Assessment of the Impact of Post-Thaw Stress Pathway Modulation on Cell Recovery following Cryopreservation in a Hematopoietic Progenitor Cell Model

The development and use of complex cell-based products in clinical and discovery science continues to grow at an unprecedented pace. To this end, cryopreservation plays a critical role, serving as an enabling process, providing on-demand access to biological material, facilitating large scale production, storage, and distribution of living materials. Despite serving a critical role and substantial improvements over the last several decades, cryopreservation often remains a bottleneck impacting numerous areas including cell therapy, tissue engineering, and tissue banking. Studies have illustrated the impact and benefit of controlling cryopreservation-induced delayed-onset cell death (CIDOCD) through various “front end” strategies, such as specialized media, new cryoprotective agents, and molecular control during cryopreservation. While proving highly successful, a substantial level of cell death and loss of cell function remains associated with cryopreservation. Recently, we focused on developing technologies (RevitalICE™) designed to reduce the impact of CIDOCD through buffering the cell stress response during the post-thaw recovery phase in an effort to improve the recovery of previously cryopreserved samples. In this study, we investigated the impact of modulating apoptotic caspase activation, oxidative stress, unfolded protein response, and free radical damage in the initial 24 h post-thaw on overall cell survival. Human hematopoietic progenitor cells in vitro cryopreserved in both traditional extracellular-type and intracellular-type cryopreservation freeze media were utilized as a model cell system to assess impact on survival. Our findings demonstrated that through the modulation of several of these pathways, improvements in cell recovery were obtained, regardless of the freeze media and dimethyl sulfoxide concentration utilized. Specifically, through the use of oxidative stress inhibitors, an average increase of 20% in overall viability was observed. Furthermore, the results demonstrated that by using the post-thaw recovery reagent on samples cryopreserved in intracellular-type media (Unisol™), improvements in overall cell survival approaching 80% of non-frozen controls were attained. While improvements in overall survival were obtained, an assessment on the impact of specific cell subpopulations and functionality remains to be completed. While work remains, these results represent an important step forward in the development of improved cryopreservation processes for use in discovery science, and commercial and clinical settings.

Cardiovascular Disease (CVD) Risk Assessment of HIV Medication Regimens using hematopoietic CD34+ progenitor cells

Background To determine the effects of integrase inhibitor (INSTI) in comparison to non INSTI based regimens such as non-nucleoside reverse transcriptase inhibitors (NNRTIs) based regimens on cardiovascular disease (CVD) risk in HIV+ patients without overt history of CVD or diabetes, with normal CD4:CD8 count. For CVD risk assessment we primarily used hematopoietic CD34+ progenitor cells, as a biomarker.Methods19 male subjects ages 32-61 years with BMI 21.0- 36.0, were enrolled. This was a single time point, cross-sectional, observational study. Subjects were enrolled under 2 groups (either on INSTI based regimen with 13 subjects or NNRTI (non-INSTI) based regimens with 6 subjects) who were taking stable doses of HAART. The medication regimens were a combination of one NRTI (typically tenofovir-emtricitabine) plus one INSTI or NNRTI. Our outcome measures were focused on cardiovascular and endothelial cell function and systemic inflammation. Our primary outcome measures were peripheral blood derived hematopoietic progenitor cell number (CD34 and CD133 positive), CD34+ cell function and gene expression studies. Our secondary outcomes were arterial stiffness measures and serum-based markers of inflammation. ResultsA significant increase in percentage number of progenitor cells, CD133+ cells (P=0.004) was noted along with an increase of double progenitor mark positive CD133+/CD34+ progenitor cell population was observed in INSTI group as compared to NNRTI group, by flow-cytometry. mRNA gene expression for antioxidant gene catalase was noted along with a trend towards a decrease in gene expression of inflammatory marker IL6 (p=0.06) was observed in CD34+ from INSTI group vs NNRTI group. The plasma IL-6 and CRP levels did not change significantly between the groups. Neutrophil-Lymphocyte ratio (NLR), an important marker of inflammation, was noted to be lower in INSTI group. A mean fasting glucose level was also lower in the INSTI group compared to NNRTI group (p=0.03). Interestingly, Urine- Microalbumin levels were higher in the INSTI group compared to NNRTI group (p=0.08), while eGFR levels were lower in the INSTI group (p=0.002). The arterial stiffness measures did not show statistically significant differences between the two groups. ConclusionWe conclude that the INSTI regimen may provide a better CVD risk profile compared to NNRTI based HAART regimen; however the increased albuminuria along with lower eGFR, noted in INSTI group is of concern. Because of the small size, these results would need replication in additional studies before changing clinical practice.Clinical Trial Registration https://clinicaltrials.gov/ct2/show/NCT03782142?cond=Hiv&spons=Sabyasachi+sen&cntry=US&state=US%3ADC&city=Washington&draw=2&rank=1ClinicalTrials.gov Identifier: NCT03782142

GATA2 deficiency elevates Interferon Regulatory Factor-8 to subvert a progenitor cell differentiation program

Cell type-specific transcription factors control stem and progenitor cell transitions by establishing networks containing hundreds of genes and proteins. Network complexity renders it challenging to discover essential versus modulatory or redundant components. This scenario is exemplified by GATA2 regulation of hematopoiesis during embryogenesis. Loss of a far upstream Gata2 enhancer (-77) disrupts the GATA2-dependent transcriptome governing hematopoietic progenitor cell differentiation. The aberrant transcriptome includes the transcription factor Interferon Regulatory Factor-8 and a host of innate immune regulators. Mutant progenitors lose the capacity to balance production of diverse hematopoietic progeny. To elucidate mechanisms, we asked if IRF8 is essential, contributory or not required. Reducing Irf8, in the context of the -77 mutant allele, reversed granulocytic deficiencies and the excessive accumulation of dendritic cell committed progenitors. Despite many dysregulated components that control vital transcriptional, signaling and immune processes, the aberrant elevation of a single transcription factor deconstructed the differentiation program.

For Better or for Worse: COVID-19 Vaccination during or Early after (Immuno-) Chemotherapy or Hematopoietic Progenitor Cell Transplantation

Background: Patients with hematologic conditions have a high mortality rate when infected with SARS-CoV-2 (Williamson, Nature 2020). Protection of this group from severe COVID-19 is therefore important. However, according to available vaccination guidelines, one should consider to postpone vaccination of patients on or early after chemotherapy, hematopoietic progenitor cell transplantation (HCT) or with graft versus host disease, because of anticipated poor efficacy. Based on previous (non-COVID-19) vaccination studies among hematology patients, we hypothesized that a significant group of patients may acquire sufficient protection following COVID-19 vaccination, despite disease and therapy related immunodeficiencies. Methods: We conducted a prospective cohort study with 17 cohorts of hematology patients of particular risk for severe COVID-19 who are considered to have no or limited benefit from vaccination. We evaluated humoral immune responses following 2 doses (28 days apart) of the mRNA-1273 vaccine (Moderna/Spikevax) in 722 patients, at baseline and 28 days after each vaccination as SARS-COV-2 S1- (spike)-specific serum IgG antibody concentrations by bead-based multiplex immune assay. The threshold for adequate antibody response is set at ≥300 binding antibody units (BAU)/ml according to the international WHO standard, and is associated with virus plaque reducing neutralization test titers of ≥40 PRNT 50. This study is registered as EudraCT 2021-001072-41, NL76768.029.21. Results: Patient cohorts and corresponding vaccine responses are depicted in Table 1. Vaccine efficacy, as measured by antibody concentration, 4 weeks after the 2 nd mRNA-1273 vaccination was available for 691 out of 722 participants. The majority of patients (389/691; 56%) obtained an S1 antibody titer that is considered adequate (≥300 BAU/ml). Twenty-nine percent of patients (198/691) did not seroconvert (S1 antibody titer <10 BAU/ml), while the remaining 15% (104/691) did seroconvert but not to sufficient levels (10-300 BAU/ml). Adequate responses were observed in the majority of patients with sickle cell disease using hydroxyurea, chronic myeloid leukemia (CML) receiving tyrosine kinase inhibitor therapy, acute myeloid leukemia (AML) on or early after high dose chemotherapy, patients with myeloproliferative disorders on ruxolitinib, patients with multiple myeloma (MM), including those on daratumumab and those early after high-dose melphalan and autologous HCT, patients with untreated chronic lymphocytic leukemia (CLL), and patients with chronic GvHD. Insufficient or absent antibody responses were observed in the majority of AML patients receiving hypomethylating agents, CLL patients on ibrutinib, patients with B-cell non-Hodgkin's Lymphoma (NHL) during or shortly after rituximab-chemotherapy or following BEAM chemotherapy and autologous HCT, allogeneic HCT recipients <6 months after transplantation, and CAR-T cell therapy recipients. However, even in these low-responder groups considerable numbers of patients did mount sufficient antibody titers. In others, titers increased after each of both vaccinations, suggesting that booster vaccination may enhance antibody titers to sufficient levels (Figure 1). Conclusion: Vaccination with mRNA-1273 had significant efficacy in severely immunocompromised hematology patients. Adequate humoral immune responses after two dose vaccination were reached in the majority of patients receiving therapy for sickle cell disease, MPD, MM, CML and AML, in patients early after HCT and in patients with GvHD. We are currently evaluating clinical and immunologic parameters that correlate with sufficient antibody responses, pseudovirus neutralization and SARS-COV-2-specific B and T cell numbers, phenotype and function. Per study design, all participants with absent or insufficient antibody responses (<300 BAU/ml) will receive a booster vaccination 5 months after initial vaccination, and antibody responses to booster vaccinations will be presented as well. Unlike currently available guidelines, COVID-19 vaccination should not be postponed. Moreover, as antibody titers increased after each of both vaccinations, booster vaccination of patients with absent or insufficient antibody responses seems warranted. Figure 1 Figure 1. Disclosures Mutsaers: AstraZeneca: Research Funding; BMS: Consultancy. Van Meerten: Janssen: Consultancy; Kite, a Gilead Company: Honoraria. Kater: BMS, Roche/Genentech: Other: Ad Board, , Research Funding; Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; Abbvie: Honoraria, Other: Ad Board, Research Funding; Genmab, LAVA: Other: Ad Board, Steering Committee. Zweegman: Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Nijhof: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene/Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

In Situ Microscopy Studies of Infused Megakaryocytes: Implications in Thrombopoiesis

The questions of whether thrombopoiesis - the release of platelets from megakaryocytes - occurs both as megakaryocytes emerge from the intramedullar space or occurs as well in the pulmonary vascular bed remains unanswered. Studies by Lefrançais E, et al, (Nature, 2017) demonstrated by in situ microcopy that perhaps 50% of all platelet release in mice occurs from megakaryocytes released from the marrow and traveled to the lungs where they undergo thrombopoiesis over a 20- to 60-minute time-period. We examined whether CD34+-derived human megakaryocytes infused into immunocompromized NSG mice would also shed platelets in the lungs in a similar fashion. We differentiated CD34+-derived hematopoietic stem-progenitors for 12 days in culture using conditions previously described (Wang Y, et al., Blood 2015). We found that unlike platelet-like-particle (PLP) formation in in vitro cultures of CD34+ hematopoietic progenitor cell (HPC)-derived (CD34+) megakaryocytes, which undergo asynchronous shedding of the PLPs, that over 95% of infused CD34+ megakaryocytes shed their platelets within the first 40 minutes much as has been observed for endogenous murine megakaryocytes. The average number of cytoplasmic extensions per megakaryocytes was ~2.7, again very similar to what was seen with endogenous murine megakaryocytes. In contrast, CD34+ cells grown in culture into megakaryocytes for a shorter period of time of only 7 days, poorly shed any cytoplasmic fragments. We also studied human megakaryocytes grown from immortalized megakaryocyte progenitor cell lines (imMKCLs) from induced pluripotent stem cells (iPSCs) generated by the Eto laboratory and kindly provided by Dr. Koji Eto, Kyoto University). These cells were grown and differentiated into terminal megakaryocytes as described (Nakamura S, Cell Stem Cell, 2014) for 4 days in culture. These cells have been proposed to be useful for large-scale preparation of PLPs in vitro for clinical use in place of donor-derived platelets. The resultant infused human imMKCL-derived megakaryocytes also synchronously shed platelets, but only 50% of the infused cells shed their cytoplasm in contrast to >95% of CD34+ megakaryocytes. Moreover, cytoplasmic extensions were decreased to an average of ~1.1 per megakaryocyte. We had proposed that in vitro-generated megakaryocytes might be directly infused into patients in place of further manipulating the megakaryocytes to release functional platelets in vitro using a bioreactor. However, such megakaryocytes will likely be contaminated with a higher level of HPCs than anticipated from in vitro-prepared platelets, and concern exists that they may lead to unacceptable graft versus host complications. We, therefore, examined whether irradiating megakaryocytes as one strategy to eliminate this concern results in megakaryocytes that are still functional and found that megakaryocytes irradiated with up to 25 Gy retain platelet yield per infused megakaryocytes with the platelets having the same half-life. If irradiated and kept in culture, these megakaryocytes begin to shed platelets and undergo apoptosis notably by 24 hours. We also examined whether the pulmonary bed differs from other vascular beds, and infused CD34+ megakaryocytes both intravenously and intra-arterially in parallel studies and found that following intra-arterial infusion, megakaryocytes were mostly entrapped in various organs, but shed few platelets. Thus, our studies suggest that the pulmonary bed is unique for platelet shedding from entrapped megakaryocytes. Whether this is due to the structural organization of the pulmonary beds, its endothelial lining, its reverse exchange in oxygen, carbon dioxide and pH from other capillary beds or the mechanical forces of inhalation and exhalation that expand and contract the capillary cross-sectional area needs to be examined. Our studies show that infused human megakaryocytes synchronously release platelets over a 40-minute window and can do so even after being irradiated and that this occurs specifically in the lungs not only has potential clinical application, but also raises biological questions about what determines thrombopoiesis-readiness and what are the features of the pulmonary bed that allows this synchronous release. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Linking GATA2 Deficiency to Dysregulated Innate Immune Signaling

Cell type-specific transcription factors governing hematopoietic stem and progenitor cell transitions establish networks containing hundreds of genes and proteins. Network complexity renders it challenging to discover essential versus modulatory or redundant components. This scenario is exemplified by GATA2 mechanisms that control hematopoiesis during embryogenesis. Loss of a far upstream Gata2 enhancer (-77) disrupts the GATA2-dependent genetic network governing hematopoietic progenitor cell differentiation (Johnson KD. et al., Sci. Adv., 2015). The aberrant network includes the transcription factor Interferon Regulatory Factor-8 and a host of innate immune regulators, including Toll-like receptors (TLRs) (Johnson KD. et al., J. Exp. Med., 2020). Mutant embryonic progenitors lose the capacity to balance production of diverse hematopoietic progeny and generate excessive monocytic progeny. As IRF8 is vitally important for monocytic and dendritic cell differentiation (Yanez A. and Goodridge H., Curr. Opin. Hematol., 2016), we asked whether IRF8 is essential, contributory, or inconsequential. Using a double-mutant genetic rescue in vivo system, we demonstrated that reducing Irf8, in the context of the -77 mutant allele, reversed granulocytic deficiencies and the excessive accumulation of dendritic cell-committed progenitors. In -77 -/- E14.5 fetal livers, monocyte progenitors (MPs) increased 2.3-fold (P = 0.006), granulocyte progenitors (GPs) decreased 2.2-fold (P = 0.003) and common dendritic cell progenitors (CDPs) increased 10.2-fold (P = 0.021) relative to wildtype littermates. Ablating Irf8 in -77 mutants (-77 -/-; Irf8-/-) restored MPs and CDPs to wildtype levels and reversed the GP deficiency; further increasing GPs 4.2-fold relative to wildtype (P = 0.0009). Despite many dysregulated components that control vital transcriptional, signaling and immune processes, the aberrant elevation of a single transcription factor deconstructed the embryonic hematopoiesis program. We analyzed the mechanistic and biological implications of IRF8 dysregulation concomitant with ectopic upregulation of other innate immune genes (including Toll-like receptors (TLRs) in GATA2-deficient embryonic progenitors. In principle, such genes might function upstream, downstream, or in parallel with IRF8. Based on TLR upregulation and TLR roles in progenitor mechanisms (Nagai Y. et al., Immunity, 2006; Schuettpelz L. et al., Leukemia, 2014; Caiado F. et al., J. Exp. Med., 2021), we tested whether GATA2 deficiency in embryonic progenitors impacts cellular responsiveness to TLR ligands. Wild type and -77 enhancer-mutant progenitors were treated with increasing concentrations of the TLR1/2 ligand Pam 3CSK 4. The mutant progenitors were hypersensitive to Pam 3CSK 4, which resulted in supra-physiological induction of Tnf expression (2.8-fold at 34 nM, P = 0.004; 3.2-fold at 68 nM, P = 0.0003). Quantitative analyses indicated that hypersensitivity reflected increased Pam 3CSK 4 efficacy, but not potency. GATA2 re-expression in the mutant progenitors attenuated the elevated IRF8 expression and TLR signaling, normalizing Tnf and Ccl3 expression to a level comparable to that of wild type progenitors. In GATA2-rescued mutant progenitors, Tnf and Ccl3 expression decreased 3.9-fold (P = 0.005) and 2.5-fold (P = 0.047), respectively. Thus, GATA2 suppresses TLR signaling in embryonic progenitors. Ongoing studies are elucidating the mechanistic interconnections between IRF8- and TLR-dependent inflammatory networks in GATA2 deficiency during embryonic and adult hematopoiesis in cell populations and single cells, relationships between murine and human mechanisms, and the impact of targeted interventions that modulate these mechanisms. Disclosures No relevant conflicts of interest to declare.

Essential role of zyxin in platelet biogenesis and glycoprotein Ib-IX surface expression

Platelets are generated from the cytoplasm of megakaryocytes (MKs) via actin cytoskeleton reorganization. Zyxin is a focal adhesion protein and wildly expressed in eukaryotes to regulate actin remodeling. Zyxin is upregulated during megakaryocytic differentiation; however, the role of zyxin in thrombopoiesis is unknown. Here we show that zyxin ablation results in profound macrothrombocytopenia. Platelet lifespan and thrombopoietin level were comparable between wild-type and zyxin-deficient mice, but MK maturation, demarcation membrane system formation, and proplatelet generation were obviously impaired in the absence of zyxin. Differential proteomic analysis of proteins associated with macrothrombocytopenia revealed that glycoprotein (GP) Ib-IX was significantly reduced in zyxin-deficient platelets. Moreover, GPIb-IX surface level was decreased in zyxin-deficient MKs. Knockdown of zyxin in a human megakaryocytic cell line resulted in GPIbα degradation by lysosomes leading to the reduction of GPIb-IX surface level. We further found that zyxin was colocalized with vasodilator-stimulated phosphoprotein (VASP), and loss of zyxin caused diffuse distribution of VASP and actin cytoskeleton disorganization in both platelets and MKs. Reconstitution of zyxin with VASP binding site in zyxin-deficient hematopoietic progenitor cell-derived MKs restored GPIb-IX surface expression and proplatelet generation. Taken together, our findings identify zyxin as a regulator of platelet biogenesis and GPIb-IX surface expression through VASP-mediated cytoskeleton reorganization, suggesting possible pathogenesis of macrothrombocytopenia.

Label-free separation of neuroblastoma patient-derived xenograft (PDX) cells from hematopoietic progenitor cell products by acoustophoresis

Background Graft-contaminating tumor cells correlate with inferior outcome in high-risk neuroblastoma patients undergoing hematopoietic stem cell transplantation and can contribute to relapse. Motivated by the potential therapeutic benefit of tumor cell removal as well as the high prognostic and diagnostic value of isolated circulating tumor cells from stem cell grafts, we established a label-free acoustophoresis-based microfluidic technology for neuroblastoma enrichment and removal from peripheral blood progenitor cell (PBPC) products. Methods Neuroblastoma patient-derived xenograft (PDX) cells were spiked into PBPC apheresis samples as a clinically relevant model system. Cells were separated by ultrasound in an acoustophoresis microchip and analyzed for recovery, purity and function using flow cytometry, quantitative real-time PCR and cell culture. Results PDX cells and PBPCs showed distinct size distributions, which is an important parameter for efficient acoustic separation. Acoustic cell separation did not affect neuroblastoma cell growth. Acoustophoresis allowed to effectively separate PDX cells from spiked PBPC products. When PBPCs were spiked with 10% neuroblastoma cells, recoveries of up to 98% were achieved for PDX cells while more than 90% of CD34+ stem and progenitor cells were retained in the graft. At clinically relevant tumor cell contamination rates (0.1 and 0.01% PDX cells in PBPCs), neuroblastoma cells were depleted by more than 2-log as indicated by RT-PCR analysis of PHOX2B, TH and DDC genes, while > 85% of CD34+ cells could be retained in the graft. Conclusion These results demonstrate the potential use of label-free acoustophoresis for PBPC processing and its potential to develop label-free, non-contact tumor cell enrichment and purging procedures for future clinical use.