The New York Blood Center (NYBC) is a community, nonprofit blood bank based in New York City. Established in 1964 by Dr. Aaron Kellner, NYBC supplies blood to approximately 200 hospitals in the Northeast United States. NYBC and its operating divisions also provide transfusion-related medical services to over 500 hospitals nationally.NYBC, along with its operating divisions Community Blood Center of Kansas City, Missouri (CBC), Innovative Blood Resources (IBR), Blood Bank of Delmarva (BBD), and Rhode Island Blood Center (RIBC), collect approximately 4,000 units of blood products each day and serve local communities of more than 75 million people in the Tri-State area (NY, NJ, CT), Mid Atlantic area (PA, DE, MD, VA), Missouri and Kansas, Minnesota, Nebraska, Rhode Island, and Southern New England. In addition to serving the New York City metropolitan area, New Jersey, Connecticut and Pennsylvania, in May 2014, NYBC merged its operations with Community Blood Center of Greater Kansas City (CBC). In February 2016, NYBC and CBC announced the creation of the Kansas City-based National Center for Blood Group Genomics, a new laboratory that will focus on precision testing of blood donor samples.NYBC maintains close relationships with both New York City's Police Department (NYPD) and Fire Department (FDNY). Among NYBC's largest donor groups is the NYPD, which donated more than 11,000 pints of blood through November 2015. At the same time, the FDNY participates with NYBC in the "Be The Match Program" operated by the National Marrow Donor Program (NMDP). More than 8,000 FDNY members are on the potential donor list, and 179 members have already given this life saving gift to those in need. FDNY members represent more than 10% of all NYBC bone marrow donors. Each year, at an annual induction ceremony hosted by FDNY and NYBC at FDNY headquarters, donors and their recipients meet for the first time. In 2016, Firefighter Mike Wilson was introduced to a recipient from Erie, Pennsylvania, who received his lifesaving bone marrow to treat her acute myeloid leukemia (AML), while Firefighter Frank Perdue met a recipient diagnosed with essential thrombocythemia, a rare chronic blood disorder. In 2015, firefighter Michael McCauley of Staten Island met his recipient, a United States Marine Sergeant who saw combat in Iraq, and who was subsequently diagnosed with acute myeloid leukemia (AML). Through FDNY’s participation in NYBC’s program, McCauley’s bone marrow is credited with saving the recipient's life.NYBC houses Lindsley F. Kimball Research Institute and the Howard P. Milstein National Cord Blood Center, a public cord blood bank named after board member Howard Milstein. The National Cord Blood Program (NCBP), directed by Dr. Pablo Rubinstein, is the oldest and largest in the world. In August 2015, the NCBP announced a new collaboration with the University of California, Davis Health System to manufacture specialized lines of highly adaptable stem cells for potential new therapies for diseases that include anemia, leukemia, lymphoma, sickle cell disease and severe combined immunodeficiency.The Lindsley F. Kimball Research Institute (LFKRI) has been awarded grants to conduct research in such areas as epidemiology and the development of HIV self-testing interventions, cellular therapy and transfusion medicine, in vitro platelet production, blood genomics, immunology, the development of infectious disease screening techniques and preventions for diseases like severe acute respiratory syndrome, Hepatitis B and Hepatitis C.
The spatial organization and transcriptional activity of transcription factors are increasingly recognized to be regulated by liquid-liquid phase separation (LLPS). Whether GATA1, the master regulator of erythropoiesis, undergoes LLPS and how this process influences erythroid development have remained unknown. Here, we show that GATA1 forms dynamic nuclear condensates in HEK293T, HUDEP2 progenitors, and erythroleukemia cells, as well as concentration-dependent droplets in vitro. These condensates exhibit hallmark properties of LLPS, including fusion behavior, sensitivity to 1,6-hexanediol, and rapid fluorescence recovery after photobleaching (FRAP). Domain deletion and mutational analyses revealed intrinsically disordered region 2 (IDR2) as the primary driver of GATA1 LLPS. Importantly, the congenital anemia-associated R307H mutation and dephosphorylation-mimicking S310A substitution within IDR2 disrupted droplet formation and abolished FRAP recovery. Mechanistically, loss of LLPS reduced GATA1 chromatin occupancy, impaired its assembly with cofactors such as FOG1 and LMO2, and abrogated activation of erythroid gene promoters in luciferase assays, leading to defective terminal differentiation. Together, these findings uncover a previously unrecognized mechanism by which GATA1 regulates erythropoiesis through LLPS and highlight modulation of its IDR2 domain and post-translational modifications as potential therapeutic strategies for erythroid-related disorders.
Immune checkpoint blockade can elicit durable antitumor responses, yet tumor heterogeneity and adaptive resistance often necessitate combination strategies that increase systemic toxicity. In vivo genome editing offers a programmable route to durable immunomodulation but remains difficult to spatially confine in solid tumors. Here we develop a magnetically gated genome-editing platform that enables spatially confined immunomodulation. The system integrates a non-replicating baculoviral vector with magnetic nanoparticles (MBV), in which magnetic activation restores viral transduction despite complement-mediated inactivation, thereby confining CRISPR activity to tumor regions. Baculoviral transduction engages antiviral innate programs that promote chemokine signaling and antigen presentation in tumors. In a syngeneic colon cancer model, MBV-mediated disruption of Pdl1 restricts checkpoint loss to tumor tissue while preserving immune activation, enhancing immune infiltration and suppressing tumor growth. Local Pdl1 editing synergizes with CTLA-4 blockade, extending survival without overt toxicity. These findings define MBV as a controllable genome-editing architecture for localized combination immunotherapy.
Mesenchymal stem cells (MSCs) were first described over 30 years ago as multipotent stromal cells that can self-renew and differentiate into bone, cartilage, and fat tissues. MSCs have numerous functions, such as modulating the immune system, regulating inflammation, promoting wound healing, improving engraftment following hematopoietic transplant, and providing a supportive microenvironment for hematopoietic stem cells. Human umbilical cord tissue, which is more easily obtained than bone marrow aspirates, contains a population of MSCs. These cells can enhance thrombopoiesis in umbilical cord blood-derived megakaryocytes, leading to the production of platelets with low baseline activation levels. Scaled-up methods to enhance platelet production in vitro while maintaining a low activation state would benefit transfusion medicine and circumvent frequent platelet donor shortages. Here, we outline a protocol to isolate and expand MSCs from human umbilical cord tissue and characterize their cell surface protein expression by flow cytometry, self-renewal potential by colony-forming assay, and tri-lineage differentiation capacity.
Intravascular hemolysis is a hallmark of sickle cell disease (SCD). While it has been well established that the hemolysis-derived products, such as hemoglobin (Hb) and free heme, exert proinflammatory and pro-oxidative effects, contributing to the vascular and tissue damage in SCD, the effects of hemolysis on erythropoiesis have not been studied. We and others have reported that hemolysis in SCD led to upregulation of type I interferon IFNα. We further documented that the ability of Townes sickle mice to increase their erythropoietic capacity to compensate for anemia was impaired. To examine whether the impaired erythropoiesis in SCD is associated with the hemolysis-driven IFNα production and to define the underlying mechanisms, we injected mice with hemin, red cell lysate, or IFNα to mimic the hemolysis status in SCD and then examined erythropoiesis by colony-forming assay, flow cytometric analysis, and western blot. We also examined the effects of hemin and IFNα on erythropoiesis using an in vitro erythroid culture system. We found that intravascular hemolysis inhibited erythropoiesis in SCD through inhibition of erythropoietin (EPO)/erythropoietin receptor (EPOR) signaling via a heme-IFNα-CISH axis. Herein, we describe how to prepare hemin, red cell lysate, and IFNα, and present examples of in vivo and in vitro assays to assess erythropoiesis. Our methods can be applied to study changes in erythropoiesis in other diseases characterized by intravascular hemolysis.
BACKGROUND ABO histocompatibility plays a critical role in red blood cell (RBC) transfusions and is of great importance in solid organ and hematopoietic stem cell transplantation. ABO is primarily typed from red blood cell samples using serological reagents. However, stem cell donors are often screened using buccal swabs which cannot be used for serologic testing. Furthermore, serologic testing can be complicated by recent transfusions, weak reactions, or unusual reaction patterns, which require resolution. ABO genotyping can help in these circumstances. Here we show the development and validation of a targeted Next Generation Sequencing (NGS) ABO genotyping assay and companion interpretive software. STUDY DESIGN AND METHODS Short-read NGS was performed on 237 samples (blood n = 237 and buccal swab n = 42) obtained for routine transplant work-up testing and 10 samples with complex and rare ABO with previous reference laboratory workups. Three samples selected to represent common heterozygous ABO allele combinations underwent long-read sequencing as a proof of principle. All data was analyzed by our bloodTyper software. RESULTS The targeted NGS results for the solid organ and stem cell donor sample set was 99.3% (n = 287/289) concordant with serology with the two discordants likely being due to an unrecognized Ael and natural tissue-specific chimerism. In the complex and rare ABO samples NGS improved characterization of the ABO gene and uncovered important changes not initially found in the previous workups. Long-read sequencing was able to fully phase the three samples tested. CONCLUSION Through use of standard NGS protocols and automated interpretive software, targeted NGS allowed for reliable genotyping of ABO allele haplotypes without subject matter expert intervention. As such, targeted ABO NGS should become the standard for ABO genotyping.
