Y Chromosome Genes May Play Roles in Directed Differentiation of Human Embryonic Stem Cells.

The human Y chromosome harbors genes that are mainly involved in the growth, development, sexual dimorphism, and spermatogenesis process. Despite many studies, the function of the male-specic region of the Y chromosome (MSY) awaits further clarication, and a cell-based approach can help in this regard. In this study, we have developed four stable transgenic male embryonic stem cell (ESCs) lines that can overexpress male-specic genes HSFY1, RBMY1A1, RPS4Y1, and SRY. As a proof of principle, we differentiated one of these cell lines (RPS4Y1 over-expressing ESCs) to the neural stem cell (rosette structure) and characterized them based on the expression level of lineage markers. RPS4Y1 expression in the Doxycycline-treated group was signicantly higher than control groups in transcription and protein levels. Furthermore, we found Doxycycline-treated group had a higher differentiation eciency than the untreated control groups. Our results suggest that the RPS4Y1 gene may play a critical role in neurogenesis. Also, the generated transgenic ESC lines can be widely employed in basic and preclinical studies, such as sexual dimorphism of neural and cardiac functions, the development of cancerous and non-cancerous disease models, and drug screening. with three biological and technical replicates (control and test). Signicant differences between groups were statistically analyzed using an ordinary one-way, two-way ANOVA tests and a two-tailed unpaired student’s t-test in Graphpad Prism 8 (Graphpad Software). Data are displayed as mean ± standard error of the mean (SEM) and ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 indicated statistically signicant in all the experiments.


Introduction
The human Y chromosome is a male sex-determination chromosome and escapes meiotic recombination. Despite its small size and limited gene content, the Y chromosome has crucial roles in development, differentiation, as well as gender-based diseases, which are often con ned to the hypothesis (Bellott et al., 2014).
The Y chromosome harbors 47 protein-coding genes, where 35 of them have protein evidence (PE) at protein level (PE1), 5 proteins at the transcript level (PE2), and 7 genes encoding uncertain proteins (PE5) (https://www.nextprot.org/about/protein-existence). Therefore, Chromosome-centric Human Proteome Project (C-HPP) was launched in 2012, to identify human missing proteins and investigate their functions. (Meyfour et al., 2019). The human Y Chromosome Proteome Project (Y-HPP) is trying to recognize the functions of the Y chromosome's proteins (Jangravi et al., 2013).
In pursuit of this goal, several studies have been undertaken, including a tumor suppressor role for KDM5D in the prostate cancer cell line (Jangravi et al., 2015) and new roles for DDX3Y in neural differentiation of NTERA-2 human embryonal carcinoma cells (NT2) , as well as TBL1Y and also KDM5D in cardiac differentiation of hESC (Meyfour et al., 2019, Meyfour et al., 2017a. Since many genes are silent in adult cells, investigating the functions of their proteins is challenging. To meet this challenge (pluripotent stem cells) PSCs might be used as a valuable tool to achieve proteins and missing proteins function, which is also a target of the Y chromosome project (Alikhani et al., 2020).
These studies highlighted the remarkable role of some genes that help to candidate the target genes for more investigations. Among genes of interest, theexpression of RPS4Y1 showed sexual dimorphism of the kidney, and SRY mediated kidney development and played a role in the blood pressure control in males (Meyfour et al., 2017b).
The expression of RBMY1, HSFY1, RPS4Y1, and SRY increased during the neural cell differentiation of the NT2 cell line . Furthermore, The overexpression of RBMY1, HSFY1, RPS4Y1, and several other MSY genes, was observed at the transcriptional level during the cardiac differentiation of hESCs (Meyfour et al., 2017a).
Genes located in the azoospermia factor (AZF) regions have a critical role in spermatogenesis and fertility, and AZF microdeletion can impair their functions. Our target genes, RBMY1A1 and HSFY1, are in the AZF region. The RBMY1 family consists of approximately 30 genes and pseudogenes in six subgroups on the Y chromosome. RBMY1A1 contains a conserved domain called the SRGY motif (serine, arginine, glycine, and tyrosine) that modulated its function. RBMY1 plays a signi cant role in cardiac development, spermatogenesis, infertility, prostate cancer, and hepatocellular carcinoma (HCC) (Meyfour The Heat Shock Factors (HSFs) are a family of transcription factors that encode chaperones (Duchateau et al., 2020). Their critical role is stress responses in abnormal conditions such as oxidative stress, thermal stress, hypoxia, and protein degradation (Chatterjee and Burns, 2017). They also participate in gametogenesis, embryonic development, and the integrity of the organ. Deregulation of HSFs could be a risk factor for reproductive failure, cancer, neurogenesis, and neurodegenerative disorders (Ma, 2000). HSFY (heat shock transcription factor, Y chromosome) is a member of HSFs and has been located in the AZFb region of the Y chromosome (Rosenfeld, 2017). This gene is associated with spermatogenic failure, infertility (Peng, 2009;Zenteno-Ruiz, 2001), varicoceles (Meyfour, 2017), maturation arrest (MA) (McCann-Crosby, 2014), sertolicell-onlysyndrome (Meyfour, 2017) and plays a crucial role in the brain and cardiac development (Meyfour et al., 2017b). SRY (sex determining region Y) encodes a transcription factor (a member of the high mobility group [HMG]-box family) (Jangravi et al., 2013) and involved in spermatogenesis, male sex determination, brain sexual differentiation, epigenetic processes, brain, cardiac and kidney development ( (Kido and Lau, 2015) and prostate cancers (Ely et al., 2010), and brain disorders (Wu et al., 2009). SRY expression and function in the human brain have been examined in several studies (Rosenfeld, 2017, Wu et al., 2009, Kido et al., 2017. SRY suppression in the human neuroblastoma cell line led to a downregulation in enzymes involved in the dopamine synthesis of males and may describe the cause of more susceptibility of males in dopaminergic-based neurological disorders (Parkinson's disease and schizophrenia) (Loke et al., 2015).
Although the SRY may be needed for the normal function of the male brain, an aberrant expression of this gene could impair neurogenesis and other disorders in mice pups (Kido et al., 2017). The SRY expression level in primed hESCs was higher than naive and embryoid bodies derived from hESCs (Taleahmad et  Each of the target genes has various molecular and biological functions in different signaling pathways and human diseases (Table 1). Although, the precise mechanisms of functions have not been entirely understood. Most of the information that researchers identi ed are speculations based on the similarity of homologous regions to other genes. Gene Ontology (GO) of target genes is listed in Table 1.
Based on the studies, we candidate HSFY1, SRY, RBMY1A1, and RPS4Y1 genes for more investigations (Skuse, 2000, Serajee and Mahbubul Huq, 2009, Lau and Zhang, 2000. To this end, we described development of the transgenic cell lines over-expressing target genes in an inducible manner. Then, they were characterized in terms of karyotype, pluripotency, and integrated gene expression level. We also discussed the applications of them in clinic and research. In this study, we used a cell-based approach, using hESCs to generate four inducible cell lines able to increase gene of interest expression. As a proof of principle, we then differentiated one of these cell lines (RPS4Y1 over-expressing ESCs) to the neural stem cells.
RPS4Y1 is expressed during prenatal and infancy; indicating, it could play a critical role in early brain development (Meyfour et al., 2017b). Our results indicate a possible sex-dependent regulation of neural development that might underlie sexual dimorphism of human brain.

Cell line generation and characterization
We cloned the genes of interest by gateway technology and veri ed them with DNA sequencing. After the production of the lentiviral particles, the transduced cells were titrated using puromycin treatment until all of the un-transduced cells (negative control) were died ( Supplementary Fig 1. A Before cell transduction, we required to know about the multiplicity of infection (MOI) of the target cells. We found out, at MOI of 40, almost 30% of cells were transduced. (Supplementary Fig 3. A-D). Furthermore, the cellular toxicity of polybrene was assessed. The optimal polybrene concentration for hESC-RH6 was 6 µg/ml ( Supplementary Fig 3. E, F).
Following 10 to14 days, the stem cell colonies were formed and were transferred into the new plates ( Fig  2. A). Morphological characteristics of transgenic cell lines were observed using light microscopy. Then their pluripotency was con rmed by alkaline phosphatase activity assay. The expression level of the pluripotency markers (OCT4, NANOG, and SOX2) were evaluated by qRT-PCR (Fig 1. A, B) and there were no signi cant differences between over-expressing cell lines and hESC-RH6 in pluripotency markers. The qRT-PCR results were statistically analyzed by t-test and SD's and showed a signi cant rise (P < 0.05). Giemsa banding pattern was shown a normal karyotype for new stable transgenic cell lines ( Supplementary Fig 4. A).
The mock cell line (RH6-GFP) was characterized based on morphological characteristics. Doxycyclineinduced expression of the mock promoter was observed by uorescence microscopy. Purple dye detection in alkaline phosphatase staining con rmed the pluripotency state (Fig 1. C).
For the characterization of transgenic stable cell lines, PCR performed using puromycin primers and the accuracy of gene integration was veri ed. After that, the DNA band size of the puromycin resistance gene (383 bp) was con rmed. In the following comparison between the three cell groups, we have shown differences between our target genes expression in hESC-RH6 and untreated transgenic cell lines (as

Characterization based on qRT-PCR, immunocytochemistry assay and western blotting
After the development of stable transgenic cell lines, "RPS4Y1 over-expressing ESCs" were differentiated to neural stem cells. Following the RPS4Y1 overexpression, the Rosette structures in the test group (treated by DOX) were more elegant and more than control groups (wild-type and untreated transgenic cell lines), especially on the last day (Fig 3. A, B).
The results of the immuno uorescence test showed an increase in RPS4Y1 protein expression in the treated group compared to untreated group in the process of neural differentiation (Fig 3, C). Moreover, the rosette markers (SOX1 and NESTIN) were increased in the treated group versus untreated group using immunostaining (Fig 3, D).
Based on Western blot data, RPS4Y1 upregulation in ESC-RH6 at pluripotent state (Day 0) was observed, and then RPS4Y1 downregulation during neural differentiation (Day 3) was seen. In the end, RPS4Y1 expression was re-increased (Day 8) (Fig 3.E). The transgenic-untreated group (DOX negative) was shown the same trend, but doxycycline treatment changed that in the treated group in comparison with the control groups (Fig 3.E).
To prove the neural differentiation, we showed overexpression of SOX1, PAX6, and NESTIN in all three groups (test and controls) by qRT-PCR. An increasing trend in all groups was observed, but in the treated group (DOX positive), neural lineage markers (SOX1, PAX6, and NESTIN) upregulated signi cantly compare to the control groups (Fig 3, F-H).
In the pluripotent state (ESC-RH6 on day 0), we observed RPS4Y1 upregulation, and after inducing neural differentiation, RPS4Y1 expression decreased (day 3) and on the last day (Day 8) re-increased. The same trend was in the transgenic cell lines; however, the difference in RPS4Y1 expression of the treated group (DOX positive) was signi cantly more than the control groups (Fig 3. I).

Discussion
Although much of our current knowledge of the primary cell lines are derived from mouse models, the mice have weak similarities to the human in the developmental process ( The importance of balance in the expression of genes is obvious, and any alterations in copy number or expression level of a wild-type gene can lead to mutated phenotypes (Kido and Lau, 2015).
In this regard, the researchers investigated the functions of some MSY genes by knocking down gene expression. Knockdown of RPS4Y1, KDM5D DDX3Y TBL1Y, and SRY expression studied in human or mice models, and thereby the novel roles for these genes discovered in the brain, cardiac, carcinogenesis, pluripotency, etc. Overexpression experiments may be a powerful tool for linking genes to biological pathways. Generally, overexpression of individual genes performs for two purposes: desired amounts of gene products are obtained that can be used in drug screening and other studies. On the other hand, possible biological functions of target gene products can be determined (Prelich, 2012).
One of the most validated ways toward gain-of-function studies is to generate stable cell lines, and the best technique for gene integration in hESCs is transduction by lentiviruses (Magrin et al., 2019, Milone and O'Doherty, 2018). After the generation of over-expressing stable cell lines, monitoring of the gene integration e ciency and the induced target gene overexpression during the differentiation is essential. Moreover, maintenance of pluripotency, viability, and normal morphology of the cells should be considered.
In addition to many roles, we have already mentioned for the Y chromosome, genes on this chromosome play crucial roles in brain function. Gender differences in brain development and prevalence of neuropsychiatric disorders such as depression, autism, parkinson, schizophrenia, and attentionde cit/hyperactivity disorder (ADHD) were reported. Expression of sex chromosome genes may contribute to gender differences in brain functions, and act independently of gonadal hormones (Liu et al., 2017, Kopsida et al., 2009).
One study investigated the effects of increasing in the copy number of genes. Data showed that increased doses of Y chromosome genes are associated with the risk of autism-related behaviors and ADHD symptoms in male with XYY syndrome. In this study, NLGN4Y expression level, through the effects on the synaptic function, is related to more severe degrees of the autism (Ross et al., 2015). Another study reported a set of preeclampsia molecular risk factors, which may lead brain development toward an inclusive risk of autism. One of the these stimuli, is the gene encoded by the Y chromosome; RPS4Y1, a STAT3 signaling inhibitor, that acts as a sex-based differential factor in male dominance in Autism (Xie et al., 2020).
In the current study, stable transgenic cell lines, were generated using lentivectors. First, we cloned target genes, produced lentiviral particles, and transduced ESC-RH6 cells via lentivectors. Ultimately, we generated four stable transgenic cell lines contain genes of interest (HSFY1, SRY, RBMY1A1, and RPS4Y1), and one as the mock cell line containing the same vector but expressing GFP reporter instead of the target gene. These transgenic cell lines, in addition to displaying the characteristics of the hESCs could overexpress the target genes by doxycycline treatment. The accuracy of the gene of interest integration was con rmed at the DNA level. The karyotype of the transgenic cell lines was normal, and also we showed the pluripotency potential in the test and control groups. Furthermore, we reported differences in mRNA expression level between test and control populations (DOX positive and negative) and proved that the mRNA expression level in the test group (DOX positive) was signi cantly higher than controls. Additionally, we showed these signi cant changes at the protein expression level between the test and control groups.
As a proof of principle, to investigate the overexpression effects in the differentiation process, the RPS4Y1 over-expressing cell line, was differentiated to neural stem cells based on the literature. Then, we evaluated the expression of neural and pluripotency markers, as well as RPS4Y1 on days 0, 3, and 8 in both test and control groups. The morphological and molecular changes of the test and controls were monitored during neural differentiation up to the formation of rosette structures. In general, following the RPS4Y1 overexpression, differentiation e ciency increased and the number of Rosette structures in the treated group was more than control groups, especially on the last day. On the other hand, the RPS4Y1 expression pattern at the transcription and protein levels indicates a decrease in the expression of this gene in all groups on day 3, followed by an increase in expression on day 8 of neural differentiation, compared to the undifferentiated state.
Therefore, it could be concluded that in the natural state, the RPS4Y1 gene increases the e ciency of neural differentiation and may contribute to create Rosette structures. Accordingly, RPS4Y1, as a structural protein of the ribosome, may play a vital role in the development of the brain and nerve cells.
Moreover, these stable transgenic cell lines are valuable tools for the gain-of-function studies in Y chromosome-linked genes. Inducible overexpression of target genes during differentiation into the desired cell fate may lead to changes in the e ciency or morphology of the differentiated cells. These changes could indicate a possible modi cation in signaling pathways. Further analysis is required to identify a protein interaction network of target genes. The generated cell lines could be widely used in basic and preclinical studies, such as sexual dimorphism of neural and cardiac functions, the cancerous and noncancerous modeling, and drug screening. These stable cell lines would provide a step forward in the identi cation of MSY genes functions and their network of protein interactions. Inducible overexpression of the MSY genes in female ESCs can use to further understanding of their roles and consider as promising progress in C-HPP goals.

Production of lentiviral particles.
We used a second-generation packaging vector including 5µg packaging vectors (psPAX2 and pMD2.G from Sigma-Aldrich, Germany) and 5µg inducible vector contains target genes (pLIX-403 vector -Tet-On) for lentiviral particles production. On the rst day, the HEK293T cells were cultured at 5×10 5  The optimal concentration is the lowest amount that kills around 100% of un-transduced cells versus transduced cells in 3-4 day (Duchateau, 2020). Before titration and antibiotic selection, the optimal antibiotic concentration was estimated by GFP labeled lentiviral particles tittering with ow cytometry (Dasari, 2001). These viral particles (GFP labeled) were used to determine the Multiplicity of infection (MOI) and also to produce a mock cell line (Supplementary Method 1) (Ma et al., 2003).

Cell culture and transduction.
After some passages, approximately 1×10 5 single hESCs were seeded in two tests, and one control well of a six-well Matrigel-coated plate. hESC medium with 10 mM Rho-associated protein kinase inhibitor (ROCKi; Sigma-Aldrich, Germany) was used for that. 24 hours after seeding, the cells were transduced with lentiviral particles that contained 6 μg/ml polybrene and incubated in the condition mentioned above (Supplementary Method 2).

Antibiotic selection and colony pickup.
The medium was refreshed 24 hours after transduction with the hESC medium. For the cell line selection, transduced cells were treated by the optimal dosage of puromycin for around 4-5 days, and the medium was refreshed every day until the stable colonies were observed. However, due to the toxicity of puromycin, an appropriate dosage needed to be determined (Supplementary Method 3). Consequently, the colonies were picked up, and after some sub-culturing, were treated with 5 μg/mL Doxycycline (Takara Bio USA) for 24-65 hours. The new colonies called HSFY1, RBMY1A1, RPS4Y1, and SRY overexpressing transgenic cell lines.

Neural differentiation
We induced feeder-free hESC-RH6 and RPS4Y1 over-expressing cell line that was differentiated for eight days into the neural stem cell in the Dulbecco's modi ed Eagle's medium and Ham's F-12 (Invitrogen, USA), 5% knockout serum replacement (KOSR; Gibco BRL, Paisley, UK), 1% nonessential amino acids (NEAAs; Invitrogen, USA), l-glutamine ( The pluripotency, karyotype, and mycoplasma contamination of over-expressing cell lines were investigated by alkaline phosphatase activity assay (Sigma-Aldrich, 86R, USA), G-banding chromosome analysis, and PCR assay, respectively. These experiments were performed based on the manufacturer's protocols.
4.8 Molecular con rmation of genomic insertion.
The genomic insertion of target genes in hESCs-RH6 is required to con rm by the PCR. Therefore, genomic DNA from resistant colonies was isolated manually (Supplementary Method 4). We used primer sequences of the puromycin resistance gene as follows: (Forward: 5'-GGTCACCGAGCTGCAAGAAC -3', Reverse: 5'-GCTCGTAGAAGGGGAGGTTG -3'). 150 ng of the DNA template was pooled in a 20 μl total reaction volume containing the primers and Taq master mix 2x (Ampliqon, Herlev, Denmark). The cycling program and subsequent processes were provided in Supplementary Methods. nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) based on the manufacturer's protocol (Sigma-Aldrich, Germany), and cells were observed under the uorescence microscope.

Statistical analysis.
Analysis by the qRT-PCR and western blot was performed with three biological and technical replicates (control and test). Signi cant differences between groups were statistically analyzed using an ordinary one-way, two-way ANOVA tests and a two-tailed unpaired student's t-test in Graphpad Prism 8 (Graphpad Software). Data are displayed as mean ± standard error of the mean (SEM) and * p < 0.05, * * p < 0.01, * * * p < 0.001, and * * * * p < 0.0001 indicated statistically signi cant in all the experiments.
Declarations Table  Table 1.   hESCs. An insert checking PCR for a fragment of puromycin resistance DNA was carried out. The mRNA expression levels of target genes. The differences between the expression level of genes of interest in hESC-RH6 (wild-type group) and DOX negative (untreated group) with DOX positive (treated group) lines shown by qRT-PCR. Data expressed as mean ± SEM. * p < 0.05, * * p < 0.01, * * * p < 0.001 and * * * * p < 0.0001 (ordinary one-way ANOVA test). (D) The protein expression levels of target genes. The differences between the expression level of target proteins in DOX negative compare with DOX positive groups was shown by western blot assay. -actin is used as loading control. Figure 3