In vitro characterization of 3D culture-based differentiation of human liver stem cells.

Introduction: The lack of functional hepatocytes poses a significant challenge for drug safety testing and therapeutic applications due to the inability of mature hepatocytes to expand and their tendency to lose functionality in vitro. Previous studies have demonstrated the potential of Human Liver Stem Cells (HLSCs) to differentiate into hepatocyte-like cells within an in vitro rotary cell culture system, guided by a combination of growth factors and molecules known to regulate hepatocyte maturation. In this study, we employed a matrix multi-assay approach to comprehensively characterize HLSC differentiation. Methods: We evaluated the expression of hepatic markers using qRT-PCR, immunofluorescence, and Western blot analysis. Additionally, we measured urea and FVIII secretion into the supernatant and developed an updated indocyanine green in vitro assay to assess hepatocyte functionality. Results: Molecular analyses of differentiated HLSC aggregates revealed significant upregulation of hepatic genes, including CYP450, urea cycle enzymes, and uptake transporters exclusively expressed on the sinusoidal side of mature hepatocytes, evident as early as 1 day post-differentiation. Interestingly, HLSCs transiently upregulated stem cell markers during differentiation, followed by downregulation after 7 days. Furthermore, differentiated aggregates demonstrated the ability to release urea and FVIII into the supernatant as early as the first 24 h, with accumulation over time. Discussion: These findings suggest that a 3D rotation culture system may facilitate rapid hepatic differentiation of HLSCs. Despite the limitations of this rotary culture system, its unique advantages hold promise for characterizing HLSC GMP batches for clinical applications.

Quantitation of endogenous GnRH by validated nano-HPLC-HRMS method: a pilot study on ewe plasma.

Gonadotropin-releasing hormone isoform I (GnRH), a neuro-deca-peptide, plays a fundamental role in development and maintenance of the reproductive system in vertebrates. The anomalous release of GnRH is observed in reproductive disorder such as hypogonadotropic hypogonadism, polycystic ovary syndrome (PCOS), or following prenatal exposure to elevated androgen levels. Quantitation of GnRH plasma levels could help to diagnose and better understand these pathologies. Here, a validated nano-high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) method to quantify GnRH in ewe plasma samples is presented. Protein precipitation and solid-phase extraction (SPE) pre-treatment steps were required to purify and enrich GnRH and internal standard (lamprey-luteinizing hormone-releasing hormone-III, l-LHRH-III). For the validation process, a surrogate matrix approach was chosen following the International Council for Harmonisation (ICH) and FDA guidelines. Before the validation study, the validation model using the surrogate matrix was compared with those using a real matrix such as human plasma. All the tested parameters were analogous confirming the use of the surrogate matrix as a standard calibration medium. From the validation study, limit of detection (LOD) and limit of quantitation (LOQ) values of 0.008 and 0.024 ng/mL were obtained, respectively. Selectivity, accuracy, precision, recovery, and matrix effect were assessed with quality control samples in human plasma and all values were acceptable. Sixteen samples belonging to healthy and prenatal androgen (PNA) exposed ewes were collected and analyzed, and the GnRH levels ranged between 0.05 and 3.26 ng/mL. The nano-HPLC-HRMS developed here was successful in measuring GnRH, representing therefore a suitable technique to quantify GnRH in ewe plasma and to detect it in other matrices and species.

Development and application of high resolution mass spectrometry analytical method to study and identify the photoinduced transformation products of environmental pollutants.

Terpenes are among the major causes of pleasant or unpleasant odors close to active or inactive landfills. We studied R-limonene and p-cymene environmental degradation products using the heterogeneous photocatalysis mediated by titanium dioxide to explore the odor pollution. The aim of the study was the development of mass spectrometry based methods both hyphenated with GC and HPLC to identify and characterize transformation products (TPs) derived from photodegradation of R-limonene and p-cymene. With the GC-MS method we identified three TPs for R-limonene and two for p-cymene comparing the obtained mass spectra with those in the NIST library. While with HPLC-MS method, thanks to the use of the high resolution of MS tool, we recognized four and five TPs for R-limonene and p-cymene respectively. No p-cymene was detected as R-limonene transformation product. The methods developed were then applied to real environmental samples coming from landfills active (Lan1) or inactive (Lan2 and Lan3) located in northern Italy. R-limonene was detected in the active landfill (Lan1 at the concentration of 2.35 µg/mL) together with one of its TPs and one TP derived from p-cymene. p-Cymene was detected in the other two inactive landfills (Lan2 and Lan3 concentrations 0.025 and 0.15 µg/mL, respectively) together with one of its TP and two TPs coming from R-limonene photodegradation. The finding of TPs together with R-limonene and p-cymene both in active and inactive landfills point out the attention on the reduction of these molecules in the environment to reduce pollution and human risks.

Quali-Quantitative Characterization of Volatile and Non-Volatile Compounds in Protium heptaphyllum (Aubl.) Marchand Resin by GC-MS Validated Method, GC-FID and HPLC-HRMS2.

Protium heptaphyllum (Aubl.) Marchand (PH) trees are endemic to the tropical region of South America, mostly Brazil. Antibacterial, antinociceptive, anti-inflammatory, anxiolytic, antidepressant and anti-hyperlipidemic/anti-hypercholesterolemic effects were reported for its resinous exudate Protiumheptaphyllum resin (PHR). This work aims to provide a qualitative and quantitative consistent chemical profiling of the major constituents of this resin and two extracts enriched in acid (acidic triterpene concentrated extract, ATCE) and neutral triterpenes (α and ß-amyrin concentrated extract, AMCE). GC-MS/GC-FID was used for volatile terpene fraction, a validated GC-MS method was developed for quantification of neutral α and ß-amyrin and HPLC-APCI HRMS2 was used for acidic triterpenes analysis. The chemical investigation reported 29 molecules, including 14 volatile terpenes, 6 neutral triterpenes and 11 acid triterpenes. The most abundant compounds were α-amyrin (251.28 g kg-1, 123.98 g kg-1 and 556.82 g kg-1 in PHR, ATCE and AMCE, respectively), ß-amyrin (172.66 g kg-1, 95.39 g kg-1 and 385.58 g kg-1 in PHR, ATCE and AMCE, respectively), 3-oxo-tirucalla-7,24-dien-21-oic acid (80.64 g kg-1, 157.10 g kg-1 and 15.31 g kg-1 in PHR, ATCE and AMCE, respectively) and 3α-hydroxy-tirucalla-8,24-dien-21-oic acid (77.71 g kg-1, 130.40 g kg-1 and 11.64 g kg-1 in PHR, ATCE and AMCE, respectively). Results showed specific enrichment of acidic and neutral triterpenoids in the two respective extracts.