Oral Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Introduction. The regenerative potential of mesenchymal stromal cells (MSCs) has been the focus of numerous clinical trials to treat degenerative disc disease (DDD). Although, intradiscal MSC injections improved pain and disability in patients with DDD, disc regeneration was not achieved. The immunosuppressive capacity of MSCs are thought to be responsible for the favorable outcomes. Pro-inflammatory, hypoxic, and 3D culture priming of MSCs are common strategies to enhance their immunosuppressive potency, but the underlying mechanisms are not well understood. Therefore, this study aimed to i) compare the effects of these priming strategies on the in vitro immunosuppressive potential of MSCs, ii) assess if priming effects are stable under standard and translationally relevant culture conditions, and iii) investigate if these priming strategies engage the same immunosuppressive mechanisms.

Methods. Commercial human iliac crest-derived bone marrow MSCs were used (Figure 1). Functional in vitro T cell suppressive potency measurements were conducted to assess the impact of pro-inflammatory, hypoxic, and 3D culture priming on the immunosuppressive potential of MSCs. Primed MSCs were either cultured under standard cell culture conditions or translationally relevant culture conditions, and their transcriptomic adaptations were monitored over time. The translationally relevant conditions were priming approach-specific, i.e. following hypoxic priming, MSCs were cultured under a low oxygen concentration (5% O₂) typical for IVDs; following pro-inflammatory priming, MSC were cultured under inflammation levels found in degenerating IVDs (50 pg/mL TNF-α); and following 3D culture priming, MSC were kept in spheroids, to mimic intra-discal spheroid administration. Next-generation sequencing was performed to assess if different priming strategies activate distinct immunosuppressive mechanisms.

Results. i) All priming strategies induced profound transcriptomic changes in MSCs (Figure 2). The in vitro T cell proliferation was suppressed by pro-inflammatory (-99% ± 11%, p < 0.001) and 3D culture primed MSCs (-35% ± 11%, p = 0.03), but not by hypoxic primed MSCs (+21% ± 14%, p = 0.40) (Figure 3a). ii) Priming effects rapidly faded under standard cell culture conditions but were partially preserved under translationally relevant conditions (Figure 3b). Inflammatory disc condition (50 pg/mL TNF-α) after pro-inflammatory priming partially preserved the T cell suppressive capacity (day 4: ‑35% ± 11%, p = 0.04, day 8: ‑22% ± 11%, p = 0.46). Continuous spheroid culture completely rescued the enhanced immunosuppressive priming effects from fading (day 4: ‑27% ± 11%, p = 0.21, day 8: ‑41% ± 11%, p = 0.007). iii) Pro-inflammatory priming upregulated genes involved in immune system responses and defense response, 3D culture priming caused transcriptomic changes involving the cytoskeleton and cellular signaling (Figure 4).

Discussion. This study demonstrated that i) pro-inflammatory, hypoxic, and 3D culture priming result in different immunosuppressive potencies. ii) Priming effects were transient and rapidly lost, yet continuous 3D culture was able to maintain the immunosuppressive potential. iii) The cellular mechanisms leading to T cell suppression were different in pro-inflammatory and 3D culture priming. Continuous 3D culture could act as a functionalized formulation, supporting the administration of MSC spheroids for a sustainably improved immunosuppressive potency.