Global Gene Expression Analysis During Stem Cell Differentiation and Dedifferentiation
If the dedifferentiated cells had regained the potential of hMSCs, it was reasonable to predict that they would share a similar gene expression profile to primitive stem cells. Thus, the differentiation and dedifferentiation culture system employed here provided a useful platform to identify genes that control stem cell self-renewal and differentiation. Global gene expression levels were determined and compared in undifferentiated hMSCs, differentiated osteoblasts, chondrocytes, and adipocytes, as well as dedifferentiated cells from osteoblasts, chondrocytes, and adipocytes. Genes were categorized into two groups according to their expression patterns (Fig. 2A). “Differentiation genes” included genes that showed increased expression during differentiation but decreased expression during dedifferentiation. On the other hand, “stemness genes” included genes that were downregulated during differentiation but upregulated during dedifferentiation. As expected, dedifferentiated cells exhibited a gene expression profile similar to that of undifferentiated hMSCs, and both groups of cells were distinct from differentiated cells, as analyzed by PCA (Fig. 2B). Within the differentiated groups, cells from the individual lineages exhibited different expression profiles, indicating the intrinsic uniqueness of each cell type.
Figure Figure 2.. Identification of genes regulating stem cell self-renewal and multilineage differentiation. (A): Schematic diagram depicting the experimental design. Genes were selected based on their expression change during differentiation and dedifferentiation processes. (B): Principal component analysis of global gene expression data. Differentiated cells (i.e., AD, OS, and CH) exhibited a significantly different global gene expression profile compared with undifferentiated human mesenchymal stem cells (hMSCs) and dedifferentiated cells (De-AD, De-CH, and De-OS). On the other hand, undifferentiated and dedifferentiated cells shared a similar global gene expression profile. (C): Venn diagrams showing the genes that changed their expression levels during differentiation and dedifferentiation processes by at least twofold (p < .05). (D): Real-time reverse transcription-polymerase chain reaction analysis of expression levels of five selected genes in undifferentiated hMSCs, differentiated as well as dedifferentiated OS, AD, and CH. Three genes, AFAP, PTPRF, and RAB3B, decreased their expressions during differentiation and increased their expressions during dedifferentiation in all three lineages. The other two genes, DKK3 and FZD7, exhibited an expression pattern similar to that of the others during osteogenesis and adipogenesis but a different expression pattern during chondrogenesis. Abbreviations: AD, adipocytes; AFAP, actin filament-associated protein; CH, chondrocytes; De-AD, dedifferentiated adipocytes; De-CH, dedifferentiated chondrocytes; De-OS, dedifferentiated osteoblasts; DKK3, dickkopf 3; hMSC, human mesenchymal stem cell; MSC, mesenchymal stem cell; OS, osteoblasts; PTPRF, protein tyrosine phosphatase receptor F.
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As shown in Figure 2, there are 460 genes in the stemness genes group, with more than twofold significant decrease during differentiation and increase during dedifferentiation, in at least one lineage. Among these, 62 genes exhibited similar expression patterns in two lineages, and 11 genes in all three lineages (supplemental online Table 2). In the differentiation genes group, 456 genes were upregulated during differentiation and downregulated during dedifferentiation significantly, by more than twofold in at least one lineage, with 12 genes shared by all three lineages and 40 genes by two lineages (supplemental online Table 1). Six genes from differentiation genes group that are typical markers of individual lineage were selected for quantitative RT-PCR analysis, including BSP and OC for osteoblasts, LPL and FABP4 for adipocytes, and COMP and MMP13 for chondrocytes. Although they confirmed the GeneChip data, the quantitative PCR results revealed much higher fold changes in gene expression during both differentiation and dedifferentiation processes in all six genes (supplemental online Table 3). In addition, the fold change between dedifferentiated and undifferentiated cells was close to one, suggesting that the two cell populations are similar at the transcription level as indicated by PCA. Taken together, these results confirmed our hypothesis and proved the effectiveness of our system to identify candidate genes controlling stem cell self-renewal and multipotency.
In general, 37% of differentially expressed genes belong to one of eight major canonical pathways: integrin signaling (RHOJ, ITGA10, FN1, RAP2B, ITGA7, RHOU, COL4A1, ITGA11, LAMB3, LAMA4, PIK3R1, AKT3, MAPK3, COL4A2, LAMA2), IGF-1 signaling (YWHAH, IGF1, IGFBP5, IGFBP4, IGFBP3, IGFBP7, PIK3R1, AKT3, PRKAR2B, MAPK3, FOXO1A), G-protein-coupled receptor signaling (NFKBIA, PDE3A, PDE1C, RGS2, EDNRB, PIK3R1, AKT3, PRKAR2B, RGS4, MAPK3, AGTR1), IL-6 signaling (NFKBIA, HSPB1, IL6, IL1R1, interleukin 1 receptor type II [IL1R2], LBP, JAK2, TNFAIP6, MAPK3, MAP2K3), insulin receptor signaling (PPP1CB, JAK2, PIK3R1, PTPRF, AKT3, PRKAR2B, MAPK3, SGK, FOXO1A, NCK1), pyrimidine metabolism (UPP1, TXNRD1, POLE4, REV3L, ENTPD1, NME1, TRUB2, NT5E, DUT, DPYSL3), nuclear factor-γB signaling (NFKBIA, TNFSF11, NGFB, IL1R2, IRAK3, PIK3R1, AKT3, IL1R1, TLR2), and Wnt/β-catenin signaling (SFRP4, ACVR2, FZD1, FZD7, WNT5A, LEF1, TCF3, AKT3, CDH2, DKK3).
The identified stemness genes and differentiation genes are involved in different cellular processes and functions. For example, a large percentage of stemness genes are involved in signaling pathways, such as IGF-1 signaling (YWHAH, IGF1, IGFBP4, IGFBP5, IGFBP3, AKT3, MAPK3), JAK/Stat signaling (STAT1, STAT4, SOCS2, SOCS5), TGF-β signaling (INHBA, SMURF2, SMAD3, SERPINE1, ACVR2), and Wnt/β-catenin signaling (SFRP4, WNT5A, FZD7, CDH2, TCF3, DKK3). On the other hand, the differentiation genes group contains genes involved largely in metabolism. For instance, GCLC, GLUL, and GSS in glutamate metabolism and GPX3, ANPEP, GCLC, and GSS in glutathione metabolism. Genes in nuclear factor κB (NF-κB) signaling (NFKBIA, IL1R2, IRAK3, PIK3R1, TLR2) and death receptor signaling (TNFSF1, BIRC3) are also significantly represented in the differentiation genes group. Among the genes that shared expression pattern in more than two lineages, those involved in organ morphology, renal and urological disease, amino acid metabolism, dental disease, organismal survival, and free radical scavenging are highly represented in the differentiation genes group, whereas the stemness genes group contains genes that are primarily involved in cell morphology, cancer, cell-to-cell signaling and interaction, cellular growth and proliferation, nervous system development and function, tissue development, and tumor morphology.
Ninety-one genes in the stemness genes group encode proteins that are cell surface proteins and/or receptors, including 20 genes shared by at least two lineages (Table 1). Except for a few genes whose functions are unknown, the majority of these genes function in defined cellular processes, such as metabolism (ATPase and solute carrier proteins), carcinogenesis and metastasis (Tetraspanin family members), cell growth and survival and senescence (AXL, TNFRSF10D), development (NOTCH2, NUMB, JAG1), and signal transduction (PTPRF, FZD7, ICAM1).
Table Table 1.. Selected cell surface proteins and receptors
Functional Analysis of Genes Involved in Stem Cell Self-Renewal and Multipotency
As proof of principle, five genes from the stemness genes group were selected based on their unique expression pattern in individual lineage and their cellular function. By reducing their expression level using siRNA, their effects on hMSC expansion and multilineage differentiation were accessed. Initially identified from GeneChip data, PTPRF exhibited similar expression patterns in all three lineages, AFAP and RAB3B in two lineages, and FZD7 and DKK3 in only one lineage. However, quantitative RT-PCR analysis demonstrated that AFAP and RAB3B also shared a pattern similar to that of PTPRF in all three lineages (Fig. 2D; supplemental online Table 3), whereas FZD7 and DKK3 appeared to behave differently in chondrogenic lineage than in osteogenic and adipogenic lineages (Fig. 2D; supplemental online Table 3). Using gene-specific siRNA, we successfully reduced the gene expression level by 60%–80% compared with transfection controls (Fig. 3A). Protein levels were also reduced for all five genes as confirmed by Western analysis (Fig. 3B) and immunofluorescence staining (Fig. 3C), and inactivation of these genes was sustained for at least 7 days post-siRNA transfection.
Figure Figure 3.. Reduction of gene expression using siRNA transfection. (A): Real-time reverse transcription-polymerase chain reaction analysis of transcript levels of five selected genes 7 days post-transfection. On average, expression of selected genes was significantly reduced by 60%–80% of untransfected and transfection controls. Values are mean ± SD (n = 3). *, p < .05. (B): Western analysis of protein levels 7 days post-siRNA transfection. (C): Immunofluorescence staining of proteins in human mesenchymal stem cells post-transfection. Protein reduction was sustained 7 days post-siRNA transfection. Bar = 10 μm. Abbreviations: AFAP, actin filament-associated protein; DKK3, dickkopf 3; FZD7, frizzled 7; PTPRF, protein tyrosine phosphatase receptor F.
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When cultured in basal medium, hMSCs with reduced level of AFAP, PTPRF, RAB3B, FZD7, or DKK3 exhibited a slower growth rate up to 9 days post-transfection, with a more dramatic difference observed in cells with inactivated DKK3 and PTPRF, but little difference after 9 days, except in DKK3-inactivated cells, compared with controls (Fig. 4A). These results suggest that these genes have little effect on stem cell proliferation when functioning alone. In addition, reduction of each of these five genes dramatically increased the percentage of cells undergoing apoptosis (Fig. 4C), suggesting that these genes function as cell survival protectors.
Figure Figure 4.. Effect of gene reduction by siRNA transfection on human mesenchymal stem cell proliferation and cellular viability. (A): Growth curves showing cell proliferation over 14 days post-siRNA transfection. Cells exhibited a slightly slower rate of proliferation from day 1 to day 8 but a similar rate after day 8, compared with both untransfected control and transfection control. Values are mean ± SD (n = 3). (B): Gene knockdown by siRNA transfection increased the percentage of apoptotic cells 3 days post-transfection. Mean values are presented (n = 2). Abbreviations: AFAP, actin filament-associated protein; DKK3, dickkopf 3; FZD7, frizzled 7; PTPRF, protein tyrosine phosphatase receptor F.
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hMSCs with reduced expression of these five genes were challenged to undergo differentiation into three mesenchymal lineages. As shown in Figure 5, cells with inactivated AFAP, DKK3, FZD7, PTPRF, or RAB3B were able to differentiate into osteoblasts with enhanced ALP activity (Fig. 5A). Reduction of AFAP, FZD7, or RAB3B enhanced ALP transcription significantly (Fig. 5B). Furthermore, inactivation of FZD7 significantly increased OC expression, whereas others appeared to have little difference compared with controls (Fig. 5C). Unlike the uniform enhancement on osteogenesis, inactivation of each of these five genes exhibited a unique impact on chondrogenesis. As shown in Figure 6, reduction of DKK3 increased the production of Alcian Blue-stained sulfated proteoglycan but decreased the collagen type II. Inactivation of AFAP and RAB3B had an indiscernible effect on sulfated proteoglycan but a dramatic increase on collagen type II deposition. On the other hand, reduction of either FZD7 or PTPRF decreased both proteoglycan and collagen type II synthesis. Contrary to the enhancement of osteogenesis and varied effects on chondrogenesis, inactivation of any of these five genes suppressed adipogenesis, demonstrated by the reduction in the number of Oil Red O-positive adipocytes (Fig. 6).
Figure Figure 5.. Osteogenic differentiation potential of human mesenchymal stem cells was altered by siRNA-mediated reduction of gene expression. (A): Alkaline phosphatase (ALP) staining of transfected cells cultured in the absence or presence of osteogenic induction medium for 14 days. Compared with controls, transfected cells showed enhanced ALP staining at the cellular level in osteogenic medium (bottom row). In addition, the number of ALP-positive cells increased in four transfected cell populations (AFAP, FZD7, PTPRF, and RAB3B), even in the absence of osteogenic stimuli (top row). (B): ALP expression levels analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Reduction of AFAP, FZD7, and RAB3B significantly increased ALP transcription. (C): Osteocalcin (OC) expression levels analyzed by quantitative RT-PCR. Reduction of FZD7 expression level increased OC transcription significantly, whereas other genes had little effect on OC level. For (B) and (C), all values are mean ± SD (n = 3). *, p < .05. Bar = 10 μm. Abbreviations: AFAP, actin filament-associated protein; DKK3, dickkopf 3; FZD7, frizzled 7; PTPRF, protein tyrosine phosphatase receptor F; TC, transfected control; UC, untransfected control.
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Figure Figure 6.. Chondrogenic and adipogenic differentiation of human mesenchymal stem cells (hMSCs) was altered by siRNA gene reduction. Chondrogenesis of hMSCs was assessed on the basis of Alcian Blue staining of sulfated proteoglycan matrix and collagen type II immunostaining after cells were induced in chondrogenic medium for 21 days post-transfection. Sections from two individual pellets are shown for each staining. The level of sulfated proteoglycan was increased in cells transfected with DKK3 siRNA and decreased in those transfected with FZD7 or PTPRF siRNA. The sulfated proteoglycan level was not significantly changed in cells transfected with AFAP or RAB3B siRNA. On the other hand, reduction of AFAP or RAB3B increased collagen type II production, whereas reduction of DKK3, FZD7, or PTPRF decreased it. Adipogenesis was detected by the presence of Oil Red O-stained neutral lipids in the cytoplasm. All transfected cells exhibited fewer adipocytes after a 21-day induction. Bar = 10 μm. Abbreviations: AFAP, actin filament-associated protein; DKK3, dickkopf 3; FZD7, frizzled 7; PTPRF, protein tyrosine phosphatase receptor F; TC, transfected control; UC, untransfected control.
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