MSCs from different anatomical locations exhibit varying differentiation potential. First, to verify that the cells used in this study have multipotency to differentiate into several cell types, we performed differentiation assays for osteogenesis and adipogenesis. Alizarin red S staining showed that all types of MSCs could differentiate into osteoblasts and form mineralized nodules, but the degree of mineralization was lower for Mx-MSCs than for other MSCs (Fig. 1A). Adipogenic studies found that I-MSCs had the greatest potential for adipogenic differentiation compared with Mx-MSCs and Md-MSCs (Fig. 1B). In the calcium content assay, I-MSCs and Md-MSCs produced more Ca than Mx-MSCs when the cells were cultured in ODM for 4 weeks (Fig. 1C). Moreover, Ca levels in I-MSCs were significantly higher than in Mx-MSCs when the cells were cultured in ODM for 6 weeks (Fig. 1C).
The general profile of RNA-seq data.
The Ion Proton system generated 31.5–52.8 million single-ended 50–200 bp reads from nine samples (Fig. 2A). Unmapped reads were reduced to less than 5% by removing low-quality reads. Genes with low expression (FPKM < 1.0) were removed from the three groups, leaving a total of 12,676 genes. Most non-coding RNAs (long intergenic noncoding RNAs, microRNAs, small nuclear RNAs, small nucleolar RNAs, and pseudogenes) had a FPKM < 1 in all samples (Fig. 2B). Using genes expressed in at least one of the groups with FPKM ≥ 1, we detected 10 different gene clusters in MSCs that exhibited distinct expression patterns (Fig. 2C). The genes in each cluster were significantly enriched by specific GO terms. For instance, GO terms associated with the cell cycle and cell adhesion detected from cluster 7 consisted of genes up-regulated in Mx-MSCs compared with other MSCs, while GO terms associated with cell migration detected from cluster 2 consisted of genes up-regulated in I-MSCs compared with other MSCs. These results suggested that the cell growth and migration properties of each cell type were different.
GO term enrichment analysis reveals differences between I-MSCs and Mx-/Md-MSCs.
To detect potential key regulators of each sample, we investigated DEGs between Mx-MSCs vs. I-MSCs, Md-MSCs vs. I-MSCs, and Mx-MSCs vs. Md-MSCs, and identified 973, 365, and 602 DEGs, respectively (Fig. 3A). To investigate differences in skeletal development and endochondral and intramembranous ossification, GO term enrichment analysis for I-MSC-specific DEGs was performed. A total of 140 DEGs were up-regulated in I-MSCs compared with Mx- and Md-MSCs (U-DEGs) (Fig. 3B). DAVID annotation of these DEGs revealed that most of the top 10 enriched GO terms were involved in development. A total of 96 DEGs were down-regulated in I-MSCs compared with Mx- and Md-MSCs (D-DEGs), and most of the top 10 enriched GO terms were also involved in development (Fig. 3C). These results indicate that I-MSCs and Mx-/Md-MSCs are regulated by genes involved in development.
Whole transcriptome analysis shows that MSCs derived from tissue in the maxillofacial region are HOX-negative.
Based on GO term analysis, significant DEGs between I-MSCs and MSCs derived from jaw bone (Mx-/Md-MSCs) were selected in order of the largest fold change. The top 20 up-regulated DEGs in I-MSCs compared with Mx- or Md-MSCs revealed that most genes specifically expressed in I-MSCs were from the HOX gene family although WISP3 was also specifically expressed in I-MSCs (Tables 1 and 2).
Table 1
The top 20 up-regulated DEGs in I-MSCs compared with Mx-MSCs
| | | |
| FPKM (Mean) | FC (log2) |
| I-MSCs | Mx-MSCs. |
HOXC6 † | 30.353 | < 0.0001 | 18.211 |
HOXC10 † | 22.068 | < 0.0001 | 17.752 |
HOXC8 † | 12.962 | < 0.0001 | 16.984 |
HOXB4 † | 12.954 | < 0.0001 | 16.983 |
HOXC9 † | 12.075 | < 0.0001 | 16.882 |
HOXA3 † | 1.255 | < 0.0001 | 16.487 |
HOXA11 † | 2.189 | < 0.0001 | 14.912 |
HOXD4 † | 2.538 | < 0.0001 | 14.631 |
HOXA5 † | 3.083 | < 0.0001 | 14.528 |
HOXA7 † | 7.18 | < 0.0001 | 14.347 |
HOXA4 † | 2.084 | < 0.0001 | 14.345 |
HOXC-AS1 †§ | 2.071 | < 0.0001 | 14.338 |
HOXC-AS2 †§ | 1.64 | < 0.0001 | 14.001 |
HOXC11 † | 1.627 | < 0.0001 | 13.99 |
HLA-DQB1 | 0.81 | < 0.0001 | 13.982 |
HOXA6 † | 4.767 | < 0.0001 | 13.616 |
HOXA-AS2 †§ | 2.081 | < 0.0001 | 13.366 |
WISP3 | 1.056 | < 0.0001 | 13.331 |
HOXA1 † | 2.363 | < 0.0001 | 12.984 |
HOTAIRM1 †§ | 9.188 | < 0.0001 | 12.278 |
†: HOX gene family, §: non-coding RNAs, FC: Fold change of FPKM value between two samples (log2 of FC) | |
Table 2
The top 20 up-regulated DEGs in I-MSCs compared with Md-MSCs
| | | |
| FPKM (Mean) | FC (log2) |
| I-MSCs | Md-MSCs. |
VTRNA1-1 § | 336.731 | < 0.0001 | 21.693 |
HOXC10 † | 22.068 | < 0.0001 | 17.752 |
HOXA1 † | 2.363 | < 0.0001 | 14.528 |
HOXA4 † | 2.084 | < 0.0001 | 14.347 |
HOXA-AS2 †§ | 2.081 | < 0.0001 | 14.345 |
HOXC-AS2 †§ | 1.64 | < 0.0001 | 14.001 |
HOXA3 † | 1.255 | < 0.0001 | 13.616 |
WISP3 | 1.056 | < 0.0001 | 13.366 |
HOXC6 † | 30.353 | 0.214447 | 7.145 |
CACNG8 | 10.729 | 0.145148 | 6.208 |
HOXC9 † | 12.075 | 0.179618 | 6.071 |
LOC400043 § | 11.018 | 0.28332 | 5.281 |
CELSR3 | 7.617 | 0.223098 | 5.093 |
HOXB7 † | 16.183 | 0.494379 | 5.033 |
CHI3L1 | 18.989 | 0.660538 | 4.845 |
ROR2 | 1.729 | 0.0724027 | 4.577 |
HIST1H2BH | 13.291 | 0.606649 | 4.454 |
UNC5C | 1.071 | 0.0503351 | 4.412 |
TENM2 | 1.548 | 0.0745513 | 4.376 |
HOXA11 † | 2.189 | 0.106172 | 4.366 |
†: HOX gene family, §: non-coding RNAs, FC: Fold change of FPKM value between two samples (log2 of FC) | |
Next, we further evaluated the expression profile of all HOX genes among the three samples. We obtained HOX FPKM values, and analyzed the degree and distribution of HOX gene expression levels (Supplemental Figs. 1A and 1B). I-MSCs were found to have a HOX-positive profile, while MSCs derived from jaw bones (Mx-/Md-MSCs) had HOX-negative profiles. To investigate the gene expression profiles of other cell types, we obtained RNA-seq data from human embryonic stem cells (ESCs), human ESC-derived MSCs (ES-MSCs), and human bone marrow-derived MSCs (BM-MSCs) from the Sequence Reads Archive (DDBJ accession number: SRA245478) and previous RNA-seq data owned by Tokyo Women’s Medical University (TWMU) from human periodontal ligament-derived MSCs (PDL-MSCs), BM-MSCs (TWMU-BMMSCs), and BM-MSCs cultured in ODM (ODMSCs). Figure 4a and 4b show that ECSs and PDL-MSCs also possessed a HOX-negative profile, similar to Mx- and Md-MSCs. Interestingly, although ESCs had a HOX-negative profile, the expression patterns of HOX genes changed to HOX-positive after the cells differentiated into MSCs. There was less change in the expression profile of HOX genes between TWMU-BMMSCs and ODMSCs, suggesting that HOX mRNA expression may not be affected by ODM.
Characteristics of gene expression patterns in different types of tissue-derived MSCs and ESCs.
To investigate whether HOX-negative MSCs such as PDL-MSCs, Mx-MSCs, and Md-MSCs showed similar gene expression patterns, U-DEG and D-DEG mRNA expression was analyzed among nine samples. U-DEG and D-DEG expression patterns in PDL-MSCs were similar to those in Mx-/Md-MSCs, while U-DEG and D-DEG expression patterns in MSCs derived from bone marrow (BM-MSCs, TWMU-BMMSCs, and ODMSCs) and ES-MSCs were similar to those in I-MSCs (Supplemental Figs. 2A and 2B). These results revealed the similarity of U-DEGs and D-DEGs in HOX-negative MSCs, except for ESCs, and in HOX-positive MSCs.
Next, we investigated the specific up-regulated DEGs in maxillofacial region-derived MSCs (Mx-MSCs, Md-MSCs, and PDL-MSCs; Mfr-MSCs) compared with the others. We identified eight genes with FPKM levels in each Mfr-MSC sample that were twice as high as in the other six samples: MSX1, NCAM1, LHX8, BARX1, FOXF1, S100A4, ZNF185, and NPTX1 (Fig. 5). The expression levels of LHX8, BARX1, FOXF1, and NCAM1 were quite low (FPKM < 1.0) in non Mfr-MSCs, indicating that they could be used as specific marker genes for Mfr-MSCs.
Gene expression profiles of CD antigens in each sample.
Figure 6 shows the gene expression profile of CD molecules in each type of MSC and ESC. The mRNA expression of positive markers for MSCs, such as CD105, CD73, and CD44, was significantly higher (FPKM > 50) in all MSCs compared with ESCs (FPKM < 3.0). CD90, an MSC-positive marker, was highly expressed (FPKM > 100) in both MSCs and ESCs. The mRNA expression of MSC-negative markers, such as CD14, CD34, and CD45, showed low levels (FPKM < 2.0) in MSCs and ESCs. CD106 and CD270 were not expressed (FPKM = 0) in ESCs, although these genes demonstrated high or low expression in MSCs. The expression patterns of all CD antigen genes were similar among different type of MSCs, except for ESCs.