Towards the Standardization of Mesenchymal Stem Cell Secretome-Derived Product Manufacturing for Tissue Regeneration.

Mesenchymal stem cell secretome or conditioned medium (MSC-CM) is a combination of biomolecules and growth factors in cell culture growth medium, secreted by mesenchymal stem cells (MSCs), and the starting point of several derived products. MSC-CM and its derivatives could be applied after injuries and could mediate most of the beneficial regenerative effects of MSCs without the possible side effects of using MSCs themselves. However, before the clinical application of these promising biopharmaceuticals, several issues such as manufacturing protocols and quality control must be addressed. This review aims to underline the influence of the procedure for conditioned medium production on the quality of the secretome and its derivatives and highlights the questions considering cell sources and donors, cell expansion, cell passage number and confluency, conditioning period, cell culture medium, microenvironment cues, and secretome-derived product purification. A high degree of variability in MSC secretomes is revealed based on these parameters, confirming the need to standardize and optimize protocols. Understanding how bioprocessing and manufacturing conditions interact to determine the quantity, quality, and profile of MSC-CM is essential to the development of good manufacturing practice (GMP)-compliant procedures suitable for replacing mesenchymal stem cells in regenerative medicine.

The Secretome of Human Dental Pulp Stem Cells and Its Components GDF15 and HB-EGF Protect Amyotrophic Lateral Sclerosis Motoneurons against Death.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable paralytic disorder caused by the progressive death of upper and lower motoneurons. Although numerous strategies have been developed to slow disease progression and improve life quality, to date only a few therapeutic treatments are available with still unsatisfactory therapeutic benefits. The secretome of dental pulp stem cells (DPSCs) contains numerous neurotrophic factors that could promote motoneuron survival. Accordingly, DPSCs confer neuroprotective benefits to the SOD1G93A mouse model of ALS. However, the mode of action of DPSC secretome on motoneurons remains largely unknown. Here, we used conditioned medium of human DPSCs (DPSCs-CM) and assessed its effect on survival, axonal length, and electrical activity of cultured wildtype and SOD1G93A motoneurons. To further understand the role of individual factors secreted by DPSCs and to circumvent the secretome variability bias, we focused on GDF15 and HB-EGF whose neuroprotective properties remain elusive in the ALS pathogenic context. DPSCs-CM rescues motoneurons from trophic factor deprivation-induced death, promotes axon outgrowth of wildtype but not SOD1G93A mutant motoneurons, and has no impact on the spontaneous electrical activity of wildtype or mutant motoneurons. Both GDF15 and HB-EGF protect SOD1G93A motoneurons against nitric oxide-induced death, but not against death induced by trophic factor deprivation. GDF15 and HB-EGF receptors were found to be expressed in the spinal cord, with a two-fold increase in expression for the GDF15 low-affinity receptor in SOD1G93A mice. Therefore, the secretome of DPSCs appears as a new potential therapeutic candidate for ALS.

Dental stem cell-conditioned medium for tissue regeneration: Optimization of production and storage.

BACKGROUND: Mesenchymal stem cells (MSC) effects on tissue regeneration are mainly mediated by their secreted substances (secretome), inducing their paracrine activity. This Conditioned medium (CM), including soluble factors (proteins, nucleic acids, lipids) and extracellular vesicles is emerging as a potential alternative to cell therapy. However, the manufacturing of CM suffers from variable procedures and protocols leading to varying results between studies. Besides, there is no well-defined optimized procedure targeting specific applications in regenerative medicine. AIM: To focus on conditioned medium produced from dental MSC (DMSC-CM), we reviewed the current parameters and manufacturing protocols, in order to propose a standardization and optimization of these manufacturing procedures. METHODS: We have selected all publications investigating the effects of dental MSC secretome in in vitro and in vivo models of tissue regeneration, in accordance with the PRISMA guidelines. RESULTS: A total of 351 results were identified. And based on the inclusion criteria described above, 118 unique articles were included in the systematic review. DMSC-CM production was considered at three stages: before CM recovery (cell sources for CM), during CM production (culture conditions) and after production (CM treatment). CONCLUSION: No clear consensus could be recovered as evidence-based methods, but we were able to describe the most commonly used protocols: donors under 30 years of age, dental pulp stem cells and exfoliated deciduous tooth stem cells with cell passage between 1 and 5, at a confluence of 70% to 80%. CM were often collected during 48 h, and stored at -80 °C. It is important to point out that the preconditioning environment had a significant impact on DMSC-CM content and efficiency.

Identification of secreted factors in dental pulp cell-conditioned medium optimized for neuronal growth.

With their potent regenerative and protective capacities, stem cell-derived conditioned media emerged as an effective alternative to cell therapy, and have a prospect to be manufactured as pharmaceutical products for tissue regeneration applications. Our study investigates the neuroregenerative potential of human dental pulp cells (DPCs) conditioned medium (CM) and defines an optimization strategy of DPC-CM for enhanced neuronal outgrowth. Primary sensory neurons from mouse dorsal root ganglia were cultured with or without DPC-CM, and the lengths of ßIII-tubulin positive neurites were measured. The impacts of several manufacturing features as the duration of cell conditioning, CM storage, and preconditioning of DPCs with some factors on CM functional activity were assessed on neurite length. We observed that DPC-CM significantly enhanced neurites outgrowth of sensory neurons in a concentration-dependent manner. The frozen storage of DPC-CM had no impact on experimental outcomes and 48 h of DPC conditioning is optimal for an effective activity of CM. To further understand the regenerative feature of DPC-CM, we studied DPC secretome by human growth factor antibody array analysis and revealed the presence of several factors involved in either neurogenesis, neuroprotection, angiogenesis, and osteogenesis. The conditioning of DPCs with the B-27 supplement enhanced significantly the neuroregenerative effect of their secretome by changing its composition in growth factors. Here, we show that DPC-CM significantly stimulate neurite outgrowth in primary sensory neurons. Moreover, we identified secreted protein candidates that can potentially promote this promising regenerative feature of DPC-CM.

Effects of green light photobiomodulation on Dental Pulp Stem Cells: enhanced proliferation and improved wound healing by cytoskeleton reorganization and cell softening.

Photobiomodulation (PBM) has been shown to improve cell proliferation and cell migration. Many cell types have been investigated, with most studies using deep penetrating red light irradiation. Considering the interest of surface biostimulation of oral mesenchymal cells after surgical wound, the present study aimed to assess green light irradiation effects on Dental Pulp Stem Cells' (DPSC) proliferation and migration. To understand the mechanisms underlying these effects, we investigated cytoskeleton organization and subsequent cell shape and stiffness. A 532-nm wavelength Nd:YAG laser (30 mW) was applied between 30 and 600 s on DPSC in vitro. Cell proliferation was analyzed at 24, 48, and 72 h after irradiation, by cell counting and enzymatic activity quantification (paranitrophenylphosphate phosphatase (pNPP) test). A wound healing assay was used to study cell migration after irradiation. Effects of PBM on cytoskeleton organization and cell shape were assessed by actin filaments staining. Elasticity changes after irradiation were quantified in terms of Young's modulus measured using Atomic Force Microscopy (AFM) force spectroscopy. Green light significantly improved DPSC proliferation with a maximal effect obtained after 300-s irradiation (energy fluence 5 J/cm2). This irradiation had a significant impact on cell migration, improving wound healing after 24 h. These results were concomitant with a decrease of cells' Young's modulus after irradiation. This cell softening was explained by actin cytoskeleton reorganization, with diminution of cell circularity and more abundant pseudopodia. This study highlights the interest of green laser PMB for the proliferation and migration of mesenchymal stem cells, with encouraging results for clinical application, especially for surgical wound healing procedures.