A pre-clinical trial using bio-material-based delivery of in vitro-derived mesenchymal stem cells to effect cartilage repair in a canine model of osteoarthritis

Abstract

With a view towards harnessing the therapeutic potential of canine mesenchymal stem cells (cMSCs) as modulators of inflammation, immune response and tissue regeneration candidates, and to avoid the issues of the variable quality and quantity of harvested MSCs, we have examined the immunomodulatory and anti-inflammatory properties of cMSCs produced from canine induced pluripotent stem cells (ciPSC.MSCs), and compared them to cMSCs harvested from canine adipose tissue (cAT.MSC) and bone marrow (cBM.MSC). ciPSC.MSCs constitutively express inducible nitric oxide synthase (iNOS), galectin-9 (GAL-9), cyclooxygenase-2 (COX-2), transforming growth factor-β1 (TGF-β1), prostaglandin receptor-2α (PTGER-2α) and indoleamine 2, 3 dioxygenase (IDO). When stimulated with the pro-inflammatory cytokines, canine tumor necrosis factor-α (cTNF-α), canine interferon-γ (cIFN-γ), or combination of both, ciPSC.MSCs upregulated their expression of iNOS, GAL-9, TGF-β1, PTGER-2α, IDO and COX-2.In another study, we examined the osteogenesis and chondrogenesis capability of ciPSC.MSCs in comparison to native canine MSCs, cAT.MSCs and cBM.MSCs, the MSCs cultured with chondrogenic and osteogenic differentiation media and also were seeded in decellularized extracellular matrix (DECM) bio-scaffolds. The culture of ciPSC.MSCs and harvested MSCs with chondrogenic and osteogenic factors associated with the expression of “cartilage-specific” markers including SOX-9, collagen type II (COL II), AGGRECAN, cartilage oligomeric matrix protein (COMP) and also “bone-specific” marker, collagen type I (COL I), in higher or comparable level in ciPSC.MSCs than cAT.MSCs and cBM.MSCs. Interestingly, the osteogenic potential of ciPSC.MSCs was confirmed by immunofluorescence detection of hydroxyapatite and also staining of calcium compounds in DECM bio-scaffolds seeded with ciPSC.MSCs with osteogenic differentiation factors. Also, the staining of seeded DECMs bio-scaffolds with ciPSC.MSCs in combination with chondrogenic factors showed the regeneration of cartilage tissue confirmed by uniformed and intense glycosaminoglycan (GAGs) staining. These findings totally can develop into the introduction of commercially available ‘off-the-shelf” iPSC.MSCs as anti-inflammatory supplements and regenerative therapy for the treatment of diseases like canine bowel syndrome and osteoarthritis.We also generated canine induced pluripotent stem cell (ciPSC) from female adult dog BM.MSCs by using a novel Sendai virus-mediated re-programming method. The re-programmed cells were maintained in culture with leukaemia inhibitory factor (LIF) or in a combination of LIF and basic fibroblast growth factor (bFGF), which are thought to support either naive or primed pluripotent states, respectively. Both types of generated cells expressed endogenous pluripotency markers including canine NANOG, OCT-4, SOX-2, DAX-1, REX-1 and TERT. Both groups formed embryoid bodies containing derivatives of the three germ layers as determined by the expression of the germ layer-specific markers CXC chemokine receptor 4 (CXCR-4) and alpha-fetoprotein (AFP) (endoderm), collagen, type II, alpha (COLL-IIA) and GATA-2 (mesoderm), and βIII-TUBULIN, ENOLASE and NESTIN (ectoderm). Most importantly, ciPSCs maintained with LIF/bFGF showed expression of XIST indicating the inactivation of X chromosome which is typically indicative of a primed state of pluripotency. In contrast, ciPSCs maintained with LIF did not show XIST expression which reflects the presence of two active X chromosomes and therefore a more naive state of pluripotency. Also, the expression of REX-1 and NR5A2 in ciPSCs cultured with LIF further confirmed the more naive state of our maintained ciPSCs with LIF in comparison to those cultured with LIF/bFGF. However, the RNA-Seq analysis showed the exclusive transcription of naive-specific markers including ESRRB, DAPP4, DAPP2 and PRDM14 in lentiviral re-programmed ciPSCs while these markers have been absent in Sendai re-programmed ciPSCs, maintained with LIF and also LIF/bFGF . In addition, the confirmed presence of two active X chromosome (XaXa) and the mRNA expression of REX-1 in lentiviral re-programmed ciPSCs has further shown that lentiviral re-programmed ciPSCs are in fully naive state of pluripotency than Sendai re-programmed ciPSCs maintained with LIF and also LIF/bFGF. Studies in mouse and human have shown that the state of pluripotency is an important consideration when modelling cellular functions and disease states; our data suggests that similar considerations of pluripotent state should be made when using canine pluripotent cells for future studies.