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The Application of Pharmacogenetic Markers of The Response to Vincristine, Methotrexate and Thiopurine Therapies in Children with Acute Lymphoblastic Leukemia
No full textAkutna limfoblastna leukemija (ALL) je najčešća maligna bolest kod dece i lečenje obolelih od ALL predstavlja
veliki izazov u pedijatrijskoj onkologiji. Iako se upotrebom savremenih protokola lečenja postiže
visoka stopa preživljavanja, postoji značajna varijabilnost u odgovoru na terapiju i pojavi neželjenih efekata
na lekove. Individualni odgovor na terapiju varira, između ostalog, i usled genetičkih faktora. Farmakogenetika
pruža mogućnost prilagođavanja terapije individualnim genetičkim profilima pacijenata, čime se
može poboljšati efikasnost lečenja i smanjiti toksičnost.
Okosnicu svih savremenih protokola lečenja dečje ALL čine citotoksični lekovi vinkristin, metotreksat i
tiopurinski lekovi. Dosadašnjim farmakogenetičkim istraživanjima tražena je veza između povećane
toksičnosti ili smanjene efikasnosti terapije ovim lekovima i varijanti u brojnim genima. Nedvosmisleno je
pokazano da određene varijante u genu za tiopurin S-metiltransferazu mogu dovesti do ozbiljne
mijelotoksičnosti usled primene tiopurina. Nedavno je pokazano da sličan efekat mogu imati i varijante u
genu NUDT15, mada se one predominantno javljaju u azijskim populacijama. Što se uticaja genetičkih varijacija
na efekte terapije vinkristinom i metotreksatom tiče dosadašnja istraživanja su bila manje
jednoznačna. Kao potencijalni farmakogenetički markeri odgovora na terapiju vinksristinom izdvajaju se
varijante u genima CEP72 i CYP3A5, dok se u slučaju metotreksata najviše studija bavilo varijantama u genima
MTHFR i SLCO1B1.
Očekuje se da će dalji napredak farmakogenetike, usled primene najsavremenijih metoda, omogućiti individualizaciju
terapijskih protokola, smanjenje učestalosti terapijskih komplikacija i poboljšanje kvaliteta
života pacijenata. Uvođenje farmakogenetičkih testiranja u rutinsku kliničku praksu predstavlja važan
korak ka personalizovanoj medicini u lečenju ALL kod dece, jer povećava šanse za dobar odgovor na terapiju
i dugoročno preživljavanje obolelih.Acute lymphoblastic leukemia (ALL) is the most common malignant disease in children and represents a
significant challenge in pediatric oncology. Although modern treatment protocols achieve high survival
rates, there is considerable variability in treatment response and the occurrence of adverse effects. The
individual response to therapy varies, among other factors, due to genetic influences. Pharmacogenetics
offers the possibility of tailoring therapy to the individual genetic profiles of patients, thereby improving
treatment efficacy and reducing toxicity.
The cornerstone of all modern treatment protocols for pediatric ALL consists of cytotoxic drugs such as
vincristine, methotrexate, and thiopurine drugs. Previous pharmacogenetic studies have sought to identify
the connection between increased toxicity or reduced efficacy of these drugs and variants in various genes.
It has been unequivocally demonstrated that certain variants in the TPMT gene can lead to severe
myelotoxicity due to the use of thiopurines. Recently, it has been shown that similar effects can be caused
by variants in the NUDT15 gene, although these are predominantly found in Asian populations. Regarding
the impact of genetic variations on the effects of vincristine and methotrexate therapy, research to date
has been less conclusive. Potential pharmacogenetic markers for vincristine response include variants in
the CEP72 and CYP3A5 genes, while in the case of methotrexate, most studies have focused on variants in
the MTHFR and SLCO1B1 genes.
It is expected that further advances in pharmacogenetics, driven by the application of the state-of-the-art
methods, will enable the individualization of therapeutic protocols, reduce the incidence of therapeutic
complications, and improve the quality of life for patients. The introduction of pharmacogenetic testing
into routine clinical practice represents an important step toward personalized medicine in the treatment
of ALL in children, as it increases the chances for optimal response to drug treatment and long-term
survival of affected patients
Isolation of bacteria and characterization of their enzymes to develop biocatalysts for plastic biodegradation
No full textUsled masovne proizvodnje i neadekvatnog korišćenja plastike došlo je do rastućeg globalnog problema akumulacije plastičnog otpada. Različite strategije su primenjene kako bi se ovaj problem rešio, ali upotreba mikroorganizama i njihovih enzima za razgradnju plastičnih polimera bio je fokus istraživača u proteklih nekoliko decenija. Glavni ciljevi ove doktorske disertacije bili su izolacija i karakterizacija bakterijskih sojeva koji razgrađuju plastiku, prevashodno poli(etilentereftalat) (PET) i poli(uretane) (PU), njihov dalji razvoj u efikasne biokatalizatore, identifikacija enzima odgovornih za navedenu aktivnost i istraživanje potencijalnih metoda biološke valorizacije plastičnog otpada.Ispitivanjem laboratorijske kolekcije mikroorganizama tri bakterijska soja su odabrana kao potencijalni biokatalizatori za razgradnju plastike. Bacillus subtilis BPM12 efikasno je formirao biofilm na PET plastici i pokazao izuzetnu aktivnost na bis(2-hidroksietil) tereftalatu i različitim supstratima, strukturno sličnim PET oligomerima i monomerima. Kroz heterolognu i homolognu ekspresiju, potvrđeno je da je enzim Bpm12CE ključan za ovu aktivnost. Delovanjem više različitih enzima, Amycolatopsis mediterranei ISP5501 razgrađivao je različite vrste PU, a upotrebom model jedinjenja PU-7 potvrđena je i hidroliza uretanske veze. Streptomyces sp. PU10 pokazao se kao jedan od najaktivnijih, do sada prijavljenih, sojeva za hidrolizu PU disperzije – Impranil sa preko 96% razgradnje za 72 h. Na osnovu rezultata kvantitativne proteomike zaključeno je da su u razgradnju PU uključene esteraza, amidaza i oksidaza ovog soja, dok se produkti razgradnje inkorporiraju u primarni metabolizam Streptomyces sp. PU10 i usmeravaju ka biosintezi poliketida. Konačno, uspostavljena je i strategija za konvertovanje biorazgradivih skrob/poli(vinil-alkohol) filmova u jedinjenja sa dodatom vrednošću: biopolimer poli(hidroksibutirat) i biološki aktivni biopigment undecilprodigiozin.Due to mass production and inadequate plastic usage, a growing issue of global plastic waste accumulation has emerged. Various strategies have been employed to address this problem, but using microorganisms and their enzymes for plastic polymer degradation has captivated researchers' attention in recent decades. The primary goal of this doctoral dissertation was the isolation and characterization of bacterial strains capable of degrading plastics, mainly poly(ethylene terephthalate) (PET) and poly(urethanes) (PU), and their further development into efficient biocatalysts, the identification of enzymes responsible for this activity and exploration of potential methods for the biological valorization of plastic waste.From a laboratory collection of microorganisms, three bacterial strains were selected as potential biocatalysts for plastic degradation. Bacillus subtilis BPM12 efficiently formed biofilm on PET plastic, exhibiting exceptional activity on bis(2-hydroxyethyl) terephthalate and structurally similar PET oligomers and monomers. Through heterologous and homologous expression, the enzyme Bpm12CE was confirmed to be crucial for this activity. Amycolatopsis mediterranei ISP5501 degraded various types of PU using multiple enzymes, and the hydrolysis of the urethane bond was confirmed using the model compound PU-7. Streptomyces sp. PU10 demonstrated high activity, with over 96% degradation of PU dispersion - Impranil within 72 hours. Quantitative proteomics results indicated that an esterase, amidase, and oxidase of this strain were involved in PU degradation, with the degradation products being incorporated into the primary metabolism of Streptomyces sp. PU10 and directed towards polyketide biosynthesis. Finally, a strategy was established for converting biodegradable starch/poly(vinyl alcohol) films into value-added compounds: the biopolymer poly(hydroxybutyrate) and the biologically active biopigment undecylprodigiosin.Datum odbrane: 14-06-202
Inhibition of Aβ Peptide Aggregation by Late Embryogenesis Abundant Proteins: A New Approach for Alzheimer’s Disease Treatment
Get PDFAlzheimer’s disease (AD), is the most common neurodegenerative disorder, sharing unclear
pathophysiology and massive social costs. Today, more than 55 million people have been diagnosed
with AD, and this number is forecast to increase more than twice by 2050. AD is tightly associated with
the presence of amyloid beta (Aβ) deposits, organised into insoluble, amyloid fibrils. Despite numerous
contemporary studies focused on the Aβ aggregation reduction, the cure for AD has not been found yet.
Our Project intends to implement the elements of molecular mechanisms underlying the remarkable
phenomenon of plant desiccation tolerance and develop a new Aβ anti-aggregation strategy. Late
Embryogenesis Abundant proteins (LEAPs) are markedly induced upon desiccation and can stabilise the
native structure of proteins and membranes by a mechanism that is not fully understood.
The primary Project aim is to investigate the structural properties of Ramonda serbica LEAPs and their
interactions with Aβ. Firstly, we will recombinantly produce LEAPs with the highest potential to inhibit
Aβ aggregation. Further, we will analyse LEAPs’ secondary structure under different conditions, focusing
on their order-to-disorder transitions. The final aim is to identify Aβ/LEAPs interactions and assess the
Aβ anti-aggregation potential of LEAPs in vitro, which will have an impact on developing new strategies
for AD treatment. Moreover, the results of our project will be important for cryopreservation,
biotechnology, plant physiology, and agriculture. The partner groups involved in this project (Slovak: IEP
SAS) and Serbian: IMGGE) were selected based on their expertise in protein production and structure
analysis. The expertise transfer between partner laboratories will be ensured by mutual training of the
involved PhD and postdoc researchers.Principal Investigator: Dr Marija Vidović, IMGGEDuration period: 2024-202
Chemical engineering analysis of hydrodynamic conditions in a biomimetic 3D in vitro osteosarcoma model
No full textCreating an appropriate 3D in vitro microenvironment for tumour cells is a complex task, which
requires knowledge of biology, medicine and engineering. The application of chemical engineering
principles helps assessing hydrodynamic and mass transport conditions, which directly affect
cultured cells. In this work chemical engineering calculations were applied in the analysis of
hydrodynamic conditions in a 3D in vitro osteosarcoma model based on 2 wt.% Ca-alginate hydrogel
scaffolds with 2 wt.% particulate hydroxyapatite and perfusion bioreactors (“3D Perfuse”, Innovation
Center of the Faculty of Technology and Metallurgy, Belgrade, Serbia). The scaffolds (~9.5 mm in
diameter, ~4.5 mm thick disks) were manually seeded with K7M2-wt murine osteosarcoma cells
(15x106
cells/ml scaffold volume) and cultivated in perfusion bioreactors at continuous medium flow
(superficial velocity: 40 µm/s), while cultures under static conditions served as a control. After 7
days, the cells stayed viable, metabolically active and spontaneously formed spheroid-like structures
within the scaffold pores under both culture conditions. However, as revealed by histological
analyses, the aggregates (d~150 µm) under perfusion conditions were larger as compared to the
static control (d~90 µm), indicating positive effects of fluid flow on cell aggregation. In order to
explain the obtained results, two chemical engineering approximations were applied. Firstly, the
fluid flow through the pores of the scaffolds was considered as laminar flow through a cylindrical
tube yielding estimated shear stresses on the walls of the pores of up to 5 mPa. Then, each
aggregate was considered as a round object in laminar flow and the solution of the Stokes equation
revealed that aggregates were subjected to shear stresses up to 20 mPa. The obtained values could
be used for assessment of mechanisms of fluid flow effects on osteosarcoma cells, as a guideline in
designing future experiments and a starting point in setting up more complex CFD models.Conference Programme Book:Tumour Microenvironment Meeting & 3D Model Workshop, 6-8 September 202
Lung microbiota changes during pulmonary Aspergillus fumigatus infection in rats
No full textSince the realization that the lungs are not sterile but are normally inhabited by various bacterial species, studies have been conducted to define healthy lung microbiota and to investigate whether it changes during lung diseases, infections, and inflammation. Using next-generation sequencing, we investigated bacterial microbiota from whole lungs in two rat strains (previously shown to differ in gut microbiota composition) in a healthy state and during pulmonary infection caused by the opportunistic fungus Aspergillus fumigatus. No differences in alpha diversity indices and microbial composition between DA and AO rats before infection were noted. Fungal infection caused dysbiosis in both rat strains, characterized by increased alpha diversity indices and unchanged beta diversity. The relative abundance of genera and species was increased in DA but decreased in AO rats during infection. Changes in lung microbiota coincided with inflammation (in both rat strains) and oxidative stress (in DA rats). Disparate response of lung microbiota in DA and AO rats to pulmonary fungal infection might render these two rat strains differentially susceptible to a subsequent inflammatory insult
Gut microbial dysbiosis occurring during pulmonary fungal infection in rats is linked to inflammation and depends on healthy microbiota composition
Get PDFWhile the effect of gut microbiota and/or inflammation on a distant body
site, including the lungs (gut–lung axis), has been well characterized, data about the
influence of lung microbiota and lung inflammation on gut homeostasis (lung–gut
axis) are scarce. Using a well-characterized model of pulmonary infection with the
fungus Aspergillus fumigatus, we investigated alterations in the lung and gut microbiota
by next-generation sequencing of the V3–V4 regions of total bacterial DNA. Pulmo-
nary inflammation due to the fungus A. fumigatus caused bacterial dysbiosis in both
lungs and gut, but with different characteristics. While increased alpha diversity and
unchanged bacterial composition were noted in the lungs, dysbiosis in the gut was
characterized by decreased alpha diversity indices and modified bacterial composition.
The altered homeostasis in the lungs allows the immigration of new bacterial species of
which 41.8% were found in the feces, indicating that some degree of bacterial migration
from the gut to the lungs occurs. On the contrary, the dysbiosis occurring in the gut
during pulmonary infection was a consequence of the local activity of the immune
system. In addition, the alteration of gut microbiota in response to pulmonary infection
depends on the bacterial composition before infection, as no changes in gut bacterial
microbiota were detected in a rat strain with diverse gut bacteria. The data presented
support the existence of the lung–gut axis and provide additional insight into this
mechanism.
IMPORTANCE Data regarding the impact of lung inflammation and lung microbiota
on GIT are scarce, and the mechanisms of this interaction are still unknown. Using a
well-characterized model of pulmonary infection caused by the opportunistic fungus
Aspergillus fumigatus, we observed bacterial dysbiosis in both the lungs and gut that
supports the existence of the lung–gut axis.
KEYWORDS fungal lung infection, gastrointestinal microbiota, lung microbiota,
lung-gut axis, rats
B
acteria inhabit every part of the human body, but most of them are found in the gut.
Gut microbiota are responsible for many functions, including nutrient metabolism,
immunomodulation, maintenance of host physiology, and protection against pathogen
overgrowth (1). To date, numerous scientific studies confirm the important role of
gut bacteria in health and disease. This microbial community impacts not only local
immunity but also a distant body site, such as the lungs. Disturbances in gut bacterial
composition have been linked to asthma (2), chronic obstructive pulmonary disease
(3), cystic fibrosis (4), and lung cancer (5). Furthermore, pulmonary involvement was
noted in inflammatory gastrointestinal disease characterized by microbial dysbiosis (6),
Month XXXX Volume 0 Issue 0 10.1128/spectrum.01990-23 1
Editor Agostinho Carvalho, University of Minho,
Braga, Portugal
Address correspondence to Maja Tolinacki,
[email protected].
The authors declare no conflict of interest.
See the funding table on p. 15.
Received 11 May 2023
Accepted 25 July 2023
Published 25 August 2023
Copyright © 2023 Popovic et al. This is an open-
access article distributed under the terms of the
Creative Commons Attribution 4.0 International
license. Downloaded from https://journals.asm.org/journal/spectrum on 09 October 2023 by 147.91.199.205.
supporting the existence of a gut–lung axis. The gut bacterial microbiota or some of
their constituents impact the immune response in the lungs against viruses (7–9),
bacteria (10–13), fungi (14), and allergic airway inflammation (15) mainly through the
effect of the gut microbiota (or their metabolites) on the immune cell activity.
While the gut–lung axis is well characterized, the influence of the lung microbiota
as well as lung inflammation on gut homeostasis has attracted much more attention in
recent years. The first indication of the lung–gut axis was a higher prevalence (compared
to healthy subjects) of gastrointestinal symptoms in patients with asthma (16) and
chronic obstructive pulmonary disease (17). The existence of gastrointestinal symptoms
in patients with pulmonary virus infection has also been documented (18). Gastrointesti-
nal symptoms (abdominal pain, nausea, vomiting, and diarrhea) were noted in 11.6%
of children with influenza infection (18), and a later study showed a decrease in alpha
diversity in the feces of influenza-infected patients compared to healthy controls (19).
Fecal bacterial samples from patients with COVID-19 infection were shown to cluster
separately from those in healthy controls as well, but in the majority of these patients,
SARS-Cov-2 could be detected in the feces (20). Experimental studies in mice confirmed
the occurrence of gut dysbiosis following respiratory influenza virus infection (21–25)
and respiratory syncytial virus infection (24), despite the fact that the virus has not
been detected in the gut (21, 22, 24, 25). It has been shown that the alteration of gut
microbiota is a consequence of infection with live virus particles, as administration of an
attenuated influenza vaccine had no effect on the microbiota (24).
Bacterial dysbiosis in the gut also occurs following pulmonary bacterial infection. A
decrease in alpha diversity indices and differential relative abundance of fecal microbiota
were noted in patients with pulmonary tuberculosis (26, 27) and in mice infected with
Mycobacterium tuberculosis (28) and Klebsiella pneumoniae (29). Even administration of
the major component of the outer membrane of Gram-negative bacteria, lipopolysac-
charide, to the lungs caused gut bacteria dysbiosis (30).
In addition to pulmonary infections caused by viruses or bacteria, alteration of the gut
microbiota was noted in mice exposed to hyperoxia (31) and in patients with lung cancer
(compared to healthy individuals) (32) indicating that pulmonary inflammation/injury
affects the gut microbiota regardless of its origin. Despite a growing body of evidence
for interaction between the lungs and gut, there is still a lot of work to be done to
understand this crosstalk. There are virtually no data regarding gut microbiota changes
during pulmonary infection caused by fungi. Our previous study showed an alteration
in immune-mediated homeostasis of the gut in a rat model of sublethal pulmonary
infection with A. fumigatus (33). Using the same experimental model of infection in Dark
Agouti (DA) rat strain, we aimed to investigate changes in the lung and gut microbiota
by next-generation sequencing of the V3–V4 regions of total bacterial DNA in these
two organs. Possible mechanisms of lung–gut communication were also investigated. In
addition, to examine whether gut dysbiosis is a general characteristic during pulmonary
fungal infection, we analyzed feces from infected Albino Oxford (AO) rats, a strain that
develop quantitatively different immune response to fungus A. fumigatus (34) and whose
gut microbiota was previously shown to respond differently to oral cadmium administra-
tion (35) compared to DA rats
Reconstitution of non-carrier, heterozygous and homozygous prothrombin belgrade mutation carrier plasma using recombinant proteins
Get PDFIntroduction: The prothrombin Belgrade variant (c.1787G>A, p.Arg596Gln) is a rare mutation found in
Serbia, Japan, China, America, India and leads to antithrombin resistance. Prothrombin Belgrade mutation influencesthrombin-antithrombin interactions and leadsto impaired inactivation of mutated thrombin. Also, it affectssodium binding site in thrombin, which isimportant forswitching from fast thrombin
configuration (coagulant properties) to slow configuration (anticoagulant properties). It has only been
found in a heterozygous state, which could mean that homozygous carriers are incompatible with life.
By using prothrombin (FII) deficient plasma, we could reconstitute plasma of wild type, heterozygous and
homozygous carrier, which could give more insight into the mechanism of this mutation.
Methods: Recombinant wild type and mutated prothrombin were generated by transient transfection
in HEK293T cell line. Western blot analysis was performed to test the efficiency of transfection. Human
Prothrombin ELISA (Nordic BioSite, Sweden) was used in order to measure recombinant prothrombin
concentration. Overall Hemostasis Potential (OHP) assay was performed to assess recombinant protein
activity. Recombinant wild type and mutated prothrombin were added to FII deficient plasma (Siemens,
Germany) in order to create reconstituted plasma, in the final concentration of 0.1 mg/mL, as it is approximately the level of prothrombin in human plasma.
Results: Reconstituted plasma samples that correspond to non-carrier, heterozygous carrier, and homozygous mutation carrier plasma were reconstructed. Recombinant proteinstested by OHP assay were
functional.
Conclusion: Reconstituted plasma samples allow us to examine the mechanism of prothrombin Belgrade mutation in various assays and in homozygous form as well
Electronic cigarette vapour condensate affects mitochondrial potential in BEAS2B cell
Get PDFIntroduction: Cigarette smoke exposure is a known risk factor for development of lung diseases and
electronic cigarettes(e-cigarettes) were introduced as a popular and safer alternative to combustible tobacco products. Increasing number of studies are reporting their adverse biological effects both in vivo
and in vitro. Aim of this study was to evaluate the effect of e-cigarettes on mitochondrial function in
lung bronchial epithelial cells.
Methods: Electronic cigarette vapor condensate (ECC) was generated using an e-cigarette device on a
suction trap cooled in a dry ice/ethanol bath.We used unflavoured and flavoured e-cigarette liquids with
and without nicotine. Human bronchial epithelial BEAS2B cells were seeded in 96well plates and treated
with 2% e-cigarette vapour condensate for 24h. Mitochondrial membrane potential was measured using
50nM TMRE (Tetramethyl rhodamine ethyl ester) and cells were visualized on ImageXpress® Pico Automated Cell Imaging System (Molecular Devices, San Jose, CA, USA) with a 10x objective.
Results: We found a significant reduction of TMRE fluorescence in treated cells compared to the control. Imaging of treated cells also revealed changes in cell morphology and the presence of mitochondria in TNT-like structures.
Conclusion: Mitochondrial dysfunction has been associated with various pathological conditions including lung diseases such as asthma, COPD and lung cancer. Due to their relative novelty, the role of
electronic cigarette use in development of chronic lung diseasesisstill relatively unknown. Our findings
contribute to the growing list of studies pointing to their adverse biological effects and imply their involvement in processes contributing to chronic lung diseases
Transcriptional profiling of PHF19 gene in colon cancer cell lines cultivated in 3D
Get PDFIntroduction: A recent comprehensive pan-cancer transcriptome analysis revealed differential activity
of two alternative promoters of the gene PHF19 in malignant and non-malignant gut mucosa. Transcription from the promoter which is up-regulated in colorectal cancer results in the synthesis of transcript PHF19-207. This finding indicates that transcript PHF19-207 could potentially be used as a
biomarker for this disease. Our study aimed to assess the expression profile of the PHF19 gene in colon
cancer.
Methods: Immortalized colonic epithelial cell line isolated from healthy tissue (HCEC-1CT) as well as a
set of colon cancer cell lines (DLD1, SW620, HCT116) were used for transcriptional profiling of PHF19 in
cells cultivated in 3D. The transcriptional profile was obtained using RNA sequencing and the function
of transcript PHF19-207 was evaluated using in silico tools.
Results: Our analysis confirmed the up-regulation of transcript PHF19-207 in all malignant cell cultures
in comparison to the healthy cell line HCEC-1CT. The expression of transcript PHF19-207 was more notable in cell lines that originated from colon cancer in later stages. Coding Potential Calculator tool classifies this transcript as non-coding, with a probability of 0.2. Annolnc tool shows the up-regulation of
thistranscript in colorectal cancer cell lines and its down-regulation in healthy samples. Also, thistool predicts that transcript PHF19-207 localizes in the nucleus.
Conclusion: We conclude that transcript PHF19-207 could serve as a biomarker for colorectal cancer.
Also, we hypothesize that thistranscript is a lncRNA with a role in gene expression regulation and could
be linked to oncogenesis
Immobilized NT2/D1 cells in alginate fibers: a promising 3D model system for investigating human neurogenesis and screening the effect of drugs and bioactive compounds
Get PDFIntroduction: The NT2/D1 embryonal carcinoma cell line represents a well-established in vitro model of
human neurogenesis. It’s widely used for studying neurodevelopmental processes, neurotoxicity, and
neurodegenerative disorders. The utilization of alginate fibers as a 3D cell culture system offers a biocompatible and structurally supportive environment for neural differentiation and maturation of cells,
making it a suitable tool for investigating neurodevelopmental processes.
Methods: In thisstudy, we evaluated the alginate microfibers as a 3D modelsystem for in vitro neural differentiation of NT2/D1 cells.We described the immobilization of NT2/D1 cellsin alginate microfibers and
the effect of propagation in this 3D model on morphological features, viability, and proliferation of immobilized cells. We also assessed the RA-induced initiation of neural differentiation of NT2/D1 cellsin alginate microfibers by comparison with the initiation of neural differentiation in adherent 2D cell culture.
Results: Our results showed that immobilized NT2/D1 acquired morphological features characteristic
of cells propagated in 3D model systems and retain viability, proliferative capacity, and ability to attach
to adherent surfaces. In addition, immobilized NT2/D1 cells preserved neural differentiation capacity.
Upon RA induction we detected a marked decrease in the expression of specific pluripotency-maintaining markers, SOX2, OCT4, and NANOG. Consecutively, the expression of early neural markers, SOX3,
PAX6, and miR219 was significantly increased.
Conclusion: Neural differentiation of NT2/D1 cellsimmobilized within alginate fibersrepresents a highly
promising 3D modelsystem forstudying human neurogenesis and offers a valuable platform forscreening the effect of drugs and bioactive compounds on human neural differentiation