Downregulation of Oct-4 and Nanog in late-passage primary mesenchymal stem
cells
Embryonic transcription factors, such as Oct4 and Nanog, normally expressed in early embryos and ESCs, inhibit tissue-specific genes and enhance self-renewal and pluripotency (Boiani and Scholer, 2005). To evaluate whether loss of pluripotency occurred during normal passage of hMSCs, we examined the expression of Oct4 and Nanog in primary hMSCs. Semiquantative RT-PCR and real-time RT-PCR analysis revealed higher mRNA levels of Oct4 and Nanog at passage 3 (P3) than at passage 10 (P10) (Fig. 1A), indicating loss of pluripotency during expansion of primary hMSCs.
ESCs, a powerful tool to study mammalian development, form embryoid bodies (EBs) and express a panel of developmental markers upon removal of feeder layer or leukemia inhibitory factor. To evaluate whether spontaneous differentiation with the expression of developmental markers occurred during normal passage of primary hMSCs, we examined the expression levels of ectoderm (Pax6 (Hill et al., 1991)), primitive endoderm (Gata4 and Gata6 (Fujikura et al., 2002)) and definitive endoderm (Sox17 and FoxA2 (Kubo et al., 2004)) markers by RT-PCR. The expression levels of Pax6, Gata4 and FoxA2 were higher at P10 than at P3 (Fig. 1Ba). We next looked at the expression of germline markers (Clark et al., 2004), and found the expression levels of Stella, Dazl,
Vasa and Scp3 were higher at P10 (Fig. 1Bb). Finally, we examined two lineage-specific markers expressed in EBs, the neural (Nestin) and cardiac specific genes (Nkx 2.5 and cTn1) and found P10 had higher expression of Nestin and cTn-1 (Fig. 1Bc). These results point to upregulation of developmental markers and lineage-specific genes in late-passage primary hMSCs.
Transient upregulation of Oct4 and Nanog during early differentiation of
hTERT-transfected hMSC
Since differentiation and cellular senescence are associated with loss of telomere length (Baxter et al., 2004; Sharpless and DePinho, 2004), and telomerase is highly expressed in ESCs (Amit et al., 2000; Thomson et al., 1998), we hypothesized overexpression of hTERT would enhance pluripotency and overcome spontaneous differentiation. Because of the difficulties in bypassing cellular senescence (Akimov et al., 2005; Okamoto et al., 2002), selecting and expanding single cell clones by expressing hTERT alone in primary hMSCs and the fact both hTERT and E6E7 are necessary to circumvent cellular
senescence thoroughly (Akimov et al., 2005; Okamoto et al., 2002), we avoided transferring hTERT to primary hMSCs, but rather to E6E7-transfected hMSCs- the KP cells(Hung et al., 2004) and monitored transgene expression by a fluorescent reporter-
green fluorescence protein (eGFP). Several single-cell derived clones were isolated and one of the clones, 3A6 was used for further analysis. 3A6 grown in monolayer in
DMEM-LG supplemented with 10% FBS had a remarkably shorter population doubling time (1.9 days) compared with the parental KP cells (3.0 days). RT- PCR revealed the expression of hTERT and eGFP in 3A6. Flow cytometry also demonstrated 3A6 has a normal surface protein profile like the normal hMSCs (Supplementary Fig. 1).
Besides the increase in proliferation rate, the ectopic expression of hTERT might also affect pluripotency. Therefore, we compared the expression levels of Oct4 and Nanog between KP and 3A6. Unexpectedly, RT-PCR and real-time RT-PCR unraveled the downregulation of both Oct4 and Nanog in 3A6 compared with KP (Fig. 2A).
Downregulation of the embryonic transcription factors such as Oct4 and Nanog is associated with differentiation of neural stem cells, hematopoietic stem cells and MSCs.
However, an increase in Oct4 expression in ESCs causes differentiation into primitive endoderm (Niwa et al., 2000), mesoderm (Niwa et al., 2000) and early cardiac lienage (Zeineddine et al., 2006). Overexpression of Nanog also drives the expression of ectoderm markers (Zeineddine et al., 2006). The expression pattern of Oct4 and Nanog during differentiation is completely different between ESCs and adult stem cells such as MSCs, and should serve as an indicator to discriminate ESCs from MSCs (Darr et al., 2006; Niwa et al., 2000; Zeineddine et al., 2006). We therefore induced 3A6 to undergo
osteogenic and neural differentiation and examined the expression of Oct4 and Nanog.
During osteogenic differentiation, we noticed a continuous upregulation of Oct4 and Nanog until day 7 followed by downregulation of both genes at day 14 (Fig. 2Ba).
Similarly, during neural differentiation, the upregulation of Oct4 and Nanog was observed during early differentiation (Fig. 2Bb). These results indicated 3A6 has a differential gene expression of embryonic markers similar to the early differentiation of ESCs, suggesting the most primitive state of hTERT-transfected hMSCs
Downregulation of developmental markers and lineage-specific genes in hTERT-transfected hMSCs
To clarify the blocking of spontaneous differentiation by ectopic expression of hTERT in hMSCs, we compared the expression of developmental markers and lineage-specific genes between 3A6 and KP by performing RT-PCR for trophoectoderm (CDX2 and CGß), germline (Dazl, Vasa and Scp3), osteogenic (BSP, Bone Sialoprotein and OCN, Osteocalcin) and neural (Pax6 and Nestin) specific markers. We noted a general downregulation of expression for all these genes at 3A6 compared with KP (Fig. 2C), indicating the ability of hTERT to block spontaneous differentiation and maintain 3A6 in an undifferentiated state.
Transient upregulation of Oct-4 and Nanog upon early differentiation of
hTERT-expressed hBMSC
Downregulation of the embryonic markers such as Oct-4 and Nanog is associated with differentiation of neural stem cells, haematopoietic stem cells and MSCs, nevertheless, upregulation of OCT-4 and Nanog was observed during the formation of embryonic body (Darr et al., 2006; Zeineddine et al., 2006)- the early differentiation of embryonic stem cells, and overexpression of Oct-4 was also proved to driving primitive endoderm (Niwa et al., 2000), primitive mesoderm (Niwa et al., 2000) and early cardiac (Zeineddine et al., 2006) differentiation of embryonic stem cells. Since 3A6 was developed on the purpose to have a BMSC cell line with the potential of ESCs, we then investigated whether 3A6 has a differential expression pattern of Oct-4 and Nanog similar to that of ESCs during the process of early differentiation. To illustrate the relationship between expression profile of Oct-4 and Nanog and early differentiation state of 3A6, we exploited osteogenic and neuronal differentiation as models in search of transient expression alteration of Oct-4 and Nanog during early differentiation. With induction of osteogenic differentiation, quantitative real-time PCR revealed continuous upregulation of Oct-4 and Nanog until day 7 which were followed by the downregulation of both genes thereafter (Fig. 2Ba). Similarly, during neuronal differentiation, the upregulation of Oct-4 and Nanog expression in early differentiation state were noeted (Fig. 2Bb). These results
indicated that 3A6 has a differential gene expression of embryonic markers similar to the early differentiation of ESCs, suggesting the most primitive state of hTERT-expressed BMSCs
Downregulation of developmental markers and lineage-specific genes in
hTERT-expressed hBMSCs
To further clarify the reversion of stemness, that is, the recovery to a more primitive state after ectopic expression of hTERT at 3A6, we compared the relative expression level of developmental markers and lineage-specific genes between 3A6 and KP, which would be another indicator of the primitive state of stem cells besides embryonic markers, Oct-4 and Nanog. Among these genes, we choose trophoectoderm specific markers, Cdx2 and CGß; germ-line specific markers, Dazl, Vasa and Scp3; osteogenic specific genes, Bsp
and Ocn; neuronal specific genes, Pax6 and Nestin as targets of comparison. The RT-PCR results (Fig. 3A) disclosed generalized downregulation of these developmental markers and lineage-specific genes at 3A6 than that at KP.
Improvement of differentiation potential by introducing hTERT to hMSCs After characterization of 3A6 and unraveling its relative quiescent state, it is of great interest if the differentiation potential of 3A6 would be sustained, enhanced and reversed to a considerably primitive state, as with ESCs. We first examined if 3A6 sustained the normal capabilities of hMSCs, such as mesenchymal (osteogenic, adipogenic and chondrogenic) and non-mesenchymal (neural) differentiation and hematopoietic
supporting potential (cobblestone forming). 3A6 had normal or elevated osteogenic and chondrogenic differentiation potential compared with one KP-derived single cell clone, whereas 3A6 had decreased adipogenic differentiation potential (Fig. 3A). These data are consistent with previous studies that overexpression of hTERT increased osteogenic potential and the inverse relationship between osteogenic and adipogenic differentiation.
For neural differentiation, 3A6 adopted the typical morphology of neural progenitor cells, including bipolar elongated cell processes and retracted cell bodies, and expressed neural lineage specific markers, such as Nestin and Pax6 on stimulation with noggin in serum free conditions for 14 days (Fig. 3B). For co-cultured CD34+ hematopoietic stem cells with 3A6 cells, we noted the formationof cobblestone areas from hematopoietic cells that transmigratedbeneath the layer of 3A6 cells (Fig. 3C).
Previously, only ESCs has proven to be able to successfully differentiate toward trophoectoderm (Xu et al., 2002) and germline (Clark et al., 2004) in vitro, but Johnson
and others (Johnson et al., 2005) detected the expression of germline markers in bone marrow and peripheral blood, and Nayernia and others (Nayernia et al., 2006) further implied the germline differentiation potential of mouse MSCs. No literature so far, however, has revealed the differentiation potential of MSCs toward trophoectoderm. To test the most versatile differentiation potential of hMSCs after ectopic expression of hTERT, we directed 3A6 towards trophoectoderm and germline differentiation upon stimulation with BMP4 (Xu et al., 2002) and retinoic acid (RA) (Geijsen et al., 2004), respectively. This has been used to initiate trophoblast and germline differentiation in human ESCs. As demonstrated by RT-PCR, 3A6 started to express the trophoectoderm specific markers, such as CDX2 and CGß (Fig. 3D), and germline specific markers
(Clark et al., 2004), such as Stella, Dazl, Vasa, and Scp3 (Fig. 3E) after differentiation.
These results together suggest 3A6 not only sustained normal potential as hMSCs, but also adopted the potential that was previously exclusive to ESCs.
Enhanced differentiation efficiency of hTERT-transfected hMSCs
Besides the differentiation potential, another significant issue would be the differentiation efficiency of 3A6. Spontaneous differentiation, noted during expansion of primary hMSCs and KP, might hamper differentiation efficiency because less uncommitted cells
differentiation efficiency because of its less committed state. To clarify this hypothesis, we directed KP and 3A6 toward osteogenic or neural lineage and compared their
differentiation efficiency by histochemical staining and lineage-specific gene expression.
We observed 3A6 had higher alkaline phosphatase and Alizarin Red S staining compared with KP no matter at day 3, day 7 or day 14 of osteogenic differentiation (Fig. 4A). The expression levels of osteogenic markers- BSP and OCN were also elevated in 3A6 compared with KP during osteogenic differentiation. The expression levels of neural markers- Nestin and Pax6 were also elevated in 3A6 during neural differentiation (Fig.
4B).
Global hypomethylation of development and differentiation associated genes in
hTERT-transfected hMSCs
To prove the recovery of pluripotency after hTERT transfection might be attributed to epigenetic remodeling, we conducted a genome-wide analysis of DNA methylation between 3A6 and KP cells, which contained about 240000 probes for 24000 CpG islands.
The average methylation level of 3A6 (1.630 ± 9.456) was significantly lower than KP
(1.762 ± 17.187 ) (Supplementary Fig. 2). The number (percentages) of annotated genes
detected as hypermethylated by the probes were 6703 (16.2 %) and 7239 (17.6 %) for 3A6 and KP, respectively. These results are consistent with the finding CpG islands are
hypomethylated DNA(Saxonov et al., 2006) and reveal KP has greater DNA methylation level than 3A6. Since global DNA demethylation occurs immediately following fertilization and ESCs are nearly devoid of methylation markers(Li, 2002; Meshorer and Misteli, 2006), the decrease in global CpG island methylation level in 3A6 further demonstrates its primitive state.
Due to the decrease in numbers of hypermethylated genes in 3A6, we then analyzed genes demethylated after hTERT overexpression according to different gene categories using Gene Ontology (Fig. 5). Notably, the demethylated genes were highly associated with development (p value= 1.09E-16) and cellular differentiation (p value= 0.0208).
However, we didn’t find a relatively higher expression level of the demethylated genes in 3A6 than in MSCs and differentiated ESCs by comparing their transcriptome microarrays (data not shown), suggesting the hypomethylated state didn’t actually assure the gene expression, but rather, kept these genes in a state poised for activation.
Ectopic expression of hTERT downregulated three major DNMT genes
transcription in hMSCs
Attempting to discover factors that might induce DNA demethylation in 3A6, we used real-time RT-PCR to quantify the expression level of three major DNMTs between 3A6
and KP. Surprisingly, the levels of DNMT1, DNMT3A and DNMT3B were markedly suppressed in 3A6 compared with KP (Supplementary Fig. 3A). Since DNA methylation could also be controlled by the polycomb group protein, EZH2(Vire et al., 2006), we checked the expression of EZH2 by real-time RT-PCR. The expression levels of EZH2 were not different between 3A6 and KP (Supplementary Fig. 3B). From these results, the decrease in CpG island methylation in 3A6 is associated with the decrease in DNMT gene expression.
The hTERT-transfected hMSC expression profile converges toward ESCs
To gain insight into the convergence of 3A6 toward ESCs, we compared the expression profile of 3A6 with various normal human tissues, MSCs and ESCs. This data set therefore contained different tissues from embryo, endoderm, epithelial, or mesenchymal origins. The expression profilesof each chip were compared using principal component analysis(PCA) to discover the similarityof the expression profiles within and across the cells or tissues. PCA using all probe sets showed ESC and MSC each formed a distinct group and were quite different from all the normal human tissues. Interestingly, the 3A6 expression profile located very close to ESCs rather near MSCs, signaling the expression profile of 3A6 converged toward ESCs (Figure 6).