Synergistic effect and molecular mechanism of nicotinamide and UM171 in ex vivo expansion of long-term hematopoietic stem cells.

Introduction: Several approaches to expand human hematopoietic stem cells (HSCs) have been reported, but the ability of these methods to expand long-term hematopoietic stem cells (LT-HSCs) remains to be improved, which limits the application of HSCs-based therapies. Methods: CD34+ cells were purified from umbilical cord blood using MacsCD34 beads, and then cultured for 12 d in a serum-free medium. Flow cytometry was used to detect phenotype, cell cycle distribution, and apoptosis of the cultured cells. Colony-forming cell (CFC) assays can evaluate multi-lineage differentiation potential of HSCs. Real-time polymerase chain reaction was employed to detect the expression of genes related to self-renewal programs and antioxidant activity. DCFH-DA probes were used to evaluate intracellular production of reactive oxygen species (ROS). Determination of the effect of different culture conditions on the balance of cytokine by cytometric bead array. Results: Here, we show a combination, Nicotinamide (NAM) combined with pyrimidoindole derivative UM171, can massively expand LT-HSCs ex vivo, and the expanded cells maintained the capability of self-renewal and multilineage differentiation. Additionally, our data indicated that UM171 promoted self-renewal of HSCs by inducing HSCs entry into the cell cycle and activating Notch and Wnt pathways, but the infinite occurrence of this process may lead to mitochondrial metabolism disorder and differentiation of HSCs. NAM kept HSCs in their primitive and dormant states by reducing intracellular ROS levels and upregulating the expression of stemness related genes, so we believed that NAM can act as a brake to control the above process. Conclusions: The discovery of the synergistic effect of NAM and UM171 for expanding LT-HSCs provides a new strategy in solving the clinical issue of limited numbers of HSCs.

Effects of different concentrations of nicotinamide on hematopoietic stem cells cultured in vitro.

BACKGROUND: In vitro expansion to increase numbers of hematopoietic stem cells (HSCs) in cord blood could improve clinical efficacy of this vital resource. Nicotinamide (NAM) can promote HSC expansion ex vivo, but its effect on hematopoietic stem and progenitor cells (HSPCs, CD34+CD38) and functional subtypes of HSCs - short-term repopulating HSCs (ST-HSCs, CD34+CD38CD45RACD49f+) and long-term repopulating HSCs (LT-HSCs, CD34+CD38CD45RACD49f+CD90+) is not yet known. As a sirtuin 1 (SIRT1) inhibitor, NAM participates in regulating cell adhesion, polarity, migration, proliferation, and differentiation. However, SIRT1 exhibits dual effects by promoting or inhibiting differentiation in different tissues or cells. We propose that the concentration of NAM may influence proliferation, differentiation, and SIRT1 signaling of HSCs. AIM: To evaluate the effects and underlying mechanisms of action of different concentrations of NAM on HSC proliferation and differentiation. METHODS: CD34+ cells were purified from umbilical cord blood using MacsCD34 beads, and cultured for 10-12 d in a serum-free medium supplemented with cytokines, with different concentrations of NAM added according to experimental requirements. Flow cytometry was used to detect phenotype, cell cycle distribution, and apoptosis of the cultured cells. Real-time polymerase chain reaction was used to detect the transcription levels of target genes encoding stemness-related factors, chemokines, components of hypoxia pathways, and antioxidant enzymes. Dichloro-dihydro-fluorescein diacetate probes were used to evaluate intracellular production of reactive oxygen species (ROS). Determination of the effect of different culture conditions on the balance of cytokine by cytometric bead array. RESULTS: Compared with the control group, the proportion and expansion folds of HSPCs (CD34+CD38) incubated with 5 mmol/L or 10 mmol/L NAM were significantly increased (all P < 0.05). The ST-HSCs ratio and fold expansion of the 5 mmol/L NAM group were significantly higher than those of the control and 10 mmol/L NAM groups (all P < 0.001), whereas the LT-HSCs ratio and fold expansion of the 10 mmol/L NAM group were significantly higher than those of the other two groups (all P < 0.05). When the NAM concentration was > 10 mmol/L, cell viability significantly decreased. In addition, compared with the 5 mmol/L NAM group, the proportion of apoptotic cells in the 10 mmol/L NAM group increased and the proportion of cells in S and G2 phase decreased. Compared with the 5 mmol/L NAM group, the HSCs incubated with 10 mmol/L NAM exhibited significantly inhibited SIRT1 expression, increased intracellular ROS content, and downregulated expression of genes encoding antioxidant enzymes (superoxide dismutase 1, peroxiredoxin 1). CONCLUSION: Low concentrations (5 mmol/L) of NAM can better regulate the balance between proliferation and differentiation, thereby promoting expansion of HSCs. These findings allow adjustment of NAM concentrations according to expansion needs.

Synergistic effect and molecular mechanism of PVA and UM171 in ex vivo expansion of primitive hematopoietic stem cells.

Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells (HSCs) used for transplantation; the number of cells in a single UCB is too small to quickly establish bone marrow (BM) implantation, and ex vivo expansion of HSCs has the potential to overcome this limitation. The purpose of this study is to explore the culture conditions conducive to the maintenance and expansion of hematopoietic stem and progenitor cells (HSPCs) and long-term hematopoietic stem cells (LT-HSCs) derived from human umbilical cord blood, compare the different effects of albumin (HSA) and polyvinyl alcohol (PVA), optimize the culture system using UM171 and investigate the molecular mechanism of PVA and UM171 promoting the expansion of primitive hematopoietic stem cells. CD34+ cells were purified from UCB using MacsCD34 beads, and then cultured in serum-free medium supplemented with cytokines for 12 days, with PVA or UM171 added according to experimental requirements; the relative percentage of different HSCs subsets after culture were detected by flow cytometry; CFU Assay Setup for detecting the multilineage differentiation potential of HSCs; RT-PCR detection of gene expression levels; reactive oxygen detection assessment of intracellular ROS levels. (1) The conditions of 20 ng/mlSCF, 100 ng/mlTPO, and 5% oxygen concentration are conducive to the maintenance of LT-HSCs. (2) Compared with HSA, PVA significantly increased the proportion of HSPCs and LT-HSCs, as well as dramatically promoted the expression of antioxidant enzymes and reduced the production of reactive oxygen species (ROS). (3) After adding UM171 to PVA-based medium, the proportion of HSPCs and LT-HSCs further increased, and downstream genes of Notch and Wnt pathways were selectively activated. (1) PVA may inhibit ROS production by upregulating the expression of antioxidant enzymes, which is beneficial for maintaining stemness and inhibiting differentiation of HSCs. (2) The antioxidant properties of PVA can delay differentiation, while UM171 can promote self-renewal by regulating the stem cell pathway, and the combination of them is beneficial for the maintenance and expansion of HSCs in vitro.

Expansion strategies for umbilical cord blood haematopoietic stem cells in vitro.

Haematopoietic stem cell transplantation (HSCT) is considered an effective treatment for some haematopoietic malignancies, haematopoietic failure and immunodeficiency. Compared with bone marrow and mobilized peripheral blood, cord blood has the advantages of easy access, being harmless to donors and low requirement for HLA matching. In addition, umbilical cord blood transplantation (UCBT) has achieved remarkable clinical success in the past 30 years due to the low recurrence rate of malignancies treated by UCBT, mild degree of chronic graft-versus-host disease (GVHD) and good quality of life for patients after transplantation. However, the number of cells in a single cord blood is too small for rapid bone marrow implantation. We summarize the various factors involved that need to be considered in the expansion of haematopoietic stem cells (HSCs) in vitro, which all avoid complex operations, such as vector construction and virus transfection. We also found it necessary to identify a new molecule as the carrier of HSCs cultured in vitro, which not only would provide a three-dimensional structure conducive to the self-renewal of HSCs but also prevent their differentiation.

Nitrogen conversion in relation to NH3 and HCN during microwave pyrolysis of sewage sludge.

The nitrogen conversions in relation to NH3 and HCN were investigated during microwave pyrolysis of sewage sludge. The nitrogen distributions and evolution of nitrogen functionalities in the char, tar, and gas fractions were conducted. The results suggested that the thermal cracking of protein in sludge produced three important intermediate compounds, including the amine-N, heterocyclic-N, and nitrile-N compounds. The deamination of amine-N compounds resulted from labile proteins cracking led to the formation of NH3 (about 7.5% of SS-N) between 300 and 500 °C. The cracking of nitrile-N and heterocyclic-N compounds in the tars from the dehydrogenation and polymerization of amine-N generated HCN (6.6%) from 500 to 800 °C, respectively. Moreover, the ring-opening of heterocyclic-N in the char and tar contributed to the release of NH3 accounting for about 18.3% of SS-N with the temperature increasing from 500 to 800 °C. Specifically, the thermal cracking of amine-N, heterocyclic-N and nitrile-N compounds contributed to above 80% of the total (HCN+NH3) productions. Consequently, it might be able to reduce the HCN and NH3 emissions through controlling the three intermediates production at the temperature of 500-800 °C.

Key intermediates in nitrogen transformation during microwave pyrolysis of sewage sludge: a protein model compound study.

The nitrogen transformations with attention to NH3 and HCN were investigated at temperatures of 300-800°C during microwave pyrolysis of a protein model compound. The evolution of nitrogenated compounds in the char, tar and gas products were conducted. The amine-N, heterocyclic-N and nitrile-N compounds were identified as three important intermediates during the pyrolysis. NH3 and HCN were formed with comparable activation energies competed to consume the same reactive substances at temperatures of 300-800°C. The deamination and dehydrogenation of amine-N compounds from protein cracking contributed to the formation of NH3 (about 8.9% of Soy-N) and HCN (6.6%) from 300 to 500°C. The cracking of nitrile-N and heterocyclic-N compounds from the dehydrogenation and polymerization of amine-N generated HCN (13.4%) and NH3 (31.3%) between 500 and 800°C. It might be able to reduce the HCN and NH3 emissions through controlling the intermediates production at temperatures of 500-800°C.

Distribution variation of a metabolic uncoupler, 2,6-dichlorophenol (2,6-DCP) in long-term sludge culture and their effects on sludge reduction and biological inhibition.

Distribution variation of a metabolic uncoupler, 2,6-dichlorophenol (2,6-DCP), in long-term sludge culture was studied, and the effects on sludge reduction and biological inhibition of this chemical during the 90-day operation were established. The extracellular polymeric substance (EPS) matrix functioned as a protective barrier for the bacteria inside sludge flocs to 2,6-DCP, resulting in the transfer of 2,6-DCP from the liquid phase to the activated sludge fraction. Significant sludge reduction (about 40%) was observed after the addition of 2,6-DCP in the first 40 days, while the ineffective function of 2,6-DCP in sludge reduction (days 70-90) might be correlated to the EPS protection mechanism. The inhibitory effect of 2,6-DCP on the COD removal was extremely lower than on the nitrification performance due to the fact that 2,6-DCP was much more toxic to autotrophic microorganisms than heterotrophic microorganisms. Moreover, both of them recovered to a higher level again with the transfer potential of 2,6-DCP to sludge. Thus, the application of metabolic uncoupler for excess sludge reduction should be cautious.

[Determination of perfluorooctanoic acid and its salts in the coating layer of nonstick pan by gas chromatography/mass spectrometry coupled with accelerated solvent extractor].

A sensitive, accurate and reproducible analytical method was developed and validated to quantify perfluorooctanoic acid (PFOA) and its salts in the coating layer of nonstick pan. Scraped samples of 0. 5 gram were directly extracted with acetonitrile using an accelerated solvent extractor at 175 degrees C, 10. 3 MPa for 20 min. After rotary evaporation concentration, the PFOA was derivatized with acetyl chloride and methanol at 65 degrees C for 60 min in an air-tight container. An HP-1 MS capillary column (30 m x 0. 25 mm, 0. 33 degrees m) was used for the separation of the analyte. The procedure for the analysis of PFOA was based on gas chromatography interfaced to negative chemical ion mass spectrometry, operated in selected ion monitoring mode. Selected ions of m/z 312, 350, 378, 408, 428 were chosen for quantitation. The assay was linear over the range of 0 - 1 000 microg/L, and the detection limit of PFOA was 5 microg/kg. The precision and recovery were assessed on four different concentrations (5, 20, 100, 500 micro/kg). The recoveries were in the range of 90. 9% - 96. 2% and the relative standard deviations were 1. 37% -6. 37%. The procedure was applied to monitoring PFOA in the coating layer of nonstick pan from supermarket and no positive result was found.