Specific, Sensitive, and Stable Reporting of Human Mesenchymal Stromal Cell Chondrogenesis

Abstract

Chondrogenesis is critical to the development and repair of not only articular cartilage but also bone. Preclinical studies suggest that defects in both tissues can be repaired using culture-expanded chondroprogenitor cells, such as mesenchymal stem/stromal cells (MSCs), but directing efficient chondrogenesis by candidate cell populations is an ongoing bottleneck to their clinical application. The goal of this study was to describe a method for the molecular reporting of chondrogenic activity by primary stem/progenitor cells that can complement more labor-intensive destructive measures. A chondrogenesis-responsive promoter was generated, consisting of four repeats of a SOX9-binding enhancer sequence from the first intron of COL2A1 positioned upstream of the core COL2A1 promoter. This promoter was inserted into a lentiviral expression plasmid containing reporter genes copepod green fluorescent protein (copGFP) and firefly luciferase (fLuc), and the resulting lentiviral vector (LV) was used to transduce human MSCs derived from intramedullary reamings. To determine the specificity and stability of reporter expression, MSCs were differentiated in pellet culture for up to 4 weeks. To assess the sensitivity of reporter detection in vivo, undifferentiated and predifferentiated MSC pellets were implanted into osteochondral defects made in immune-suppressed rats. Chondrogenic differentiation of LV-transduced MSCs in pellet culture led to a strong upregulation of both copGFP and fLuc. Robust reporter activity was achieved using LV doses that did not compromise MSC chondrogenesis. Specific reporter induction was sustained over several passages post-transduction. Reporter expression levels were dependent on both pellet culture duration and TGF-b1 dose. When predifferentiated pellets were implanted into rat osteochondral defects, reporter activity was initially diminished but recovered over the following 2 weeks, suggesting acute postsurgical inflammation suppressed reporter expression. This hypothesis was supported by potent cytokine inhibition of reporter levels and glycosaminoglycan deposition within additional pellets in vitro. When combined with lentiviral transgene integration, the COL2A1-based promoter allowed specific, sensitive, and stable reporting of MSC chondrogenic activity. This promoter can be used with the extensive selection of reporter vectors now available to study different chondroprogenitor cells with promise for cartilage and bone tissue engineering and regenerative medicine.

A Transgenic Rat for Noninvasive Assessment of Chondrogenesisin Vivo

“…To evaluate chondrogenesis during articular cartilage repair, we applied a bilateral osteochondral defect (OCD) model as previously described. 19 Rats were anesthetized with isoflurane, and both hind limbs were shaved and sanitized by alternating ethanol and povidone iodine scrubs. A 2-cm skin incision was made 1 cm lateral to the knee joint, so that the wound clips used for skin closure would not interfere with bioluminescence signal at early imaging timepoints.…”

Illuminating the Regenerative Properties of Stem Cells In Vivo with Bioluminescence Imaging

“…[ 43 ] Besides, stem cells have been transduced in vitro and subsequently implanted and imaged in vivo. [ 44,45 ]…”

“…This approach is frequently employed to monitor the differentiation and stress response of stem cells. [ 36,44 ] Another method utilizes "causes recombination" (Cre) recombinase to regulate luciferase expression. The enzyme catalyzes site‐specific DNA recombination events to limit luciferase expression to cells of interest, such as endogenous stem cells.…”

“…[ 106 ] hBMSCs transduced with a Fluc gene downstream of a SOX9‐binding enhancer region were implanted in rat osteochondral defects to reveal the sensitivity of chondrogenesis to post‐surgical inflammation. [ 44 ] The stem cells were pre‐differentiated towards a chondrogenic phenotype before implantation. The ratio of the BLI signal from implants with SOX9‐responsive Fluc in one knee to that from implants with constitutive Fluc expression in the opposite knee decreased significantly the first two days after surgery but then recovered over two weeks.…”

“…A similar pattern of luminescence was induced in vitro by exposure to inflammatory cytokines, suggesting that acute post‐surgical inflammation suppressed chondrogenesis. [ 44 ]…”