Synergistic work between MFSelector and TO-GCN
MFSelector and TO-GCN were the two methods of data analysis used in this study. These methods worked together synergistically to provide a deeper understanding of MSC differentiation into mesangial cells. MFSelector determined the degree of monotonicity for all genes during the differentiation process and it provided an estimation of the expression behaviour of the gene during differentiation. TO-GCN used co-expression relationship to connect TF genes as pairs, in which they have similar expression patterns (i.e. significantly high Pearson correlation coefficient) over time. It inferred expression time orders for all TF genes in the network with the starting TF in the strongest descending pattern identified by MFSelector. By applying this method to time-series experiments, TO-GCN provided the time order information of gene regulations in developmental processes. The data obtained from both methods was further used to identify the TF-key genes at specific time points to the TO-GCN at different levels. This helped to elucidate the network interaction between TF-TF and TF-key genes at each level of TO-GCN.
In this study, the FOSL1 gene, expressed in the strongest monotonic descending pattern, was the initial node to generate all time-ordered levels of nodes in the GCN by the breadth-first search algorithm. As the network was constructed based on co-expression, TFs in the same or next levels of FOSL1 in TO-GCN would be also in a descending patterns. This was consistent with the genes in descending pattern identified by MFSelector. Lower DE values (stronger monotonic pattern) of descending pattern genes appeared in early levels from L1 to L3 (green nodes in Fig. 2). The ascending genes with higher monotonicity (lower DE value) appeared later at the levels from L10 to L13 (purple nodes in Fig. 2). Genes with a weak monotonic pattern (either descending or ascending) were located in between the descending and ascending high monotonicity pattern genes.
The key genes and MSC biomarkers were down-regulated during the differentiation process
MSC biomarkers such as ANPEP and LIF were down-regulated during the differentiation process. ANPEP, also called CD13, is well known as an MSC marker. On the other hand, LIF, another well-established MSC marker, has been reported to affect cell growth by inhibiting differentiation but maintaining the stemness of the stem cell. When LIF levels drop, the cells will start the process of differentiation [20]. Meanwhile, depletion of AURKA, known for stem cell renewal, resulted in compromised self-renewal and consequent differentiation [21].
In this study, many genes related to cell cycle regulation (CDK1, CCNB1 and GNL3) and DNA replication (CDC6) were down-regulated. CDK1 is a key regulator of mitosis. High levels of CDK1 are associated with the pluripotency stage of embryonic stem cells (ESC). Decreased CDK1 activity to a level without perturbing the cell cycle is sufficient to induce differentiation [22]. Meanwhile CCNB1 gene expression are increased during G2/M phase and decreased during terminal differentiation [23]. GNL3, also known as nucleostemin, regulates the cell cycle and affects cell differentiation; the amount of GNL3 decreases as differentiation progresses. GNL3 is also a biomarker for many stem cells and cancer cells [24]. CDC6 is an essential regulator of DNA replication in eukaryotic cells. Down regulation of CDC6 will lead to a drop of DNA replication before differentiation can take place [25, 26]. Even though these genes regulate the cell cycle or DNA replication, all findings show that when these genes are down-regulated in stem cells, differentiation will start.
By referring to the TFBS database, key gene LIF, a MSC marker, was regulated by TF MEOX2. Another MSC marker, key gene ANPEP, was regulated by TFs SOX9 and HMGA1. These 3 TFs (MEOX2, SOX9 and HMGA1) are known as regulators of the stem cell state through transcriptional networks that induce pluripotency. Theodorou et al. reported that neuronal differentiation in ESC was inhibited when MEOX2 overexpressed [27]. Shah et al. did a study showing that when ESC differentiation was induced, there was a decreased expression of HMGA1 which was also observed in other pluripotency factors. Conversely, forced expression of HMGA1 blocked the differentiation of ESC [28]. Meanwhile for SOX9, upon the differentiation of MSC into hepatocytes, SOX9 expression was down-regulated [29].
Biomarkers contribute to mesangial cell characteristics and functions
Eleven mesangial cell key genes with DE≤4 were selected for further analysis. The majority of these key genes are reported as biomarkers for mesangial cells or related to the functions of mesangial cells. TAGLN, or SM22-alpha, is expressed in smooth muscle cells. It is known as one of the earliest commitment biomarkers of differentiated smooth muscle cells and has been suggested to regulate their contractile functions [30]. This gene has a role in generating committed progenitor cells from undifferentiated hMSC by regulating cytoskeleton organization. TAGLN in the kidney is up-regulated in repopulating mesangial cells in vivo. Meanwhile SERPINE2 and IGFBP5 are reported to be expressed in mesangial cells [31, 32] and MYOM1 is known to be expressed in smooth muscle cells [33].
ACTA2 and MYH9 play an important role in regulating both smooth muscle and non-muscle cell contractile activity [31, 34]. Another contraction related gene is PTGIS, also known as prostacyclin synthase. PTGIS is the final committed enzyme in the metabolic pathway leading to prostaglandin I2 (PGI2) production and PGI2 is needed to mediate mechanism of vascular contraction [35]. PGFRB is needed for stimulation of contraction and chemotaxis [36]. PYGM encodes a muscle enzyme that is involved in glycogenolysis.
Mesangial cells are phagocytic cells and expression of ITGA8 in mesangial cells facilitates phagocytosis. About 15% of the total mesangial cell population in the glomerulus is capable of exhibiting immunological function such as phagocytosis [37]. Another phagocytic related gene is MFGE8 which is robustly expressed in mesangial cells. This gene has many functions, including promoting the phagocytic removal of apoptotic cells [31, 38].
By referring to the TFBS database, TFs SRF and TEAD3 were found to regulate 3 key genes each. This shows that TF SRF and TEAD3 play an important role in mesangial cells. SRF is a ubiquitous expressed TF that drives smooth muscle cell-specific gene expression and is necessary for contractile and cytoskeletal functions [39, 40]. TEAD3 has been reported to abolish myocardin function and is consistently expressed in smooth muscle cells [41].
Pathway enrichment analysis on each TO-GCN level
Proliferation and differentiation processes are two distinct and mutually exclusive processes during development. To initiate stem cell differentiation, certain cell proliferation related genes or pathways have to be down-regulated. In TO-GCN analysis, it was observed that pathways for cell cycle, DNA replication and ribosome biogenesis in eukaryotes were enriched in the early levels. Estefanía et al. has reported that terminal differentiation is the process by which dividing cells stop proliferating in order to start new specific functions, which means that DNA replication fades as cells advance in their commitment to terminal differentiation [42]. This observation not only occurred in cell differentiation, but also happened in organogenic processes where cells must exit the mitotically active state before entering terminal differentiation [43, 44]. Therefore, these early levels can be classified as differentiation preparation.
From L4 to L7, pathways involved in regulating or triggering differentiation were enriched. Pathways like RNA degradation, phosphatidylinositol signaling system, ubiquitin mediated proteolysis, circadian rhythm, thyroid hormone signaling pathway, and mRNA surveillance pathway were enriched in this stage. Lou et al. showed that RNA degradation drives stem cell differentiation [45]. They discovered that the steady-state level of RNAs is dictated by their decay rate and this specific RNA decay such as Nonsense-mediated mRNA decay (NMD) have a role in promoting differentiation mechanisms [46, 47]. NMD is a surveillance pathway and its main function is as a quality control pathway to reduce errors in gene expression by eliminating mRNA transcripts that contain premature stop codons [48]. During the differentiation, NMD elicits the decay of specific subsets of mRNAs and promotes the decay of mRNAs encoding pluripotency factors [45]. Meanwhile, phosphatidylinositol signaling system, ubiquitin mediated proteolysis and thyroid hormone signaling pathway have been reported to take part in initialization or to modulate the differentiation [49, 50].
From L8 to L14, pathways involved in mesangial cell maturation were enriched. Some of the enriched pathways are related to characteristics and functions of mesangial cells. Mesangial cells are modified smooth muscle cell, which means these cells can contract upon stimulation. Therefore, pathways related to contraction such as oxytocin signaling pathway are expected to be enriched in these cells, where oxytocin is required to stimulate prostaglandin (PG) production before PG causes the contraction [51]. As mesangial cells are specialized renal pericytes that have phagocytosis ability [3], it is not surprising to find the phagosome pathway was enriched. In between L13 to L14, PI3K-Akt signaling pathway, cGMP-PKG signaling pathway, and ECM-receptor interaction were enriched. These pathways have been reportedly activated in mesangial cells [52-54].
The strongest evidence that the co-cultured MSC have differentiated into mesangial cells is by confirming the pathway enrichment of vascular smooth muscle contraction [55]. As mesangial cells are modified smooth muscle cells, we have further conducted a wet lab contraction functional validation. Results showed that the differentiated cells can contract and have proven that the cells have fully matured in their differentiation in which the cells now possess mesangial cell functions. On the other hand, pure MSC failed to exhibit contraction ability.
Construction of muscle contraction-specific gene network
In transcriptional regulatory networks or TFBS databases, usually only TF-TF or TF-key gene relationship, a single stage relationship is recorded. However, in this study, TF-TF-key gene relationships as a multiple-stages relationship between TFs and key genes were linked together based on co-expression. This gives us a broad inference of the TF-TF-key genes relationship for the specific function.
In this study we have shown that differentiated mesangial cells have contraction ability. With this specific biological function, a co-expression gene network related to muscle contraction was constructed. This network has illustrated the relationship between key genes and its upstream regulators or TFs. To our knowledge, such function-specific TF-TF-muscle contraction-related key gene network has not been reported before. This indicates that a mathematically calculated co-expression network can provide us with a first step or hints for further wet lab validation before full biological TF-TF-key gene relationship is fully uncovered. Other biological functions such as phagocytosis of mesangial cells can also be explored and constructed using the methods presented herein.