The Contribution of Y Chromosome Genes to Spontaneous
Differentiation of Human Embryonic Stem Cells into
Embryoid Bodies In Vitro
Sexual dimorphism in mammals can be described as subsequent transcriptional differences from their distinct sex chromosome complements. Following X inactivation in females, the Y chromosome is the major genetic difference between sexes. In this study, we used a male embryonic stem cell line (Royan H6) to identify the potential role of the male-specific region of the Y chromosome (MSY) during spontaneous differentiation into embryoid bodies (EBs) as a model of early embryonic development.
Materials and Methods
In this experimental study, RH6 cells were cultured on inactivated feeder layers and Matrigel. In a dynamic suspension system, aggregates were generated in the same size and were spontaneously differentiated into EBs. During differentiation, expression patterns of specific markers for three germ layers were compared with MSY genes.
Spontaneous differentiation was determined by downregulation of pluripotent markers and upregulation of
fourteen differentiation markers. Upregulation of the ectoderm markers was observed on days 4 and 16, whereas
mesoderm markers were upregulated on the 8th day and endodermic markers on days 12-16. Mesoderm markers
correlated with 8 MSY genes namely
We found a significant correlation between spontaneous differentiation and upregulation of specific MSY genes. The expression alterations of MSY genes implied the potential responsibility of their gene co-expression clusters for EB differentiation. We suggest that these genes may play important roles in early embryonic development.
Immediately after fertilization, the sex of the human embryo is determined by the spermatozoon carrying either a Y or an X chromosome (1). The sex chromosomes induce specific aspects of organ development in the absence of gonadal sex hormones (2). There are fundamental metabolic differences between female and male preimplantation embryos (3, 4). Briefly, three main aspects of sexual dimorphism have been observed including gene expression profiles, kinetics of growth, and embryonic mortality (5). Male embryos have a greater number of cells and metabolic activities than females with a significantly faster development (6-8).
Sexual dimorphisms are genetically initiated very early in embryonic development (9, 10); however the exact molecular mechanisms leading these differences remain to be comprehended. The sex chromosomes have conserved the essential sex-specific genes on a set of ancestral autosomes (11). Different chromosomal complements can display sexual dimorphism due to the different expression patterns of genes during preimplantation development (12, 13). The X chromosome is inactivated in the differentiated state of human embryonic stem cells (hESCs), causing the same content of the X chromosome in both sexes (14). Otherwise, it typically results in premature abortion and fetal death (15). The Y chromosome is the major genetic difference between sexes and plays an important role in male embryos especially at the preimplantation stage of early fetus development. The Y chromosome size is approximately 60 Mb containing two distinct segments. The male-specific region of the Y chromosome (MSY) contains genes specific to sexual dimorphism and undergoes no meiotic crossing over with a homolog. Two pseudo-autosomal regions flank the MSY on both sides and frequently undergo X−Y crossing over at male meiosis. (16). There are 47 genes on the MSY region as described in NeXtProt, of which 26 genes are validated at protein level (PE1), 11 genes at transcript level (PE2), 3 genes at homology base (PE3) and 7 genes at uncertain level (PE5) (www.nextprot.org, v2.23.1).
The Chromosome-Centric Human Proteome Project (C-HPP) has been established to identify all
proteins encoded by each human chromosome (17, 18). The Y-Chromosome Human Proteome Project
(Y-HPP), as part of C-HPP, identifies and annotates protein products of the Y chromosome
genes using many methods including the cellbased approache, as one of the most important
approaches (19). By taking advantage of hESCs, we can show how Y-HPP has been conducted to
gain a rich understanding of the MSY genes during development. Two individual
characteristics of hESCs make them well-matched for this kind of studies. First, hESCs
provide a unique self-renewal capacity and an abundant source for proteomics analysis.
Second, hESCs offer an interesting opportunity for simulating human embryonic development
In hESCs, a range of tissue-specific differentiation is initiated via the formation of
tissue-like spheroids called embryoid bodies (EBs) (21). EBs are 3-dimensional ESC
aggregates that can determine the major genes involved in early embryogenesis following the
lineage events to form three germ layers (mesoderm, endoderm, and ectoderm) (21-23). The
lineage-specific differentiation of EBs
Materials and Methods
This experimental study was carried out in accordance with the guide for the care and use of laboratory animals and approved by the Local Ethical Committee of Royan Institute for Stem Cell Biology and Technology with a code number IR.ACECR.ROYAN.REC.1396.15.
In this study, Royan H6 (RH6), a human embryonic stem cell line, was cultured on a mouse embryonic fibroblast (MEF) feeder layer. MEFs were mitotically inactivated prior to the addition of the RH6 cells by adding mitomycin C (10 µg/mL, Sigma, Netherlands). The base media for hESC was prepared with a combination of DMEM / F12 (Gibco) supplemented with 20% knockout serum replacement (KOSR, Gibco), 1% nonessential amino acids (Gibco), 1% insulin-transferrin-selenium (ITS, Invitrogen), 0.1mM beta-mercaptoethanol (Sigma, Germany), and 100 units/mL penicillin and 100μg / mL streptomycin (Gibco). Human recombinant bFGF (Basic fibroblast growth factor) (Royan Biotech, Iran) was added to the hESC media (final concentration, 12 ng/ml) at the seeding time. The cell cultures were incubated at 37˚C in a 5% CO2 atmosphere with daily media changes. The cells were passaged upon reaching 70% confluence. Then, RH6 cells were cultured on a thin Matrigel layer in hESC media containing 100 ng/ml bFGF free of any feeder cells for induction of an efficient differentiation. Freshly coated-Matrigel plates were prepared at least 2 hours prior to seeding the cells, according to manufacturer’s instructions. Briefly, for a 6-well plate, 500 μL of diluted Matrigel solution was used per well and incubated at 37˚C to be polymerized. RH6 cells were directly seeded on the wet Matrigel coated plate and allowed to settle for 30-90 minutes in an incubator (5% CO2, 37˚C) before flooding them with culture media. The hESC media was carefully added to each sample well. The cultures were maintained for 7 days, with daily media changes to form the RH6 colonies.
Dynamic suspension of expanded RH6
After two passages on Matrigel, the RH6 cells were transferred to 125 mL spinner flask (Cellspin; Integra Biosciences AG, Switzerland) at a 40rpm agitation rate. For large-scale expansion, a 100-ml working volume was used as previously described (29). Briefly, undifferentiated RH6 cells were cultured with the optimal starting concentration of 2−3×105 cells/mL at the hESC media, which was conditioned by MEFs, fresh 10 mM Rhoassociated kinase inhibitor (ROCKi; Sigma, Netherlands) and 100 ng/mL bFGF. The spinner flask was placed on a magnetic stir plate in an incubator at 37˚C and 5% CO2 without changing media during the first two days. RH6 cells were expanded in a 3D-dynamic suspension culture after 4 days.
Spontaneous differentiation of RH6 into EBs
In the current study, RH6 cells were grown on inactivated feeder layers to gain the growth factors, cytokines and
nutrients required for maintaining pluripotency. The cells
were then transferred onto Matrigel (Sigma, Germany)
to be free of any feeder cells and were prepared for a
successful differentiation. The same size aggregates were
generated from single cells in a dynamic suspension system
and spontaneously differentiated into three embryonic
germ layers of EBs. RH6 3D aggregates were formed in
controlled sizes and shapes by optimizing the agitation
speed, the impeller type and the incubation density for 4
days. The homogeneously sized colonies (175 ± 25 μm,
approximately) were used to generate EBs by inducing
spontaneous differentiation in static suspension condition
for 20 days. The EB differentiation media consisted of
KnockOut DMEM/F-12 base media, supplemented with
20% fetal bovine serum (FBS; Hyclone), 0.05 mM betamercaptoethanol, 1% glutamine (Gibco), 1% essential amino
acids, 100 units/mL penicillin and 100 μg/mL streptomycin.
For spontaneous differentiation, RH6 aggregates were
cultured as a static suspension system in a 6-cm ultra-low
attachment dish containing 5 ml of bFGF-free media for
8 days. The culture media was changed every 2 days. On
day 8, RH6 aggregates were transferred into 0.1% gelatincoated plates to maintain spontaneous differentiation in a 2D
cell culture system for 12 days, hence undergoing a 20-day
differentiation. Samples were collected at several time points
(0, 4, 8, 12, 16 and 20 days) for expression analysis of the
pluripotency and differentiation markers in comparison to the
MSY genes in early embryonic development (Table 1,, See
supplementary online information at
Ribonucleic Acid Isolation and Quantitative RealTime PCR (qRT-PCR)
According to the manufacturer’s protocol, total Ribonucleic acid (RNA) was isolated using
TRIzol reagent (Invitrogen, USA). The purified RNA was reverse-transcribed into cDNA.
Quantitative real-time PCR (qRT-PCR) was performed in the Rotor Gene 6000 (Corbett,
Statistical analysis was performed for three biological replicates of each gene. Data are presented as mean ± SEM. Statistical significance was detected using a twoway ANOVA (∗ P<0.05) in Graphpad Prism software (Graphpad Software, USA). The relative expressions were compared to D0. Heatmap was generated using the heatmap.2 and g-plots libraries in the statistical software R (http://www.r-project.org). Heatmap was used to generate gene co-expression clusters based on pairwise Spearman correlations. Each square determined the correlation value between expression profiles of two genes. According to matched expression profiles, hierarchical clustering trees of the genes were shown in the top and left sides. The circo map was created with circos software (http://www. circos.ca).
Generation of the three embryonic germ layers
To study the role of MSY genes in early embryonic development, RH6 cells were induced to differentiate spontaneously into the three embryonic germ layers of EBs. Stem cells were initially cultured on MEFs and Matrigel as a feeder layer and complex protein matrix, respectively, to maintain self-renewal and pluripotency (Fig .2A,). Then, in a 3D dynamic suspension culture, RH6 single cells formed colonies with the same size and retained the characteristics of an undifferentiated hESC. Stem cell aggregates grew as a homogenous population of small cells forming spheroid clumps with distinct borders (Fig .2B,). Differentiation was spontaneously induced through two sequential steps. At first, the aggregates with equal sizes made distinct cystic structures in a static suspension culture and closely compacted as a dark cavity in the center of the spheroid clumps like a solid ball. Therefore, EBs were well-organized with 3 germ layers which enlarged several times (Fig .2C,). In the next step, EBs were cultured on a gelatin-coated plate as 2D culture systems to sequentially generate endodermal and ectodermal layers (Fig .2D,).
Expression of pluripotency and layer-specific markers during differentiation
We investigated the expression of some specific markers to evaluate cellular
pluripotency and spontaneous differentiation at several time points (0, 4, 8, 12, 16 and
20 days). QRT-PCR was used to investigate the expressions of pluripotency markers
For assessment of spontaneous differentiation, we also compared the expression of
pluripotency and layer-specific markers in all samples. Although layerspecific markers
showed very low expression levels in undifferentiated cells, they increased during RH6
differentiation (Fig .3A,). Spearman correlation was applied by Heatmap to identify clusters
with highly similar temporal expression patterns at several time points. Our analysis
showed four distinct marker clusters (Fig .3B,). The first cluster consisted of pluripotency
The expression pattern of MSY genes in EB
The X-degenerate, X-transposed and ampliconic segments are euchromatic sequences of the
MSY region of the Y chromosome . The
We investigated the expression pattern of Y
chromosome genes to determine the genes involved
in early EB differentiation. In general, our analysis
was performed for 24 genes at protein evidence level
(PE1), 8 genes at transcript evidence (PE2), 3 genes
inferred from homology levels (PE3), as well as 3
genes at uncertain protein level (PE5), that have been
demonstrated in Figure S1,, See supplementary online
MSY genes showed altered expression levels during EB differentiation, as summarized in
Figure 4. The expression pattern of 38 MSY genes was compared by Circos map at several
time points (0, 4, 8, 12, 16 and 20 days, Fig .4,). The inner colored segments were
representative for each specific gene and the outer segments demonstrated the
differentiation time points. The green segment, for example, was related to the
The expression of MSY genes and EB markers were compared by Spearman’s Heatmap. The
results detected four clusters, which contained highly correlated genes (Fig .5,). The
mesoderm markers showed high correlation with 8 MSY genes including
The Y-HPP was instituted to achieve a complete knowledge about the function,
quantification, subcellular localization, and expression pattern of human Y chromosome
protein and genes, especially during embryonic development. In the direction of one of the
Y-HPP goals, we analyzed the expression pattern of most MSY genes in the process of
male-ESCs (RH6) spontaneous differentiation into EBs as an
Ronen and colleagues (2014) suggested that the MSY genes, including
Petropoulos and colleagues (2016) indicated that the expression of Y chromosome genes
increased in male embryos since day 8, whereas the X chromosome genes were more expressed in
female embryos on days 3 and 4. Therefore, X genes were gradually downregulated after 5 days
in return for the upregulation of Y genes starting on day 8. Petropoulos’ study has shown
that 10 of the Y chromosome genes,
Torres and colleagues (2013) showed that
The present study is the first report to genetically investigate MSY genes during spontaneous differentiation of RH6 into EBs. Using Spearman’s Heatmap we identified distinct gene co-expression clusters to validate the correlation of MSY genes with each germ layer. The expression alterations characterized the potential responsibilities of each cluster for the differentiation of mesoderm, ectoderm and endoderm layers. We suggest that these genes may play important roles in early embryonic developments of males. Our results, along with future studies on directed differentiations, are potentially essential for a better understanding of gender-specific factors in embryonic developmental differences.
This work was financially supported by a grant (Code:95000131) from Royan Institute for Human Y Chromosome Proteome Project (Y-HPP) and the National Institute of Genetic Engineering & Biotechnology (NIGEB). There is no conflict of interest in this study
S.N.D., F.K.; Contributed to all experimental work, data and statistical analysis, and interpretation of data. S.N.D.; Wrote the manuscript. S.N.H.; Provided scientific advice throughout the project and performed cell culture. H.R.S-L., G.H.S; Supervised the project scientifically and contributed to establishing the main idea of the presented work and designing the experimental study. H.R.S.L., G.H.S, H.B, S.N.H.; Contributed to financial support and final approval of the manuscript. All authors have read and approved the final version of this manuscript.