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.

Advances in Model Systems for Human Cytomegalovirus Latency and Reactivation

Human cytomegalovirus (HCMV) is a highly prevalent beta-herpesvirus and a significant cause of morbidity and mortality following hematopoietic and solid organ transplant, as well as the leading viral cause of congenital abnormalities. A key feature of the pathogenesis of HCMV is the ability of the virus to establish a latent infection in hematopoietic progenitor and myeloid lineage cells.

Reduced expression of hematopoietic progenitor kinase 1 in T follicular helper cells causes autoimmunity of systemic lupus erythematosus

Backgroud T follicular helper (Tfh) cells have been discovered to be the main CD4+ T cells assisting B cells to produce antibody. They are over activated in patients with systemic lupus erythematosus (SLE) and consequently lead to excessive immunity. Hematopoietic progenitor kinase 1 (HPK1) negatively regulates T cell-mediated immune responses and TCR signal. This study aimed to investigate the roles of HPK1 in SLE Tfh cells. Methods HPK1 mRNA and protein levels in Tfh cells were measured by real-time quantitative PCR and western blot analysis, respectively. The production of IL-21, B cell−activating factor (BAFF), interferon γ (IFNγ), IL-17A, IgM, IgG1, IgG2, and IgG3 were analyzed using enzyme linked immunosorbent assay. Tfh cells proliferation was evaluated with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results HPK1 mRNA and protein levels were significantly reduced in SLE Tfh cells, and negatively correlated with SLE disease activity index (SLEDAI) and Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) Damage Index for SLE (SDI). Knocking down HPK1 with siRNA in normal Tfh cells greatly elevated Tfh cells proliferation and secretions of IL-21, BAFF, IFNγ, IgG1, IgG2, and IgG3. There were no marked alterations in IL-17A and IgM productions. The opposite effects were observed in SLE Tfh cells transfected with HPK1 overexpressing plasmid: Tfh cells proliferation and productions of IL-21, BAFF, IFNγ, IgG1, IgG2, and IgG3 were all alleviated. And there were no significant changes in IL-17A and IgM levels. Conclusion Our results suggest for the first time that inhibited expression of HPK1 in SLE Tfh cells leading to Tfh cells overactivation and B cells overstimulation, subsequently, the onset and progression of SLE.

Modeling human yolk sac hematopoiesis with pluripotent stem cells

In the mouse, the first hematopoietic cells are generated in the yolk sac from the primitive, erythro-myeloid progenitor (EMP) and lymphoid programs that are specified before the emergence of hematopoietic stem cells. While many of the yolk sac–derived populations are transient, specific immune cell progeny seed developing tissues, where they function into adult life. To access the human equivalent of these lineages, we modeled yolk sac hematopoietic development using pluripotent stem cell differentiation. Here, we show that the combination of Activin A, BMP4, and FGF2 induces a population of KDR+CD235a/b+ mesoderm that gives rise to the spectrum of erythroid, myeloid, and T lymphoid lineages characteristic of the mouse yolk sac hematopoietic programs, including the Vδ2+ subset of γ/δ T cells that develops early in the human embryo. Through clonal analyses, we identified a multipotent hematopoietic progenitor with erythroid, myeloid, and T lymphoid potential, suggesting that the yolk sac EMP and lymphoid lineages may develop from a common progenitor.

Extramedullary Hematopoiesis of the Liver and Spleen

Hematopoiesis is the formation of blood cellular components and, consequently, immune cells. In a more complete definition, this process refers to the formation, growth, maturation, and specialization of blood cells, from the hematopoietic stem cell, through the hematopoietic progenitor cells, to the s pecialized blood cells. This process is tightly regulated by several elements of the bone marrow microenvironment, such as growth factors, transcription factors, and cytokines. During embryonic and fetal development, hematopoiesis takes place in different organs: the yolk sac, the aorta–gonad mesonephros region, the lymph nodes, and not lastly, the fetal liver and the spleen. In the current review, we describe extramedullary hematopoiesis of the spleen and liver, with an emphasis on myeloproliferative conditions.

Competition between hematopoietic stem and progenitor cells controls hematopoietic stem cell compartment size.

Cellular competition for limiting hematopoietic factors is a physiologically regulated but poorly understood process. Here, we studied this phenomenon by hampering hematopoietic progenitor access to Leptin receptor+ mesenchymal stem/progenitor cells (MSPCs) and endothelial cells (ECs). We show that HSC numbers increased by 2-fold when multipotent and lineage-restricted progenitors failed to respond to CXCL12 produced by MSPCs and ECs. HSCs were qualitatively normal, and HSC expansion only occurred when early hematopoietic progenitors but not differentiated hematopoietic cells lacked CXCR4. Furthermore, the MSPC and EC transcriptomic heterogeneity was remarkably stable, suggesting that it is impervious to dramatic changes in hematopoietic progenitor interactions. Instead, HSC expansion was caused by increased availability of membrane-bound stem cell factor (mSCF) on MSPCs and ECs due to reduced consumption by cKit-expressing hematopoietic progenitors. These studies revealed an intricate homeostatic balance between HSCs and proximal hematopoietic progenitors regulated by cell competition for limited amounts of mSCF.

Arsenite exposure inhibits the erythroid differentiation of human hematopoietic progenitor CD34+ cells and causes decreased levels of hemoglobin

Arsenic exposure poses numerous threats to human health. Our previous work in mice has shown that arsenic causes anemia by inhibiting erythropoiesis. However, the impacts of arsenic exposure on human erythropoiesis remain largely unclear. We report here that low-dose arsenic exposure inhibits the erythroid differentiation of human hematopoietic progenitor cells (HPCs). The impacts of arsenic (in the form of arsenite; As3+) on red blood cell (RBC) development was evaluated using a long-term culture of normal human bone marrow CD34+-HPCs stimulated in vitro to undergo erythropoiesis. Over the time course studied, we analyzed the expression of the cell surface antigens CD34, CD71 and CD235a, which are markers commonly used to monitor the progression of HPCs through the stages of erythropoiesis. Simultaneously, we measured hemoglobin content, which is an important criterion used clinically for diagnosing anemia. As compared to control, low-dose As3+ exposure (100 nM and 500 nM) inhibited the expansion of CD34+-HPCs over the time course investigated; decreased the number of committed erythroid progenitors (BFU-E and CFU-E) and erythroblast differentiation in the subsequent stages; and caused a reduction of hemoglobin content. These findings demonstrate that low-dose arsenic exposure impairs human erythropoiesis, likely by combined effects on various stages of RBC formation.

EVI1-Positive AML Cells Show Distinct Features and High Dependency on ETS Transcription Factor ERG

Inappropriate expression of Ecotropic viral integration site 1 (EVI1) has been associated with dismal clinical outcomes in acute myeloid leukemia (AML), while EVI1 is indispensable for normal hematopoiesis. We have previously reported that EVI1 expression is restricted to hematopoietic stem cell fraction and EVI1-expressing cells show robust long-term reconstitution capacity using Evi1-IRES-GFP knock-in (EVI1-GFP) mice, which enable us to track Evi1 expression on a single cell basis. In this study, we tried to elucidate the functional implication of EVI1 expression in AML using these mice. We generated murine EVI1-GFP AML model by retrovirally transducing MLL-AF9 or -ENL fusion gene into Lineage- Sca-1+ c-kit+ (LSK) cells from EVI1-GFP mice followed by transplantation into lethally irradiated syngeneic mice. Clonogenic and leukemogenic potentials of AML cells, especially leukemic cells with a granulocyte-macrophage progenitor phenotype (L-GMPs) from these mice, were compared according to GFP expression. Remarkably, GFP-positive L-GMPs tended to show lower colony-forming activity in semi-solid media and lower leukemia-initiating potential than GFP-negative L-GMPs. GFP-positive L-GMPs, however, induced a more aggressive form of AML, characterized by shorter survival in the secondary transplantation model. When EVI1-GFP AML mice underwent cytotoxic chemotherapy with cytarabine, the GFP-positive fraction was enriched during myelosuppression, indicating the survival advantage of EVI1-positive cells. To investigate the downstream target of EVI1, we employed murine EVI1-AML model, where murine hematopoietic cells exogenously expressing 3×FLAG-tagged EVI1 were transplanted into syngeneic mice. Using EVI1-AML cells, we performed chromatin-immunoprecipitation coupled to next-generation sequencing (ChIP-seq) by anti-FLAG tag antibody. To identify leukemia-specific targets of EVI1, the result was compared with the result of ChIP-seq obtained from 32D-cl3 murine hematopoietic progenitor cells with 3×FLAG tag inserted into 3'-end of the coding region of the EVI1 gene. Gene ontology analysis revealed that genes involved in immune processes are explicitly enriched in the leukemia samples. Among the list of EVI1-bound genes, we tried to refine functional downstream targets of EVI1, which are upregulated in murine EVI1-AML cells and of which expressions are positively correlated with EVI1. By combining the ChIP-seq data with murine transcriptome data that compare hematopoietic progenitor cells expressing empty-vector and EVI1+ AML cells, and public gene expression datasets of human AML (Valk et al. NEJM 2004), we picked out 18 genes as candidate EVI1 downstream genes. Functional screening using EVI1-AML cells and shRNAs against these genes revealed that silencing of ETS transcription factor ERG (ETS-related gene) markedly suppressed proliferation and colony-forming activity of EVI1-AML cells, as well as rendered them vulnerable to cytotoxic agents. Normal c-kit+ hematopoietic progenitor cells were less affected by shRNAs against ERG. By comparing MLL-ENL immortalized murine hematopoietic cells with high and low EVI1 expression, EVI1-high MLL-ENL cells showed higher ERG dependency than EVI1-low MLL-ENL cells. Pharmacological inhibition of ERG also led to marked inhibition of EVI1-AML cells and EVI1-high MLL-ENL cells. Finally, knockdown of ERG remarkably delayed AML development in bone marrow transplantation model of EVI1-AML and EVI1-expressing MLL-ENL AML. Our data suggest that EVI1-positive AML cells are characterized by an aggressive nature and resistance to cytotoxic agents, as well as low leukemia stem cell capacity. ERG would be a novel downstream target of EVI1, on which survival of EVI1-expressing AML cells depends. Disclosures Masamoto: Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Chugai Pharmaceutical Company: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; MSD K.K.: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; SymBio Pharmaceuticals: Speakers Bureau. Kurokawa: Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau.