Silencing of Antichondrogenic MicroRNA-221 in Human Mesenchymal Stem Cells Promotes Cartilage Repair In Vivo

Abstract

There is a growing demand for the development of experimental strategies for efficient articular cartilage repair. Current tissue engineering-based regenerative strategies make use of human mesenchymal stromal cells (hMSCs). However, when implanted in a cartilage defect, control of hMSCs differentiation toward the chondrogenic lineage remains a significant challenge. We have recently demonstrated that silencing the antichondrogenic regulator microRNA-221 (miR-221) was highly effective in promoting in vitro chondrogenesis of monolayered hMSCs in the absence of the chondrogenic induction factor TGF-b. Here we investigated the feasibility of this approach first in conventional 3D pellet culture and then in an in vivo model. In pellet cultures, we observed that miR-221 silencing was sufficient to drive hMSCs toward chondrogenic differentiation in the absence of TGF-b. In vivo, the potential of miR-221 silenced hMSCs was investigated by first encapsulating the cells in alginate and then by filling a cartilage defect in an osteochondral biopsy. After implanting the biopsy subcutaneously in nude mice, we found that silencing of miR-221 strongly enhanced in vivo cartilage repair compared to the control conditions (untreated hMSCs or alginate-only). Notably, miR-221 silenced hMSCs generated in vivo a cartilaginous tissue with no sign of collagen type X deposition, a marker of undesired hypertrophic maturation. Altogether our data indicate that silencing miR-221 has a prochondrogenic role in vivo, opening new possibilities for the use of hMSCs in cartilage tissue engineering. STEM CELLS 2016;34:1801-1811 SIGNIFICANCE STATEMENTWe demonstrated here the effectiveness of an innovative approach based on transient transfection of inhibitor of antichondrogenic miR-221 to direct human mesenchymal stromal cells (hMSCs) toward chondrogenesis both in vitro and in vivo, without exposure to the chondrogenic inducer TGF-b. miR-221 knockdown was sufficient to repair an osteochondral defect subcutaneously implanted in mice, promoting the production of newly formed tissue expressing Collagen type II but not Collagen type X. It is well known that the expression of Collagen type X, a marker for chondrocyte hypertrophy, is an undesired outcome and remains an issue to be solved in the cell-based approach for cartilage regeneration. Therefore, the ability of miR-221 depleted hMSCs to downregulate Collagen type X, both in vitro and in vivo, represents a crucial event on formation of cartilaginous repair tissue and a promising approach to translate into the clinic.

Scaffold‐Based microRNA Therapies in Regenerative Medicine and Cancer

“…[102,106] Whether one of the routes may be better suited for clinical translation remains to be elucidated, although in situ transfecting scaffolds hold greater potential to exist as "off-the-shelf" products. [107] Many distinct scaffold types have been tuned to serve the purpose of miRNA delivery, including several hydrogels, [58,92,106,[108][109][110][111][112][113][114] electrospun fibers, [63,[115][116][117][118] and more prolifically porous or spongy scaffolds. [46,47,50,54,55,96,112,[119][120][121][122][123][124][125][126][127][128][129][130] Before reviewing the details of their applications, summarized in Table 2 and described further in the next section, we will discuss the key characteristics making these materials amenable to miRNA delivery.…”

“…[138] To diminish these undesired effects, it is frequently combined with natural polymers such as hyaluronic acid (HyA), as in the commercial HyStem-HP and Glycosan HyStem. Other natural polymers included in miRNA eluting hydrogels are alginate, [112] silk fibrin, [111] fibrinogen, thrombin, and Matrigel. [92,110] Of note, hydrogels are the first biomaterials used successfully for the delivery of naked miRNA therapeutics, [106,108,109] that is, without the addition of a delivery vector.…”

“…Lolli et al [112] recently published a body of work showing silencing of antichondrogenic miR-221 in hMSCs promotes cartilage repair in vivo. This group previously demonstrated that silencing of the antichondrogenic regulator miR-221 resulted in enhanced chondrogenic marker expression in monolayer BM-MSCs cultured without TGF-β.…”

“…[161] This study was validated here both in 3D pellet culture and additionally in vivo. [112] Lipofectamine RNAiMAXmiR-221 inhibitor was used to transfect monolayer BM-MSCs and showed higher than 95% miR-221 knockdown before resuspension in alginate. This alginate-cell suspension was then added to simulated osteochondral defects and implanted subcutaneously in nude mice.…”

Gene therapy for chondral and osteochondral regeneration: is the future now?

“…MiR-181b, whose targets are different proteins of hippo pathways [87] implicated in chondrocyte regulation, is a negative regulator of chondrocyte differentiation and cartilage development and its synthesis upregulates MMPs increasing the degradation of ECM [88]. MiR221 silencing shows a pro-chondrogenic role in vivo [89] inducing an increase of chondrogenic markers (e.g. collagen type II), and of positive chondrogenic transcription factor Sox9 and Tricho-rhino-phalangeal syndrome 1 protein (TRPS1).…”