Sherilyn A. Gross

Increased expansion and differentiation of cord blood products using a two-step expansion culture

OBJECTIVE [corrected] The use of allogeneic cord blood (CB) products as a source of cellular support for patients receiving high-dose chemotherapy has been limited primarily to smaller children due to the low numbers of cells in a CB unit. Ex vivo expansion of CB cells has been proposed as a method to increase the number of cells available for transplantation. Following high-dose chemotherapy administration, we transplanted adult patients with CB expanded in static culture for 10 days, in DM containing stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), and megakaryocyte growth and development factor (MGDF). Patients achieved neutrophil engraftment in a median of 26 days (range 15 to 45). In an attempt to hasten the time to neutrophil engraftment, we developed a two-step culture system that results in increased expansion of total nucleated cells and further maturation of neutrophil precursors. MATERIALS AND METHODS CD34(+) cells isolated from CB products were cultured for 7 days at 37 degrees C in 100-mL Teflon culture bags containing 50 mL of DM containing SCF, G-CSF, and MGDF (100 ng/mL). The cells were harvested from these bags after 7 days of incubation at 37 degrees C and transferred to 1-L Teflon bags containing 1 L of DM plus SCF, G-CSF, and MGDF. After a second culture period of 7 days, the cells were harvested, washed, and assayed for mature (granulocyte-macrophage colony-forming cells [GM-CFC]) and primitive progenitor cells (high proliferative potential colony-forming cells [HPP-CFC]). RESULTS The two-step cultures resulted in a median total nucleated cell expansion of 438-fold (range 286 to 952, N = 11); the original one-step cultures resulted in a median expansion of 98-fold (range 59 to 350, N = 5). Equivalent expansion of committed progenitor cells (GM-CFC) and primitive progenitor cells (HPP-CFC) was obtained. CD34(+) cells were expanded a median of 29-fold in the two-step cultures (N = 11). The two-step culture contained more mature neutrophil cells, by morphologic examination, than the one-step cultures, similar to ex vivo expanded peripheral blood progenitor cells (PBPC). CONCLUSION The two-step ex vivo expansion conditions described for CB resulted in increased numbers of total nucleated cells, GM-CFC, HPP-CFC, and CD34(+) cells and morphologically resembled ex vivo expanded PBPC, which have been shown to provide more rapid neutrophil engraftment than unexpanded PBPC. We propose that the availability of increased numbers of expanded CB cells may result in more rapid engraftment of neutrophils following infusion to transplant recipients.

Cryopreservation of Stem Cells Using Trehalose: Evaluation of the Method Using a Human Hematopoietic Cell Line

While stem cell cryopreservation methods have been optimized using dimethylsulfoxide (DMSO), the established techniques are not optimal when applied to unfertilized human embryonic cells. In addition, important questions remain regarding the toxicity and characteristics of DMSO for treatment of stem cells for clinical use. The objective of this study was to establish an optimal method for cryopreservation of stem cells using low concentrations (0.2 M) of trehalose, a nontoxic disaccharide of glucose, which possesses excellent protective characteristics, in place of current methods utilizing high concentrations (1-2 M) of DMSO. A human hematopoietic cell line was used in this investigation as a surrogate for human stem cells. Trehalose was loaded into cells using a genetically engineered mutant of the pore-forming protein alpha-hemolysin from Staphylococcus aureus. This method results in a nonselective pore equipped with a metal-actuated switch that is sensitive to extracellular zinc concentrations, thus permitting controlled loading of trehalose. Preliminary experiments characterized the effects of poration on TF-1 cells and established optimal conditions for trehalose loading and cell survival. TF-1 cells were frozen at 1 degrees C/min to -80 degrees C with and without intra- and extracellular trehalose. Following storage at -80 degrees C for 1 week, cells were thawed and evaluated for viability, differentiation capacity, and clonogenic activity in comparison to cells frozen with DMSO. Predictably, cells frozen without any protective agent did not survive freezing. Colony-forming units (CFU) generated from cells frozen with intra- and extracellular trehalose, however, were comparable in size, morphology, and number to those generated by cells frozen in DMSO. There was no observable alteration in phenotypic markers of differentiation in either trehalose- or DMSO-treated cells. These data demonstrate that low concentrations of trehalose can protect hematopoietic progenitors from freezing injury and support the concept that trehalose may be useful for freezing embryonic stem cells and other primitive stem cells for therapeutic and investigational use.

Analysis of BTEX groundwater concentrations from surface spills associated with hydraulic fracturing operations

Concerns have arisen among the public regarding the potential for drinking-water contamination from the migration of methane gas and hazardous chemicals associated with hydraulic fracturing and horizontal drilling. However, little attention has been paid to the potential for groundwater contamination resulting from surface spills from storage and production facilities at active well sites. We performed a search for publically available data regarding groundwater contamination from spills at U.S. drilling sites. The Colorado Oil and Gas Conservation Commission (COGCC) database was selected for further analysis because it was the most detailed. The majority of spills were in Weld County, Colorado, which has the highest density of wells that used hydraulic fracturing for completion, many producing both methane gas and crude oil. We analyzed publically available data reported by operators to the COGCC regarding surface spills that impacted groundwater. From July 2010 to July 2011, we noted 77 reported surface spills impacting the groundwater in Weld County, which resulted in surface spills associated with less than 0.5% of the active wells. The reported data included groundwater samples that were analyzed for benzene, toluene, ethylbenzene, and xylene (BTEX) components of crude oil. For groundwater samples taken both within the spill excavation area and on the first reported date of sampling, the BTEX measurements exceeded National Drinking Water maximum contaminant levels (MCLs) in 90, 30, 12, and 8% of the samples, respectively. However, actions taken to remediate the spills were effective at reducing BTEX levels, with at least 84% of the spills reportedly achieving remediation as of May 2012. Our analysis demonstrates that surface spills are an important route of potential groundwater contamination from hydraulic fracturing activities and should be a focus of programs to protect groundwater. Implications: While benzene can occur naturally in groundwater sources, spills and migration of chemicals used for hydraulic fracturing activities have recently been thought to be a main source of benzene contamination in groundwater. However, there is little scientific literature to support that claim. Therefore, we accessed a publically available database and tracked the number of reported surface spills with potential groundwater impact over a 1-year period. Although the number of surface spills was minimal, our analysis provides scientific evidence that benzene can contaminate groundwater sources following surface spills at active well sites. Supplemental Materials: Supplemental materials are available for this paper. Go to the publishers online edition of the Journal of the Air & Waste Management Association for an illustration of the average concentration of each BTEX chemical from pooled sample measurements, and various metrics from all 77 spills analyzed in this study.

Aggressive natural killer cell leukemia : report of a Chinese series and review of the literature

Aggressive natural killer cell leukemia (ANKL) is a rare Epstein-Barr virus (EBV)-associated fulminating disease that is widely disseminated at diagnosis. Because of its typically extranodal presentation, differing degrees of NK cell involvement, and varying bone marrow pathology, ANKL can be confused with a reactive process. These features, coupled with a rapidly fatal course, have hampered systematic study of the pathogenesis of ANKL. Nine cases of ANKL were diagnosed and characterized by a single laboratory over a 2-year period. Constant features at presentation included disseminated disease, high fever, bone marrow involvement, and a high lactate dehydrogenase index. All cases were positive for EBV early region protein and negative for latent membrane protein 1, and all had a germline T-cell receptor gene configuration. Peripheral blood counts were variable, with severe thrombocytopenia being the most frequently encountered abnormality (7 of 9 cases). Hematophagocytosis, dyserythropoiesis, and stromal degeneration were the most frequent findings in the bone marrow. Neo-plastic cells in the bone marrow were consistently CD2+, CD56+, CD45+, CD34-, CD117-, CD4-, and surface CD3-. Most cases were HLA-DR+ (8/9) and CD8- (8/9). Complex clonal cytogenetic abnormalities were found in 8 of 9 cases. Because of its aggressive course, rapid and accurate diagnosis of ANKL is essential for a better understanding of the etiology, pathogenesis, and treatment of the disease.

Benzene and human health: A historical review and appraisal of associations with various diseases

Over the last century, benzene has been a well-studied chemical, with some acute and chronic exposures being directly associated with observed hematologic effects in humans and animals. Chronic heavy exposures to benzene have also been associated with acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS) in humans. Other disease processes have also been studied, but have generally not been supported by epidemiologic studies of workers using benzene in the workplace. Within occupational cohorts with large populations and very low airborne benzene exposures (less than 0.1–1.0 ppm), it can be difficult to separate background disease incidence from those occurring due to occupational exposures. In the last few decades, some scientists and physicians have suggested that chronic exposures to various airborne concentrations of benzene may increase the risk of developing non-Hodgkin’s lymphoma (NHL) (, Am J Ind Med 31:287–295; , Cancer Epidemiol Biomarkers Prev 16:385–391), multiple myeloma (MM) (, Ann NY Acad Sci 609:225–230; , Ann NY Acad Sci 1076:90–109), and various other hematopoietic disorders. We present a state-of-the-science review of the medical and regulatory aspects regarding the hazards of occupational exposure to benzene. We also review the available scientific and medical evidence relating to benzene and the risk of developing various disorders following specific levels of exposure. Our evaluation indicates that the only malignant hematopoietic disease that has been clearly linked to benzene exposure is AML. Information from the recent “Benzene 2009,” a symposium of international experts focusing on the health effects and mechanisms of toxicity of benzene, hosted by the Technical University of Munich, has been incorporated and referenced.

A prospective study of 728 cases of non-Hodgkin lymphoma from a single laboratory in Shanghai, China

The frequency of subtypes of lymphoid neoplasms was determined in a prospective series of 831 patients presenting at 29 Shanghai hospitals over a 4-year period. Diagnosis and classification was established in a single laboratory according to the 2001 WHO classification system. The frequency of non-Hodgkin lymphoma was 87.6% (n = 728) and Hodgkin lymphoma was 12.4% (n = 103). The most prevalent NHL subtypes diagnosed using WHO criteria were diffuse large B cell lymphoma (DLBCL), precursor B lymphoblastic leukemia/lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Although a low incidence has been reported in some Asian populations, CLL/SLL was commonly encountered, indicating that chronic lymphoid neoplasms are not rare in Shanghai. Consistent with previous reports, our findings indicate a decrease in the frequency of follicular lymphoma and an increase in T cell neoplasms compared to the West. Precursor T lymphoblastic leukemia/lymphoma, anaplastic large T cell lymphoma, aggressive NK cell leukemia, angioimmunoblastic T cell lymphoma and peripheral T cell lymphoma were prominent subtypes of T cell NHL.

Prospective study of 174 de novo acute myelogenous leukemias according to the WHO classification: subtypes, cytogenetic features and FLT3 mutations

Abstract:  We report a prospective study of 174 unselected adult de novo acute myeloid leukemia (AML) cases diagnosed using the WHO classification. Of those, 57 (33%) were AML with recurrent cytogenetic abnormalities, 41 were (24%) AML with multilineage dysplasia, 74 (42%) were AML not otherwise categorized, and two were acute leukemias of ambiguous lineage. Clonal cytogenetic abnormalities were detected in 64% of the WHO AML cases with t(15;17) (15%), t(8;21) (12%), +8 (11%), −7/del7q (8%) and del9q (5%) being the most common ones. The FLT3/ITD mutations (FMS‐like tyrosine kinase 3/internal tandem duplication) were observed in 12% of the WHO AML cases, which is much lower than ones in the literature, while the 6% incidence of the FLT3‐activating loop mutations (either FLT3/D835 or FLT3/I836) was comparable with others. Both mutations were associated with leukocytosis. Our study also suggests that the FLT3 mutations are biomarkers independent of cytogenetic characteristics.

Inhibition of NF-κB by hydroquinone sensitizes human bone marrow progenitor cells to TNF-α-induced apoptosis

Suppression of hematopoiesis is an important mechanism governing blood cell formation. Factors such as tumor necrosis factor alpha (TNF-alpha) inhibit proliferation and colony-forming activity of bone marrow cells and activate nuclear factor kappa B (NF-kappaB) in multiple cell types. Activated NF-kappaB is required for many cells to escape apoptosis, including hematopoietic progenitor cells (HPC). The benzene metabolite hydroquinone (HQ) alters cytokine response and induces cell death in HPC, and inhibits NF-kappaB activation in T and B cells. Therefore, we studied the potential role of HQ-induced NF-kappaB inhibition in a hematopoietic cell line (TF-1) and primary HPC in rendering these cells susceptible to TNF-alpha-induced apoptosis. We demonstrate in both cell types that TNF-alpha activates NF-kappaB, and HQ exposure inhibits activation of NF-kappaB by TNF-alpha in a dose dependent manner. We further investigated the ability of HQ to potentiate TNF-alpha-induced apoptosis in these cells, and found that HQ sensitized the cells to the pro-apoptotic effect of TNF-alpha. These results suggest that NF-kappaB plays a key role in HPC survival, and that HQ-induced inhibition of NF-kappaB leaves these cells susceptible to cytokine-induced apoptosis. These effects may play a role in the suppression of hematopoiesis seen in some benzene exposed individuals.

Integrating WHO 2001-2008 criteria for the diagnosis of Myelodysplastic Syndrome (MDS): a case-case analysis of benzene exposure.

We characterized the prevalence of hematopoietic and lymphoid disease for 2923 consecutive patients presenting at 29 hospitals from August 2003 to June 2007. Diagnoses were made in our laboratory using WHO criteria based on morphologic, immunophenotypic, cytogenetic, FISH and molecular data. A total of 611 subjects (322 males/289 females) were prospectively diagnosed with MDS using WHO (2001) criteria. Update and re-evaluation of cases using MDS (2008) criteria resulted in 649 MDS cases. Using WHO (2008) criteria, refractory cytopenia with multilineage dysplasia (RCMD) accounted for 68% of total cases, refractory anemia with excess blasts (RAEB), 16.3%; refractory anemia (RA), 6.5%; refractory cytopenia with unilineage dysplasia (RCUD), 4%; and MDS-unclassifiable (MDS-U), 4.5%. Subjects were administered questionnaires and information on previous disease, work histories and exposures to potential etiologic agents such as benzene (BZ) was obtained. A total of 80/649 (13.2%) were determined to have some BZ exposure. The frequency of clonal cytogenetic abnormalities in all MDS was 30%, the most common being +8>del(20)q>del(7q)>del(5q), while the analogous frequency in BZ-exposed cases was only 24%. To further investigate the characteristics of MDS associated with BZ, we identified a subset of cases with high BZ exposure. These BZ signal cases were each matched by age and gender to two cases with no known BZ exposure. When contrasting BZ signal cases vs matched cases with no BZ exposure, we found a high odds ratio (OR) for the WHO subtype MDS-U (OR=11.1), followed by RAEB and RCUD (OR=1), RA (OR=0.7) and RCMD (OR=0.6). Multilineage dysplasia with abnormal eosinophils (MDS-Eo) was strongly associated with BZ exposure, whereas the relative risk of clonal cytogenetic abnormalities was reduced for high BZ-exposed cases (OR=0.5). These findings are strongly indicative that MDS subtypes are influenced by BZ exposure, and taken together with previous studies, the features of MDS-Eo suggest that altered immune regulation plays a major role in the pathogenesis of MDS following chronic exposure to BZ.

Characterization and phenotypic analysis of differentiating CD34+human bone marrow cells in liquid culture

Abstract:  Our current understanding of human haematopoietic stem cell biology is based in part on the characterization of human CD34+ bone marrow cell differentiation in vitro. CD34 is highly expressed on early stem cells and haematopoietic progenitor cells with clonogenic potential and is gradually lost during differentiation and commitment. However, CD71 (transferrin receptor) is expressed at low levels on early stem cells and generally increases during haematopoietic progenitor cell proliferation. We reasoned that the combination of these surface markers would provide a useful framework for the simultaneous analysis of multiple lineage differentiation of CD34+ haematopoietic progenitor cells in liquid culture. In this report, we identify the phenotype of distinct subpopulations of myeloid, erythroid and lymphoid cells in liquid suspension culture using differential expression of CD34 vs. CD71 in combination with specific lineage markers. Freshly isolated human CD34+ bone marrow cells were introduced into suspension culture and monitored over a 6‐d period using 3‐colour flow cytometry. This is the first demonstration that differential expression of CD34 vs. CD71 can be used to simultaneously monitor differentiation of multiple haematopoietic cell lineages in liquid suspension culture, facilitating the study of cytokine‐, drug‐ or chemical‐induced alterations in haematopoietic progenitor cell differentiation in vitro.