Granulocyte-macrophage Colony-forming Units Research Articles

EFFICIENCY OF BONE MARROW PRECURSOR CELL COLONY-FORMING AS A PREDICTOR OF DISEASE COURSE IN PLASMA CELL MYELOMA PATIENTS WITH A HISTORY OF RADIATION EXPOSURE.

Assessment of role of the bone marrow colony-forming efficiency in plasma cell myeloma patients at different stages of treatment as a prognostic criterion for the disease course. The colony forming efficiency (CFE) was assayed in stage I-II plasma cell myeloma (PCM)patients (n = 37) aged 42-73, namely in patients survived after the Chornobyl NPP accident (n = 21) and persons notexposed to ionizing radiation (n = 16). There were 11 males exposed to ionizing radiation and having got stage I PCM,9 males and 3 females exposed and having got stage II PCM, 3 males and 3 females not exposed and having got stageI PCM, 6 males and 2 females not exposed and having got stage II PCM. Healthy persons (n = 20) were included in thecontrol group. Number of the bone marrow (BM) granulocyte-macrophage colony-forming units (CFU-GM) in both exposedand not exposed PCM patients depended on a disease stage. CFU-GM was (16.7 ± 1.2) in the stage I PCM patients vs.(11.1 ± 1.1) in the stage II PCM ones both being lower (p < 0.05) compared to control (64.5 ± 2.2). Changes in cluster formation were similar, i.e. (37.7 ± 1.6) and (19.4 ± 1.3) correspondingly in the stage I and stage II PCM patients.Respective values in control were (89.8 ± 3.6). The CFE in stage I and stage II PCM patients at the time of diagnosiswas lower (5.7 ± 1.5 and 2.4 ± 1.1 respectively) vs. control (39.5 ± 1.51, p < 0.05), but has increased in remission upto (29. 6 ± 1.8) and (13.8 ± 1.2) respectively. There was no difference at that between the irradiated and non-irradiated patients. Number of the fibroblast colony-forming units (CFU-F) in the stage I and stage II PCM patients duringdiagnosis, namely (43.9 ± 5.4) and (22.5 ± 3.7), was lower (p < 0.05) vs. control (110.5 ± 4.9). Upon reaching remission the CFU-F value increased significantly (p < 0.05), reaching (87.4 ± 4.2) and (55.6 ± 2.7) correspondingly in thestage I and stage II PCM patients. Dependence of the BM cell CFE on the stage of PCM and presence or absence of remission was established. Prognostic value of the CFE of BM CFU-GM in terms of life span of patients was shown (Ro Spearm = 0.39,p < 0.02), namely in case of CFE > 20 before the polychemotherapy administration the life span of PCM patients wassignificantly longer vs. cases of CFE < 20.

Breakthroughs in Preclinical Development of Ataluren (PTC124) As Therapeutic Option for Patients Affected By Shwachman-Diamond Syndrome: Towards the First Clinical Trial

Shwachman-Diamond syndrome (SDS) is one of the more common inherited bone marrow failure syndromes (IBMFS). Almost 90% of patients with SDS present mutations in the Shwachman-Bodian-Diamond syndrome gene (SBDS) which encodes for the homonymous small protein involved in ribogenesis. SDS is a multiple-organ disease mostly characterized by exocrine pancreas insufficiency, bone malformations, and more importantly bone marrow failure. Most patients with SDS present severe neutropenia, whereas thrombocytopenia and anemia are less frequent. Furthermore, 15-20% of patients develop myelodysplastic syndrome with high risk of acute myeloid leukemia (AML). STAT3 pathway is upregulated both in primary SDS leukocytes and immortalized B cells. Being STAT3 a key regulator of interleukin-6 (IL-6), we postulated that STAT3 hyper-activation could lead to a dysregulation of the IL-6 signaling cascade. Increased levels of IL-6 have been found in pediatric patients with AML and it has been associated with poorer outcomes in these patients, highlighting IL-6 as a cytokine potentially involved in the development of AML. Thus, our hypothesis is that STAT3-IL6 axis may contribute to leukemogenesis in SDS. Almost 55% of patients with SDS carry a specific nonsense mutations, namely the c.183-184TA&amp;gt;CT, which cause a premature termination codon (PTC). Ataluren (PTC124, PTC Therapeutics Inc, NJ) is a small PTC suppressor molecule already approved by the European Medicines Agency as a therapeutic option for Duchenne muscular dystrophy. Interestingly, we recently reported that ataluren can restore SBDS expression in bone marrow progenitors and in peripheral blood mononuclear cells isolated from patients with SDS. Moreover, we have shown that ataluren can reduce mTOR hyper-phosphorylation and excessive apoptotic rate observed in SDS leukocytes. More importantly, we reported that ataluren can improve myeloid differentiation in a small cohort of patients (Bezzerri et al, Am J Hematol 2018). In this further analysis considering an enlarged cohort of 20 SDS patients carrying nonsense mutations we found the following: Ataluren can significantly improve both myeloid colony-forming unit-granulocyte/macrophage (CFU-GM) and colony-forming unit granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM) generation from bone marrow mononuclear stem cells obtained from an enlarged cohort of 20 patients with SDS carrying nonsense mutations. Ataluren indeed almost doubled the number of CFU-GM and CFU-GEMM after 7 and 14 days of treatment.Colony-forming unit erythroid (CFU-E) generation was not affected by the treatment.Ataluren induces neutrophil maturation in SDS bone marrow mononuclear stem cells (mean increase of 61% CD16+ CD11b+ cells over untreated controls) after 24-48 hours of treatment.Consistently with STAT3 hyper-activation observed in SDS cells, here we show that patients with SDS present a significantly increased level of IL-6 in plasma (4.3-fold higher expression than the healthy control group). Also lymphoblastoid cell lines (LCL) and primary bone marrow mesenchymal stromal cells (MSC) obtained from patients with SDS show increased IL-6 release in culture supernatants compared to healthy controls (2.5-fold and 6.8-fold higher levels, respectively).Of note, ataluren can reduce IL-6 expression in SDS cells restoring normal levels both in LCL and MSC. In conclusion, these new data support the enrollment of patients for the first clinical trial for this drug in SDS. Furthermore, this study could pave the way for the use of ataluren for other nonsense mutation-mediated IBMFS where STAT3-IL6 axis and similar pro-leukemic pathways are involved. Disclosures Bezzerri: Marco Cipolli, Valentino Bezzerri, Baroukh Maurice Assael: Patents &amp; Royalties: WO2018/050706 A1 "Method of treatment of Shwachman-Diamond syndrome". Cipolli:Marco Cipolli, Valentino Bezzerri, Baroukh Maurice Assael: Patents &amp; Royalties: WO2018/050706 A1 "Method of treatment of Shwachman-Diamond syndrome".

Abstract 2913: Serpinb3a-/- mice are radioresistant

Abstract Serpins are a superfamily of serine protease inhibitors that regulate proteolytic pathways by utilizing a conformational change to bind to and inhibit their target peptidases. Currently 36 human serpins have been identified, but the biological roles of most are not fully understood (Silverman et al., J Biol Chem. 285(32):24299-305, 2010). Serpinb3a (the mouse orthologue of human SERPINB3 and -B4) is an intracellular serpin and inhibits both serine and cysteine peptidases invitro. We now report radioresistance of Serpinb3a-/- mice and their hematopoietic progenitor cells. Serpinb3a-/- and control Balb/c mice were irradiated to a dose of 8.0 Gy total body irradiation and followed for the development of the hematopoietic syndrome. Long Term Bone Marrow Cultures (LTBMCs) were established from the bone marrow flushed from the femurs and tibias of Serpinb3a-/- and Balb/c mice. Irradiation survival curves were performed on bone marrow stromal cell lines established from the LTBMCs and on fresh bone marrow isolated from the femurs of sacrificed Serpinb3a-/- and Balb/c mice. Colony forming unit granulocyte-macrophage (CFU-GM) colonies of 50 cells or greater were counted. The data from 3 experiments were analyzed using linear quadratic and single-hit, multi-target models. Serpinb3a-/- mice showed increased survival following 8.0 Gy TBI compared to Balb/c mice (p &amp;lt; 0.0001). CFU-GM from Serpinb3a-/- were radioresistant compared to Balb/c marrow (ň = 4.9 ± 1.4 and 1.4 ± 0.1, p = 0.0451, respectively). Serpinb3a-/- bone marrow stromal cells were radioresistant with an increased Do (1.97 + 0.11 Gy) compared to bone marrow stromal cells from Balb/c mice (Do = 1.45 + 0.03 Gy, p = 0.0113). Loss of Serpinb3a provides a radiation survival advantage, increases radioresistance of both hematopoietic progenitor cells and marrow stromal cells, and may lead to identification of new targets for design of radiation mitigator drugs. This research was supported by grant NIAID/NIH U19-A168021. Citation Format: Stephanie Thermozier, Michael Epperly, Darcy Franicola, Xichen Zhang, Renee Fisher, Donna Shields, Hong Wang, John Willis, Cliff Luke, Gary Silverman, Joel Greenberger. Serpinb3a-/- mice are radioresistant [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2913.

Ganoderma spore lipid protects mouse bone marrow mesenchymal stem cells and hematopoiesis from the cytotoxicity of the chemotherapeutic agent.

Cancer chemotherapeutic agents are frequently toxic to bone marrow and impair bone marrow functions. It is unclear whether ganoderma spore lipid (GSL) can protect bone marrow cells from the cytotoxicity of chemotherapy. To investigate the protective effects of GSL on bone marrow mesenchymal stem cells (MSCs) and hematopoiesis, we examined the effects of GSL on MSCs in vitro and hematopoiesis in vivo after treatment with the chemotherapeutic agent cyclophosphamide. MSCs and peripheral blood cells were isolated and counted from the bone marrow of normal mice were pre-treated with GSL before CTX treatment or co-treated with GSL and CTX, followed by examining the changes in phenotype, morphology, proliferation, apoptosis, and differentiation potentials. The results showed that GSL could reduce the CTX-induced changes in the phenotype of MSCs and maintain the elongated fibroblast-like morphology. MTT and annexin V/propidium iodide (PI) analyses found that GSL pre-treatment and co-treatment increased the proliferation and decreased the apoptosis in CTX-treated MSCs. Furthermore, GSL improved the osteogenic and adipogenic differentiation potentials of CTX-treated MSCs. In vivo, GSL treatment increased the number of peripheral blood cells including white blood cells (WBC) and platelets (PLT) in the CTX-treated mice and enhanced the in vitro formation of hematopoietic lineage colonies (erythrocyte colony forming unit, CFU-E; erythroid burst-forming units, BFU-E; and granulocyte macrophage colony-forming units, CFU-GM) from bone marrow cells in these mice. These findings suggest GSL could protect MSCs and hematopoiesis from the cytotoxicity of CTX and might become an effective adjuvant to attenuate side effects of chemotherapy during cancer treatment.

Cotargeting the JAK/STAT signaling pathway and histone deacetylase by ruxolitinib and vorinostat elicits synergistic effects against myeloproliferative neoplasms.

The majority of patients with Philadelphia-negative myeloproliferative neoplasms (MPNs) harbor a gain of function mutation V617F in Janus kinase (JAK) 2. Although JAK2 inhibitors such as ruxolitinib have been shown to be clinically efficacious, the hematological toxicity and eventual drug resistance limit its use as monotherapy. Other gene mutations or dysregulation correlated with the disease phenotype and prognosis have been found to contribute to the complexity and heterogeneity of MPNs, giving rise to an increasing demand for combination therapies. Here, we combine ruxolitinib and the histone deacetylase inhibitor vorinostat as a rational combination strategy for MPNs. We tested the combination of ruxolitinib and vorinostat in cells with the JAK2V617F mutation, such as HEL cells, c-Kit+ cells from JAK2V617F transgenic mice and bone marrow mononuclear cells (BMMNCs) from patients with MPN. Our results showed significant synergistic effects of this combination strategy. Cotreatment with ruxolitinib and vorinostat synergistically induced apoptosis, cell cycle arrest and inhibition of the colony-forming capacity of HEL cells by attenuating the JAK/signal transducer and activator of transcription (STAT) and protein kinase-B (AKT) signaling pathways. In particular, cotreatment with ruxolitinib and vorinostat prevented the formation of large colonies of colony-forming unit-granulocyte/erythroid/macrophage/megakaryocytes (CFU-GEMMs) and colony-forming unit-granulocyte/macrophages (CFU-GMs) derived from the BMMNCs of patients with MPN. Taken together, these data provided preclinical evidence that the combination of ruxolitinib and vorinostat is a potential dual-target therapy for patients with MPN.

Osteoclastogenesis from bone marrow cells during estrogen-induced medullary bone formation in Japanese quails.

Osteoclasts are differentiated from hematopoietic mononuclear cells by regulation of the receptor activator of nuclear factor kappa-B (RANK)/receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) system. Medullary bone (MB) that forms in the bone marrow of female birds is remodeled under the control of circulating estrogen (E2) during the laying period. Although the osteoclasts of MB are differentiated from mononuclear cells, the mechanism of osteoclastogenesis is not known. We investigated whether MB osteoclastogenesis is regulated by the RANK/RANKL/OPG system using MB from male quails induced with E2. Bone marrow cells (BMCs) differentiate into osteoclasts that have the ability of bone resorption via stimulation of RANKL/M-CSF, but this ability is suppressed by OPG and differentiation is inhibited by calcinurin inhibitors. We found that BMCs at 3days after E2 administration had high bone osteoclastogenesis ability and colony forming unit-granulocyte/macrophage (CFU-GM)/colony forming unit-macrophage (CFU-M) formation abilities. We conclude that MB osteoclasts are differentiated from BMCs by the RANK/RANKL/OPG system, and that precursor cells of osteoclasts are increased during MB formation.

Cryopreservation and thawing of hematopoietic stem cell CD34-induced apoptosis through caspase pathway activation: Key role of granulocytes

IntroductionCell damage inescapably occurs during both the freezing and the thawing graft processes for autologous hematopoietic stem cell (HSC) transplantation. To estimate HSC injury, a quality control is performed including: (i) CD34+ quantification; (ii) percentage of CD34+ viability and (iii) evaluation of HSC functional ability to form colony forming unit–granulocyte macrophage (CFU-GM). Apoptosis involves complex pathways such as caspase enzymes. Here, we assess the extent of apoptosis that is caspase-dependent before and after cryoconservation of CD34+, using a Fluorescent Labeled Inhibitor of CAspases (FLICA). MethodsCaspase pathway activation status was evaluated in 46 patients (multiple myeloma [n = 24], lymphoma [n = 22]), by flow cytometry, using a 7-aminoactinomycin-D (7AAD)/FLICA staining test, in CD34+, CD3+, CD14+ and CD56+ cells. Viable 7AAD−/FLICA+ cells were then correlated with various parameters. ResultsWe showed a significant caspase pathway activation, with 23% CD34+/7AAD−/FLICA+ cells after thawing, compared with the 2% described in fresh CD34+ cells (P < 0.0001). Moreover, caspase pathway was significantly activated in thawing CD3+, CD56+ and CD14+ cells. We also report a significant correlation between the rate of CD34+/7AAD−/FLICA+ cells and post-thawing granulocytes count (P = 0.042) and their potential to be differentiated into CFU-GM (P = 0.004). DiscussionOur results show substantial cell death, induced by the increase of caspase pathway activation, secondary to the thawing process, and across all study cell types. This observation may affect the immune response quality during recipient aplasia, without detecting a clinical impact. Moreover, caspase pathway activation through CD3+ and CD56+ subpopulations could modify the therapeutic result of donor lymphocytes infusion (DLI).

Alterations of Hematologic and Hematopoietic Parameters in Mice Exposed to Pulsed Electromagnetic Field.

Effects of pulsed electromagnetic field (PEMF) on hematology and hematopoiesis might vary with different PEMF parameters. The purpose of this study was to evaluate the possible effects of PEMF exposure at different pulses on hematologic and hematopoietic parameters in mice. Groups of male BALB/c mice were whole body exposed or were sham exposed (control) to PEMF at 100, 1000, and 10000 pulses. After PEMF exposure, blood samples and bone marrow cells of mice were collected for hematologic examinations, bone marrow nucleated cell counting, colony-forming units of granulocyte-macrophage (CFU-GM) colony assay, and serum granulocyte-macrophage colony-stimulating factor (GM-CSF) assay. Compared with the control group, white blood cells (WBC) and lymphocytes (LYM) in the 100 and 1000 pulses exposed groups were significantly increased but not changed in the 10000 pulses exposed group. Red blood cells (RBC), hemoglobin (HGB), and platelets (PLT) were not changed in all exposed groups. There was no significant difference in mouse bone marrow nucleated cell number between the control group and each exposed group 7 days after PEMF exposure. The CFU-GM clone number of bone marrow cells and serum GM-CSF level were significantly increased in the 100 and 1000 pulses exposed group but not changed in the 10000 pulses exposed group. Our results indicated that the PEMF exposure at fewer pulses may induce statistically significant alterations in some hematologic and hematopoietic parameters of mice but no changes can be found in the more pulses PEMF-exposed groups.

Reduction of CFU-GM and circulating hematopoietic progenitors in a subgroup of children with chronic neutropenia associated with severe infections and delayed recovery.

Myelopoiesis was evaluated in 66 pediatric patients with chronic neutropenia who were positive for anti-neutrophil antibodies (median age at diagnosis: 11 months, median neutrophil count at diagnosis: 419/μl). Other causes of neutropenia were excluded. Bone marrow morphology, clonogenic tests and/or the peripheral blood CD 34+ cell count, and apoptotic rate were evaluated in 61 patients with neutropenia lasting > 12 months or severe infections. The peripheral blood CD 34+ cell count and apoptotic rate were evaluated in five patients with shorter neutropenia. The median follow-up time was 29 months (range 7–180 months). Forty-seven patients (71.2%) had a spontaneous recovery after 7–180 months (median 29 months). The group of patients younger than 24 months at diagnosis (n = 50) had a higher probability of recovery (40/50 vs. 7/16 χ2 p<0.01) with a shorter period of neutropenia (median 26 versus 47 months, Kaplan-Meier analysis p = 0.001). The colony-forming units–granulocyte-macrophage (CFU-GM) were significantly decreased in 26/35 patients (74%) evaluated for clonogenic tests. All patients with normal CFU-GM recovered (9/9 patients); whereas, neutropenia persisted in 12/26 patients with reduced CFU-GM (46%, Pearson χ2 p = 0.02). In 36/55 (65%) patients evaluated by flow cytometry we observed reduced circulating CD34+ cells compared with controls of the same age. An increase in the circulating CD34+ cell apoptotic rate was observed in 28/55 patients (51%). Infections requiring hospitalization were observed in 9/18 (50%; Pearson χ2, p = 0.03) patients with both decreased circulating CD34+ cells and increased CD34+ apoptotic rates. In the group aged < 24 months, we observed a significant correlation between the persistence of neutropenia and decreased circulating CD34+ cells (Pearson χ2 p = 0.008). In conclusion, reduced CFU-GM and circulating hematopoietic progenitors were observed in a subgroup of children with chronic neutropenia who were positive for anti-neutrophil antibodies and had a higher incidence of severe infections and delayed spontaneous remission.

Compatibility effects of ginseng and Ligustrum lucidum Ait herb pair on hematopoietic recovery in mice with cyclophosphamide-induced myelosuppression and its material basis

BackgroundGinseng (G) and Ligustrum lucidum Ait (LLA) are core traditional Chinese medicines in treating myelosuppression formula. The present study was designed to profile effect of G and LLA herb pair (G-LLA) on myelosuppressed mice. MethodsThe mice myelosuppression model was established by intraperitoneal (i.p.) injection of cyclophosphamide (Cy). Hematopoietic function of bone marrow was measured by hemopoietic progenitor cell culture and peripheral blood count, and serum hemopoietic factors were tested by enzyme-linked immunosorbent assay. Bone marrow cell cycle was performed by flow cytometry. HPLC was used to measure 20 potential chemical components related to myelosuppression, including ginsenoside Rg1, Re, Rb1, Rc, Rb2, Rb3, Rd, Rk3, Rh4, 20 (S)-Rg3, 20 (R)-Rg3, Rk1, Rg5, salidroside, and so on. ResultsG, LLA, and G-LLA improved the amount of peripheral blood cells and bone marrow cells of myelosuppressed mice (P < 0.01). They significantly increased the colony quantity of colony-forming unit–granulocyte macrophage, burst-forming unit−erythroid, colony-forming unit−erythroid, and colony-forming unit−megakaryocyte and amount of G2/M and S phase cells (P < 0.01). They also significantly decreased the amount of hematopoiesis-related cytokines (P < 0.01). The content of chemical components in G-LLA changed, and the change of rare saponin was the most obvious. ConclusionThese results show that G-LLA herb pair might produce synergistic or complementary compatibility effects on bone marrow suppression after chemotherapy. It suggests that the substance basis of G-LLA for treating bone marrow suppression may be effective chemical components.

Effect of salidroside on bone marrow haematopoiesis in a mouse model of myelosuppressed anaemia.

This study was designed to investigate the effect of salidroside (SAL) on bone marrow haematopoiesis in a mouse model of myelosuppressed anemia. After the mouse model was established by 60Co γ irradiation and cyclophosphamide, pancytopenia and a sharp reduction in bone marrow stromal cells and bone marrow haematopoietic stem cells (lineage−Sca1+c-kit+) were observed. This was greatly alleviated by SAL (25 mg/kg, 50 mg/kg, 100 mg/kg) in a dose-dependent manner (50% effective dose value of 35.7 mg/kg and 61.2 mg/kg, respectively), followed by a distinct increment in anti-apoptotic protein Bcl-2. For cell culture in vitro, treatment with SAL resulted in a significant recovery of burst-forming unit–erythroids, and colony-forming unit–granulocyte macrophages on Day 7, and colony-forming unit–erythroids on Day 3, dose-dependently, but not of colony-forming unit–megakaryocyte macrophages. Inoculation of bone marrow cells derived from SAL-administrated donor mice resulted in a 60% survival of recipient mice at the high dose of 100 mg/kg SAL at 2 months after surgery. SAL appeared to be able to stimulate the restoration of bone marrow haemopoietic regulation in myelosuppressed anemia. Based on the downregulation of Fas ligand associated with the expression of Caspase-3 at the protein level, it was suggested that SAL might have an anti-apoptotic effect on bone marrow cells in the Fas-apoptotic pathway of Fas/FasL–caspase-3.

Novel Bivalent and D-Peptide Ligands of CXCR4 Mobilize Hematopoietic Progenitor Cells to the Blood in C3H/HeJ Mice

The interaction of SDF-1α (also known as CXCL12) with the CXCR4 receptor plays a critical role in the retention of hematopoietic stem cells (HSCs) in bone marrow. The viral macrophage inflammatory protein-II (vMIP-II), a human herpesvirus-8 (HHV-8)-encoded viral chemokine, can bind the CXCR4 receptor and inhibit endogenous ligand-induced calcium responses and cell migration. Previously, we used the bivalent ligand approach to link synthetically two unnatural D-amino acid peptides derived from the N-terminus of vMIP-II (DV1 and DV3, respectively) to generate a dimeric peptide, DV1-K-(DV3) (also named HC4319), which shows very high affinity for CXCR4. Here, we studied the biological effects of this dimeric peptide, HC4319, and its monomeric counterpart, DV1, on SDF-1α-induced signaling in CXCR4- or CXCR7-transfected Chinese hamster ovary cells and mobilization of hematopoietic progenitor cells (HPCs) in C3H/HeJ mice using an HPC assay. HC4319 and DV1 inhibited significantly the phosphorylation of Akt and Erk, known to be downstream signaling events of CXCR4. This in vivo study in C3H/HeJ mice showed that HC4319 and DV-1 strongly induced rapid mobilization of granulocyte–macrophage colony-forming units (CFUs), erythrocyte burst-forming units, and granulocyte–erythrocyte–monocyte–megakaryocyte CFUs from the bone marrow to the blood. These results provide the first reported experimental evidence that bivalent and D-amino acid peptides derived from the N-terminus of vMIP-II are potent mobilizers of HPCs in C3H/HeJ mice and support the further development of such agents for clinical application.

The aldehyde dehydrogenase cord blood potency assay excludes early apoptotic cells.

Cord blood units (CBUs) are processed, frozen, and thawed before use in hematopoietic stem cell (HSC) transplantation. The manipulations affect HSC functionality, that is, induce apoptosis and reduce viability. HSC content, commonly expressed as CBU potency, that is, the expected ability of a CBU to restore hematopoiesis, is traditionally approximated through viable CD34+ cells and the colony-forming unit (CFU) cell cultivation assay. Alternative approaches, for example, the aldehyde dehydrogenase (ALDH) enzyme-based assay, are also forthcoming. We hypothesized that the ALDH assay might exclude apoptotic cells since it is based on enzyme activity. To investigate this, we designed a protocol for simultaneous staining of viable and apoptotic CD34+ and ALDH+ cells using 7-aminoactinomycin (7-AAD) and annexin V, in frozen-thawed CBUs. Results were correlated with results from the colony-forming unit-granulocyte/macrophage (CFU-GM) assay. Samples from 57 CBUs were thawed and simultaneously analyzed for CD34+ cells, ALDH+ cells, viability (7-AAD), and apoptosis (annexin V) using flow cytometry. Enumeration of CFUs was also performed. No nonviable and few apoptotic cells (mean 0.7%) were identified in the ALDH+ population compared to the viable CD34+ population (mean 3.6%). The total number of ALDH+ cells correlated better than viable CD34+ cells (r = 0. 72 vs. r = 0.66; p < 0.0001) with the results of the CFU assay. The ALDH assay excludes nonviable and apoptotic cells, and therefore correlates better with CFU enumeration compared to the number of viable CD34+ cells. We propose that the ALDH assay might replace the CFU-GM method in CBU potency measurements.

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.

Correlation of CD34+ Hematopoietic Stem Cells and CFU in Peripheral Blood Apheresis Products in Patients with Malignant Lymphoproliferative Diseases Before and After Cryopreservation Prior to auto-HSCT

Aim. To establish correlation between CD34+ autologous hematopoietic stem cell (HSC) count and colony-forming units (CFU) in the same peripheral blood apheresis product samples before and after cryopreservation in multiple myeloma and lymphoma patients, and to assess clinical value of these parameters. Materials &amp; Methods. Cell samples of peripheral blood cyta-pheresis product and cell cultures were studied before and after cryopreservation in 32 multiple myeloma and 25 lymphoma patients who underwent autologous HSC transplantation. The material was analyzed using culture technique and flow cytometry. Results. The paper provides information on the relationship between CD34+ HSC count obtained by flow cytometry, and CFU in cell culture obtained by cytapheresis of the same peripheral blood samples. A direct correlation was confirmed between CD34+ count and all the CFUs before and after cryopreservation in lymphoma patients. Correlation between CD34+ count and granulocyte-macrophage CFUs was revealed in multiple myeloma and lymphoma patients before cryopreservation. Conclusion. The parameter of colony-forming capacity used for the assessment of the functional HSC was shown to be equally reliable criterion for condition evaluation of autotransplant proliferative pool than CD34+ cells. Both methods should be applied for qualitative and quantitative evaluation of an autotransplant for multiple myeloma and lymphoma patients.

Flow Cytometric Aldehyde Dehydrogenase Assay Enables a Fast and Accurate Human Umbilical Cord Blood Hematopoietic Stem Cell Assessment.

Objective:Colony-forming units of granulocytes/macrophages (CFU-GM) analysis is the most widely used method to determine the hematopoietic stem cell (HSC) content of human umbilical cord blood (CB) for prediction of engraftment potential. The measurement of aldehyde dehydrogenase (ALDH) activity is a more recent method for HSC qualification. Our aim was to correlate phenotypic and functional assays to find the most predictive method.Materials and Methods:In this study, flow cytometric quantitation of CD34+ cells and ALDH positivity along with CFU-GM capacity were assessed in fresh and post-thaw CB units.Results:Among 30 post-processing samples, for each CB unit the mean total number of nucleated cells (TNCs) was (93.8±30.1)x107, CD34+ cells were (3.85±2.55)x106, ALDH+ cells were (3.14±2.55)x106, and CFU-GM count was (2.64±1.96)x105. Among an additional 19 post-thaw samples the cell counts were as follows: TNCs, (32.79±17.27)x107; CD34+, (2.18±3.17)x106; ALDH+, (2.01±2.81)x106; CFU-GM, (0.74±0.92)x105. Our findings showed that in fresh samples TNCs, CD34+ cells, and ALDH correlated highly with counts of CFU-GM, CFU-erythroids/granulocytes-macrophages/megakaryocytic cells (GEMM), and burst forming units of erythroids (BFU-E) as follows: TNCs, r=0.47, r=0.35, r=0.41; CD34+, r=0.44, r=0.54, r=0.41; and ALDH, r=0.63, r=0.45, r=0.6, respectively. In terms of post-thaw samples, the correlations were as follows: TNCs, r=0.59, r=0.46, r=0.56; CD34+, r=0.67, r=0.48, r=0.61; and ALDH, r=0.61, r=0.67, r=0.67, for CFU-GM, CFU-GEMM, and BFU-E, respectively. All correlations were statistically significant.Conclusion:In our experience, HSC assessment by ALDH activity yields the highest correlation with conventional analytical methods, particularly for post-thaw samples. Thus, this fast, inexpensive method has the potential to overcome the weaknesses of other techniques.

Myocardial infarction does not affect circulating haematopoietic stem and progenitor cell self-renewal ability in a rat model.

What is the central question of this study? Although peripheral blood haematopoietic stem and progenitor cells are potentially important in regeneration after acute myocardial infarction, their self-renewal ability in the post-acute phase has not yet been addressed. What is the main finding and its importance? In rat peripheral blood, we show that myocardial infarction does not negatively affect circulating haematopoietic stem and progenitor cell self-renewal ability 2 weeks after acute infarction, which suggests a constant regenerative potential in the myocardial infarction post-acute phase. Given the importance of peripheral blood haematopoietic stem and progenitor cells (HPCs) in post-acute regeneration after acute myocardial infarction (MI), the aim of the present study was to investigate the number and secondary replating capacity/self-renewal ability of HPCs in peripheral blood before and 2weeks after MI. In female Lewis inbred rats (n=9), MI was induced by ligation of the left coronary artery, and another nine underwent sham surgery, without ligation, for control purposes. Myocardial infarction was confirmed by troponinI concentrations 24h after surgery. Peripheral blood was withdrawn and fractional shortening and ejection fraction of the left ventricle were assessed before (day0) and 14days after MI or sham surgery (day14). After mononuclear cell isolation, primary and secondary functional colony-forming unit granulocyte-macrophage (CFU-GM) assays were performed in order to detect the kinetics of functional HPC colony counts and cell self-renewal ability in vitro. The CFU-GM counts and cell self-renewal ability remained unchanged (P>0.05) in both groups at day14, without interaction between groups. In the intervention group, higher day0 CFU-GM counts showed a relationship to lower fractional shortening on day14 (ρ=-0.82; P<0.01). Myocardial infarction did not negatively affect circulating HPC self-renewal ability, which suggests a constant regenerative potential in the post-acute phase. A relationship of cardiac contractile function 14days after MI with circulating CFU-GM counts on day0 might imply functional colony count as a predictive factor for outcome after infarction.

Mesenchymal Stromal Cell Irradiation Interferes with the Adipogenic/Osteogenic Differentiation Balance and Improves Their Hematopoietic-Supporting Ability

Bone marrow mesenchymal stromal cells (MSCs) are precursors of adipocytes and osteoblasts and key regulators of hematopoiesis. Irradiation is widely used in conditioning regimens. Although MSCs are radio-resistant, the effects of low-dose irradiation on their behavior have not been extensively explored. Our aim was to evaluate the effect of 2.5 Gy on MSCs. Cells from 25 healthy donors were either irradiated or not (the latter were used as controls). Cells were characterized following International Society for Cellular Therapy criteria, including in vitro differentiation assays. Apoptosis was evaluated by annexin V/7-amino-actinomycin staining. Gene expression profiling and reverse transcriptase (RT)-PCR of relevant genes was also performed. Finally, long-term bone marrow cultures were performed to test the hematopoietic-supporting ability. Our results showed that immunophenotypic characterization and viability of irradiated cells was comparable with that of control cells. Gene expression profiling showed 50 genes differentially expressed. By RT-PCR, SDF-1 and ANGPT were overexpressed, whereas COL1A1 was downregulated in irradiated cells (P = .015, P = .007, and P = .031, respectively). Interestingly, differentiation of irradiated cells was skewed toward osteogenesis, whereas adipogenesis was impaired. Higher expression of genes involved in osteogenesis as SPP1 (P = .039) and lower of genes involved in adipogenesis, CEBPA and PPARG (P = .003 and P = .019), together with an increase in the mineralization capacity (Alizarin Red) was observed in irradiated cells. After differentiation, adipocyte counts were decreased in irradiated cells at days 7, 14, and 21 (P = .018 P = .046, and P = .018, respectively). Also, colony-forming unit granulocyte macrophage number in long-term bone marrow cultures was significantly higher in irradiated cells after 4 and 5 weeks (P = .046 and P = .007). In summary, the irradiation of MSCs with 2.5 Gy improves their hematopoietic-supporting ability by increasing osteogenic differentiation and decreasing adipogenesis.

Single-stage treatment of infected tibial non-unions and osteomyelitis with bone marrow granulocytes precursors protecting bone graft.

Infected non-unions present a clinical challenge, especially with risk of recurrent infection. Bone marrow contains granulocyte precursors identified in vitro as colony forming units-granulocyte macrophage (CFU-GM) have a prophylactic action against infection. We therefore tested the hypothesis that bone marrow concentrated granulocytes precursors added to a standard bone graft could decrease the risk of recurrence of infection when single-stage treatment of infected tibial non-unions is performed with bone graft. During a single-stage procedure 40 patients with infected tibial non-union received a spongious bone graft supercharged with granulocytes precursors after debridement (study group). A control group (40 patients) was treated in a single stage with local debridement and standard bone graft obtained from the iliac crest. The antibiotic therapy protocol was the same (60days) in the two groups. CFU-GM progenitors were harvested from bone marrow aspirated on the opposite iliac crest of the site where the cancellous bone was obtained. Union (radiographs and CT scan), a recurrence of clinical infection, and need for subsequent surgery were evaluated. Thirty-eight (95%) patients who received graft supercharged with granulocytes precursors achieved successful union without recurrence of infection during the seven-year follow-up versus 28 (70%) control patients; for the control group the mean graft resorption volume was 40%, while no bone graft resorption was found for the study group. Supercharging the cancellous bone graft with bone marrow granulocytes precursors protect the site of infected non-union from recurrence of infection and bone resorption of the graft.

ExVivo Selection of Transduced Hematopoietic Stem Cells for Gene Therapy of β-Hemoglobinopathies.

Although gene transfer to hematopoietic stem cells (HSCs) has shown therapeutic efficacy in recent trials for several individuals with inherited disorders, transduction incompleteness of the HSC population remains a hurdle to yield a cure for all patients with reasonably low integrated vector numbers. In previous attempts at HSC selection, massive loss of transduced HSCs, contamination with non-transduced cells, or lack of applicability to large cell populations has rendered the procedures out of reach for human applications. Here, we fused codon-optimized puromycin N-acetyltransferase to herpes simplex virus thymidine kinase. When expressed from a ubiquitous promoter within a complex lentiviral vector comprising the βAT87Q-globin gene, viral titers and therapeutic gene expression were maintained at effective levels. Complete selection and preservation of transduced HSCs were achieved after brief exposure to puromycin in the presence of MDR1 blocking agents, suggesting the procedure’s suitability for human clinical applications while affording the additional safety of conditional suicide.