Overexpression of OCT4 Causes Changes in Morphology and Adhesion in hHFMSCs
The morphology of OCT4-reprogrammed hHFMSCs greatly changed in the process of erythropoietic differentiation, whereby the spindle-shaped cells became polygonal, and a population of small floating round or quasi-round cells emerged from the adherent polygonal cells (Fig. 1a). We then determined the relative size of these cells by flow cytometry. The data showed that the size of adherent hHFMSCsOCT4 was notably smaller than that of hHFMSCs, while floating hHFMSCsOCT4 were smaller than the adherent cells (Fig. 1b), which was consistent with what we observed under an optical microscope. At the same time, cell adhesion changed as a portion of the cells gradually became suspended in the medium, so dissociation and adhesion assays were carried out to detect cell adhesion. The percentage of single cells was higher in adherent hHFMSCsOCT4 (47.5%) than in hHFMSCs (9.4%), and the percentage was higher in floating hHFMSCsOCT4 (85%) than in adherent hHFMSCsOCT4 (Fig. 1c). In the adhesion assay, the percentage of remaining cells was lower in adherent cells (18.4%) than in hHFMSCs (70.9%), while it was lower in floating hHFMSCsOCT4 (2.3%) than in adherent hHFMSCsOCT4 (Fig. 1d). The above results validated that the morphology of OCT4-reprogrammed hHFMSCs changed and adhesion decreased. It is worth noting that it was the population of floating hHFMSCsOCT4 with low-adhesion prone to transdifferentiate towards erythroid lineage after stepwise stimulation by cocktails of hematopoietic cytokines. Accordingly, cell morphology and adhesion might be the negative factors affecting erythropoiesis.
Transcripts in Cells with Diverse Morphology and Adhesion are Quite Different
To investigate the role of cell morphology and adhesion during erythrocyte differentiation from hHFMSCsOCT4, RNA-seq was performed and the DEGs were sorted. First, to compare the similarity of samples within a group and the diversity of each group, the principle components were analyzed. As shown in the three-dimensional distribution (Fig. 2a), the closer distances of cells within each group implied good repetitiveness, and the distances between every two groups were significantly greater, especially when adherent hHFMSCsOCT4 and floating hHFMSCsOCT4 were compared with hHFMSCs, respectively. Based on these data combined with the correlation coefficient and clustering of correlation provided in Additional file 1, OCT4 induced hHFMSCs to derive novo cell populations, remarkably, cells with different morphology and adhesion might possess distinct gene transcripts.
Next, DEGs were sorted among the three groups. When we compared adherent hHFMSCsOCT4 with hHFMSCs, 2401 upregulated genes and 1882 downregulated genes were identified (Fig. 2b), and 3107 upregulated genes and 2999 downregulated genes were determined in floating hHFMSCsOCT4 compared to hHFMSCs (Fig. 2c), indicating that OCT4 conferred considerable changes of transcriptome in the whole genome to hHFMSCs. Importantly, 833 upregulated genes and 1107 downregulated genes were also identified when floating hHFMSCsOCT4 were compared with adherent hHFMSCsOCT4 (Fig. 2d). Although the number is smaller, it would definitely play a considerable role in floating cells. Venn diagram analysis revealed a total of 612 and 388 group-specific DEGs for adherent hHFMSCsOCT4 vs. hHFMSCs and floating hHFMSCsOCT4 vs. adherent hHFMSCsOCT4, respectively (Fig. 2e). In particular, 1785 group-specific DEGs were identified in floating hHFMSCsOCT4 vs. hHFMSCs, which was a much larger number than that in the other two comparison groups. This considerable number of DEGs probably bring about significant changes in biological function to OCT4-reprogrammed hHFMSCs. Especially, the group-specific DEGs may yield tremendous changes to floating hHFMSCsOCT4 when compared with hHFMSCs. In the DEGs analysis, it is likely that the common and group-specific DEGs collectively affected the morphological characteristics and subsequent transdifferentiation.
Floating Cells Lose Part of Pluripotency and Gain Hematopoietic Differentiation Potential
Transduction of the key pluripotent TF OCT4 would definitely influence the pluripotency of hHFMSCs. Consequently, we focused our analysis on the expression of related genes in cells with different morphology and adhesion. hHFMSCs expressed negligible levels of OCT4, LEFTY2, SOX18, POU3F2 and SEMA4D (Additional file 2: Table S1), and both adherent hHFMSCsOCT4 and floating hHFMSCsOCT4 expressed higher levels of pluripotent genes, including LEFTY2, KLF4, MYC, POUs, SEMAs and SOXs, than hHFMSCs (Fig. 3a). Some of the pluripotent genes, such as LEFTY2, SOX4, and SEMA6C, however, were downregulated in floating hHFMSCsOCT4 compared with adherent hHFMSCsOCT4 (Fig. 3a), which was validated by KEGG enrichment analysis as downregulated genes were enriched in the term signaling pathways regulating pluripotency of stem cells in floating hHFMSCsOCT4 vs. adherent hHFMSCsOCT4 (Fig. 3b). These results suggested that OCT4-reprogramed hHFMSCs acquired pluripotency but lost some of it after adherent cells transformed into the floating subset. The DEGs were then clustered according to their GO terms using DAVID, and the top 10 GO terms related to differentiation and development enriched with upregulated genes and downregulated genes were separately analyzed. Upregulated DEGs were involved in the terms of germ layer differentiation in adherent hHFMSCsOCT4 and floating hHFMSCsOCT4 when compared with hHFMSCs (Table 2 and Additional file 2: Table S2, S3), and the upregulated genes in floating cells were specially enriched in the terms T-helper 1 cell differentiation and regulation of erythrocyte differentiation (Table 2), implying a potential for erythropoietic differentiation.
Table 2
Differentiation- and development-related GO terms enriched of upregulated DEGs in floating hHFMSCsOCT4 versus hHFMSCs.
id | Term | P-value | Enrichment score |
GO:0046548 | retinal rod cell development | 0 | 6.255870445 |
GO:0045063 | T-helper 1 cell differentiation | 0 | 6.255870445 |
GO:0060351 | cartilage development involved in endochondral bone morphogenesis | 0 | 6.255870445 |
GO:1903225 | negative regulation of endodermal cell differentiation | 0 | 6.255870445 |
GO:0060538 | skeletal muscle organ development | 0 | 6.255870445 |
GO:0061153 | trachea gland development | 0 | 6.255870445 |
GO:1902871 | positive regulation of amacrine cell differentiation | 0 | 6.255870445 |
GO:0045646 | regulation of erythrocyte differentiation | 0 | 6.255870445 |
GO:0003431 | growth plate cartilage chondrocyte development | 0 | 6.255870445 |
GO:0045605 | negative regulation of epidermal cell differentiation | 0 | 6.255870445 |
Downregulation of the Tight Junction Pathway in Floating hHFMSCsOCT4
To further explore the role of cell morphology and adhesion during erythropoiesis in hHFMSCsOCT4, the top 10 GO terms, covering biological process, molecule function and cellular component, are displayed in Fig. 4a. Downregulated genes were obviously enriched in relevant terms,such as cell adhesion, focal adhesion, cytoskeleton and cell-cell junction in adherent hHFMSCsOCT4 compared to hHFMSCs. In addition, downregulated genes were significantly enriched in the term cell adhesion in floating hHFMSCsOCT4 relative to adherent hHFMSCsOCT4, suggesting the sharp decrease in adhesion of floating hHFMSCsOCT4. Besides, KEGG analysis revealed that downregulated genes were enriched in cell signaling pathways including regulation of actin cytoskeleton, cell adhesion molecules and focal adhesion in adherent hHFMSCsOCT4 vs. hHFMSCs, as well as pathways of regulation of actin cytoskeleton, gap junction, adherens junction and focal adhesion in floating hHFMSCsOCT4 vs. adherent hHFMSCsOCT4 (Fig. 3b). These results verified the changes in cell morphology and adhesion of hHFMSCsOCT4, which were consistent with our observations. Therefore, the morphology- and adhesion-related genes aroused corresponding alterations in OCT4-reprogrammed hHFMSCs and facilitated the switch between adherent and floating subpopulations.
Tight junctions, generally known for their fence function controlling cellular matter diffusion, can also modulate cell adhesion and the cytoskeleton (12, 22). The TJ pathway was found to be downregulated by KEGG analysis in these three comparison groups, and the TJ pathway was annotated through the KEGG database and the DEGs were annotated (Fig. 4b). There were 12 upregulated genes and 20 downregulated genes in the TJ pathway. The results clearly showed that TJ genes were dynamically expressed, and several programs, such as cell proliferation, adhesion, cytoskeleton, cell polarity, paracellular permeability and most importantly cell differentiation, were involved. Furthermore, fluctuation of one member in the TJ pathway would inevitably affect other member molecules and thereby have an impact on the biological functions of hHFMSCsOCT4 and initiate the switch between the two states characterized by different morphology and adhesion.
Gene Expression Validation by qPCR and Western blot
Cell junction molecules, including TJ members are involved in cell adhesion and could directly affect cell adhesion (11). Therefore, we performed qPCR to detect the mRNA expression levels of selected genes associated with TJs, adhesion or cytoskeleton (Fig. 5a). As expected, the expression level of the TJ member gene CLDN11 was significantly decreased in adherent hHFMSCsOCT4 and floating hHFMSCsOCT4 relative to hHFMSCs, but both CLDN6 and CLDN7 were increased. Especially, CLDN5 was downregulated in adherent hHFMSCsOCT4 and then upregulated in floating hHFMSCsOCT4. The expression levels of JAM1 and JAM3, which play an important role in the commitment of lineage specification and cellular signaling transduction in HSCs, were respectively decreased and increased in floating hHFMSCsOCT4 vs. hHFMSCs. Moreover, the expression levels of TJP1, TJP2 and TJP3, core members associated with the cytoskeleton and intracellular signaling transduction, were remarkably upregulated 5.4-fold, 59.4-fold and 7.6-fold in floating hHFMSCsOCT4. These results indicated disrupted molecular homeostasis of the TJ pathway upon OCT4 transduction. We also found that the cytoskeleton gene ACTN2 increased more than 20-fold in floating cells, and the expression of E-cadherin increased in adherent hHFMSCsOCT4 and then decreased in floating hHFMSCsOCT4, implying that the changes in adhesion of these cells might be related to calcium signals.
Changes in cell morphology and adhesion in the process of erythropoiesis could influence the biological functions of hematopoietic cells, including proliferation, self-renewal, differentiation and etc. (8), we also detected the expression levels of the terminal erythroid differentiation-related gene ROCK1 and the essential hematopoietic development gene RUNX1. Expression of these two genes were found decreased in floating cells, indicating that the state of hematopoietic program was not yet triggered, although OCT4 conferred pluripotency in this group of cells to some degree as other pluripotency genes MYC, KLF4, etc. were upregulated as shown in previous sequencing data.
Next,Western Blotting was carried out to validate the protein expression levels of TJP1, JAM1, CLDN5, CLDN11 and RUNX1. As shown in Fig. 5b, the expression tendency of these proteins was consistent with the mRNA levels. The above results corresponded to our sequencing data, except for TJP1, which was remarkably increased in adherent cells and floating cells compared to hHFMSCs but was not identified by sorting DEGs.
A Putative Regulatory Network in Floating hHFMSCsOCT4
Although pluripotency-, cell morphology- and cell adhesion-related genes have been identified, the internal correlations between these factors are still unknown. Therefore, we visualized the significant DEGs in floating hHFMSCsOCT4 vs. hHFMSCs and constructed a network (Fig. 6a). This network suggested that pluripotency-related genes in hHFMSCs, such as OCT4, SOX2, c-MYC (MYC) and KLF4, were regulated by genes related to cell adhesion, cell junction, and cytoskeleton, including TJP1, TJP2, FN1 (fibronectin 1), CTNNB1 (β-catenin), CDH1 (E-cadherin), ACTB (β-actin) and ACTG1 (γ-actin 1). In addition, there were interactions within the TJ pathway, as well as interplay between the TJ pathway, cell adhesion- and cytoskeleton-related molecules, such as PTK2 (FAK), CDH1, CTNNB1, ACTB, and ACTG1. Hematopoietic genes, such as, CD44, CD117 (KIT), RUNX1 and ROCK1 could similarly bind to or interact with cell adhesion and junction molecules. These results strongly imply that dynamic expression of genes related to adhesion, cytoskeleton and junctions results in the low-adhesion and different morphology of hHFMSCsOCT4, whereas floating cells lost some of the pluripotency and became more prone to differentiation into blood cells. Furthermore, TJP1 uniquely links pluripotency, hematopoiesis and cytoskeleton with the TJ pathway through KLF4, RUNX1, ACTB and ACTG1. Fluctuation of one member in TJ pathway would inevitably affect other member molecules, thereby impacting the biological functions of hHFMSCsOCT4 and initiating the transform between the two states of different morphology and adhesion characteristics.
In summary, transduction of OCT4 brought about great differences in morphology and adhesion to hHFMSCs, whereby two subsets of cells appeared and gained pluripotency, with floating cells losing some of their pluripotency. The dynamically expressed TJ pathway before erythropoietic inducement might act as a pivotal point of changes in cell morphology and adhesion, resulting in damaged pluripotency in floating cells and probable constructed hematopoietic capacity in a TJP1-dependent way.