Development of quality controls to assess the potency of Human Regenerative Macrophages (REMaST®) for clinical use

We have recently developed a novel cell-based therapy consisting of autologous adult Regenerative Macrophages (REMaST®) for nervous tissue regeneration. Strong preclinical evidence has shown the effect of REMaST® on nerve growth and spinal cord injury (SCI) healing. Multiple administrations of REMaST® starting from the subacute phase following severe (complete) compressive-contusive SCI, improved motor recovery and rewired the SCI hostile microenvironment, reducing the fibrotic scar, increasing vasculature and tissue oxygenation and dampening the chronic inflammation. Hemera is now developing the protocol to generate REMaST® for clinical use. In this contest, we developed ad hoc quality controls (QCs) to test the potency of REMaST®. Based on the major mechanisms of action observed in preclinical studies, we developed QCs to assess the neuroprotective, chemotactic, and immunomodulatory properties. To test the direct role of REMaST® on neural cell survival, we co-cultured them with motor neurons (MNs) derived from human induced Pluripotent Stem Cells (iPSCs) in condition of oxygen and glucose deprivation (OGD). Subsequently, as a measure of cellular damage, we quantified LDH release through a biochemical colorimetric assay. REMaST® co-cultured with human MNs were able to decrease neuron death up to 78% compared to MNs baseline control (MNs 1±0.76; REMaST®: 0.21±0.24; p=0.039,). REMaST® migrate towards the site of injury in response to specific stimuli, including SDF-1. To test REMaST® chemotactic property, we performed the boyden chamber assay, exposing REMaST® to the chemoattractant SDF-1, compared to canonical anti-inflammatory (M2) macrophages. Quantification of migrated DAPI-positive cells showed that REMaST® are 60% more responsive to SDF-1 as compared to M2 (REMaST®: 17.9600±1.7710; M2: 10.9600±0.8302, p<0.05). REMaST® immunomodulatory properties were investigated by quantifying the M1 iNOS expression. In M1-polarized macrophages iNOS is typically upregulated during inflammatory reactions. By using flow cytometry analysis, we measured M1 iNOS expression, in M1 cultured both alone and with REMaST®. We found that REMaST® co-culture downregulates up to 15% M1 macrophages iNOS expression, with respect to M1 baseline control (M1: 7.99±0.66; 1:10 REMaST®/M1: 6.84±0.58; p=0.024). These QCs capable to test REMaST® potency will be used for the development of the protocol to generate REMaST® for clinical use.