (CFU) assay, delivers results that are difficult to standardize and requires a substantial amount of time (up to 14 days) for cellular growth. Recently, the authors developed a rapid (<24 h) flow cytometry assay based on the measurement of intracellular phosphorylated STAT5 (pSTAT5) in CD34+ cord blood stem and progenitor cells in response to IL-3 stimulation. The present work presents a novel adaptation of the protocol for use with autologous peripheral blood stem cells (PBSCs) and a performance comparison with the CFU assay. METHODS: The flow cytometry intracellular staining assay was optimized for PBSCs, and patient samples were analyzed using the PBSC-IL-3-pSTAT5 and CFU assays. Warming events were also simulated to emulate impaired potency products. RESULTS: Optimization led to minor protocol adjustments, such as removal of the red blood cell lysis step, the addition of a formaldehyde fixation step and an increase in anticoagulant concentration. The PBSC-IL-3-pSTAT5 assay discriminated between normal and impaired samples and identified 100% (18 of 18) of the impaired samples, thus showing better specificity than the CFU assay. CONCLUSIONS: The updated IL-3-pSTAT5 potency assay has several important advantages, such as accelerating the release of autologous stem cell products and enabling the detection of potentially impaired products. The assay could also be used to rapidly assess the potency of any cryopreserved allogeneic stem cell product, such as those processed during the coronavirus disease 2019 pandemic.

Preparation and growth factor characterization of cord blood-derived plasma, serum, growth factor-rich plasma and induced serum.

BACKGROUND: In recent years, there has been a significant decline in the demand for cord blood units (CBUs). This trend has led to cord blood banks (CBBs) exploring complementary uses of CBUs in order to exploit the full potential of this unique, valuable, and readily available product. The aim of this study was to establish standardized protocols for the preparation of four cord blood (CB) derivatives: plasma (CB-P), serum (CB-S), induced-serum (CB-IS) and growth factor-rich plasma (GFRP); to measure their respective growth-factor content and their potential as cell culture supplements, and finally, to identify the characteristics of each derivative beyond their growth-factor composition, in the context of optimizing CBBs resources. STUDY DESIGN AND METHOD: To this end, CB plasma and serum were prepared and their concentrations for ten growth factors (TGF-ß1, TGF-ß2, TGF-ß3, EGF, HGF, PDGF-BB, VEGF-A, VEGF-D, IGF-I, IGF-II) were determined using a multiplex bead array, and compared to adult plasma and serum. Protocols for the preparation of CB-IS and GFRP with reverse anticoagulation using CaCl2·2H2O were also established, and all four derivatives were compared for their EGF and TGF-ß1 content by enzyme-linked immunosorbent assay (ELISA), and also for their cell culture supplement capacity. RESULTS: CB plasma was shown to be richer than adult plasma for TGF-ß2 and TGF-ß3, with a lower concentration of IGF-I and IGF-II. CB serum was shown to be richer than adult serum for TGF-ß2, TGF-ß3, EGF, HGF, PDGF-BB, and VEGF-A and D, and poorer in IGF-I and II. The measure of EGF and TGF-ß1 concentrations has shown that CB serum is the most concentrated (1874 pg/ml and 41 094 pg/ml) of the CB derivatives, followed by induced-serum (405 pg/ml and 16 735 pg/ml), growth factor-rich plasma (131 and 7947 pg/ml) and plasma (8 and 2845 pg/ml). All four CB derivatives were shown to be superior to FBS in sustaining cell growth at low doses. CONCLUSION: Our study shows that four CB derivatives can be easily prepared and pooled to provide significant volume of products that vary in their growth factor composition. A cord blood bank interested in introducing such manufacturing will need to evaluate the financial and processing characteristics of each derivative. The use of standardized manufacturing protocols such as the ones we suggest could help research initiatives exploring the potential therapeutic uses of such rich and high-quality starting material.

Intravenous immunoglobulin modulates the expansion and cytotoxicity of CD8+ T cells.

Intravenous immunoglobulin (IVIg) is successfully used in the treatment of autoimmune diseases involving self-reactive CD8(+) T cells. However, its direct influence on the cytotoxic response remains unknown. Using an antigen cross-presentation assay and a mouse model of ovalbumin (OVA) immunization, we showed that IVIg decreases the in vitro activation, proliferation and cytokine secretion of OVA-specific CD8(+) T cells (OT-I), as well as the in vivo generation of OVA-specific CD8(+) T cells. In addition, IVIg significantly decreases the proportion of perforin- and CD107a-expressing CD8(+) T cells, and inhibits the cytotoxic activity of OVA-activated OT-I cells. The interference of IVIg with the CD8(+) T-cell response is associated with T-cell receptor blockade, therefore reducing the interaction between effector and target cells. A similar blockade is observed on human CD8(+) T cells, suggesting that the observations reported here could apply to the IVIg-mediated improvement of CD8(+) T-cell-mediated autoimmune conditions in human patients.

Prevention of T cell activation by interference of internalized intravenous immunoglobulin (IVIg) with MHC II-dependent native antigen presentation.

Activation of self-reactive CD4(+) T cells plays a central role in the initiation and maintenance of autoimmune diseases. We recently reported that intravenous immunoglobulin (IVIg) inhibits the MHC II-restricted CD4(+) T cell activation induced by the presentation of immune complexes. Because native antigens can also play a role in the induction of several autoimmune diseases, we determined whether IVIg could also affect CD4(+) T cell activation following presentation of native antigens by APCs. Here we report that IVIg significantly reduces the activation of CD4(+) T cells by native ovalbumin. The inhibitory effect is FcγR-independent and occurs following internalization of IVIg inside APCs, where it interferes with the intracellular events leading to MHC II-dependent antigen presentation. The effect of IVIg on native antigen presentation could therefore contribute to dampen the autoimmune reaction by reducing CD4(+) T cell activation and the subsequent inflammatory response induced by these cells.