Unit Assay Research Articles

Elucidating Lineage-Specific Myelotoxicity and Chromosomal Aberration Status in Hydroquinone- Exposed Hematopoietic Stem / Progenitor cells

Benzene exposure has been associated with hematotoxicity and leukemogenicity. However, the impact of benzene exposure on complex microenvironment of Hematopoetic Stem Cells (HSCs) niche, comprising of HSCs and lineage-specific progenitors remains elusive. Thus, a study on benzene-targeting HSCs niche could uncover mechanism linking benzene to HSCs niche alteration. This study evaluates the lineage-specific responses following exposure to a benzene metabolite, namely hydroquinone (HQ) in targeting HSCs and myeloid-committed progenitors. Freshly isolated murine bone marrow cells (BMCs) were exposed to HQ at series of concentrations (0 – 50 µM) for 24 hours; followed by cell viability analysis using MTT assay. Chromosomal aberration (CA) status was determined using karyotyping analysis. Expression of surface antigen for HSCs (Sca-1) was confirmed by flow cytometer. Lineage-specific myelotoxicity was studied using the colony-forming unit (CFU) assay for the following myeloid progenitors: CFU granulocyte /erythrocyte /macrophage /megakaryocyte (CFU-GEMM), CFU-granulocyte/macrophage (CFU-GM), CFU-granulocyte (CFU-G), CFU-macrophage (CFU-M), CFU-erythroid (CFU-E) and Burst-forming unit erythroid (BFU-E). HQ reduced (p<0.05) viability of BMCs at 25 µM and 50 µM with the IC10, IC25, and IC50 were at 17 µM, 23 µM and 35 µM, respectively. Increased (p<0.05) frequency of CA was observed in HQ-treated group as compared to control. Reduced (p<0.05) Sca-1+ cells at 17 µM, 23 µM and 35 µM indicates cytotoxic effect of HQ targeting HSCs population. Reduced (p<0.05) total colony counts were noted following HQ exposure at 6.25 µM and a complete inhibition of colony growth were observed at higher concentration (12.5 µM). HQ reduced (p<0.05) the growth of CFU-GEMM, CFU-GM and CFU-G at 6.25 µM, while the growth of CFU-M, CFU-E and BFU-E were not remarkably affected at lower concentrations (1.56 µM and 6.25 µM). Conclusively, HQ induces chromosomal aberration and cytotoxicity on HSCs with notable lineage-specific responses in governing benzene-mediated myelotoxicity targeting HSCs niche.

Thioredoxin mitigates radiation-induced hematopoietic stem cell injury in mice

BackgroundRadiation exposure poses a significant threat to public health. Hematopoietic injury is one of the major manifestations of acute radiation sickness. Protection and/or mitigation of hematopoietic stem cells (HSCs) from radiation injury is an important goal in the development of medical countermeasure agents (MCM). We recently identified thioredoxin (TXN) as a novel molecule that has marked protective and proliferative effects on HSCs. In the current study, we investigated the effectiveness of TXN in rescuing mice from a lethal dose of total body radiation (TBI) and in enhancing hematopoietic reconstitution following a lethal dose of irradiation.MethodsWe used in-vivo and in-vitro methods to understand the biological and molecular mechanisms of TXN on radiation mitigation. BABL/c mice were used for the survival study and a flow cytometer was used to quantify the HSC population and cell senescence. A hematology analyzer was used for the peripheral blood cell count, including white blood cells (WBCs), red blood cells (RBCs), hemoglobin, and platelets. Colony forming unit (CFU) assay was used to study the colongenic function of HSCs. Hematoxylin and eosin staining was used to determine the bone marrow cellularity. Senescence-associated β-galactosidase assay was used for cell senescence. Western blot analysis was used to evaluate the DNA damage and senescence protein expression. Immunofluorescence staining was used to measure the expression of γ-H2AX foci for DNA damage.ResultsWe found that administration of TXN 24 h following irradiation significantly mitigates BALB/c mice from TBI-induced death: 70% of TXN-treated mice survived, whereas only 25% of saline-treated mice survived. TXN administration led to enhanced recovery of peripheral blood cell counts, bone marrow cellularity, and HSC population as measured by c-Kit+Sca-1+Lin– (KSL) cells, SLAM + KSL cells and CFUs. TXN treatment reduced cell senescence and radiation-induced double-strand DNA breaks in both murine bone marrow lineage-negative (Lin–) cells and primary fibroblasts. Furthermore, TXN decreased the expression of p16 and phosphorylated p38. Our data suggest that TXN modulates diverse cellular processes of HSCs.ConclusionsAdministration of TXN 24 h following irradiation mitigates radiation-induced lethality. To the best of our knowledge, this is the first report demonstrating that TXN reduces radiation-induced lethality. TXN shows potential utility in the mitigation of radiation-induced hematopoietic injury.

Aqueous extract of Phragmitis rhizoma ameliorates myelotoxicity of docetaxel in vitro and in vivo

BackgroundA variety of anticancer chemotherapeutics induce adverse side effects including myelotoxicity. Dried roots of Phragmites communis Trinius, Phragmitis rhizoma, have been clinically used in traditional folk medicine to relieve various symptoms like fever. In this study, we evaluated the protective effect of the aqueous extract of Phragmitis rhizoma (EPR) against docetaxel-induced myelotoxicity in vitro and in vivo.MethodsThe in vitro myelo-protective effect of EPR was evaluated using the colony forming unit (CFU) assay with hematopoietic progenitor cells. The in vivo efficacy of EPR was evaluated in myelosuppressed C57BL/6 male mice which were induced by repeated intraperitoneal injections of 30 mg/kg docetaxel for 3 times. EPR was orally administered for 4 days to docetaxel-induced myelosuppressed C57BL/6 male mice which were induced by intraperitoneal injection of 30 mg/kg docetaxel for 3 times: Group 1 (vehicle control, n = 10), Group 2 (docetaxel plus vehicle, n = 10), Group 3 (docetaxel plus EPR 30 mg/kg, n = 10), Group 4 (docetaxel plus EPR 100 mg/kg, n = 10) and Group 5 (docetaxel plus EPR 300 mg/kg, n = 10). Whole blood counts were measured automatically, and immune organs were histologically examined. Expression of immunomodulatory cytokines was measured by quantitative real-time polymerase chain reaction or enzyme-linked immunosorbent assay. The toxicity of EPR itself was evaluated in normal human cell lines including IMR-90, foreskin fibroblast and human umbilical vein endothelial cells. The hepatotoxicity of EPR was predicted by multi-parametric assays involving cell viability, caspase 3/7 activity, GSH contents and LDH leakage using the HepaRG hepatic cell line.ResultsCo-treatment of EPR or its major component, p-hydroxycinnamic acid, increased the numbers of hematopoietic CFU counts in the docetaxel-induced in vitro myelotoxicity assay system. The in vitro protective effect of EPR against docetaxel toxicity was replicated in a myelosuppressed animal model: white blood cells, neutrophils, lymphocytes and red blood cells rebounded; bone marrow niche and structural integrity of the thymus were preserved; and the expression of immune-stimulating cytokines including IL3, IL6, SCF and GM-CSF was enhanced. Furthermore, EPR and p-hydroxycinnamic acid promoted the proliferation of primary splenocytes and thymocytes. In the toxicity assays, no remarkable signs related with toxicity were observed in all tested normal human cells and HepaRG.ConclusionsEPR has the potential to ameliorate docetaxel-mediated myelotoxicity in both in vitro and in vivo models. However, the identification of the responsible active components and the precise underlying myelo-protective mechanism of EPR need to be elucidated before novel drug development using EPR can precede.

Abstract 643: CX 3 CR1 Identifies Adventitial Macrophage Progenitor Cells (AMPCS), a Local Source of Self-Renewing Macrophages in Postnatal Murine Arteries

Background: Macrophages are integral to vascular biology and disease. Although traditional paradigms assert that vascular macrophages originate from circulating monocytes, recent data suggest that some are seeded in utero from CX 3 CR1 + progenitors, and are maintained postnatally by local self-renewal. We previously identified a novel population of locally-maintained adventitial macrophage progenitor cells (AMPCs) as a source of self-renewing macrophages in adult mouse arteries. Hypothesis: AMPCs express CX 3 CR1 and do not derive from definitive hematopoiesis. Methods: Single-cell disaggregates were prepared from postnatal murine aortas. AMPC content was assessed by macrophage colony-forming unit (CFU-M) assays. Flow cytometry together with fluorescence activated cell sorting (FACS) were used to investigate the immunophenotypic profile of AMPCs and fate-mapping to assess their origins. Results: CFU-M prevalence in C57BL/6J aortic cells was highest in neonatal mice (~100 per 10 5 cells), and diminished progressively with age (~55/10 5 at 3w, ~15/10 5 at 12w, ~5/10 5 at 52w, n&gt;4, P&lt;0.01). Secondary replating of single cells from aortic CFU-M revealed striking self-renewal capacity, with 1 in 10 cells forming new CFU-M (n=8). Undifferentiated CFU-M displayed &gt;95% expression of the stem cell markers Sca-1 and c-Kit, and high levels of CX 3 CR1, but did not acquire the monocyte/macrophage markers CD11b or F4/80 until treated with macrophage-colony stimulating factor (n=6). Mice deficient in CX 3 CR1 (Cx 3 cr1 GFP/GFP ) produced fewer CFU-M than heterozygous (Cx 3 cr1 +/GFP ) littermates (8.7±0.7 vs 15.3±0.7 per 10 5 , P&lt;0.001, n&gt;7). FACS selection confirmed that CFU-M forming AMPCs were exclusively contained within a CX 3 CR1 + subpopulation that did not express either CD11b or F4/80 (n=3). Finally, CFU-M analysis from Flt3 Cre xRosa mT/mG mice demonstrated that AMPCs arise from a FLT3 -ve source, indicating that their origins are independent of definitive hematopoiesis (n=4). Conclusion: Clonogenic, self-renewing murine AMPCs express CX 3 CR1 but not the monocyte/macrophage markers CD11b and F4/80. The high prevalence of AMPCs in neonatal aorta is consistent with prenatal seeding from CX 3 CR1 + progenitors, independent of definitive hematopoiesis.

Estimation of Serum Folate and Homocysteine Levels in Oral Submucous Fibrosis-A Prospective Study

Aim & Objective: The aim of the study was to evaluate the levels of serum folate and homocysteine in clinical stages of Oral submucous fibrosis (OSF) patients. Methodology: The study sample comprised of 40 clinically and histopathologically diagnosed OSF patients those who are not undergoing treatment and 10 healthy controls. OSF was categorised by clinical staging. Serum foliate and homocysteine levels estimation was done by reverse phase high performance liquid chromatography analysis unit and chemiluminescence immuno assay, respectively. Results were statistically analysed using one-way ANOVA [Analysis of variance]. Results: Irrespective of age and gender, serum folate deficiency and high levels of homocysteine were noted in OSF patients when compared with healthy controls. Conclusion: Serum hyper homocysteinemia and folate deficiency were noted with the progression of the disease. Folate supplements can be given to decrease the levels of homocysteine, to arrest the progression of the disease. However, further longitudinal studies are needed to establish an association between serum homocysteine and serum folate.

Nuclear NFATc1 Causes Development of Fms-like Receptor Tyrosine Kinase 3 Receptor (FLT3) Internal Tandem Duplication Mutation-Positive (FLT3-ITD) Acute Myeloid Leukemia (AML) and Mediates Drug Resistance

Abstract Background: Although an internal tandem duplication (ITD) of the Fms-like receptor tyrosine kinase 3 receptor (FLT3) confers a very poor prognosis in acute myeloid leukemia (AML), it is insufficient to cause AML, suggesting important cooperating genetic events. NFATc1 is a member of the nuclear factor of activated T-cells (NFAT) transcription factor family, whose activation is dependent on nuclear translocation. NFATc1 is well known for its central role in regulating T-cell development and function, but was also shown to contribute to neoplastic transformation in diverse types of cancers. We have recently demonstrated that NFATc1 is overexpressed in primary FLT3-ITD+ AML causing resistance to the FLT3-kinase inhibitor sorafenib in vitro. Here we asked, whether NFATc1 induces AML transformation and drug resistance when expressed in the context of FLT3-ITD+ in vivo. Methods: Using the stem cell like (SCL)-promoter, Cre-recombinase inducible expression of a constitutively nuclear NFATc1 protein (cnNFATc1) was targeted to the early hematopoietic stem cell compartment. The SCL-Cre/cnNFATc1 mice were subsequently crossed with transgenic FLT3-ITD mice to yield Scl-Cre+/-/cnNFATc1+/+/FLT3-ITD+/+ mice (FCN+/+), which co-express cnNFAT and FLT3-ITD in the stem cell compartment. Results: Whereas FLT3-ITD mice developed a non-lethal, mild myeloproliferative disease, co-expression of FLT3-ITD with cnNFATc1 led to rapidly fatal AML. The median survival of FCN mice was only four months (range, 2 to 8 ). In median, FCN mice had 15-fold (range, 10 to 20) increased white blood cell count, a high proportion of circulating peripheral blasts, severe anemia and thrombocytopenia. There was also a significant B-cell developmental block. As opposed to F+/+ animals, FCN+/+ mice revealed a severe spleno- and hepatomegaly secondary to a gross leukemic infiltration, which disrupted the normal anatomical architecture of the involved organs. Fluorescence activating cell sorting and colony forming unit (CFU) assays demonstrated that cnNFATc1, when expressed in the context of FLT3-ITD, leads to a significant expansion of the lin-Sca+c-kit+ (LSK) stem cell-, progenitor cell (LK)-, the Gr-1-/CD11b+ monocytic and Gr-1low/CD11b+ immature myeloid compartments in bone marrow and spleen. FCN+/+-AML was polyclonal, and re-transplantable in secondary wild-type recipient mice. In vivo, cnNFATc1 caused not only potent FLT3-ITD inhibitor resistance to sorafenib and to the more FLT3-ITD-selective compound quizartinib, but also to chemotherapy. Comparative mRNA-sequencing of sorted LK and LSK compartments in wild-type, F+/+, C+/- N+/+, and FCN+/+ mice will reveal the genetic basis of the biological cooperativity between NFATc1 and FLT3-ITD in stem and progenitor cell compartments. Consistent with the murine data, a data set of AML patients (n=163) revealed that NFATc1 overexpression alone or when combined with FLT3-overexpression was associated with a significantly decreased overall survival (hazard ratio [HR] 1.91, CI 1.29-2.18, p=0.001 versus HR 2.57, CI 1.62-4.08, p&lt;0.0001). Conclusions: These results provide for the first time evidence for an important role of nuclear NFATc1 overexpression in the molecular pathogenesis of FLT3-ITD positive AML and the induction of drug resistance. These data also underscore that the mutational landscape in AML insufficiently describes AML biology. Disclosures No relevant conflicts of interest to declare.

Ex-vivo expansion of nonhuman primate CD34+ cells by stem cell factor Sall4B.

BackgroundHematopoietic CD34+ stem cells are widely used in the clinical therapy of complicated blood diseases. Stem cell factor Sall4B is a zinc finger transcription factor that plays a vital role in hematopoietic stem cell expansion. The purpose of our current study is to further evaluate how Sall4B might affect the expansion of CD34+ cells derived from nonhuman primates.MethodsSall4B was overexpressed in nonhuman primate bone marrow-derived CD34+ cells via a lentiviral transduction system. The granulocyte–erythrocyte–macrophage–megakaryocyte colony-forming unit (CFU) assay evaluated the differentiation potential of primate CD34+ cells that were expanded with Sall4B. Furthermore, an in-vivo murine system was employed to evaluate the hematopoietic potential of primate Sall4B-expanded CD34+ cells.ResultsOverexpression of Sall4B promoted ex-vivo nonhuman primate CD34+ cell expansion by 9.21 ± 1.94-fold on day 9, whereas lentiviral transduction without Sall4B expanded cells by only 2.95 ± 0.77-fold. Sall4B maintained a significant percentage of CD34+ cells as well. The CFU assay showed that the Sall4B-expanded CD34+ cells still possessed multilineage differentiation potential. A study using nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice in vivo revealed that Sall4B led to an increase in the number of repopulating cells and the 9-day-old Sall4B-transduced CD34+ cells still possess self-renewal and multilineage differentiation capacity in vivo, which are similar stemness characteristics to those in freshly isolated primate bone marrow-derived CD34+ cells.ConclusionsWe investigated the expansion of nonhuman primate bone marrow-derived CD34+ cells using the Sall4B lentiviral overexpression approach; our findings provide a new perspective on mechanisms of rapid stem cell proliferation. The utilization of Sall4B to expand CD34+ cells on a large scale through use of suitable model systems would prove helpful towards preclinical trials of autologous transplantation.

Abstract 013: Genetic Ablation of Mas Receptor Impairs Mobilization of Bone Marrow Progenitor Cells

Angiotensin (Ang)-(1-7)/Mas receptor (MasR) pathway accelerates vascular repair in ischemic conditions partly by stimulating the mobilization of vascular reparative bone marrow progenitor cells (BMPCs) into blood circulation. This study tested if the endogenous MasR expression is required for the mobilization of BMPCs in response to ischemic injury. Hind limb ischemia (HLI) was induced in wild type (WT) or MasR knock out mice (MasR-KO) (in C57Bl/6J background). BMPCs in the blood circulation were quantitated by flow cytometric enumeration of Lineage - , Sca-1 + and cKit + (LSK) cells in peripheral blood or by colony forming unit (CFU) assay. Subcutaneous osmotic pumps were used for continuous infusion of Ang-(1-7) at the rate of 1 μg/kg/min for four weeks. In vitro migration of LSK cells in response to hypoxia-regulated factors, stromal-derived factor (SDF) or by vascular endothelial growth factor (VEGF) were determined. In WT mice, HLI stimulated mobilization of LSK cells that reached maximum by day 2 (110±11 cells/mL blood, n=6). Ang-(1-7)-treatment potentiated the peak mobilization (206±24 cells/mL blood, n=8, P&lt;0.01 compared to the untreated). MasR-KO mice have reduced number of circulating LSKs (12±3 vs 43±9 per mL blood in WT, P&lt;0.01, n=5) (CFUs/mL blood 28±5 vs 54±8 in WT, P&lt;0.05, n=5). In MasR-KO mice, HLI did not induce mobilization, and blood flow recovery post-HLI was lower compared to WT (52±4% vs 89±6% in WT, P&lt;0.001, n=5), both of which were not improved by treatment with Ang-(1-7). Number of bone marrow-resident LSK cells was higher in MasR-KO mice compared to WT. Migration induced by SDF (84±6% vs 160±8% in WT, P&lt;0.001, n=5) or VEGF (97±4% vs 146±5% in WT, P&lt;0.001, n=4) was decreased in MasR-KO. These results suggest that MasR deficiency causes impaired mobilization of BMPCs likely by decreasing their sensitivity to hypoxia-regulated factors. Therefore endogenous MasR expression is essential for ischemia-dependent mobilization of BMPCs.

Intrauterine Nutrient Restriction Programs Impaired Endothelial Function Associated With Functional Dysfunction in Circulating Endothelial Progenitor Cells

ObjectiveThis investigation aimed to evaluate the effects of intrauterine nutrient restriction in both, vascular reactivity and functional capacity of endothelial progenitor cells (EPCs) in adult male offspring.MethodsThe procedures bellow were approved by Ethics Committee of Federal University of São Paulo (836880). Pregnant Wistar Rats received to Ad libitum or 50% of Ad libitum diet throughout gestation. At birth, pups were weighted. Adult male offspring from both groups (20 weeks of age) were submitted to femoral artery cannulation to register arterial blood pressure and heart rate. Vascular reactivity were performed by concentration‐response curves to Acetylcholine (ACh: 10−9M‐3×10−5M) and to Sodium Nitroprusside (SNP: 10−9M‐10−6M) in thoracic aorta rings. In addition, protein expression of eNOS phosphorylated in serine1177 by western blotting, and Nitric oxide concentration by NO Analyzer were investigated in thoracic aorta. Circulating EPCs number were characterized as CD34+VEGFR2+ cells in flow cytometer. The functional capacity of circulating EPCs was assessed in vitro by the colony‐forming unit assays. Results are show as mean ± EPM, test t de student were used.ResultsBody weight at birth (Control: 7.01±0.34g vs. Restricted: 4.3±0.08g), confirmed that Intrauterine nutrient restriction leads to intrauterine growth restriction. Intrauterine nutrient restriction did not programed alterations neither in mean arterial blood pressure (Control: 118±3mmHg vs. Restricted: 109±2mmHg), nor in heart rate (Control: 355±13 bpm vs. Restricted: 343±4 bpm). Restricted group exhibited reduction in maximal vascular response to ACh (Control: 92.18±2.7% vs. Restricted: 65.04±4.4%), but normal NPS‐induced vasodilation (Control: 98.45±2.38% vs Restricted: 93.97±2.64%) in aorta rings. Thoracic aorta from restricted offspring showed similar levels in protein expression of eNOS phosphorylated compared to control group (Control: 1.16±0.05 vs. Restricted: 1.05±0.01), on the order hand a reduction in NO concentration were observed (Control: 426.2±63.03μM/mg vs Restricted: 217.8±18.22 μM/mg). In addition, we found that circulating EPCs number was not altered by intrauterine nutrient restriction (Control: 0.008±0.0019 % vs. Restricted: 0.007± 0.0009%), but the number of colony‐forming unit was reduced (Control: 7.75±0.97 vs. Restricted: 3.80±0.82).ConclusionOur data suggests that intrauterine nutrient restriction programs endothelial dysfunction in adult offspring, which can be associated in part with reduction in NO concentration and impairment in EPCs functional capacity.Support or Funding InformationFAPESP

Effect of Fibroblast Growth Factor 2 on Equine Synovial Fluid Chondroprogenitor Expansion and Chondrogenesis.

Mesenchymal stem cells have been identified in the synovial fluid of several species. This study was conducted to characterize chondroprogenitor (CP) cells in equine synovial fluid (SF) and to determine the effect of fibroblast growth factor 2 (FGF-2) on SF-CP monolayer proliferation and subsequent chondrogenesis. We hypothesized that FGF-2 would stimulate SF-CP proliferation and postexpansion chondrogenesis. SF aspirates were collected from adult equine joints. Colony-forming unit (CFU) assays were performed during primary cultures. At first passage, SF-cells were seeded at low density, with or without FGF-2. Following monolayer expansion and serial immunophenotyping, cells were transferred to chondrogenic pellet cultures. Pellets were analyzed for chondrogenic mRNA expression and cartilage matrix secretion. There was a mean of 59.2 CFU/mL of SF. FGF-2 increased the number of population doublings during two monolayer passages and halved the population doubling times. FGF-2 did not alter the immunophenotype of SF-CPs during monolayer expansion, nor did FGF-2 compromise chondrogenesis. Hypertrophic phenotypic markers were not expressed in control or FGF-2 groups. FGF-2 did prevent the development of a “fibroblastic” cell layer around pellet periphery. FGF-2 significantly accelerates in vitro SF-CP expansion, the major hurdle to clinical application of this cell population, without detrimentally affecting subsequent chondrogenic capacity.

Time Course of Acquisition of a CSF3R Mutation and Subsequent Development of AML in a Patient with Cyclic Neutropenia

Acquired CSF3R (colony stimulating factor 3 receptor, granulocyte) mutations have been detected with a high frequency (17-34%) in severe congenital neutropenia (CN) patients. Moreover, in CN patients who developed overt acute myeloid leukemia (AML) the frequency of CSF3R mutations in the intracellular critical region reaches more than 80%. Up to now there was no report on the detection of acquired mutations in CSF3R or development of AML/MDS in cyclic neutropenia patients (CyN). This group of patients reveals the same genotype with mutations in the ELANE gene as CN patients. In contrast, both groups of patients have a clearly distinct disease penetration. Using ultra-deep sequencing approach we screened 22 CyN patients for the presence of genetic abnormalities in the critical region of the CSF3R gene. Interestingly, we found only in one CyN patient a clone with acquired mutation in G-CSF receptor (NP_000751.3, p.Gln741X) at 3% of allele frequency. The cycling neutrophils and platelets were well documented and verified the diagnosis of CyN. Within two years after the first detection, the mutant clone harboring the CSF3R mutation expanded and was detected in 16% of patient's bone marrow (BM) MNCs. The sequencing of the ELANE gene in the CyN patient with CSF3R mutation revealed the presence of two mutations p.Ala233Pro and p.Val235TrpfsX (NP_001963.1). Intriguingly, both ELANE mutations were absent in DNA isolated from each of parents indicating their de novo origin. Additionally, the patient was found to be heterozygous for synonymous SNP rs17216649 (dbSNP build 138) on the same ELANE exon permitting determination of the parental origin of mutations. The sub-clone analysis in E.coli showed that both mutations were always found on the paternally-derived allele. The missense mutation p.Ala233Pro (NP_001963.1) is well known to be associated with CN phenotype, whereas the single-base deletion p.Val235TrpfsX (NP_001963.1) was reported as cause of CN only once. There are no reports about detection of these mutations in CyN patients. Three years after the first detection of the CSF3R mutation the patient presented with 14% blast cells in bone marrow acquiring monosomy 7 and trisomy 21. Based on results of cDNA sequencing all CSF3R expressing cells in the sample were positive for p.Gln741X (NP_000751.3) mutation. Recently, we demonstrated acquisition of cooperative of RUNX1 and CSF3R mutations in more than 60% of CN-AML/MDS patients. We also found RUNX1 mutation p.Asp171Asn (NP_001001890.1) at 10% allele frequency in BM MNCs of CyN-AML patient. To evaluate at which stage of hematopoietic differentiation acquisition of CSF3R mutations appeared leading to the leukemogenic transformation, we performed colony forming unit (CFU) assays using patients BM MNCs. We observed the appearance of abnormal «CFU-blast» colonies after cultivation of BM MNCs in MethoCult H4434 Enriched medium supplemented with rh SCF, rh G-CSF, rh IL-3, rh IL-6 and rh EPO (72,2% of CFU-blasts (n=232), 21,8% of CFU-G (n=70), 5% of CFU-GM (n=16) and 1% of BFU-E (n=3)). Interestingly, we found that all sequenced CFU-G (n=4), CFU-GM (n=6) and «CFU-blast» colonies (n=20) were positive for p.Q741X CSF3R mutation. At the same time, no CSF3R mutations were identified in BFU-E colonies. In summary, we report a first case of acquisition of CSF3R mutation and subsequent development of AML in a CyN patient underlying the complex nature of genotype:phenotype relations in CN and CyN patients. This case also represents an example of an extremely rare situation of double de novo ELANE mutations and provides a unique opportunity to investigate pathogenic molecular mechanisms of leukemia development in CyN patients. The ultra-deep next generation sequencing (NGS) for identification of CSF3R mutations is a useful tool to study the frequency of CSF3R mutations not only in CN but also in CyN patients and is clinically important for the identification of patients with the risk of progression to leukemia. Disclosures No relevant conflicts of interest to declare.

224 CHARACTERIZATION OF EQUINE ENDOMETRIAL-DERIVED MESENCHYMAL STROMAL CELLS

Equine mesenchymal stromal cells (MSC) are mainly harvested from bone marrow and adipose tissue, requiring surgical procedures. Although human endometrium is known to harbor mesenchymal precursor cells, the presence of MSC in equine endometrium, a dynamic tissue, has not been investigated. This study reports for the first time the culture and characterisation of MSC from equine endometrium compared with equine bone marrow (BM)-derived MSC. Samples of equine endometrium (n = 6) and BM (n = 3) were collected postmortem. Endometrial tissue was digested using a dissociation medium containing collagenase I and DNase type I, and CD227 (mucin-1)-bound magnetic beads were utilised to separate epithelial (CD227+) from stromal (CD227–) cell fractions. Red blood cells from BM samples were excluded using a density gradient. All cell fractions were cultured in DMEM/F-12 containing 10% fetal bovine serum. After expansion, colony-forming unit (CFU) assay at passage 2, trilineage differentiation (adipogenic, chondrogenic, osteogenic), and flow cytometry analysis at passage 3/4 were performed for CD227– fractions and BM-MSC. Descriptive statistical analysis and 2-tailed t-test was performed with IBM SPSS Statistics 22 (SPSS Inc./IBM, Chicago, IL, USA). Both isolated cell fractions were plastic adherent and grew well under standard MSC culture conditions, although endometrial CD227– cells attached quicker to culture plasticware than did BM-MSC. The CFU assay at passage 2 showed no significant difference in cloning efficiency (CE) between BM-MSC (20.78 ± 2.86%) and CD227– (24.89 ± 3.04%) cell lines (P = 0.36). Flow cytometry showed the expression of MSC markers (CD29, CD44, CD90, CD105) and perivascular markers (CD146, NG2) but almost no expression of haematopoietic markers (CD34, CD45) in both cell lines (Table 1). No statistically relevant difference was seen except for the higher expression of NG2 in BM-MSC (P = 0.054). Trilineage differentiation was successfully induced in both cell lines. In conclusion, we showed the presence of putative MSC in equine endometrium. We successfully isolated and cultured these cells, which display comparable characteristics in MSC criteria as well-established BM-derived MSC. These endometrial-derived MSC may provide a convenient source for veterinary regenerative therapies in equine reproduction. Table 1.Flow cytometry marker expression (mean ± standard error) at passage 4

Abstract C52: PARP1 trapping activity of PARP inhibitors is associated with cytotoxicity in both cancer cells and healthy bone marrow

Abstract Background: PARP inhibitors (PARPi) are under clinical investigation as anticancer therapies in several indications with a focus on monotherapy in tumors with homologous recombination deficits and on combination regimens with DNA damaging chemotherapy. The combination activity of PARPi with DNA alkylating agents is due in part to trapping of PARP1 onto base excision repair intermediates. Recent studies in mice indicate that potent trapping activity limits the tolerability of PARPi in this setting. While trapping has not been directly demonstrated with PARP inhibitors alone, genetic and correlative data suggest that it may be important in the monotherapy setting as well. The objective of this study was to further delineate the role of trapping in single-agent cytotoxicity of PARPi in cancer cells and healthy bone marrow. Methods: Cytotoxic potency was determined for cancer cells using CellTiter-Glo and for erythroid and myeloid progenitors using colony forming unit (CFU) assays. PARP1 trapping was evaluated by cellular fractionation, TR-FRET and BLI. PARPi binding affinities and off-rates were determined via SPR. Cellular PAR levels were determined by ELISA. Results: Talazoparib, rucaparib and the novel PARPi A-934935 bound to PARP1 with similar affinities and off-rates and inhibited cellular PAR synthesis with identical potencies. Despite these similarities, talazoparib was a more potent trapping agent. Consistent with the increased trapping activity, talazoparib was &amp;gt;10-fold more potent than rucaparib and A-934935 at inhibiting proliferation of erythroid progenitors and &amp;gt;50-fold more potent in myeloid progenitors. Similar differences were observed in a panel of human cancer cell lines including BRCA−/− breast cancer and small cell lung cancer. Conclusions: All NAD+-competitive PARPi elicit PARP1 trapping, but different inhibitors display varying degrees of resolution between trapping and cellular PAR depletion. Understanding the relative importance of these mechanisms to the tolerability and efficacy of PARP inhibitors is of critical importance. Myelosuppression is a commonly observed toxicity in PARPi clinical trials. In the present study we used ex vivo bone marrow CFU assays to model myelosuppression. The IC50s observed were within the range of exposures observed at recommended phase II doses in monotherapy clinical trials, highlighting the clinical relevance of this experimental approach. Our data reveal that compounds inhibiting cellular PAR synthesis with equal potency span a broad range of potency in BM-CFU assays in a manner predicted by PARP1 trapping activity. These results suggest that PARP1 trapping is a primary driver of myelosuppression by PARP inhibitors. Our observation that trapping drives cytotoxicity to a similar extent in bone marrow and cancer cells sensitive to single-agent PARP inhibition suggests that PARP1 trapping activity is not advantageous with respect to the therapeutic index of PARPi in a monotherapy setting. This is consistent with observations related to the therapeutic index of PARP inhibitors in the clinic. These results also have important implications for selection of a PARPi for combination regimens where trapping is not required for efficacy and in which myelosuppression may be a concern, such as those containing platinum drugs or topoisomerase I inhibitors. Citation Format: Todd A. Hopkins, Julie L. Wilsbacher, Enrico L. DiGiammarino, Sanjay C. Panchal, Gui-Dong Zhu, Thomas D. Penning, Eric F. Johnson, David Maag. PARP1 trapping activity of PARP inhibitors is associated with cytotoxicity in both cancer cells and healthy bone marrow. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C52.

Abstract B42: Nelfinavir potentiates the anti-cancer efficacy of curcumin by subverting endoplasmic reticulum stress toward apoptosis: A promising chemoprevention approach

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related deaths among men in the United States. Despite the availability of treatments against localized PCa, the clinical management of advanced PCa remains a significant challenge in relation to both patient morbidity and escalating healthcare costs. Therefore, the ability to suppress cancer growth using safe natural compounds as chemopreventive agents, e.g. Curcumin (CUR), is emerging as a promising and cost-effective approach. The anti-cancer effects of CUR have been associated with its ability to suppress crucial survival pathways in cancer cells, e.g. PI3K/Akt and NF-κB signaling. However, clinical trials investigating the anti-cancer potential of CUR have clearly indicated that this phytochemical will be of limited efficacy when used alone. Aggressive cancer cells utilize multiple master regulatory pathways to dictate their growth, survival and death decisions, which need to be simultaneously targeted towards an effective anti-cancer approach. Indeed, recent findings demonstrate that the control of cell survival and apoptosis is exquisitely regulated via the endoplasmic reticulum (ER) stress cascades. Numerous recent studies have shown that the clinically approved HIV-1 protease inhibitor drug, Nelfinavir (NFV) is an well-tolerated oral drug with promising anti-cancer properties. The antitumor effects of NFV is believed to be via the suppression of PI3K/Akt and the induction of ER-stress. Therefore, we hypothesized that the constitutive ER-stress in aggressive cancer cells can be subverted towards apoptosis by chronic exposure to CUR and NFV, which will be a novel chemoprevention approach. In vitro studies (MTT cytotoxicity-assays) in two aggressive PCa cell lines, i.e. C4-2B and PC-3, showed significant (p&amp;lt;0.05) increases in cytotoxicity following coexposure to physiologically achievable concentrations of CUR &amp; NFV (5 μM each). However, this drug combination did not show comparable toxicity in the non-tumorigenic RWPE-1 cell line, or in the primary prostate epithelial cells (PrEC) and bone-marrow mesenchymal stem cells (MSC). Western immunoblot studies showed that combined exposure to CUR &amp; NFV acted in a synergistic manner (combination index&amp;lt;1) to suppress Akt activation (phosphorylated form) and significantly (p&amp;lt;0.05) increased the levels of early ER-stress markers, Grp78 and phospho-eIF2α. The ER-stress induced pro-apoptotic markers, i.e. ATF4, CHOP, and TRIB3 were also upregulated following acute (12 hr) exposure to CUR &amp; NFV combination. More importantly, chronic exposure (3 wks; replenished every third day) to a much lower concentration of CUR &amp; NFV combination (0.5 μM each) was able to drastically reduce the clonogenic ability of C4-2B and PC-3 cells, as evident from colony forming unit (CFU) assays. Therefore, our in vitro studies showed that NFV can potentiate the anti-cancer effects of CUR and this combination may be a promising approach against PCa growth. In vivo studies using mice containing C4-2B tumor xenografts (&amp;lt;50 mm) are currently in progress to document the safety and anti-tumor efficacy of chronic treatment with the CUR &amp; NFV combination. Initial evidence of the in vitro efficacy of this combination suggests a safe chemoprevention strategy that specifically targets the constitutive ER-stress in aggressive cancer cells. Citation Format: Aditi Mathur, Mikhail L. Kostochka, Asim B. Abdel-Mageed, Debasis Mondal. Nelfinavir potentiates the anti-cancer efficacy of curcumin by subverting endoplasmic reticulum stress toward apoptosis: A promising chemoprevention approach. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B42.

Comparative Characterization of Canine and Human Mesenchymal Stem Cells Derived from Bone Marrow

Bone marrow is a major source of mesenchymal stem cells (MSC), which are used in tissue engineering and other autologous stem cell therapies. Studies designed to use large canine models and translate the results to human practice must take into account the ex vivo and in vitro differences in the bone marrow samples. A set of morphological and physiological markers was used to compare MSC derived from canine and human bone marrow. Despite anticlotting treatment, frequent bone marrow clotting was a problem with canine samples, so we developed a protocol for enzymatic digestion of undesirable clots. We compared colony forming units (CFU) assay, population doubling time (PDT), senescenceassociated β-galactosidase (SA-β-Gal) activity, as well as the ability of cells to differentiate to osteogenic, adipogenic and chondrogenic phenotypes. Urokinase digestion resulted in recovery of MSC: 4-fold more CFU from canine and 1.6-fold more from human samples when compared to untreated samples. Canine MSC were less robust in vitro – they divided actively only for four weeks in culture, while human cells divided for longer than eight weeks. After six weeks in culture, canine MSC underwent 17 population doublings of while human cells reached 26. The percentage of senescent cells increased linearly with time, but with a faster rate in canine MSC. Human and canine MSC underwent differentiation to all lineages; however canine MSC had generally lower differentiation potential. In conclusion, the discrepancy between canine and human cultures must be considered in future MSC-based therapies based on dogs as animal model.

Spontaneous and continuous anti-virus disinfection from nonstoichiometric perovskite-type lanthanum manganese oxide

Viral pathogens have threatened human being׳s health for a long time, from periodically breakout flu epidemics to recent rising Ebola virus disease. Herein, we report a new application of nonstoichiometric Perovskite-type LaxMnO3 (x=1, 0.95, and 0.9) compounds in spontaneous and continuous disinfection of viruses. Perovskite-type LaxMnO3 (x=1, 0.95, and 0.9) is well-known for their catalytic properties involving oxidization reactions, which are usually utilized as electrodes in fuel cells. By utilizing superb oxidative ability of LaxMnO3 (x=1, 0.95, and 0.9), amino acid residues in viral envelope proteins are oxidized, thus envelope proteins are denatured and infectivity of the virus is neutralized. It is of great importance that this process does not require external energy sources like light or heat. The A/PR/8/34H1N1 influenza A virus (PR8) was employed as the sample virus in our demonstration, and high-throughput disinfections were observed. The efficiency of disinfection was correlated to oxidative ability of LaxMnO3 (x=1, 0.95, and 0.9) by EPR and H2-TPR results that La0.9MnO3 had the highest oxidative ability and correspondingly gave out the best disinfecting results within three nonstoichiometric compounds. Moreover, denaturation of hemagglutinin and neuraminidase, the two key envelope proteins of influenza A viruses, was demonstrated by HA unit assay with chicken red blood cells and NA fluorescence assay, respectively. This unique disinfecting application of La0.9MnO3 is considered as a great make up to current sterilizing methods especially to photocatalyst based disinfectants and can be widely applied to cut-off spread routes of viruses, either viral aerosol or contaminated fluid, and help in controlling the possibly upcoming epidemics like flus and hemorrhagic fever.

139 CD45−CD34+ Circulating Progenitor Cells Levels Are Associated With the Presence of Coronary Artery Disease (CAD) in Patients With Nonalcoholic Fatty Liver Disease (NAFLD)

Background and Aim: Angiogenic growth factors (AGF) play a crucial role in regulating hematopoietic stem and progenitor cell homing and differentiation. Patients with NAFLD and CAD have lower levels of AGF (Elsheikh et al. DDW 2014). Thus, in this study we investigated the level and functions of circulating progenitor cells (CPC) in patients with NAFLD and CAD. Methods: We prospectively enrolled 82 patients undergoing elective coronary angiography. Each patient had fasting serum, clinical data and abdominal ultrasound (US). All US were read centrally using a standard protocol. NAFLD was defined as radiologic fatty liver in the absence of other causes of liver disease and excessive alcohol use. Patients were divided into NAFLD with CAD (n=24), NAFLD without CAD (n=13), only CAD (n= 31) and Non-NAFLD and Non-CAD (n=14). Circulating progenitor cells (CPC) were quantified by flow cytometry based on the expression of (CD34+, CD133+, CD34+ CD133+) in presence or absence of the hematopoietic marker (CD45). To study the functional capacity of CPC, colony-forming unit (CFU) assay was used. These include erythroid progenitors (burst-forming unit erythroid (BFU-E)); granulocyte/macrophage progenitors (CFU-granulocyte, macrophage (CFU-GM); and multi-potential progenitors (CFU-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM)). Results: The levels of the CD45-CD34+, CD45CD133+ and BFU-E were higher in NAFLD patients with CAD (median, 15%, 2% and 60 colonies, respectively) than NAFLD patients without CAD (median, 9%, 1% and 37 colonies , respectively, all p-values≤0.05). We also found that the levels of the CD45-CD133+ were higher in NAFLD patients with CAD (median, 15%) than patients with only CAD (median, 11%, p 25th quartile of CD45-CD34+, CD45-CD133+ and total CFU levels had the highest risk for CAD [OR: 7.00 (1.34-36.68), 5.00 (1.07-23.46) and 7.00 (1.10-44.61) respectively]. After age adjustment, only CD45-CD34+ circulating progenitor cells remain associated with increased risk of CAD in patients with NAFLD [OR: 8.71 (1.21-62.51)]. Conclusions: Our results indicate that, levels of circulating progenitor cells CD45-CD34+ may be associated with increased risk of CAD in NAFLD patients. Increased circulating progenitor cells levels could be due to the low levels of AGF leading to impaired homing and differentiation capacity.

Abstract 1030: Are endothelial cells essential for the development of leukemia

Abstract Background The most pronounced micro-environmental changes observed in the bone marrow of leukemia patients are endothelial proliferation and angiogenesis. The molecular basis underlying how blood vessels sustain/initiate leukemia remains poorly understood. However, this may explain why anti-angiogenic agents are effective treatments for leukemia. Aim To define whether modification of the endothelium initiates and/ or perpetuates leukemia and determine the genes responsible. Methods Sleeping Beauty (SB) is a system of insertional mutagenesis that randomly integrates transposons into genomic DNA. Endothelial-specific SB activation was achieved by driving CRE-recombinase expression via the Tie-2 promoter/enhancer. Tissues were analyzed for histology, immune profiles and genes with insertional mutations. Colony forming unit (CFU) assays were performed on magnetically selected CD45-/CD31+ endothelial cells and CD45-/CD31-/Sca1+ hematopoietic stem cells isolated from bone marrow of CRE+ and CRE- mice and the resulting colonies analyzed by flow cytometry for CD3 (lymphoid) and CD14 (myeloid) populations. Results Over 50 weeks all CRE+ mice (n=106), but none of the WT mice (n=35), became moribund and were sacrificed. At necropsy, CRE+ mice displayed peripheral leukocytosis and gross anatomical/histological modifications in spleens, livers and thymus. Molecular analyses showed amplifications of either T cell (CD3; 49%) or macrophage (CD14; 28%) populations or both (23%) in the spleen and bone marrow of CRE+ mice. These leukocytes were SB positive indicating they originated from endothelial cells, and not hematopoietic stem cells. This was confirmed by CFU assays in which elevated CD3+ and CD14+ populations were found arising from the CD45-/CD31+ cells but not CD45-/CD31-/Sca1+ cells from CRE+ mice relative to WT mice. Targeted deep sequencing identified genes related to cytokine mediated signaling and transcriptional control. Many of the identified genes are already implicated in human leukemia. Conclusions Targeting SB insertional mutagenesis to the endothelium resulted in hematological malignancy in mice indicating direct control over both myeloid and lymphoid differentiation by endothelial cells. Targeted deep sequencing identified changes to known mediators in hematological lineage specification, validating our model. Also identified were novel candidates that may contribute to the development of leukemia and offer new therapeutic targets to treat this malignancy. These data, for the first time, provide evidence for a role of the endothelium in the regulation of post-natal hematopoiesis and provide a rationale for the use of anti-angiogenic therapy in the treatment of leukemia. Citation Format: Indhu Subramanian, Emily S. Fuller, Katie Powell, Rolfe Howlett, Anthony W. Ashton, Viive M. Howell. Are endothelial cells essential for the development of leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1030. doi:10.1158/1538-7445.AM2014-1030

VSTAT120 REDUCES THE INNATE INFLAMMATORY RESPONSE TO INFECTION AND ENHANCES ONCOLYTIC VIRAL THERAPY FOR GLIOBLASTOMA

BACKGROUND: A major barrier to oncolytic virus (OV) efficacy for the treatment of Glioblastoma is the innate immune response; this response limits virus replication and reduces tumor cell destruction. Our lab developed a novel OV, RAMBO, which expresses the anti-angiogenic fragment Vasculostatin (Vstat120). In addition to its anti-angiogenic effects, we observe the RAMBO virus replicates more efficiently and persists longer in vivo than a control virus (rHSVQ1). The primary objective of this study is to investigate the effects RAMBO directed Vstat120 expression on microglia/macrophage activation and OV efficacy. METHODS: Mice bearing intracranial tumors were treated with RAMBO, rHSVQ1, or PBS, and flow cytometry utilized to examine monocyte/microglia activation/recruitment. Coculture of i9nfected glioma cells with microglia were utilized in in vitro experiemnts. Cytokines were analyzed via qPCR and ELISA. Virus replication was determined by plaque forming unit (PFU) assays. RESULTS: Treatment of intracranial tumors with OV resulted in significant monocyte infiltration into the tumor. Surprisingly, this infiltration was reduced 4.5 fold in tumors treated with the RAMBO virus as compared to rHSVQ1 (p < 0.05). Infiltrating monocytes from RAMBO treated tumors had significantly less MHCII, Ly6C, and CD86 than rHSVQ1 treated tumors (p < 0.05). The microglia of RAMBO treated tumors had significantly less MHCII and CD206 expression than those treated with rHSVQ1 (p< 0.05). To further examine the interaction of Vstat120 with microglia/monocytes we developed a coculture system with murine microglia and infected human glioma cells. Glioma cells were treated with PBS, rHSVQ1, or RAMBO and cocultured with microglia. We observed a 75-fold induction of TNF-α expression in microglia cultured with rHSVQ1 infected glioma cells compared to untreated microglia (p < 0.05). Interestingly, we observed a 3.4-fold reduction in microglia TNF-α expression in RAMBO treated cocultures compared to rHSVQ1 cocultures (p < 0.01). Consistent with this ELISA confirmed a 6.9 fold decrease in microglia TNF-α secretion (p < 0.01), and virus titers revealed a 2.6 fold increase in infectious virus in RAMBO Vs eHSVQ1 treated cocultures (p < 0.01). Cocultures treated with a microglia (murine) specific TNF-α blocking antibody rescued the differences in viral replication between rHSVQ1 and RAMBO virus titers. There was no difference in virus replication of glioma cells or microglia alone. These results suggest Vstat120 reduces inflammation and enhances OV replication in part through the suppression of TNF-α expression/secretion on microglia/monocytes. This transient suppression of the microglia/monocyte inflammatory response enhances OV replication and promotes anti-tumor efficacy. CONCLUSIONS: Vasculostatin affects macrophage recruitment and activity to improve viral replication and efficacy. SECONDARY CATEGORY: Immunobiology & Immunotherapy.

Comparision Of Different Cryoprotective Solution - Albumin Vs Autologous Plasma - Its Effect On Cell Recovery and Clonogenic Potential Of Cryopreserved Peripheral Blood Hematopoietic Stem and Progenitor Cells

Background Peripheral blood is a preferable source of hematopoietic stem and progenitor cells (HSPCs) used for autologous transplantation. HSPCs are mobilized to peripheral blood and collected by leukapheresis. Prior to cryopreservation the cells need to be processed including the addition of cryoprotective mixture as dimethyl sulfoxide (DMSO) prediluted in human serum albumin solution (HSAS). In Europe there is no commercially available albumin manufactured in packs with tubing which would enable the use of sterile tubing welder. Alternatively cryoprotective solution can be prepared using autologous plasma (AP) obtained during the same leukapheresis, allowing for the preparation of HSPCs in a completely closed system and hence to reduce the risk of contamination. The goal of our study was to test if the HSAS may be replaced by autologous plasma without negative impact on cell recovery and clonogenicity. Methods Samples were prospectively collected from 18 patients with multiple myeloma (n=13) and lymphomas (n=5) mobilized with chemotherapy combined with G-CSF. Small volumes (1.5 ml) of cell suspensions obtained from the leukapheresis products were divided into 2 parts (0,5ml) placed in separate small vials, each containing different cryoprotective mixture - 5% HSAS or AP with a final 7.5% DMSO concentration. The final volume of cell suspensions equaling 1 ml, the cell concentration (0.7–1.5 × 108 /ml). The cells were frozen in IceCube, using a computer controlled cooling program and stored in liquid nitrogen for 2 - 4 months. Concentration of total protein and individual electrophoretic fractions of plasma proteins were measured. The quality of cryoprotective mixtures was evaluated by cell recovery and clonogenic potential. The recovery was determined by comparing number of living cells before and after cryopreservation, using trypan blue staining. Clonogenic potential was carried out by colony forming unit (CFU) assays. Depending on CD34+ percentage, 5-40 × 103 living cells were plated (in triplicates) in MethoCult medium and cultured for 14 days. Results The median recovery of nucleated cells for AP was 68.3% (range 40.6-96.1) and was similar to HSAS 68.5%, (41.7-100); (p=0.3; Wilcoxon matched pairs test). The number of CFUs calculated per 100 000 cryopreserved cells did not differ significantly between tested cryoprotective mixture: 187.3 (11.3-806.3) for albumin, 130.5 (15-924.2) for autologous plasma (p=0.5; Wilcoxon matched pairs test). No significant differences were observed when the number of specific types of CFUs were compared. Neither total protein nor albumin concentration of plasma correlated with the clonogenic potential of the leukapheresis product cryopreserved in AP when samples from patients with higher concentration than median were compared with the other (Mann-Whitney U test). Between January and July 2013 more than 50 successful transplants of autologous HSPCs cryopreserved with 7.5% DMSO prediluted in autologous plasma were performed in our Department. Conclusions Commercially available human serum albumin can be replaced by autologous plasma in procedure of HSPCs cryopreservation. The use of autologous plasma for cryoprotective mixture preparation does not appear to negatively affect cell recovery and clonogenic potential of leukapheresis product. The advantage of such solution is possibility of HSPCs preparation in closed system to reduce risk of auto-HSCT product contamination to the minimum. Disclosures: No relevant conflicts of interest to declare.