MANF-Knockout Human Embryonic Stem Cells React to Oxidative Stress Differently than Wild-Type Cells Master’s Programme in Neuroscience, Study Track: Neuroscience Master's thesis Author: Anu Haapala Supervisors: Dr Vassilis Stratoulias Amanda Sandelin 24.4.2023 Helsinki Faculty: Biological and Environmental Sciences Degree programme: Master’s Programme in Neuroscience Study track: Neuroscience Author: Anu Haapala Title: MANF-Knockout Human Embryonic Stem Cells React to Oxidative Stress Differently than Wild-Type Cells Level: Master’s Thesis Month and year: April 2023 Number of pages: 31 Keywords: human embryonic stem cells, MANF, oxidative stress Supervisors: Vassilis Stratoulias, Amanda Sandelin Where deposited: Helsinki University Library Additional information: - Abstract: Introduction: Oxidative stress occurs in cells when reactive oxygen species are generated as a by-product of oxygen metabolism and start to accumulate excessively. While extensive oxidative stress is highly detrimental to the cells, trophic factors help them survive. Trophic factor MANF has interested especially Parkinson’s disease researchers, but recent findings suggest that MANF plays a role in many diseases, also ones with an early childhood-onset. For this reason, it is important to investigate MANF function in different cell types. We have studied how MANF-knockout human embryonic stem cells react to oxidative stress compared to wild-type human embryonic stem cells, by exposing the cells to hydrogen peroxide and ethanol. Results: MANF-knockout human embryonic stem cells were more sensitive to oxidative stress than wild-type cells, but the variation between measurements was remarkable and the differences were statistically insignificant. We found that a transcription factor of our interest localized in the cell nuclei of MANF-knockout cells upon oxidative stress exposure. Such a nuclear translocation did not occur in wild-type cells. Moreover, we found that high concentrations (>2%) of ethanol reduced the viability of cells in only four hours. Discussion: Our findings suggest that MANF-knockout human embryonic stem cells react to oxidative stress differently than wild-type cells. Additional studies are necessary to clarify whether MANF-knockout human embryonic stem cells are indeed more sensitive to oxidative stress than wild-type cells. In the future, it would be interesting to inspect whether MANF protects human embryonic stem cells when the cells are exposed to physiologically relevant ethanol concentrations for longer periods of time. Tiedekunta: Bio- ja ympäristötieteellinen tiedekunta Koulutusohjelma: Neurotieteen maisteriohjelma Opintosuunta: Neurotiede Tekijä: Anu Haapala Työn nimi: Ihmisalkion kantasolut, jotka eivät eritä MANF-proteiinia, reagoivat oksidatiiviseen stressiin eri tavalla kuin tavalliset kantasolut Työn laji: Maisterintutkielma Kuukausi ja vuosi: Huhtikuu 2023 Sivumäärä: 31 Avainsanat: alkiokantasolut, MANF, oksidatiivinen stressi Ohjaajat: Vassilis Stratoulias, Amanda Sandelin Säilytyspaikka: Helsingin yliopiston kirjasto Muita tietoja: - Tiivistelmä: Johdanto: Solut kokevat oksidatiivista stressiä, kun happiaineenvaihdunnan sivutuotteena muodostuvia reaktiivisia happiyhdisteitä kertyy liikaa. Korkea oksidatiivinen stressi on soluille haitallista, mutta tietyt proteiinit auttavat niitä selviytymään. Tällaisiin proteiineihin lukeutuva MANF on kiinnostanut etenkin Parkinsonin tautia tutkivia, mutta MANF:lla näyttäisi olevan rooli monissa, myös varhaislapsuudessa puhkeavissa sairauksissa. Siksi on tärkeää tutkia sen toimintaa eri solutyypeissä. Tutkimme, miten MANF:ia erittämättömät ihmisalkion kantasolut reagoivat oksidatiiviseen stressiin tavallisiin ihmisalkion kantasoluihin verrattuna altistamalla niitä vetyperoksidille ja etanolille. Tulokset: MANF:ia erittämättömät solut olivat herkempiä oksidatiiviselle stressille kuin tavalliset solut. Tuloksissa oli kuitenkin paljon vaihtelua eivätkä erot olleet tilastollisesti merkitseviä. Tutkimamme transkriptiotekijä siirtyi MANF:ia erittämättömien solujen solulimasta tumaan, kun solut altistettiin oksidatiiviselle stressille. Vastaavaa ei tapahtunut tavallisissa soluissa. Altistuminen korkeille (>2 %) etanolipitoisuuksille vähensi solujen elinvoimaisuutta jo neljässä tunnissa. Pohdinta: Tulostemme perusteella MANF:ia erittämättömät ihmisalkion kantasolut reagoivat oksidatiiviseen stressiin eri tavoin kuin tavalliset ihmisen kantasolut. Lisätutkimuksia tarvitaan, jotta voidaan varmistua siitä, ovatko MANF:ia erittämättömät solut todella herkempiä oksidatiiviselle stressille kuin tavalliset solut. Tulevaisuudessa olisi myös mielenkiintoista tutkia, auttaako MANF suojelemaan ihmisalkion kantasoluja, jos solut altistetaan fysiologisesti relevanteille, matalille etanolipitoisuuksille mutta pitkäkestoisemmin. Table of contents List of Abbreviations …………………………………………………………………………………… 5 Introduction ………………………………………………………………………………………………. 6 Aims of the Study ……………………………………………………………………………………….. 8 Materials and Methods ……………………………………………………………………………….. 11 Results ………………………………………………………………………………………………………. 16 Discussion ……………….…………………………………………………………………………………. 23 Acknowledgements …………………………………………………………………………………….. 26 References …………………………………………………………………………………………………. 27 5 List of Abbreviations EtOH Ethanol hESC Human Embryonic Stem Cell H2O2 Hydrogen Peroxide MANF Mesencephalic Astrocyte-Derived Neurotrophic Factor PD Parkinson’s Disease ROCK Rho-Associated Protein Kinase ROS Reactive Oxygen Species TF Transcription Factor ZSCAN Zinc Finger and SCAN Domain-Containing (Transcription Factor Family) ZSCAN2 Zinc Finger and SCAN Domain-Containing 2 (Transcription Factor) 6 1.1. Introduction Oxidative stress is a state that occurs in cells when reactive oxygen species (ROS) are generated as a by-product of oxygen metabolism and start to accumulate excessively. ROS can be free radicals or other types of molecules, such as hydrogen peroxide (H2O2), and most of them are generated in the mitochondria, the cell power houses. While ROS emerge because of completely normal, necessary, and inevitable metabolic reactions, extensive oxidative stress can be severely detrimental to the cells. They can, for example, damage the DNA and disturb mitochondrial function, and even cause apoptosis (Andersen, 2004; Chen et al., 2006; Misrani et al., 2021; Ionescu-Tucker & Cotman, 2021). Oxidative stress levels can be influenced by environmental factors and lifestyle choices: for example, antioxidant intake can reduce it, while exposure to ethanol (EtOH) increases it (Ionescu-Tucker & Cotman, 2021; Mahdinia et al., 2021). There are at least two reasons why EtOH induces oxidative stress. Firstly, the activation of cytochrome P-450 – a key enzyme involved in alcohol metabolism – induces excessive ROS generation. Secondly, alcohol can cause inflammation that can indirectly lead to increased generation of ROS. (Rumgay et al., 2021) The deteriorating effects of oxidative stress are especially prevalent in the brain, and oxidative stress is likely a key element causing brain ageing (Andersen, 2004; Ionescu-Tucker & Cotman, 2021). Previous research has shown that the brains of people with different neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease (PD), are exposed to high levels of oxidative stress, and the functionality of their cell mitochondria is impaired (Andersen, 2004; Chen et al., 2006; Liu et al., 2018b; Misrani et al., 2021). Whether it is the cause or consequence remains unknown. In many diseases, oxidative stress is connected to endoplasmic reticulum stress, which occurs when an excessive number of misfolded proteins accumulate in the endoplasmic reticulum, an organelle that plays a crucial role in, for example, protein biosynthesis and modifications taking place post-translationally (Chong et al., 2017). Chronic endoplasmic reticulum stress can increase oxidative stress, and vice versa (Back & Kaufman, 2012; Chong et al., 2017). While oxidative stress can severely damage and even kill cells, proteins called trophic factors can help them survive (Lindholm & Saarma, 2010). Both the developing nervous system and the adult nervous system need trophic factors to function normally. For example, during development, only neurons that are adequately supported by trophic factors can stay alive and 7 keep their synaptic connections with target tissues. The loss of trophic factors has been associated with developmental disorders as well as with neurodegeneration in the adult nervous system (Blesch, 2006; Lindholm & Saarma, 2010). A couple of decades ago, Petrova and colleagues (2003) described a then novel trophic factor, mesencephalic astrocyte-derived neurotrophic factor (MANF), suggesting that MANF protects dopaminergic neurons selectively (Petrova et al., 2003). Very recently it was suggested that, instead of protecting naïve dopaminergic neurons, MANF in fact starts protecting cells only in the presence of endoplasmic reticulum stress (Eesmaa et al., 2021). Over the years, especially researchers focusing on PD have been interested in MANF as a potential treatment candidate (e.g., Voutilainen et al., 2009; Voutilainen et al., 2011), as one of the key characteristics of PD neuropathology is the remarkable loss of dopaminergic neurons in the substantia nigra pars compacta (Surmeier, 2018). Interestingly, it has been suggested that MANF protects an in vitro model of PD from mitochondrial damage and oxidative stress (Liu et al., 2018b). MANF’s treatment potential may thus rely at least partly on its ability to reduce oxidative stress. Despite the particular interest in MANF by researchers studying PD and neurodegeneration, MANF has been studied also in the context of several other diseases, and its role in early development has interested researchers as well. Recently, loss of MANF has been associated with, for example, childhood-onset hearing loss and diabetes (Ikäheimo et al., 2021; Montaser et al., 2021). Considering the important role MANF seems to have throughout the lifespan, it is interesting to study its function in different cell types including human embryonic stem cells (hESCs), which our study focuses on. It is also interesting to investigate how different transcription factors (TFs) are expressed in hESCs and whether oxidative stress plays a role in TF expression. TFs are proteins that regulate gene expression and hence the signaling between DNA, mRNA and proteins (Bemer et al., 2017). TFs are often located in the cell cytoplasm, but once activated by signals from the cell membrane, nuclear translocation occurs, i.e., the TFs move from the cytoplasm to the cell nucleus (Liu et al., 2018a). We are interested in a TF called zinc finger and SCAN domain-containing 2 (ZSCAN2), as based on our bulk RNA sequencing data, ZSCAN2 is expressed highly in both wild-type and MANF-knockout hESC (unpublished data). However, an in-silico analysis of our collaborators (unpublished, confidential data) indicated that ZSCAN2 regulates expression of the most significantly downregulated genes in MANF- knockout hESCs. 8 ZSCAN2 is part of the zinc finger and SCAN domain-containing (ZSCAN) TF family. All members of the ZSCAN TF family have a similar physical structure that includes a DNA- binding zinc finger domain and a protein-interacting SCAN domain, but the biological functions of the different family members seem to vary significantly. The ZSCAN TF family has been associated with, for example, tumor progression, but interestingly, the different family members appear to contribute to tumor progression in different, even opposing ways. Some of the family members have been studied extensively, while the functions of others remain a mystery. (Huang et al., 2019) ZSCAN2 belongs to the latter group, and to date, very little is known about its roles and functions. One published article associated ZSCAN2 with spermatogenesis (Lord et al., 2022), but to date and to our best knowledge, other relevant studies have not been published. This master’s thesis project is part of a larger research project on the trophic factor MANF in Professor Mikko Airavaara’s research group at the University of Helsinki. I worked in the group from September 2022 until February 2023, and this report describes only cell culture work and experiments which I have personally carried out. 1.2. Aims of the Study Our research group recently discovered that MANF-knockout hESCs fail to express Catalase (A. Sandelin, personal communication, September 5, 2022). Catalase is an enzyme that catalyzes a chemical reaction in which H2O2, causing oxidative stress, is decomposed into water and oxygen (figure 1). Previous findings have shown that Catalase effectively reduces oxidative stress (Andersen, 2004; Ionescu-Tucker & Cotman, 2021), and therefore, our group’s recent findings seem to suggest that MANF-knockout hESCs could be more sensitive oxidative stress than wild-type hESCs. Therefore, the first line of interest of this master’s thesis project was to investigate how MANF-knockout hESCs differ from wild-type hESCs in terms of a) MANF protein and Catalase expression, b) their sensitivity to oxidative stress. We hypothesized that a) the wild- type hESCs express MANF and Catalase but the MANF-knockout hESCs do not, and that b) the MANF-knockout hESCs are more sensitive to oxidative stress than wild-type hESCs indicated by lower cell viability after exposing the cells to induced oxidative stress. 9 Sensitivity to oxidative stress can be studied using assays that detect cell viability. Several such assays that can be used on different detection platforms (e.g., fluorescence microscopy, microplate readers) are commercially available. Kamiloglu et al. (2020) have reviewed the most used assays and divided them into five groups: 1) dye exclusion, 2) colorimetric, 3) fluorometric, 4) luminometric, and 5) flow cytometric assays. Each assay type comes with advantages and disadvantages. For example, while they are easy and fast to use, Trypan Blue solution – commonly used in dye exclusion testing – and the fluorometric MTT assay are both highly toxic to cells, and their cell counting results may be unreliable if, for instance, the cells have formed clumps. Fluorometric assays (e.g., resazurin-based solutions such as AlamarBlue and PrestoBlue) are similarly easy and fast and thus handy to use, but one of their disadvantages is that compounds tested in the experiment can bias the fluorescence signal. Luminometric assays are also easy and fast to use, and they allow monitoring cell viability in real time. However, these assays are highly sensitive to pipetting errors and other technical errors, and the incubation time needs to be carefully optimized for each tested cell type separately to reach reliable results. Finally, flow cytometric assays are useful, for example, because the dyes used for these assays (Annexin V and F2N12S) do not require permeabilization of cell membranes and they also can be used together with other markers. However, these assays are sensitive to light and some compounds that are commonly used in cell culture such as EDTA and trypsin. When selecting the most suitable cell viability assay, these pros and cons should naturally be considered, and most importantly, the gained results should be confirmed using another type of an assay. (Kamiloglu et al., 2020). As a second line of interest, we have investigated ZSCAN2 TF expression in wild-type and MANF-knockout hESCs under typical conditions and after exposure to oxidative stress. More specifically, we wanted to investigate whether ZSCAN2 cellular localization is different between MANF wild-type and MANF-KO hESC cells. Such a phenotype could explain the discrepancy between the in silico and the transcriptomic data and provide strong support for follow-up studies between MANF and ZSCAN2. We hypothesized that ZSCAN2 is expressed in the cell nucleus and cytoplasm of MANF-knockout hESCs but predominantly in the nucleus of the wild-type hESCs and that exposure to oxidative stress does not change this ZSCAN2 expression. 10 Figure 1. Mechanism of action for Catalase and illustration of our first hypothesis. a) Catalase breaks down two hydrogen peroxide molecules into two water molecules and one oxygen molecule. b) Wild-type hESCs express Catalase but MANF-knockout hESCs do not. Therefore, we have hypothesized that MANF-knockout hESCs are more sensitive to oxidative stress than wild-type hESCs. 11 2. Materials and Methods 2.1. Cell Culture The H1 hESCs, MANF-knockout and isogenic control (wild-type), were provided by Timo Otonkoski (for details regarding the generation of the knockout cell line, see Montaser et al., 2021). Cells were grown on Matrigel-coated (356231; Corning) 100 mm Petri dishes. Matrigel-coating for the dishes was prepared by diluting (1:200) ice cold Matrigel hESC- Qualified Matrix in cold (4°C) Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12 (DMEM/F-12) (21331-020; Gibco). Matrigel-coated dishes were kept at 4°C and stored for a maximum of two weeks. Upon media change, cells were washed once using DMEM/F-12. Cells were grown in 10 ml of Essential 8 Medium (E8) (A1516901; Gibco), supplemented with Essential 8 Supplement (50X) (A1517101; Gibco), according to manufacturer’s instructions. Penicillin-streptomycin (15140122; Gibco) at low concentration (1:1000) was used. Media was changed daily, and cells were typically split twice a week. Essential 8 Flex Medium (A2858301; Gibco), supplemented with Essential 8 Flex Supplement (50X) (A2858401; Gibco) and penicillin-streptomycin, was used over the weekends (20 mL for a 100 mm Petri dish), as this medium supports the culture of stem cells without the need for weekend feeding. Media were warmed up at room temperature for an hour prior to starting the work in cell culture. 2.2. Immunofluorescent Staining of Cells on Coverslips: SOX2, MANF, and Catalase Expression For this experiment, wild-type and MANF-knockout cells were seeded on coverslips at the density of 10x104 per coverslip. They were fixed using 4% PFA once confluency of at least 80% was reached. On day 1, the cells on coverslips were washed five times with 1 x PBS and permeabilized with 0.3% PBT (Triton). The cells were incubated in 5% normal donkey serum (017000121; Jackson ImmunoResearch) diluted in 0.3% PBT for 60 minutes at room temperature. Goat anti-SOX2 (AF2018; R&D Solutions; dilution 1:200), rabbit anti-MANF (HPA011175; Sigma; dilution 1:200), and rabbit anti-catalase (12980S; Bionordika; dilution 1:100) primary 12 antibody solutions were prepared in the 5% normal donkey serum. The cells were incubated in primary antibody solutions overnight at 4°C. A control coverslip was incubated in the 5% donkey serum without primary antibodies. On day 2, the cells were washed five times with PBS. The cells were incubated in anti-goat (A11057; Invitrogen; dilution 1:500) and anti-rabbit (A21206; Invitrogen; dilution 1:500) secondary antibody solution diluted in 0.3% PBT for 60 minutes at room temperature. The cells were washed four times with PBS and incubated in DAPI (D9542; Sigma-Aldrich; dilution 1:1000) diluted in PBS for 20 minutes at room temperature. The cells were washed three times with PBS. The coverslips were mounted in 8 μL of Flourmount-G (010001; SouthernBiotech) and stored at 4°C overnight. On day 3, the coverslips were imaged using light microscopy. 2.3. Cell Vulnerability to H2O2 Using PrestoBlue Cell Viability Reagent We selected a cell viability assay that made use of resazurin-based PrestoBlue Cell Viability Reagent. PrestoBlue Cell Viability Reagent gives an indication of cell viability by quantitatively measuring cell proliferation using the living cells’ reducing power. Metabolically active cells reduce resazurin to resorufin. Consequently, the reagent color changes and becomes highly fluorescent. This makes the measurement of fluorescence signal, an indicator of cell viability, possible by using a microplate reader. Wild-type and MANF-knockout hESCs were seeded in a 96-well Matrigel-coated (dilution 1:200) plate with 90 μL of E8 media with different concentrations (0 μM, 20 μM, 50 μM, 100 μM, 200 μM) of H2O2 (216763, Sigma-Aldrich) and Rho-associated protein kinase inhibitor (ROCK inhibitor Y-27632, dilution 1:1000) (126830382; Hello Bio), at the density of 7.5x104 cells per well. Five to seven samples per treatment condition per cell line were included. To correct for background fluorescence, only E8 medium and H2O2 without cells was added in the wells of the first row of the 96-well plate. The cells were incubated at 37°C and 5% CO2 for 20 hours. PrestoBlue Cell Viability Reagent (P50200; ThermoFisher) was added in wells according to the manufacturer’s instructions. The cells were incubated with the reagent for 10 minutes at 37°C and 5% CO2. Cell viability was measured by reading the fluorescence signal using Varioskan multimode microplate reader (ThermoFisher). 13 Figure 2. Timeline of the cell vulnerability to H2O2 experiment. 2.4. Cell Vulnerability to EtOH The experiment protocol was inspired by two previously published papers studying different cell types (Chandramouleeswaran et al., 2020; Huang et al., 2007). On day 1, 100 μL of E8 medium with ROCK inhibitor (dilution 1:1000) was added in each well of a Matrigel-coated (dilution 1:200) 96-well microplate. The first time, the cells were seeded at the density of 6x104 cells per well in each well. To correct for background fluorescence, only E8 medium was added the first row of the plate. In the follow-up experiments, the cells were seeded at the density of 7.5x104 cells per well. Half of the wells were reserved for wild-type hESCs and the rest for MANF-knockout hESCs. The cells were incubated at 37°C and 5% CO2 for 20 hours. On day 2, the E8 medium with ROCK inhibitor was removed. The cells were washed once using DMEM/F-12 (60 μL per well). 90 μL of E8 medium with different EtOH concentrations (0%, 0.25%, 0.5%, 1%, 2%, 5% in the first two experiments; 0%, 2%, 5%, 7.5% and 10% in a follow-up experiment) was added in all wells (i.e., one column reserved for an experimental condition). The microplate was coated tightly with Parafilm to prevent EtOH evaporation. The cells were incubated at 37°C and 5% CO2 for four hours. 10 μL of PrestoBlue™ Cell Viability Reagent (P50200; ThermoFisher) was added in each well. The cells were incubated with the reagent for 10 minutes at 37°C and 5% CO2. Cell viability was measured by reading the fluorescence signal using Varioskan multimode microplate reader. 14 Figure 3. Timeline of the cell vulnerability to EtOH experiment. 2.5. Cell Vulnerability to H202 Using Incucyte S3 Live-Cell Analysis Instrument On day 1, cells were seeded in a Matrigel-coated (dilution 1:200) 48-well plate in 300 μL of E8 medium with ROCK inhibitor (dilution 1:1000) at the density of either 5x104 (half of the rows) or 10x104 (the other half of the rows) per well. Half of the wells were reserved for wild- type hESCs and the other half for MANF-knockout hESCs. The cells were incubated at 37 C and 5% CO2 for 20 hours. On day 2, the cells were washed once using DMEM/F-12 (200 μL per well) and fresh E8 medium (300 μL per well) was added in all wells. The cells were incubated at 37°C and 5% CO2 for 23 hours. On day 3, the plan was to wash the cells once using DMEM/F-12 (200 μL per well), remove the old E8 medium, and replace it with fresh E8 medium with different concentrations of H2O2 (0 μM, 50 μM, 100 μM, 200 μM). The plate would have been incubated at 37°C and 5% CO2 for 24 hours. During this time, the cell viability would have been observed and analyzed using Incucyte S3 Live-Cell Analysis Instrument (Sartorius). This experiment was seized after day 2 because of a defect in the live-cell analysis instrument. No results will be reported here but other group members will utilize the experiment protocol later. 2.6. Immunofluorescent Staining of Cells on Coverslips: Zinc Finger and SCAN-Domain Containing 2 (ZSCAN2) Expression Wild-type and MANF-knockout cells per well were seeded at the density of 10x104 on Matrigel-coated (dilution 1:50) coverslips in a 48-well plate. Some of the cells were exposed to a 24-hour (50 µM, first part of the experiment) or 6-hour (100 and 200 µM, second part of 15 the experiment) H2O2 treatment. Some cells were treated with MANF protein for six hours (second part of the experiment). The coverslips were fixed with 4% PFA when confluency of at least 80% was reached. The immunofluorescent staining protocol has been described above (see section 2.2.). A control sample without incubation in a primary antibody solution was included in the experiment. The used primary antibody was a rabbit anti-ZSCAN2 antibody (HPA024331; Sigma-Aldrich; dilution 1:250) and the secondary antibody anti-rabbit antibody (A21206; Invitrogen; dilution 1:500). The samples were imaged by Amanda Sandelin using a confocal microscope. 2.7. Statistical Analysis Graphs were built, and statistical analyses were carried out using IBM SPSS Statistics (Version: 28.0.0.0). A p-value of <0.05 was considered statistically significant. 2.8. Consideration of Research Ethics This study has been conducted on hESCs. hESCs are, to some extent, irreplaceable in biomedical research that aims to increase our understanding of different diseases and help develop better therapies for them. The use of hESCs has many advantages. They can differentiate into any cell type of the body, making the research on different tissues and diseases possible, and they proliferate quickly and are durable. The hESCs used in this project have been handled with utmost care and respect, following all local and EU-wide laws and regulations related to stem cell research. The cells have been utilized for other experiments within our group, including ones in which they have been differentiated to other cell types. This has ensured that we minimize the need for early-stage embryos in our research. 16 3. Results 3.1. Wild-type and MANF-knockout hESCs express SOX2, but MANF-knockout hESCs do not express MANF or Catalase The aim of this experiment was to confirm our group’s earlier findings according to which a) the both cell lines we worked with are indeed stem cells and they express the stem cell marker SOX2 (Arnold et al., 2011), b) the MANF-knockout hESCs do not express MANF, to confirm that this cell line is indeed a knockout line, c) the MANF-knockout hESCs do not express Catalase. The experiment was conducted twice to investigate MANF and SOX2 expression, and once to investigate Catalase and SOX2 expression. The results of this experiment confirm that both cell lines we were working with are indeed stem cells, indicated by a clear expression of SOX2 TF (Figure 4a). The results also confirmed that the MANF-knockout hESC line is indeed a knockout line and does not express MANF, while the wild-type line clearly expresses MANF (figure 4a). Moreover, in line with our group’s previous findings, the results suggest that while the wild-type hESCs express Catalase, the MANF-knockout hESCs fail to do so (figure 4b). 17 Figure 4. SOX2, MANF and Catalase expression in wild-type and MANF-knockout hESCs. a) Wild-type and MANF-knockout hESCs express stem cell marker SOX2 (red fluorescence), but MANF-knockout hESCs do not express MANF (green fluorescence). b) Wild-type hESCs express Catalase (green fluorescence) but MANF-knockout hESCs do not express Catalase. 3.2. MANF-knockout hESCs are more sensitive to oxidative stress than wild-type hESCs but the differences are statistically insignificant 18 This experiment was carried out nine times because the results showed great variation between measurements. The first measurements included also a 20 μM H2O2 treatment condition in addition to the treatment conditions reported here, but we decided to leave this condition out of further measurements after the first analyses showed that the cell viability was consistently as high in the 20 μM H2O2 concentration as in the control condition without any H2O2 treatment. Moreover, the results of two measurements were left out from further analyses, because the results seemed highly unreliable (e.g., all cells died when treated with 50 μM H2O2 but not when treated with 100 μM H2O2). The raw data provided by the microplate reader was normalized by first calculating the average cell viability within one condition and then comparing the average viability in control condition (i.e., 1.0) to average viability in each experimental condition. Put together, the results of this experiment may suggest that the MANF-knockout hESCs may be more sensitive to oxidative stress than wild-type cells (figure 5). However, the results from individual measurements showed that cell viability in different H2O2-treatment conditions varied a lot between the different times when the experiment was carried out. To test whether the differences in cell viability between the two cell lines were statistically significant, independent samples t-tests were performed for each treatment condition separately. None of the differences were statistically significant. In the 50 μM H2O2 concentration, the viability of the MANF-knockout cells (M = 0.82, SEM = 0.05) was the same as the viability of the wild-type cells (M = 0.94, SEM = 0.05), t(7) = 1.47, p = 0.577. In the 100 μM H2O2 concentration, the viability of the MANF-knockout cells (M = 0.73, SEM = 0.11) was equal to the viability of the wild-type cells (M = 0.78, SEM = 0.10), t(7) = 0.327, p = 0.509. Finally, also in the 200 μM H2O2 concentration, the viability of the MANF-knockout cells (M = 0.19, SEM = 0.08) was similar to that of the wild-type cells, (M = 0.29, SEM = 0.13) although these differences reached a near-to-significant level, t(7) = 0.687, p = 0.06. 19 Figure 5. Cell sensitivity to H2O2-induced oxidative stress. MANF-knockout hESCs seem more sensitive to oxidative stress than wild-type hESCs when the cells are exposed to oxidative stress at the same time with ROCK inhibitor, but the differences are not statistically significant (all p-values >0.05) and variation between measurements was high as indicated by error bars (+/- 2 SE [Standard Error]). 3.3. A four-hour treatment with EtOH decreases viability of hESCs at high EtOH concentrations As the previous experiment yielded results with a high variation, the aim of this experiment was to improve the previous experimental protocol. We decided to use another compound, EtOH, to induce oxidative stress to see whether the previously seen variation between measurements might be specific to H2O2, as it is possible that the tested compounds influence the fluorescence signal (Kamiloglu et al., 2020). While the final aim was to design an experiment that would produce robust and replicable results, equally important goals were to find out at which EtOH concentration the cell viability of wild-type and MANF-knockout hESCs starts to notably decrease and whether a treatment time of only four hours (instead of the 20-hour H2O2 treatment time used in our previous experiment) is enough to decrease cell viability in hESCs. 20 This experiment was carried out three times, and the experiment was slightly modified each time to test the effects of different EtOH concentrations on hESCs and also different cell seeding concentrations. Taken together, the results from this experiment suggest that an EtOH treatment of four hours decreased the viability of both wild-type and MANF-knockout hESCs when the EtOH concentration was high (>2%) (figure 6). Otherwise, the results between different measurement points showed an inconsistent pattern in terms of cell viability. In the final experiment (figure 6c), we also noticed that the cell viability after the four-hour EtOH treatment was low in both cell lines when the cells were observed under the microscope (not reported) even though the raw data from the cell viability measurements suggested a relatively high viability. For these limitations, the normalization of the data would bias the interpretation of the results and therefore the data from these measurements is presented only as raw data. 21 22 Figure 6. Cell vulnerability to EtOH-induced oxidative stress (raw data). a.) Cells were seeded at the density of 6x104 per well. The results suggest that the overall viability of MANF- knockout hESCs is low and that the viability of wild-type hESCs decreases in a dose- dependent manner when treated with EtOH. b.) Cells were seeded at the density of 7.5x104 per well. The results suggest that the viability of wild-type hESCs starts decreasing in a dose- dependent manner when the EtOH concentration is 2% or higher. The same may be true for MANF-knockout cells but the trend is less clear. c.) Cells were seeded at the density of 7.5x104 per well. The results suggest that the cell viability of both cell lines decreases in a dose-dependent manner when the EtOH concentration is 2% or higher. 3.4. Nuclear translocation of ZSCAN2 takes place in MANF-knockout hESCs but not in wild-type hESCs when the cells are exposed to oxidative stress The first part of this experiment was repeated twice. The results suggest that ZSCAN2 TF is expressed predominantly in the cell cytoplasm of both wild-type and MANF-knockout hESCs. However, when the cells are exposed to a 50 μM H2O2 treatment, nuclear translocation takes place in MANF-knockout hESCs but not in wild-type hESCs (figures 7a & 7b). In the second part of this experiment, we wanted to investigate whether nuclear translocation takes place in wild-type hESCs when the cells are exposed to higher concentrations of H2O2 (100 µM, 200 µM) and whether a treatment with MANF protein influences ZSCAN2 expression. Unfortunately, this part of the experiment was only partially successful, as imaging the coverslips showed that some staining was unsuccessful and some coverslips with MANF-knockout cells contained only very few cells, making imaging very difficult. Therefore, not all results can be reported. However, the ones that can be reported suggest that nuclear translocation does not take place in wild-type hESCs even after as high a H2O2 treatment as 200 μM treatment. The DAPI staining shows that at this high concentration the cell nuclei have experienced damage illustrating that this treatment has deteriorated the cells (figure 7c). 23 Figure 7. ZSCAN2 expression in wild-type and MANF-knockout hESCs in control condition and after exposure to oxidative stress. a) ZSCAN2 expression (red fluorescence) is predominantly cytoplasmic in all conditions except in MANF-knockout hESCs after a 24-hour treatment with 50 μM H2O2 treatment. For MANF-knockout hESCs without a H2O2 treatment, the staining was unsuccessful. b) ZSCAN2 expression is predominantly cytoplasmic in MANF- knockout hESCs without a H2O2 treatment but nuclear in MANF-knockout treated with 50 μM H2O2 for 24 hours. c) ZSCAN2 expression is predominantly cytoplasmic in wild-type hESCs after a six-hour 200 μM H2O2 treatment. 4. Discussion Here, we have investigated how MANF-knockout hESCs differ from wild-type hESCs in terms of how sensitive they are and how they react to oxidative stress. Our findings suggest that MANF-knockout hESCs react to H2O2-induced oxidative stress differently than wild-type 24 hESCs. Once the cells were exposed to oxidative stress (a 50 μM H2O2 treatment for 24 hours), ZSCAN2 TF translocated in the nuclei of the MANF-knockout hESCs suggesting that the TF becomes active and hence influences the expression of its target genes. We did not detect this kind of nuclear translocation in the wild-type hESCs even when the wild-type cells were exposed to very high levels of oxidative stress (a 200 μM H2O2 treatment for six hours). To our knowledge, this finding has not been reported before, and it increases our understanding of both MANF and the highly understudied ZSCAN2, suggesting that MANF plays a role in ZSCAN2 expression in hESCs. Considering that, to date, only very little is known about the physiological role of ZSCAN2 and how it regulates genes, we can only speculate what this finding means. It might, however, suggest that MANF protects the cells from the detrimental effects that follow ZSCAN2 activation. The other findings of this master’s thesis project confirm our group’s earlier findings according to which MANF-knockout hESCs do not express Catalase that decomposes H2O2 into water and oxygen. Therefore, one of the most central aims of this study was to investigate whether the MANF-knockout hESCs are more sensitive to oxidative stress than wild-type hESCs utilizing H2O2 and EtOH treatments. Based on our results, there are signs of such a phenomenon, but the differences failed to reach a statistically significant level. Importantly, there was a lot of variation between different measurements, for which reason the results should be interpreted very cautiously and critically. Our results suggest that, when treated with EtOH, a four-hour treatment is enough to reduce the viability of hESCs and cell viability clearly decreases when the EtOH concentration is 2% or higher. However, this EtOH concentration is physiologically very high, and for example one previously published study suggested that a four-hour treatment with 0.3% EtOH is enough to cause apoptosis and deteriorate cell proliferation in mouse embryonic stem cells (Huang et al., 2007). Similar findings have been made also with hESCs, but the EtOH treatment times have been significantly longer, days instead of hours (Nash et al., 2012; Palmer et al., 2012). This study had several limitations. In the experiment in which we exposed the hESCs to H2O2-induced oxidative stress, the cells were exposed to oxidative stress at the same time with a ROCK inhibitor, which is used to promote stem cell survival and inhibit apoptosis in stem cell culture (Watanabe et al., 2007). In other words, this experiment was in fact investigating whether a ROCK inhibitor can rescue the hESCs that are exposed to oxidative stress without using a relevant control condition. Therefore, it is likely that this design biased 25 the results as the cells were treated with two opposing reagents: H2O2, which induces oxidative stress, and ROCK inhibitor, which promotes cell survival. Another significant limitation has to do with the fact that, while culturing the two studied cell lines (i.e., wild-type and MANF-knockout hESCs), we have noticed that the cells proliferate at a different pace: indeed, the proliferation of the MANF-knockout hESCs is slower than that of the wild-type hESCs. This quality makes designing experiments more challenging and has likely influenced the results reported here, as the used cell viability reagent PrestoBlue measures cell proliferation quantitatively. A minor limitation is that the PrestoBlue cell viability reagent we used had been stored opened at 4°C for two years or over, while according to the manufacturer, the reagent’s shelf life is up to 20 months. Also, it has been suggested that this standard PrestoBlue cell viability may not be as sensitive as possible in detecting cell viability especially if it used to investigate cells with an impaired metabolism (Luzak et al., 2022). It is certainly plausible to assume that the induced oxidative stress has impaired the metabolism of the hESCs studied in our experiments even though this wasn’t tested. However, to minimize the influence of some other disadvantages of the selected cell viability assay, only E8 media and oxidative stress-causing compounds were added in some of the cells to control for background fluorescence and two different compounds (H2O2 and EtOH) were tested to see if a certain compound biases the fluorescence signal. One more limitation is that, when exposing the hESCs to EtOH-induced oxidative stress, we noticed that, at times, also the cultured cells (one cell line at a time) were proliferating poorly. Therefore, an unidentified third factor may have influenced the results, as the overall viability wasn’t comparable between the cell lines. Put together, these limitations may have influenced the inconsistency and thus the reliability of the cell viability results we have reported here. To conclude, our results suggest that MANF-knockout hESCs react to oxidative stress differently than wild-type hESCs. Especially our finding according to which ZSCAN2 TF translocated to cell nuclei in MANF-knockout hESCs after exposing the cells to H2O2 is a very interesting finding, as it may have revealed something new about the functioning of both MANF and ZSCAN2. These findings need to be confirmed using a method that quantifies the ZSCAN2 expression levels across experimental conditions (e.g., cellular fractionation with Western blot). Due to time constraints, it was not possible to perform this type of an experiment during my time in our research group, but I have contributed to the future experiments by collecting and freezing cell pellets that will be utilized by other group members while performing follow-up experiments. Future studies should also validate the 26 anti-ZSCAN2 primary antibody that was used here and consider replicating the experiment using another relevant antibody. Whether the MANF-knockout hESCs are more sensitive to oxidative stress than wild-type hESCs is still under debate and needs to be confirmed in future studies. The findings reported here help design future experiments as the results, despite their limitations, have suggested, for example, that a four-hour exposure to EtOH is enough to reduce viability of hESCs. In the future, it would be particularly interesting to investigate whether we could increase the survival of stressed MANF-knockout hESCs’s by using, for example, antioxidants that have been shown to make stem cells more resilient toward oxidative stress and support their viability (Shaban et al., 2017). Moreover, it would be interesting to study whether a longer EtOH exposure with lower, physiologically relevant EtOH concentrations influences the viability of wild-type and MANF-knockout hESCs differently. This would be relevant to investigate, as it might provide new information on the effects of maternal alcohol use on early-stage embryos and whether MANF helps protect these cells from the oxidative stress caused by EtOH. 6. Acknowledgements I want to express my gratitude to the whole Airavaara lab for the warm welcome in the group. I want to thank especially Dr. Vassilis Stratoulias for offering this once-in-a-lifetime learning opportunity; Amanda Sandelin and Jenni Montonen for the countless hours they have dedicated in teaching and helping me; Professor Mikko Airavaara for giving valuable feedback on the manuscript; and Arianna Arbona for the irreplaceable peer support. Additionally, I would like to thank Dr. Eva Ruusuvuori for her support and kindness, and for always being available when I had questions or concerns during my master’s thesis project. Last but definitely not the least, I want to thank my dearest and nearest for all the practical and emotional support they have offered over the years. Most importantly, I want to thank Antti for being there for me, supporting me, and encouraging me throughout my student years that quite often felt never-ending. 27 References Andersen, J. K. (2004). Oxidative stress in neurodegeneration: cause or consequence? Nature Medicine, 10(S7), S18–S25. https://doi.org/10.1038/nrn1434 Arnold, K., Sarkar, A., Yram, M., Polo, Jose M., Bronson, R., Sengupta, S., Seandel, M., Geijsen, N., & Hochedlinger, K. (2011). Sox2+ Adult Stem and Progenitor Cells Are Important for Tissue Regeneration and Survival of Mice. Cell Stem Cell, 9(4), 317– 329. https://doi.org/10.1016/j.stem.2011.09.001 Back, S. H., & Kaufman, R. J. (2012). Endoplasmic Reticulum Stress and Type 2 Diabetes. Annual Review of Biochemistry, 81, 767–793. https://doi.org/10.1146/annurev- biochem-072909-095555 Bemer, M., van Dijk, A. D. J., Immink, R. G. H., & Angenent, G. C. (2017). Cross-Family Transcription Factor Interactions: An Additional Layer of Gene Regulation. Trends in Plant Science, 22(1), 66–80. https://doi.org/10.1016/j.tplants.2016.10.007 Blesch, A. (2006). Neurotrophic Factors in Neurodegeneration. Brain Pathology, 16(4), 295– 303. https://doi.org/10.1111/j.1750-3639.2006.00036.x Chandramouleeswaran, P. M., Guha, M., Shimonosono, M., Whelan, K. A., Maekawa, H., Sachdeva, U. M., Ruthel, G., Mukherjee, S., Engel, N., Gonzalez, M. V., Garifallou, J., Ohashi, S., Klein-Szanto, A. J., Mesaros, C. A., Blair, I. A., Pellegrino da Silva, R., Hakonarson, H., Noguchi, E., Baur, J. A., & Nakagawa, H. (2020). Autophagy mitigates ethanol-induced mitochondrial dysfunction and oxidative stress in esophageal keratinocytes. PloS one, 15(9), e0239625. https://doi.org/10.1371/journal.pone.0239625 28 Chen, F., Liu, Y., Wong, N.-K., Xiao, J., & So, K.-F. (2017). Oxidative Stress in Stem Cell Aging. Cell Transplantation, 26(9), 1483–1495. https://doi.org/10.1177/0963689717735407 Chong, W., Shastri, M., & Eri, R. (2017). Endoplasmic Reticulum Stress and Oxidative Stress: A Vicious Nexus Implicated in Bowel Disease Pathophysiology. International Journal of Molecular Sciences, 18(4), 771. https://doi.org/10.3390/ijms18040771 Eesmaa, A., Yu, L. Y., Göös, H., Nõges, K., Kovaleva, V., Hellman, M., Zimmermann, R., Jung, M., Permi, P., Varjosalo, M., Lindholm, P., & Saarma, M. (2021). The cytoprotective protein MANF promotes neuronal survival independently from its role as a GRP78 cofactor. The Journal of biological chemistry, 296, 100295. https://doi.org/10.1016/j.jbc.2021.100295 Huang, L.-H., Shiao, N.-H., Hsuuw, Y.-D., & Chan, W.-H. (2007). Protective effects of resveratrol on ethanol-induced apoptosis in embryonic stem cells and disruption of embryonic development in mouse blastocysts. Toxicology, 242(1-3), 109–122. https://doi.org/10.1016/j.tox.2007.09.015 Huang, M., Chen, Y., Han, D., Lei, Z., & Chu, X. (2019). Role of the zinc finger and SCAN domain-containing transcription factors in cancer. American Journal of Cancer Research, 9(5), 816–836. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6556609/ Ikäheimo, K., Herranen, A., Iivanainen, V., Lankinen, T., Aarnisalo, A. A., Sivonen, V., Patel, K. A., Demir, K., Saarma, M., Lindahl, M., & Pirvola, U. (2022). MANF supports the inner hair cell synapse and the outer hair cell stereocilia bundle in the cochlea. Life Science Alliance, 5(2), e202101068. https://doi.org/10.26508/lsa.202101068 29 Ionescu-Tucker, A., & Cotman, C. W. (2021). Emerging roles of oxidative stress in brain aging and Alzheimer's disease. Neurobiology of aging, 107, 86–95. https://doi.org/10.1016/j.neurobiolaging.2021.07.014 Kamiloglu, S., Sari, G., Ozdal, T., & Capanoglu, E. (2020). Guidelines for cell viability assays. Food Frontiers, 1(3), 332–349. https://doi.org/10.1002/fft2.44 Lindholm, P., & Saarma, M. (2010). Novel CDNF/MANF family of neurotrophic factors. Developmental neurobiology, 70(5), 360–371. https://doi.org/10.1002/dneu.20760 Liu, Y., Li, P., Fan, L., & Wu, M. (2018a). The nuclear transportation routes of membrane- bound transcription factors. Cell communication and signaling : CCS, 16(1), 12. https://doi.org/10.1186/s12964-018-0224-3 Liu, Y., Zhang, J., Jiang, M., Cai, Q., Fang, J., & Jin, L. (2018b). MANF improves the MPP+/MPTP-induced Parkinson’s disease via improvement of mitochondrial function and inhibition of oxidative stress. American Journal of Translational Research, 10(5), 1284–1294. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992546/ Lord, T., Law, N. C., Oatley, M. J., Miao, D., Du, G., & Oatley, J. M. (2022). A novel high throughput screen to identify candidate molecular networks that regulate spermatogenic stem cell functions. Biology of Reproduction, 106(6), 1175–1190. https://doi.org/10.1093/biolre/ioac048 Luzak, B., Siarkiewicz, P., & Boncler, M. (2022). An evaluation of a new high-sensitivity PrestoBlue assay for measuring cell viability and drug cytotoxicity using EA.hy926 endothelial cells. Toxicology in Vitro, 83, 105407. https://doi.org/10.1016/j.tiv.2022.105407 Mahdinia, R., Goudarzi, I., Lashkarbolouki, T., & Elahdadi Salmani, M. (2021). Maternal ethanol exposure induces behavioral deficits through oxidative stress and brain- derived neurotrophic factor interrelation in rat offspring. International Journal of 30 Developmental Neuroscience: The Official Journal of the International Society for Developmental Neuroscience, 81(8), 717–730. https://doi.org/10.1002/jdn.10148 Misrani, A., Tabassum, S., & Yang, L. (2021). Mitochondrial Dysfunction and Oxidative Stress in Alzheimer’s Disease. Frontiers in Aging Neuroscience, 13. https://doi.org/10.3389/fnagi.2021.617588 Montaser, H., Patel, K. A., Balboa, D., Ibrahim, H., Lithovius, V., Näätänen, A., Chandra, V., Demir, K., Acar, S., Ben-Omran, T., Colclough, K., Locke, J. M., Wakeling, M., Lindahl, M., Hattersley, A. T., Saarimäki-Vire, J., & Otonkoski, T. (2021). Loss of MANF Causes Childhood-Onset Syndromic Diabetes Due to Increased Endoplasmic Reticulum Stress. Diabetes, 70(4), 1006–1018. https://doi.org/10.2337/db20-1174 Nash, R., Krishnamoorthy, M., Jenkins, A., & Csete, M. (2012). Human embryonic stem cell model of ethanol-mediated early developmental toxicity. Experimental Neurology, 234(1), 127–135. https://doi.org/10.1016/j.expneurol.2011.12.022 Palmer, J. A., Poenitzsch, A. M., Smith, S. M., Conard, K. R., West, P. R., & Cezar, G. G. (2012). Metabolic biomarkers of prenatal alcohol exposure in human embryonic stem cell-derived neural lineages. Alcoholism, Clinical and Experimental Research, 36(8), 1314–1324. https://doi.org/10.1111/j.1530-0277.2011.01732.x Petrova, P., Raibekas, A., Pevsner, J., Vigo, N., Anafi, M., Moore, M. K., Peaire, A. E., Shridhar, V., Smith, D. I., Kelly, J., Durocher, Y., & Commissiong, J. W. (2003). MANF: a new mesencephalic, astrocyte-derived neurotrophic factor with selectivity for dopaminergic neurons. Journal of Molecular Neuroscience: MN, 20(2), 173–188. https://doi.org/10.1385/jmn:20:2:173 Rumgay, H., Murphy, N., Ferrari, P., & Soerjomataram, I. (2021). Alcohol and Cancer: Epidemiology and Biological Mechanisms. Nutrients, 13(9), 3173. https://doi.org/10.3390/nu13093173 31 Shaban, S., El-Husseny, M. W. A., Abushouk, A. I., Salem, A. M. A., Mamdouh, M., & Abdel-Daim, M. M. (2017). Effects of Antioxidant Supplements on the Survival and Differentiation of Stem Cells. Oxidative medicine and cellular longevity, 2017, 5032102. https://doi.org/10.1155/2017/5032102 Surmeier, D. J. (2018). Determinants of dopaminergic neuron loss in Parkinson’s disease. The FEBS Journal, 285(19), 3657–3668. https://doi.org/10.1111/febs.14607 Voutilainen, M. H., Bäck, S., Peränen, J., Lindholm, P., Raasmaja, A., Männistö, P. T., Saarma, M., & Tuominen, R. K. (2011). Chronic infusion of CDNF prevents 6- OHDA-induced deficits in a rat model of Parkinson’s disease. Experimental Neurology, 228(1), 99–108. https://doi.org/10.1016/j.expneurol.2010.12.013 Voutilainen, M. H., Bäck, S., Pörsti, E., Toppinen, L., Lindgren, L., Lindholm, P., Peränen, J., Saarma, M., & Tuominen, R. K. (2009). Mesencephalic astrocyte-derived neurotrophic factor is neurorestorative in rat model of Parkinson’s disease. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 29(30), 9651–9659. https://doi.org/10.1523/JNEUROSCI.0833-09.2009 Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J. B., Nishikawa, S., Nishikawa, S., Muguruma, K., & Sasai, Y. (2007). A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnology, 25(6), 681–686. https://doi.org/10.1038/nbt1310