Genetic and metabolic studies towards personalized conditioning regimen prior to stem cell transplantation

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for several malignant and non- malignant diseases. The busulphan (Bu)/cyclophosphamide (Cy) combination is one of the most common conditioning regimens given prior to HSCT. The general aim of the present thesis is to investigate the molecular mechanisms underlying the metabolism of the Bu/Cy conditioning regimen in order to personalize the treatment and improve the clinical outcome. To follow the metabolic pathway of busulphan, a new gas chromatography-mass spectrometry (GC-MS) method was developed and validated for the quantification and detection of busulphan and its four major metabolites. Incubation of the first core metabolite of busulphan, tetrahydrothiophene (THT), with human liver microsomes or recombinant enzymes has resulted in the formation of subsequent metabolites. The highest initial THT disappearance rate and the highest CLint value were observed with FMO3 followed by several CYPs indicating that FMO3 and, to a lesser extent, CYPs are involved in the metabolic pathway of busulphan. Moreover, FMO3 inhibition significantly (P < 0.05) affected Bu and THT kinetics in mice. In patients, FMO3 expression was significantly (P < 0.05) up-regulated during Bu treatment. In order to personalize oral Bu dosage, a reliable limited sampling model was developed and evaluated in both adult and pediatric patients. To understand the role of cyclophosphamide in the conditioning regimen, the gene expression profile over two days of Cy treatment was investigated, where 299 genes were found to be specifically affected by the treatment. Cyclophosphamide down-regulated the expression of several genes mapped to immune/autoimmune activation and graft rejection including CD3, CD28, CTLA4 and IL2R, and up-regulated immune-related receptor genes, e.g. IL1R2, IL18R1, and FLT3. Significant (P < 0.01) up-regulation, with high inter-individual variation, of the cytochrome P450 oxidoreductase (POR) gene was also observed during Cy treatment. In vitro, different batches of CYP2B6.1, with different ratios of POR/CYP, showed positive correlation between the intrinsic clearance (Vmax/Km) and the POR/CYP ratio for the Cy 4-hydroxylation. Further analysis of the above mentioned patients, prior to Cy treatment, revealed that CYP2J2 mRNA expression was significantly (P < 0.01) higher compared to healthy controls. CYP2J2 expression was further up-regulated during Cy treatment, with high inter-individual variation. Repeated treatment with Cy resulted in an increased 4-OH-Cy/Cy ratio, indicating auto-induction of Cy-metabolism. The viability of HL-60 cells, lacking CYP2B6 but expressing CYP2J2, was reduced after incubation with Cy. Inhibition of CYP2J2 reduced 4-OH-Cy formation and improved HL-60 cell survival. Cy incubation with recombinant CYP2J2 confirmed that CYP2J2 is involved in Cy bioactivation. In summary, the present results have improved our understanding of the Bu/Cy metabolism. This knowledge may help to interpret several interactions, high inter-individual variability, adverse effects and unexpected toxicity observed during and/or after the conditioning regimen. This certainly will help in developing new strategies for personalized medicine and thus improve clinical outcome

Obligations of Contracts: Intent and Distortion

<p><b>Introduction:</b> Treosulfan is an alkylating agent that is used for the treatment of ovarian cancer and for conditioning prior to stem cell transplantation. It is a prodrug that is activated non-enzymatically to two active epoxides.</p> <p><b>Objectives:</b> To optimize a protocol for both <i>in vivo</i> samples handling and <i>in vitro</i> drug preparation. Treosulfan stability was tested in biological fluids at different conditions as well as for its cytotoxicity on cell lines.</p> <p><b>Results:</b> Plasma samples can be safely frozen for a short period up to 8 h, however; for longer periods, samples should be acidified. Urine samples and cell culture media can be safely frozen regardless their pH. For <i>in vitro</i> investigations, incubation of treosulfan at 37 °C for 24 h activated 100% of the drug. Whole blood acidification should be avoided for the risk of hemolysis. Finally; treosulfan cytotoxicity on HL-60 cells has increased following pre-incubation for 24 h at 37 °C compared to K562 cell line.</p> <p><b>Conclusion:</b> The stability profiling of treosulfan provided a valuable reference for handling of biological samples for both <i>in vivo</i> and <i>in vitro</i> studies. These results can be utilized for further investigations concerning the drug kinetics and dynamics in addition to the development of new pharmaceutical formulations.</p

A Systematic Review of Keratinocyte Secretions : A Regenerative Perspective

Cell regenerative therapy is a modern solution for difficult-to-heal wounds. Keratinocytes, the most common cell type in the skin, are difficult to obtain without the creation of another wound. Stem cell differentiation towards keratinocytes is a challenging process, and it is difficult to reproduce in chemically defined media. Nevertheless, a co-culture of keratinocytes with stem cells usually achieves efficient differentiation. This systematic review aims to identify the secretions of normal human keratinocytes reported in the literature and correlate them with the differentiation process. An online search revealed 338 references, of which 100 met the selection criteria. A total of 80 different keratinocyte secretions were reported, which can be grouped mainly into cytokines, growth factors, and antimicrobial peptides. The growth-factor group mostly affects stem cell differentiation into keratinocytes, especially epidermal growth factor and members of the transforming growth factor family. Nevertheless, the reported secretions reflected the nature of the involved studies, as most of them focused on keratinocyte interaction with inflammation. This review highlights the secretory function of keratinocytes, as well as the need for intense investigation to characterize these secretions and evaluate their regenerative capacities.Funding Agencies|Centre for Advanced Medical Products, Sweden; Hand and Plastic Surgery Department, Linkoping University Hospital, Region Ostergotland, Sweden</p

Biomaterials as a Vital Frontier for Stem Cell-Based Tissue Regeneration

Biomaterials and tissue regeneration represent two fields of intense research and rapid advancement. Their combination allowed the utilization of the different characteristics of biomaterials to enhance the expansion of stem cells or their differentiation into various lineages. Furthermore, the use of biomaterials in tissue regeneration would help in the creation of larger tissue constructs that can allow for significant clinical application. Several studies investigated the role of one or more biomaterial on stem cell characteristics or their differentiation potential into a certain target. In order to achieve real advancement in the field of stem cell-based tissue regeneration, a careful analysis of the currently published information is critically needed. This review describes the fundamental description of biomaterials as well as their classification according to their source, bioactivity and different biological effects. The effect of different biomaterials on stem cell expansion and differentiation into the primarily studied lineages was further discussed. In conclusion, biomaterials should be considered as an essential component of stem cell differentiation strategies. An intense investigation is still required. Establishing a consortium of stem cell biologists and biomaterial developers would help in a systematic development of this field

Non-additive effect of the DNA methylation inhibitor, 5-Aza-dC, and glass as a culture surface on osteogenic differentiation

The clinical need for bone regenerative solutions is expanding with increasing life expectancy and escalating incidence of accidents. Several strategies are being investigated to enhance the osteogenic differentiation of stem cells. We previously reported two different approaches for this purpose, in monolayer and three-dimensional cell culture. The first approach was based on pretreating cells with 5-Aza-dC, a DNA methylation inhibitor, before the applying the differentiation media. The second approach was based on culturing cells on a glass surface during differentiation. In this study, we investigated the potential effect of combining both methods. Our results sug-gested that both approaches were associated with decreasing global DNA methylation levels. Cells cultured as a monolayer on glass surface showed enhancement in alkaline phosphatase activity at day 10, while 5-Aza-dC pretreatment enhanced the activity at day 5, irrespective of the culture surface. In three-dimensional pellet cul-ture, 5-Aza-dC pretreatment enhanced osteogenesis through Runx-2 and TGF-beta 1 upregulation while the glass surface induced Osterix.Furthermore, pellets cultured on glass showed upregulation of a group of miRNAs, including pro-osteogenesis miR-20a and miR-148b and anti-osteogenesis miR-125b, miR-31, miR-138, and miR-133a. Interestingly, 5-Aza-dC was not associated with a change of miRNAs in cells cultured on tissue culture plastic but reverted the upregulated miRNAs on the glass to the basal level. This study confirms the two approaches for enhancing osteogenic differentiation and contradicts their combination.Funding Agencies|University of Sharjah; [180-1090-127P]</p

Investigating the role of the histone deacetylases-inhibitor suberanilohydroxamic acid in the differentiation of stem cells into insulin secreting cells

Introduction: The United Arab Emirates has the second incidence of diabetes in the world. The current diabetes management plans are associated with many complications and do not provide a cure. Stem cells offer hope for permanent alleviation of this health problem through the possible differentiation into insulin-secreting cells. The current methods for the differentiation do not produce homogeneous beta-cell populations. Histone deacetylation is an epigenetic silencing mechanism that can render many genes irresponsive to the induction protocols. This study aimed at investigating the effect of the histone deacetylase inhibitor suberanilohydroxamic acid (SAHA) on the production of functional beta cells, based on a mesenchymal stem cells model. Methods: MG63 cells were treated for three consecutive days with SAHA, followed by a three-steps of beta cells differentiation protocol, with media-contained retinoic acid, epidermal growth factor, nicotinamide and exendin-4 at different stages. Then, glucose-stimulated insulin secretion was conducted to assess the functional state of the differentiated cells. Results: Pretreating the cells with SAHA enhanced the insulin production and secretion in comparison to the control (PBS) and the vehicle dimethyl sulfoxide, as shown by the immunofluorescence detection of insulin and the transcription factor “PDX1”, as well as an increase in insulin secretion in the media. Gene expression analysis showed that SAHA pretreated cells had more induction of the studied markers when challenged with high glucose level. Conclusion: Such findings indicate a novel approach to enhance the ability of stem cells to differentiate into insulin-producing cells with potential therapeutic implications

The effect of N-acetyl-l-cysteine (NAC) on liver toxicity and clinical outcome after hematopoietic stem cell transplantation

Busulphan (Bu) is a myeloablative drug used for conditioning prior to hematopoietic stem cell transplantation. Bu is predominantly metabolized through glutathione conjugation, a reaction that consumes the hepatic glutathione. N-acetyl-l-cysteine (NAC) is a glutathione precursor used in the treatment of acetaminophen hepatotoxicity. NAC does not interfere with the busulphan myeloablative effect. We investigated the effect of NAC concomitant treatment during busulphan conditioning on the liver enzymes as well as the clinical outcome. Prophylactic NAC treatment was given to 54 patients upon the start of busulphan conditioning. These patients were compared with 54 historical matched controls who did not receive NAC treatment. In patients treated with NAC, aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) were significantly (P amp;lt; 0.05) decreased after conditioning compared to their start values. Within the NAC-group, liver enzymes were normalized in those patients (30%) who had significantly high start values. No significant decrease in enzyme levels was observed in the control group. Furthermore, NAC affected neither Bu kinetics nor clinical outcome (sinusoidal obstruction syndrome incidence, graft-versus-host disease and/or graft failure). In conclusion: NAC is a potential prophylactic treatment for hepatotoxicity during busulphan conditioning. NAC therapy did not alter busulphan kinetics or affect clinical outcome.Funding Agencies|Swedish Cancer Society [CAN2011/595, CAN2014/759]; Swedish Childhood Cancer Foundation [PR2017-0083]; Radiumhemmets [161082]</p

Cytochrome P450 Oxidoreductase Influences CYP2B6 Activity in Cyclophosphamide Bioactivation.

Cyclophosphamide is commonly used as an important component in conditioning prior to hematopoietic stem cell transplantation, a curative treatment for several hematological diseases. Cyclophosphamide is a prodrug activated mainly by cytochrome P450 2B6 (CYP2B6) in the liver. A high degree of inter- and intra-individual variation in cyclophosphamide kinetics has been reported in several studies.Hydroxylation of cyclophosphamide was investigated in vitro using three microsomal batches of CYP2B6*1 with different ratios of POR/CYP expression levels. Twenty patients undergoing hematopoietic stem cell transplantation were also included in the study. All patients received an i.v. infusion of cyclophosphamide (60 mg/kg/day, for two days) as a part of their conditioning. Blood samples were collected from each patient before cyclophosphamide infusion, 6 h after the first dose and before and 6 h after the second dose. POR gene expression was measured by mRNA analysis and the pharmacokinetics of cyclophosphamide and its active metabolite were determined.A strong correlation between the in vitro intrinsic clearance of cyclophosphamide and the POR/CYP ratio was found. The apparent Km for CYP2B6.1 was almost constant (3-4 mM), while the CLint values were proportional to the POR/CYP ratio (3-34 μL/min/nmol CYP). In patients, the average expression of the POR gene in blood was significantly (P <0.001) up-regulated after cyclophosphamide infusion, with high inter-individual variations and significant correlation with the concentration ratio of the active metabolite 4-hydroxy-cyclophosphamide/cyclophosphamide. Nine patients were carriers for POR*28; four patients had relatively high POR expression.This investigation shows for the first time that POR besides CYP2B6 can influence cyclophosphamide metabolism. Our results indicate that not only CYPs are important, but also POR expression and/or activity may influence cyclophosphamide bioactivation, affecting therapeutic efficacy and treatment related toxicity and hence on clinical outcome. Thus, both POR and CYP genotype and expression levels may have to be taken into account when personalizing treatment schedules to achieve optimal therapeutic drug plasma concentrations of cyclophosphamide