Production Of Hematopoietic Cells Research Articles

Flow Cytometry Analysis of Hematopoietic Stem/Progenitor Cells and Mature Blood Cell Subsets in Atherosclerosis.

Advancing age causesphysiologic decline in tissue function. In the hematopoietic system this manifests as a progressive reduction in blood or immune cell function and clonal hematopoiesis, where a mutated hematopoietic stem cell candominate blood cell production andconfer an increased propensity for myeloid malignancy. In the aging cardiovascular system, atherosclerosis causes an inflammatory cell- driven accumulation of lipid-derived plaques in major arteries which constrains blood flow and can lead to myocardial infarction and stroke. Clonal hematopoiesis in the elderly has recently been associated with a substantially increased risk of atherosclerosis-related cardiovascular disease. However, the direct association between deregulated hematopoiesis in clonal hematopoiesis and atherosclerosis is poorly defined. Herein, we describe a flow cytometry method to prospectively analyze the crucial hematopoietic stem/progenitor, inflammatory and lymphoid cell participants in atherosclerosis. This analysis can be applied to decipher the complex relationship between hematopoietic cell types involved in clonal hematopoiesis and atherosclerosis in mouse models.

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. V: Manufacture and quality control

Chimeric antigen receptor T cells (CAR-T), especially against CD19 marker, present in lymphomas and acute B leukemia, enabled a revolution in the treatment of hematologic neoplastic diseases. The manufacture of CAR-T cells requires the adoption of GMP-compatible methods and it demands the collection of mononuclear cells from the patient (or from the donor), generally through the apheresis procedure, T cell selection, activation, transduction and expansion ex vivo, and finally storage, usually cryopreserved, until the moment of their use. An important aspect is the quality control testing of the final product, for example, the characterization of its identity and purity, tests to detect any contamination by microorganisms (bacteria, fungi, and mycoplasma) and its potency. The product thawing and intravenous infusion do not differ much from what is established for the hematopoietic progenitor cell product. After infusion, it is important to check for the presence and concentration of CAR-T cells in the patient's peripheral blood, as well as to monitor their clinical impact, for instance, the occurrence of short-term, such as cytokine release syndrome and neurological complications, and long-term complications, which require patient follow-up for many years.

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

BackgroundGraft-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.MethodsNeuroblastoma 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.ResultsPDX 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.ConclusionThese 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.

Volume determination methods for hematopoietic progenitor cell products collected by apheresis

CD34+ cell count of hematopoietic progenitor cell products is often first determined using a product sample, with the final reported cell count obtained by multiplying cell count from the sample by the total product volume. Product volume may be determined by apheresis instruments used for collection or calculated based on specific gravity (SG). Here the authors sought to determine the discrepancies between these methods and the impact on patient care. A total of 262 products collected from 176 donors by apheresis were retrospectively reviewed. Volumes calculated by apheresis instruments were compared with volumes calculated based on SG. CD34+ cell/kg doses based on volumes from apheresis instruments and SG were also compared. Furthermore, among 42 patients who required multiple collections, the authors determined whether different calculation methods would have changed the number of procedures required. The volumes for 253 products (96.6%) and CD34+ cell/kg doses for 257 products (98.1%) generated from apheresis instruments and SG were similar. Products with discrepant volumes when calculated using different methods were not associated with delayed engraftment. Forty patients who underwent multiple collections (95.2%) were under their collection goals during their first collection and would have required multiple procedures regardless of which method was used to calculate product volumes. Volumes determined based on SG did not provide a clear benefit over volumes determined by apheresis instruments. Hence, variation in volume calculation methods across different laboratories is likely to lead to minimal impact on patient care.

Immunoexpression of CD34, CD117, and p53 in Hypocellular Bone Marrow Disorders.

Objectives Hypocellular bone marrow (BM) disorders comprise heterogeneous entities associated with peripheral cytopenias and decreased production of hematopoietic cells in BM. This study was undertaken to analyze immunohistochemical expression of CD34, CD117, and p53 in morphologically diagnosed patients of hypocellular BM (aplastic anemia [AA], hypocellular myelodysplastic syndrome [h-MDS], and hypocellular acute myeloid leukemia [h-AML]). Materials and Methods BM specimens were obtained from patients presenting with pancytopenia/bicytopenia. On 30 patients diagnosed as hypocellular BM, immunohistochemistry (IHC) for CD34, CD117, and p53 was performed. Results BM cellularity was < 30% in all (100%) patients. Blast count was increased in h-MDS and h-AML. Features of dysplasia were noted in six (20%) patients. Out of these, three patients were diagnosed as h-MDS having bilineage/trilineage dysplasia, and the other three patients were of AA (11.5% patients) displaying only dyserythropoiesis. On IHC, percentage of BM CD34+ cells was increased in h-MDS+ h-AML (3.87 ± 0.86) as compared with AA (0.19 ± 0.15) and controls (0.81 ± 0.21), p = 0.01. Percentage of BM p53+ cells was also increased in h-MDS+ h-AML (2.9 ± 2.07) as compared with AA and controls, which did not show any p53+ cells, p = 0.0. No statistically significant difference was observed in the expression of CD117 in h-MDS+ h-AML (4.95 ± 3.40) compared with AA (4.49 ± 1.07), p = 0.99. Conclusion The study demonstrates the usefulness of CD34 and p53 immunoexpression as an important ancillary method in distinguishing various hypocellular BM disorders, especially h-MDS and AA. However, the role of CD117 remains unclear and needs to be evaluated further by larger studies.

Decreased IGF-1 Expression in the Bone Marrow Microenvironment Drives Hematopoietic Aging

One factor responsible for poor hematopoietic health-span is overproduction of myeloid lineage cells at the expense of lymphoid cells, which is associated with increased risk of myeloid leukemia and reduced immune responsiveness with aging. This lineage imbalance is caused by declining ability of hematopoietic stem cells (HSC) to regenerate and sustain proper lineage-balanced production of mature hematopoietic cells. We identify the hematopoietic decline to become significant in C57BL/6 mice as young as 8 months old, suggesting that by middle age the initiation events of myeloid overproduction have already begun to occur. Placing young (2mo) long-term hematopoietic stem cells (LT-HSC) into a 2mo or middle-aged (9-12mo) bone marrow (BM) microenvironment is sufficient to induce myeloid-biased cell production (2mo into 2mo: 12.3% myeloid ± SEM (n= 3) vs. 2mo into 9-12mo: 26.5% myeloid ± SEM (n= 11) ** P < 0.01), clearly implicating components of the bone marrow microenvironment in the age-related overproduction of myeloid cells. Data utilizing RNA-Seq and ELISA analysis identify a decrease in insulin-like growth factor (IGF)-1 expression as candidate signaling factor produced by cells in the middle- aged microenvironment that drive myeloid-biased hematopoiesis from HSC. LT-HSC expression of IGF-1 signaling receptor, IGF1R, is increased with age identifying that the balance of IGF signaling is altered in the bone marrow microenvironment. In vitro studies show that the stimulation of the middle-aged (12-14mo) Lin-/c-kit+/Sca-1+(LSK) population by IGF-1 leads to decreased myeloid production, rescuing the aging phenotype (12-14mo: 126.7 CFU vs. 12-14mo + IGF1: 79.9 CFU ± SEM *** P < 0.001 by one-way ANOVA ± SEM of n= 3 replicates). Phospho-flow analysis of middle-aged LT-HSC from mice indicate increased IGF1R and AKT signaling response to IGF-1 stimulation suggesting the decrease of IGF-1 bioavailability in the bone marrow microenvironment seen during aging is influencing LT-HSC function and lineage output. Our ongoing studies aim to define how aging-induced alterations in the BM microenvironment initiate myeloid lineage-biased hematopoiesis from HSC and how this is mediated through IGF signaling. Understanding these mechanisms may enable new therapeutic approaches to improve hematopoietic health-span. DisclosuresNo relevant conflicts of interest to declare.

Generation of Fungus-Specific T Lymphocytes for Use in HSCT Patients with Invasive Fungal Disease

Invasive fungal disease (IFD) is a major cause of morbidity and mortality in patients treated for haematological malignancies. The major determinants of outcome are the degree of the patient's immune incompetence, the fungal species and the presence of drug resistance in the infecting organism. As uncommon and drug resistant strains increase in frequency, effective alternative treatment strategies are needed to improve the dismal outcomes for IFD occurring after hematopoietic stem cell transplant (HSCT). Recent data provide strong evidence that a therapeutic benefit is seen using adoptive transfer of cryopreserved, partially human leukocyte antigen (HLA) matched 3rd party T-cells for post-transplant viral infections in HSCT recipients. We propose that a similar approach may be effective in IFDs.In the present study, we developed a method for rapid manufacture of large numbers of broadly reactive and highly specific antifungal T-cells starting with a stem cell product obtained from healthy donors as part of normal allogeneic transplant procedure. Water-soluble lysates of germinated conidia of Aspergillus and Candida species were used to stimulate 60.5 ± 21.7 x 106 mononuclear cells from the hematopoietic precursor cell (HPC) product. Reactive T-cells were selected using activation marker and magnetic bead isolation and expanded in culture with cytokines. After elution from the magnet, the enriched population measured 82.5±13.3 x103 cells of which 82.3±6.9% were CD4+ (n=6). All cultures were maintained for a total of 11 to 12 days, leading to 126.8±11.9x106 fungus-specific T cells, corresponding to approximately 1000 fold expansion within 12 days of culture initiation. At the end of culture greater than 97.3±0.9% of cells were CD3+, of which 91.7±1.2% were CD4+ cells and 3.6±0.7% were CD8+ cells.Responses against fungus were measured using cytokine flow cytometry after restimulation by specific fungal lysates. 59.4±0.9% of CD4+ cells were reactive against fungus-primed monocyte-derived dendritic cells (MoDCs). Production of the pro-inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-17 and interferon (IFN)-γ following challenge showed that the expanded T-cell populations had broad reactivity against Aspergillus fumigatus / flavus / terreus, Candida albicans / krusei, Fusarium oxysporum / solani and Leptosporum (Scedosporium) prolificans (Figure 1).We previously demonstrated that antifungal reactivity is mediated through HLA-DR and can occur through a single matching HLA-DR molecule. We calculated that in our HSCT population, donors with the seven highest frequency HLA-DR molecules (DRB1 1:01, 3:01, 4:01, 7:01, 11:01, 13:01 or 15:01) could be used to generate a bank of cryopreserved fungus-specific T lymphocytes to treat IFDs in HSCT recipients. Using a bank of this size, approximately 85% of HSCT recipients would have a fungus-specific T cell product in the bank matching at least 1 HLA-DR molecule. Increasing the bank to add donors with four additional HLA-DR types (DRB1 4:04, 11:04, 13:02 and 15:02) would increase the percentage of recipients with a banked product matching at least 1 of HLA-DR molecules to almost 95%.In conclusion, we have identified a GMP compliant method to rapidly manufacture fungal T-cell products to high number with specificity against a range of clinically relevant fungal pathogens including yeasts and moulds. Using these cells, we intend to construct a bank of fungus-specific T cells from donors with common HLA-DRB1 types and to test their efficacy in HSCT recipients with IFD. If effective in restoring immunity, adoptive cell therapy using 3rd party broadly reactive antifungal T-cells could be useful in improving outcomes for HSCT patients with IFDs.Figure 1: Cytokine expression profile of reactive antifungal T cells expanded with Aspergillus and Candida lysates following rechallenge with lysates of specific fungal pathogens or a mix of all lysates. Results are means of 6 experiments (±s.e.m) and shown as percentage of CD4+ T cells. [Display omitted] DisclosuresGottlieb:Indee: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.

Progress in the production of haematopoietic stem and progenitor cells from human pluripotent stem cells

Cell therapies are currently used to treat many haematological diseases. These treatments range from the long-term reconstitution of the entire haematopoietic system using the most potent haematopoietic stem cells (HSCs) to the short-term rescue with mature functional end cells such as oxygen-carrying red blood cells and cells of the immune system that can fight infection and repair tissue. Limitations in supply and the risk of transmitting infection has prompted the design of protocols to produce some of these cell types from human pluripotent stem cells (hPSCs). Although it has proven challenging to generate the most potent HSCs directly from hPSCs, significant progress has been made in the development of differentiation protocols that can successfully produce haematopoietic progenitor cells and most of the mature cell lineages. We review the key steps used in the production of haematopoietic stem and progenitor cells (HSPCs) from hPSCs and the cell surface markers and reporter strategies that have been used to define specific transitions. Most studies have relied on the use of known markers that define HSPC production in vivo but more recently single cell RNA sequencing has allowed a less biased approach to their characterisation. Transcriptional profiling has identified new markers for naïve and committed hPSC-derived HSPC populations and trajectory analyses has provided novel insights into their lineage potential. Direct comparison of in vitro- and in vivo-derived RNA single cell sequencing datasets has highlights similarities and differences between the two systems and this deeper understanding will be key to the design and the tracking of improved and more efficient differentiation protocols.

A Case of Rare Autoimmune Pancytopenia Due to Graves’ Disease

Introduction: Graves’ disease has been associated with cytopenias, most commonly anemia. Pancytopenia is a rare complication and is not often encountered.Case Presentation: A 54-year-old woman with history of multiple sclerosis on alemtuzumab was referred for abnormal thyroid function tests. At the initial visit, the patient reported a 20-pound unintentional weight loss, tremors, sweating and heat intolerance. She was 3 years post-menopausal and had no history of recent viral illness, contrast exposure or family history of thyroid disease. Physical exam revealed an anxious appearing woman with fine tremors in both hands, hyperreflexia but no lid lag or exophthalmos. Labs included TSH <0.005 uIU/mL (0.400-4.2), free T4 2.89 ng/dL (0.80-1.50), TSH receptor binding antibody 8.24 IU/L (< 2.00), TSI 7.27 IU/L (<0.56), WBC 2900/uL (4500-11000), ANC 1746/uL (1900 - 8000), hemoglobin 11.4 g/dL (11.7-15.0) and platelet 207,000/uL (150,000-450,000). Thyroid ultrasound noted sub-centimeter hypoechoic/cystic nodules and 24-hour thyroid uptake showed 56.4% symmetric uptake (upper limit of normal 30%). Due to leukopenia, the patient was started on propranolol and underwent radioiodine ablation with 14.8 mCI. Two weeks later, she reported easily bruising and gum bleeding and her blood work was significant for pancytopenia. The patient was referred to hematology and two bone marrow samples were obtained. Though MDS/MPN was initially suspected based on atypia in both specimens, there were no immunophenotypic, cytogenetic or molecular abnormalities suggesting a neoplastic process. Instead the patient was thought to have autoimmune pancytopenia secondary to Graves’ and the atypia was believed to be secondary to peripheral consumption. She was started on prednisone 1 mg/kg/day with resolution of her cytopenias.Discussion: Graves’ disease has been associated with hematological abnormalities including isolated anemia (the most common), thrombocytopenia or leukopenia. Pancytopenia is an uncommon complication and is rarely described in the literature. The exact mechanism remains unclear, but may be related to either reduced production of hematopoietic cells from the bone marrow or increased destruction of mature hematopoietic cells due to autoantibodies. Since thyroid hormones are known to increase erythropoietin, this leads to an exaggerated consumption of iron, folic acid and vitamin B12 and can cause various forms of anemias. Leukopenia may be secondary to immunologic destruction whereas thrombocytopenia may be due to antiplatelet antibodies or increased splenic sequestration. Pancytopenia is rare. Our patient was treated with high dose prednisone with resolution of her pancytopenia, which suggests an autoimmune process as the mechanism. Our case showcases a rare complication of Graves’ disease and highlights that high dose steroid therapy may improve cytopenias associated with this condition.

Performance evaluation of a novel conceptual bioprocess for clinically-required mass production of hematopoietic cells.

The novel engineered bioprocess, which was designed and modeled to provide the clinically relevant cell numbers for different therapies in our previous work (Kaleybar et al. Food Bioprod Process 122:254-268, https://doi.org/10.1016/j.fbp.2020.04.012 , 2020), was evaluated by using U937 as hematopoietic model cells. The culture system showed a 30-fold expansion of U937 cells in one-step during a 10-day culture period. The cell growth profile, the substrate and oxygen consumptions, and byproduct formations were all in agreement with the model predications during 7 days. The cell proliferation decrease after 7 days was attributed to optional oxygen limiting condition in the last days of culture. The bioreactor culture system revealed also a slight enhancement of lactate dehydrogenase (LDH) production as compared to the 2D conventional culture system, indicating the low impact of shear stress on cellular damage in the dynamic system. The results demonstrated that the conceptual bioprocess for suspended stem cell production has a great potential in practice although additional experiments are required to improve the system.

Ex Vivo Whole-Mount Imaging of Leukocyte Migration to the Bone Marrow.

The bone marrow is the major hematopoietic organ, consisting of distinct microenvironmental niches for the production of hematopoietic cells. Advanced visualizing methods are required to define and better understand the interactions between stromal and hematopoietic cells. In this chapter, we describe an ex vivo whole-mount imaging technique of the bone marrow, which allows for a fast, high-quality, and three-dimensional visualization of different bone marrow components. We provide a guide for conducting adoptive transfer experiments of fluorescently labeled leukocytes and visualizing their location in the bone marrow with respect to the bone marrow vasculature. This method presents a quick, easy, and inexpensive approach to image the bone marrow in three dimensions.

3D Microscopy of Murine Bone Marrow Hematopoietic Tissues.

The soft marrow tissues, which are found disseminated throughout bone cavities, are prime sites for hematopoietic cell production, development, and control of immune responses, and regulation of skeletal metabolism. These essential functions are executed through the concerted and finely tuned interaction of a large variety of cell types of hematopoietic and nonhematopoietic origin, through yet largely unknown sophisticated molecular mechanisms. A fundamental insight of the biological underpinnings of organ function can be gained from the microscopic study of the bone marrow (BM), its complex structural organization and the existence of cell-specific spatial associations. Albeit the application of advanced imaging techniques to the analysis of BM has historically proved challenging, recent technological developments now enable the interrogation of organ-wide regions of marrow tissues in three dimensions at high resolution. Here, we provide a detailed experimental protocol for the generation of thick slices of BM from murine femoral cavities, the immunostaining of cellular and structural components within these samples, and their optical clearing, which enhances the depth at which optical sectioning can be performed with standard confocal microscopes. Collectively, the experimental pipeline here described allows for the rendering of single-cell resolution, multidimensional reconstructions of vast volumes of the complex BM microenvironment.

Evaluation of bone marrow in patients with pancytopenia

Introduction : Pancytopenia is common hematological term described as decreased red blood cells, white cells and platelets in blood caused by either reduced production of haematopoietic cells in bone marrow or secondary destruction or reduction by drugs, toxins, radiotherapy, chemotherapy, infection etc. The diagnosis of causes for it are difficult to decide but few minimal invasive methods like bone marrow aspiration(BMA) and biopsy (BMB)are very useful. Objectives: To evaluate the diagnostic role of BMA and BMB in pancytopenia. Materials and Methods: This was observational study conducted over a period of 36 months on 145 patients. All age patients with new onset pancytopenia fulfilling inclusion criteria were included. Exclusion criteria: Radiation/chemotherapy induced pancytopenia. BMA and BMB were done in all cases. BMA smears were air dried and stained by Romanowsky stains while biopsy was fixed in formalin and sent for paraffin sectioning. BMA and BMB were analysed and data was statistically analysed. Results: Out of 145 patients, The mean age of the patients was 46years.Pallor and generalized weakness were commonest clinical complaints. Megaloblastic anemia was the most common cause (32%) followed by hypoplastic marrow in 16% cases. Rest causes were acute leukemia, anemia, myelodysplastic syndrome, metastases etc. Conclusion: BMA is good for studying morphology of hematopoietic cells while BMB for assessing cellularity and structure of bone marrow like fibrosis, infiltration & metastasis. BMA and BMB are complimentary with each other when used together for evaluation of bone marrow in routine haematological disorders. Keywords: Bone Marrow Aspiration, Bone Marrow Biopsy, Pancytopenia.

Suppression of Hematopoietic Activity in Tenascin-C–Deficient Mice

AbstractTenascin-C (TN-C), a member of the extracellular matrix (ECM) glycoprotein family, is expressed on the surface of stromal cells in the hematopoietic system or lymphoid organs. Recently, TN-C–deficient mutant mice produced by TN-C gene targeting through homologous recombination were shown to develop normally, although TNs have been reported to play important roles in organogenesis and carcinogenesis. In the present study, we found that colony-forming capacity of bone marrow (BM) cells was considerably lower in TN-C–deficient mice (a decrease of ∼35% from control), although their mononuclear cell count and BM architecture showed no significant difference from those of normal mice. Furthermore, in long-term BM culture in vitro, hematopoietic cell production (a decrease of ∼40% in Dexter's condition and of ∼65% in Whitlock-Witte's condition from control), colony-forming capacity of the produced cells (a decrease of ∼60% from control), and longevity of the cultures were markedly lower in the TN-C–deficient mice than in control mice, whereas hematopoiesis in the TN-C–deficient mutant mice was sustained. The addition of TN-C glycoprotein to long-term BM cultures of TN-C–deficient mice clearly induced the recovery of hematopoietic cell production and colony-forming capacity of hematopoietic progenitor cells. Thus, these results provide direct evidence that an ECM glycoprotein component, TN-C, plays a relevant role in hematopoiesis through interactions between stromal cells and hematopoietic progenitor cells.

Whole-Exome Sequencing Identifies a Somatic Cell Mutation Signature That Predicts Relapse Risk and Survival Probability in Multiple Myeloma

Introduction: Multiple myeloma (MM) is a plasma cell neoplasm associated with heterogeneous somatic alterations. Despite the development of novel anti-myeloma agents that have significantly prolonged patient survival, disease relapse remains a daunting problem. Our goal was to employ whole-exome sequencing (WES) to better describe the mutational landscape in MM beyond the tumor cell and identify genomic factors that might predict relapse. WES was performed using autograft samples obtained from MM patients that were then treated with high dose melphalan and autologous hematopoietic cell transplant (HCT). We identified a panel of genes that were most frequently mutated in all patients and then identified those genes mutated with greater frequency in patients that relapsed. A relapse burden signature was generated based upon the genes that were most frequently mutated genes in relapsed patients. Finally, the relapse burden signature was correlated with patient progression-free survival (PFS) and overall survival (OS) following autologous HCT. Methods. DNA was extracted from one ml of cryopreserved, mobilized hematopoietic cell product obtained from patients (N=93) that underwent HCT and was provided by the Case Comprehensive Cancer Center Hematopoietic Biorepository Core. Targeted sequencing was performed using the Tempus xE whole exome platform (Tempus, Chicago, IL). Variants were identified using a variant allele frequency (VAF) ≥0.1 for each sample. Variants were tabulated for each gene in each patient. Patients were grouped according to their relapse status; "No Relapse" (N=39) and "Relapse" (N=54) which corresponded to their post-HCT outcome. Relapse time was defined as time from transplant to event. Variants identified in each gene and patient group were counted and ranked. A relapse burden signature was defined and included twenty-two genes over-represented in the relapse group compared to the non-relapse group by &amp;gt; 10%. Genes in the relapse burden signature were subjected to gene set enrichment analysis (GSEA) and cross referenced against Gene Ontology (GO) categories. PFS and OS were defined as the time from transplant until the event of interest, with censoring at time of last follow up. Patients were regrouped according to their mutation burden in the relapse signature genes ("High burden" defined as &amp;gt;=six signature genes with variants) and their OS and PFS were analyzed with an R package (survival) to generate Kaplan-Meier curves and statistical significance based on a Chi-square test between low and high burden patients. Results: In total, 3,523 genes were identified as containing variants. Table 1 lists the top thirty genes that were identified and ranked based upon total number of mutations (mutational count) and most frequently mutated in relapsed and non-relapsed patients (sample count). We then identified those genes that were more frequently mutated by at least 10% in relapsed patients compared to non-relapsed patients (Fig. 1A). GSEA revealed that the relapse burden gene signature was associated with protein O-linked glycosylation, glycan processing, Golgi lumen and innate immune response activating cell surface receptor signaling pathways (Table 2). Interestingly, multiple mucin family members (Muc2, Muc3A, Muc12 and Muc19) were represented in the relapse burden signature. GO analysis indicated that the individual mucin genes were associated with the same signaling pathways that had been associated with the relapse burden signature by GSEA (Table 3). Importantly, a high relapse burden signature was correlated with a statistically significant reduction in both PFS and OS (Fig. 1B, C). Conclusion: Taken together, our results support the feasibility of WES to generate a relapse burden signature that predicts the risk of MM patients for relapse following HCT. Moreover, the mutational landscape associated with relapse, i.e. the specific genes mutated, has provided insights on the mechanisms of relapse. It is noteworthy that the relapse burden signature genes identified here were mutated at a much greater frequency than genes associated with clonal hematopoiesis of indeterminate potential (CHIP). The identification of patient subgroups at heightened risk of relapse can better guide treatment decisions. Future studies will be conducted to evaluate the effect of pathways identified here on myeloma cell survival and to validate actionable therapeutic targets. Disclosures Malek: Bluespark: Research Funding; Takeda: Other: Advisory board , Speakers Bureau; Medpacto: Research Funding; Janssen: Other: Advisory board, Speakers Bureau; Sanofi: Other: Advisory board; Clegene: Other: Advisory board , Speakers Bureau; Amgen: Honoraria; Cumberland: Research Funding. Caimi:Amgen: Other: Advisory Board; Bayer: Other: Advisory Board; Verastem: Other: Advisory Board; Kite pharmaceuticals: Other: Advisory Board; Celgene: Speakers Bureau; ADC therapeutics: Other: Advisory Board, Research Funding. de Lima:Celgene: Research Funding; BMS: Other: Personal Fees, advisory board; Incyte: Other: Personal Fees, advisory board; Kadmon: Other: Personal Fees, Advisory board; Pfizer: Other: Personal fees, advisory board, Research Funding.

Cryopreservation of hematopoietic cells using a pre-constituted, protein-free cryopreservative solution with 5% dimethyl sulfoxide

Background aimsAdequate cryopreservation techniques are critical to ensure optimal recovery of functional progenitor cells in hematopoietic cell (HC) transplantation, minimize risk of contamination and prevent infusion-related adverse events (irAEs). In this article, we provide graft function and infusion safety results observed by decreasing the concentration of dimethyl sulfoxide (DMSO) in cryopreservative media and by minimizing processor-dependent formulation. MethodsTen HC products, collected after standard mobilization of multiple myeloma patients, were cryopreserved with PRIME-XV FreezIS (FreezIS) and compared with products previously cryopreserved with media formulated in-house to achieve a final DMSO concentration of 10% (Std10) and 5% (Std5). At infusion, HCs were analyzed for recovery of CD34+ cells and viability; irAEs and time to engraftment of neutrophils and platelets were also monitored. ResultsMedian CD34+ cell recovery for HC cryopreserved with Std10, Std5 and FreezIS was 38%, 78% and 68%, respectively (P = 0.0002). There were less frequent irAEs with Std5 and FreezIS (10%) compared with Std10 (80%) (P ≤ 0.0001). Median time to neutrophil engraftment was comparable (11 days) for all three groups, while platelet engraftment occurred at a median of 20, 19 and 17 days, respectively (p-values not significant). ConclusionsFreezIS, a Good Manufacturing Practice-grade, pre-constituted cryopreservative with low DMSO content, maintains functional viability of the HC product while reducing the incidence of irAEs compared with 10% DMSO solutions. The pre-constituted nature of this agent also decreases processor-dependent handling, hence decreasing the risk of variability and infection.

Validation of a flow cytometry-based method for quantitative viable lymphocyte- immunophenotyping in fresh and cryopreserved hematopoietic cellular products

Background & Aim Adoptive cellular therapy with immune effector cells (IEC) has shown remarkable toxicity against tumor cells and potential in immune regulation. Both variability in cell content of starting material and compromised cell viability during manufacturing necessitate a validated method for quantifying viable lymphocyte subtypes individually. Currently-commercialized immunophenotyping methods report cell viability in thawed products with significant error since they do not include any viability staining. We hereby report on the validation of a flow cytometry-based method for quantifying viable lymphocyte- immunophenotypes in fresh and cryopreserved hematopoietic cellular products. Methods, Results & Conclusion Using fresh or frozen cellular products and stabilized blood, we report on the validation parameters: accuracy, uncertainty, precision, sensitivity, robustness and carry-over for quantification of viable CD3+, CD4+ T cells, CD8+ T cells, CD3-CD56+CD16+/- NK cells, CD19+ B cells and CD14+ monocytes of relevance to hematopoietic products and immune effector cell (IEC) products on the Cytomics FC500 Beckman Coulter cytometer. The acceptance criteria set in the validation plan were all met. The method is able to accommodate the variability in absolute numbers of cells in starting materials collected or cryopreserved from patients or healthy donors (uncertainty of 10% at 3 different concentrations), stability over time (compliance over 3 years during regular inter-laboratory comparisons), and confidence in meaningful changes during cell processing and manufacturing (intra-assay and intermediate precision of 10% CV). Furthermore, the method can accurately report on the efficacy of cell depletion, since the lower limit of quantification was established (CD3+, CD4+ and CD8+ cells at 9, 8 and 8 cells/µl, respectively). The method complies with FACT Standards for IEC, FACT-JACIE Hematopoietic Cell Therapy Standards, ICH Q2(R1) and ISO15189 standards. Furthermore, it complies with LBAFG-AAPS, ICSH/ICCS and European Bioanalysis Forum recommendations for validating such methods. The implications of this effort include: standardization of viable cell immunophenotyping of starting material for cell manufacturing, cell selection, in-process or dosing of immune effector cells. This method also complies with all relevant standards, particularly FACT-JACIE Standards in terms of enumerating and reporting on the viability of the “clinically relevant cell populations”.

MSX2 suppression through inhibition of TGF\u03b2 signaling enhances hematopoietic differentiation of human embryonic stem cells

BackgroundStrategies of generating functional blood cells from human pluripotent stem cells (hPSCs) remain largely unsuccessful due to the lack of a comprehensive understanding of hematopoietic development. Endothelial-to-hematopoietic transition (EHT) serves as the pivotal mechanism for the onset of hematopoiesis and is negatively regulated by TGF-β signaling. However, little is known about the underlying details of TGF-β signaling during EHT.MethodsIn this study, by applying genome-wide gene profiling, we identified muscle segment homeobox2 (MSX2) as a potential mediator of TGF-β signaling during EHT. We generated MSX2-deleted human embryonic stem cell (hESC) lines using the CRISPR/Cas9 technology and induced them to undergo hematopoietic differentiation. The role of MSX2 in hematopoiesis and functional regulation of TGFβ signaling in EHT was studied.ResultsWe identified MSX2 as a novel regulator of human hematopoiesis. MSX2 deletion promotes the production of hematopoietic cells from hESCs. Functional and bioinformatics studies further demonstrated that MSX2 deletion augments hematopoietic differentiation of hESCs by facilitating EHT. Mechanistically, MSX2 acts as a downstream target of TGFβ signaling to mediate its function during EHT.ConclusionsOur results not only improve the understanding of EHT, but may also provide novel insight into the efficient production of functional blood cells from hPSCs for regenerative medicine.

BMP4 and perivascular cells promote hematopoietic differentiation of human pluripotent stem cells in a differentiation stage-specific manner

The efficient and reproducible derivation and maturation of multipotent hematopoietic progenitors from human pluripotent stem cells (hPSCs) requires the recapitulation of appropriate developmental stages and the microenvironment. Here, using serum-, xeno-, and feeder-free stepwise hematopoietic induction protocols, we showed that short-term and high-concentration treatment of hPSCs with bone morphogenetic protein 4 (BMP4) strongly promoted early mesoderm induction followed by increased hematopoietic commitment. This method reduced variations in hematopoietic differentiation among hPSC lines maintained under chemically defined Essential 8 medium compared to those maintained under less-defined mTeSR medium. We also found that perivascular niche cells (PVCs) significantly augmented the production of hematopoietic cells via paracrine signaling mechanisms only when they were present during the hematopoietic commitment phase. A protein array revealed 86 differentially expressed (>1.5-fold) secretion factors in PVC-conditioned medium compared with serum-free control medium, of which the transforming growth factor-β inducible gene H3 significantly increased the number of hematopoietic colony-forming colonies. Our data suggest that BMP4 and PVCs promote the hematopoietic differentiation of hPSCs in a differentiation stage-specific manner. This will increase our understanding of hematopoietic development and expedite the development of hPSC-derived blood products for therapeutic use.

Presence of Chip-Mutated Autologous Hematopoietic Cells in Mobilized Peripheral Blood Products Is Associated with Shorter Progression-Free Survival after Autologous Transplants for Multiple Myeloma

Multiple Myeloma (MM) remains a cancer of terminally-differentiated plasma cells that reside predominantly within the bone marrow. Malignant plasma cells are nurtured by a permissive microenvironment that favors tumor progression, drug resistance and disease relapse. Recurrent somatic mutations in hematopoietic stem cells, the source of major components in bone marrow niche, lead to age-associated clonal hematopoiesis of indetermined potential (CHIP) and has been associated with inferior survivals among individuals without malignant hematologic disorders. It is possible that these mutations in non-MM cells would affect remission time after transplant. We performed whole exome sequencing to identify CHIP-associated mutations within the autograft utilized to rescue hematopoiesis after high dose melphalan and autologous hematopoietic cell transplant (HCT). We then correlated the presence of CHIP with the outcome of MM patients (pts) following autologous HCT. Methods. MM pts (N=101) that underwent HCT between 2006 and 2017 were studied. DNA was extracted from 1 ml of cryopreserved mobilized hematopoietic cell product. Targeted sequencing was then performed using Tempus xE whole exome platform (Tempus, Chicago, IL) with variable allele frequency (VAF) ≥0.1. Progression-free survival (PFS) and overall survival (OS) were defined as the time from transplantation until event of interest, with censoring at time of last follow up. Cox regression was used for time-to-event outcomes; hazard ratios (HR) and 95% confidence intervals (CI) were reported. P-values were two-sided and those &amp;lt;0.05 were considered statistically significant. Results. The median age was 60.1 years at the time of HCT. 58 pts were male and 43 female; 29 pts were African-American, 1 Asian/Indian and 71 were Caucasian. Seventy five (75%) and 42 (43%) pts had prior exposure to lenalidomide or cyclophosphamide, respectively. Thirty two pts (32%) underwent hematopoietic cell mobilization with GCS-F and plerixafor, 38 pts (38%) with cyclophosphamide and 31 pts (31%) with GCS-F alone. Forty seven pts (47%) had at least one CHIP-associated mutation. The median number of CD34+ yield in the first day was 6.7 x 10e6/kg for pts with CHIP mutations and 5.64 x 10e6/kg for pts without any CHIP mutation (p = 0.4). The presence or absence of CHIP mutations did not impact the yield of CD34+ stem cells collected (Fig. 1). The majority of mutations were either missense variants or mRNA splicing variations (Fig. 2), most commonly SF3B1, ASXL1, TET2 and ASXL2. CHIP mutations were associated with increased age (p= 0.021), but there was no association with prior history of previous cancer, smoking, cytogenetically-defined high risk disease, pre-HCT hemoglobin level or red blood cell mean corpuscular volume. There was also no significant difference between the transplant course between the two groups in terms of neutrophil and platelet engraftment or length of hospital stay. Median follow up was 32 months. During follow up 3 pts develop MDS/AML (2 pts in the CHIP group and one in no CHIP group). Coronary artery disease was the cause of death in one patient in CHIP cohort versus none in the CHIP negative cohort. Myeloma progression was the major cause of death in CHIP and No CHIP cohorts (66% vs. 60%, p=0.48). Median PFS was 19 months in CHIP cohort vs. 39 months in No CHIP group (HR: 0.62, 95% CI: 0.51-0.92, p= 0.001). Median OS has not been reached in the No CHIP group while it was 56 months in CHIP group (HR: 0.78, 95% CI: 0.62-1.04, P=0.09). In multivariate analysis, presence of CHIP remained a significant predictor of worse PFS after adjusting for age, maintenance therapy, high risk disease, performance status, triplet vs. doublet pre-SCT therapy, melphalan dose and MM genetic risk. (Table 2). Conclusion: Here we showed high frequency of large clonal hematopoiesis clones (i.e., VAF ≥0.1) in autograft of MM pts. The presence of this mutations does not impact stem cell yield or transplant course, however it impacts PFS independent of age and other unfavorable factors. Our data highlights high frequency of SF3B1; the role of this mutant in modulating apoptotic factors in myeloma microenvironment should be investigated. Disclosures Malek: Celgene: Consultancy; Amgen: Speakers Bureau; Adaptive: Consultancy; Janssen: Speakers Bureau; Medpacto: Research Funding; Sanofi: Consultancy; Takeda: Consultancy. Caimi:ADC Therapeutics: Research Funding; Celgene: Speakers Bureau; Fate Therapeutics: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kite Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Caldwell:Tempus Inc.: Employment.