Chondrogenic effect of precartilaginous stem cells following NLS-TAT cell penetrating peptide-assisted transfection of eukaryotic hTGFβ3

Abstract

Cell penetrating peptides (CPPs) are a series of promising carriers for delivering exogenous DNA to living cells. Among them, the combination of the human immunodeficiency virus TAT protein (TAT) with the SV40 large T protein nuclear localization signal (NLS) to form NLS-TAT performs well. In the present study, we took advantage of this new carrier to deliver transforming growth factor-beta 3 (TGFβ3) genes. TGFβ3 was expressed by the pEGFP-N1 vector following transfection of rat precartilaginous stem cells (PSCs), which promoted hTGFβ3 protein self-expression. At 24, 48, 72, and 120 h after transfection, the expression levels of hTGFβ3 were found to be elevated as compared with the control. The expression of hTGFβ3 was found to mediate the chondrogenic effect of PSCs. Thus, we determined the expression of the chondrogenesis-related genes type II collagen, Sox 9, and aggrecan in PSCs at 24, 48, 72, and 120 h after transfection. We found that their transcription and translation was augmented, which indicated a trend of active chondrogenesis in the PSCs. Our results demonstrated that NLS-TAT had the ability to deliver exogenous DNA into rat PSCs and could be actively expressed. This process successfully promoted PSC chondrogenesis. Additionally, PSC, may represent a new type of stem cells, and thus show great potential in regenerative repair following cartilage injury.

Transforming Growth Factor-β1 (TGF-β1) Induces Mouse Precartilaginous Stem Cell Proliferation through TGF-β Receptor II (TGFRII)-Akt-β-Catenin Signaling

“…Similar to previous studies [3,4], the neonate C57BL/6J mice were provided by the animal center of Nanjing Medical University. The tissues located around the perichondrial mesenchyme (also termed “the La Croix rings”) were cut down and digested sequentially with Complete Trypsin Solution (Chemicon International Inc., Temecula, CA, USA) with 0.05% collagenase type I (Sigma Chemical Co., St. Louis, MO, USA).…”

“…The immuno-selected mouse precartilaginous stem cells (PSCs) were then cultured in DMEM/F12 medium (Thermo Fisher Scientific Inc., Fremont, CA, USA), supplemented with 20% fetal calf serum (FCS, Gibco, Shanghai, China), 100 units/mL penicillin and streptomycin in a 5% CO 2 /37 °C incubator. The detailed procedures have been described [3,4]. The medium was switched every 2 days.…”

“…Precartilaginous stem cells (PSCs) could self-renew or differentiate into chondrocytes to promote bone growth [2,3,4]. Robinson et al have isolated PSCs from perichondrial mesenchyme (also termed “the ring of La Croix”) of neonate rats by immunomagnetic beads through fibroblast growth factor receptor-3 (FGFR-3) antibody selection [3].…”

“…Robinson et al have isolated PSCs from perichondrial mesenchyme (also termed “the ring of La Croix”) of neonate rats by immunomagnetic beads through fibroblast growth factor receptor-3 (FGFR-3) antibody selection [3]. These PSCs have potential to proliferate and to differentiate directionally into chondrocytes [3,4]. Transforming growth factor-β1 (TGF-β1) is shown to promote adult stem cell proliferation and chondrocyte differentiation [5,6], while its role in PSC proliferation and the underlying signaling mechanisms are not studied.…”

Polyethylenimine derivate conjugated with RGD-TAT-NLS as a novel gene vector

“…It has been reported that Arginine-glycine-aspartate (RGD) peptide could target to Įvȕ3 receptors specifically [9]. Cell-penetrating peptides (CPPs) can translocate macromolecules across the plasma membrane [10]. The typical CPPs were known as TAT [11] (amino acids 49-57 of the HIV TAT protein [10]) with the sequence RKKRRQRRR.…”