A bioactive hydrogel harnessing the regenerative potential of notochordal cells serves as instructive cell carrier for nucleus pulposus repair [scRNA-seq human]

Natural biomatrices are popular owing to their ability to mimic tissue-specific biological properties. These properties are crucial for the intervertebral disc, a particularly demanding tissue whose degeneration is a major cause of chronic lower back pain. Degeneration starting within the core of the disc, the nucleus pulposus (NP), is marked by the loss of notochordal cells (NCs) and associated healthy extracellular matrix. The regulative potential of the NC-secretome was exploited through a PEG-based hydrogel formulated with decellularized porcine notochordal cell matrix (dNCM). Even under conditions which mimic the degenerate niche, the hydrogel supported maintenance of the phenotype of native porcine NCs, well-known for the difficulty in preserving their vacuolated morphology. dNCM-PEG hydrogel carrying human mesendodermal progenitors (hMEPCs; iPSC-derived and capable of differentiating into NCs), was injected into NP explants with enzymatically induced matrix degradation and subjected to dynamical loading. hMEPCs engrafted successfully and a healthy disc cell phenotype was observed. Injection of dNCM+hMEPCs into degenerated discs in a pilot experimental dog study indicated that ~7 % of the 0.5 million hMEPCs engrafted. Single cell RNAseq analysis showed over 30 % of the engrafted and recovered hMEPCs co-expressed Collagen Type II and Aggrecan consistent with a functional phenotype. No tumorigenic or systemic immunogenic side effects were observed. At the tissue level, TBXT expression, as well as matrix quality, were enhanced in the treated degenerate disc tissues. Together, this study highlights the translational potential of combining cell-based therapies with a bioactive material containing part of the NC secretome, warranting further development and validation. Overall design: A pilot study using a validated preclinical dog model was performed to explore the potency of intradical injection of hMEPCs supported by dNCM. Initially, moderate degeneration was induced by partial nuclectomy (NX) in alternate IVD levels (L1-L1, L3-L4, L5-L6, L7-S1). One month after induction, these levels were intradiscally injected with dNCM+hMEPCs. Three months after the treatment the L7-S1 disc level was processed for single cell RNA sequencing together with a disc (T12-13) in which NX was performed of a dog of the same litter that was not treated with dNCM+hMEPCs which was used as degenerated control.