Semaphorin 3A and Hypoxia Inducible Factor 1 Subunit Alpha Co-Overexpression Enhances the Osteogenic Differentiation of Induced Pluripotent Stem Cells and Mesenchymal Stem Cells In Vitro
Mesenchymal stem or stromal cells (MSCs) are a population of cells with the capability to self-renew and differentiate into varied cell lineages, which are majorly housed within the bone marrow. The culture of MSCs onto osteoconductive materials, such as hydroxyapatite (HA) scaffolds, can induce osteogenic differentiation, which is considered as a promising tissue engineering-based strategy in orthopedics. However, the limited proliferation potential of MSCs and the difficulty to prepare the bank of MSCs with uniform biological activity hinder the clinical application of MSCs. Deriving functional MSCs from induced pluripotent stem cells (iPSCs) is a potential strategy developed to address these limitations.
The aim of the present work was to evaluate whether the co-overexpression of semaphorin 3A (Sema3A) and hypoxia inducible factor 1 subunit alpha (HIF1a) can further magnify the osteoconductive effects of HA scaffolds on iPSC-MSCs. Sema3A is a secreted protein that belongs to the semaphorin family. Semaphorins display pleiotropic biologic functions through their receptors, plexins. The regulatory roles of Sema3A in the angiogenesis, myogenic regeneration, and synaptic connectivity have been reported before. Recently, this factor has been suggested as an osteoprotective factor because of its ability to reduce bone resorption and enhance bone formation, and shift adipose MSCs towards osteogenic phenotype. On the basis of these previous studies, to promote osteogenic differentiation of iPSC-MSCs, our group initially decided to modify the iPSC-MSCs by overexpressing Sema3A. It is noteworthy that, besides the pro-osteogenic role of Sema3A, Sema3A can trigger a pro-apoptotic program that sensitizes cancer cells or chondrocytes to apoptosis.
HIF1a forms a heterodimer with the b-subunit. Hypoxia stabilizes HIF1a from constitutive degradation largely by suppressing the activity of oxygen-dependent prolyl hydroxylases. In contrast to pro-apoptotic Sema3A, HIF1a is a potent pro-survival factor, although this conclusion is deduced majorly from studies on cancer. Increasing evidence also validates the pro-survival role of HIF1a in none-cancer cells, such as vascular endothelial cells. Interestingly, we noted that MSCs overexpressing HIF1a displayed stronger proliferation as evidenced by a remarkable increase in 5-Bromo-2’-deoxyuridine (BrdU) incorporation in a study from Ciria et al. Considering the potential apoptosis may result from Sema3A overexpression alone, we decided to co-overexpress HIF1a with Sema3A in iPSC-MSCs in vitro.
In the present study, iPSC-MSCs were infected with Sema3A, HIF1a, or Sema3A-HIF1a overexpression lentiviruses, and their survival and osteogenic differentiation in vitro were determined. Sema3A and HIF1a were linked together with the three (GGGGS; G, glycine; S, serine) peptide fragment, and their co-expression in iPSC-MSCs was mediated by a lentiviral vector. The fusion protein retained the immune reactivity for both Sema3A and HIF1a as determined with Western blotting. iPSC-MSCs were infected with overexpression lentivirus (oeLenti) as negative control, oeLenti-Sema3A, oeLenti-HIF1a or oeLenti-Sema3A-HIF1a lentiviruses.
Sema3A overexpression alone promoted the osteogenic differentiation of iPSC-MSCs (the activity and/or expression of osteoblast markers, such as alkaline phosphatase, osteopontin, and osteocalcin, were upregulated), and suppressed cell survival. The Sema3A-HIF1a fusion protein showed a comparable osteoconductive effect to that of Sema3A without reducing cell survival. We further seeded iPSC-MSCs modified by SemaA-HIF1a overexpression onto hydroxyapatite (HA) scaffolds, and evaluated their growth and differentiation on this three-dimensional material. Additional data indicated that, as compared to iPSC-MSCs cultured in ordinary two-dimensional dishes, cells cultured in HA scaffolds grew (blank vs. HA scaffolds: 0.83 vs. 1.39 for survival) and differentiated better (blank vs. HA scaffolds: 11.29 vs. 16.62 for alkaline phosphatase activity).
Following the methods first reported by Takahashi and Yamanaka in 2006, mouse embryo fibroblasts (MEFs) were reprogrammed into iPSCs by ectopic expression of Oct3/4, Sox2, c-Myc, and Klf4, and then differentiated into MSCs. Like the MSCs directly derived from the bone marrow, iPSC-MSCs obtained in the present work were found to be positive for CD29, CD90, CD105, and CD73, and negative for hematopoietic cell lineage-specific antigens, CD34 and CD45. We further demonstrated that co-expression of Sema3A and HIF1a into iPSC-MSCs promoted their survival and osteogenic differentiation.
Lepelletier et al have identified MSCs from bone marrow as Sema3A positive cells. Sema3A expression was also detectable, though not abundant, in iPSC-MSCs in our study. These findings suggest that Sema3A expresses in MSCs regardless of the different sources. A report from Liu et al showed that Sema3A overexpression shifted the adipose MSCs towards osteogenic phenotype, and inhibited the adipogenic differentiation. In agreement with this study, we also found that Sema3A overexpression alone or together with HIF1a upregulated the osteoblast markers, ALP, Opn, and Ocn, in iPSC-MSCs. We noted that, in Liu et al’s study, the proliferation of adipose MSCs was barely affected by Sema3A overexpression, which is inconsistent with our findings. We found that Sema3A overexpression alone significantly suppressed the proliferation of iPSC-MSCs. Although the fact that MSCs used in Liu et al’s work and ours are derived from different sources may explain this inconsistent phenomena, most previous studies showing the apoptotic role of Sema3A supported our present findings.
To counteract the pro-apoptotic effects induced by Sema3A in iPSC-MSCs, HIF1a was then infused with Sema3A via a 3(GGGGS) linker, a structure that is commonly used to combine two proteins together, and then co-infected the iPSC-MSCs. Whether the two encoding fragments combined by this linker can produce functional proteins is the key for the co-expression experiment. Western blotting analysis was first performed using total protein extraction from cells infected with Sema3A-HIF1a viruses. We found that an approximate 180,000 Da blot was probed by both Sema3A and HIF1a antibodies, suggesting that the fusion protein retained the Sema3A and HIF1a immunoreactivity. Immune reactivity of a protein does not equal to its activity or function. Nonetheless, the immune reactivity of cells infected with SemaA-HIF1a to both Sema3A and HIF1a antibodies at least indicated that the antigenic structure of SemaA-HIF1a protein was not impaired.
HIF1a overexpression alone significantly promoted the proliferation of iPSC-MSCs, which confirmed its pro-survival role stated by previous studies. Interestingly, the Sema3A-HIF1a fusion products enhanced the proliferation of iPSC-MSCs, though were less significant than HIF1a overexpression alone. The stronger proliferative ability of iPSC-MSCs induced by Sema3A-HIF1a viruses also indicated that the fusion products were functional.
Nanocrystalline HA materials are widely used as the scaffold in bone repair. HA is composed of Ca10(PO4)6(OH)2, the major inorganic components of natural bone tissue. HA scaffolds provide a three-dimensional microenvironment that allows cells to attach onto, grow and differentiate. Implantation of MSCs onto three-dimensional scaffolds shows a great promise in bone repair. We next implanted iPSC-MSCs expressing Sema3A-HIF1a onto HA scaffolds, and evaluate their growth and differentiation on this scaffold. We found that the modified iPSC-MSCs could attach successfully onto the HA scaffold, suggesting that the adhesion was not affected by Sema3A-HIF1a forced overexpression. Additionally, the growth and osteogenic differentiation of iPSC-MSCs cultured on the HA scaffolds were also better than those cultured in two-dimensional dishes.
The timely formation of blood vessels in the fracture callus is a key component to accelerate bone regeneration. Besides the anti-survival role of Sema3A, its anti-angiogenesis role shall be taken into serious consideration. Of note, the pro-angiogenesis role of HIF1a has been widely reported. Hypoxia-mimicking agents, such as dimethyloxalylglycine, can protect HIF1a from degradation, and promote the secretion of vascular endothelial growth factor-A in vivo. Sole overexpression of Sema3A in adipose MSCs promoted the bone regeneration mediated by poly (lactic-co-glycolic acid) scaffolds. As the growth and osteogenic differentiation of iPSC-MSCs overexpressing Sema3A-HIF1a were better, it is anticipated that these cells may have a superior effect to those only overexpressing Sema3A in promoting bone formation in vivo. This hypothesis shall be validated in animals with bone defect injury. Whether the angiogenesis is involved in Sema3A-HIF1a overexpression-mediated bone repair in vivo shall be evaluated.
In summary, the present work shows that Sema3A-HIF1a co-overexpression augments the survival of iPSC-MSCs, and promotes their osteogenic differentiation. Modifying iPSC-MSCs with pro-survival and pro-osteogenic factors may represent a promising strategy to optimize tissue engineering-based strategy in bone repair.
doi.org/10.1097/CM9.0000000000000612
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