6-hydroxydopamine affects multiple pathways to induce cytotoxicity in differentiated LUHMES dopaminergic neurons.

The debilitating effects of Parkinson's disease (PD) progress over time and are pathophysiologically characterized by the formation of Lewy bodies due to the accumulation of α-synuclein aggregates resulting in the death of dopaminergic neurons. In the present study, we determined cell death pathways activated by acute exposure to 6-hydroxydopamine (6-OHDA) in differentiated LUHMES cells empirically followed by a 24 h toxin free interval, henceforth termed as washout/recovery period. Acute 6-OHDA exposure led to morphological changes in LUHMES cells and resulted in significant loss of neurite length and neurite thickness. Generation of reactive oxygen species and loss of mitochondrial membrane potential in the neuronal processes were persistent even after the recovery period. Our results show that 6-OHDA exposure leads to significant reduction in expression of mitochondrial OXPHOS complexes I, II, and IV and activation of caspase mediated apoptotic cell death cascade as observed by enhanced protein expression of cleaved-PARP-1 and cleaved-Caspase-3. Immunofluorescence microscopy approach confirmed that cell death occurs independent of the AIF translocation to the nucleus. Our experimental model, led to a ∼5-fold lower α-synuclein monomer expression and, interestingly, resulted in loss of protein ubiquitination in whole cell lysates. Altogether, this work provides evidence of multiple pathways targeted by 6-OHDA in differentiated LUHMES cells and expands research avenues for addressing the knowledge gap regarding the effect of 6-OHDA in the ubiquitin proteasome system for PD therapies.

Characterization of an umbilical cord blood sourced product suitable for allogeneic applications.

Aim: Umbilical cord blood (UCB) sourced allografts are promising interventions for tissue regeneration. As applications of these allografts and regulations governing them continue to evolve, we were prompted to identify parameters determining their quality, safety and regenerative potential. Materials & methods: Flow-cytometry, mass-spectrometry, protein multiplexing, nanoparticle tracking analysis and standard biological techniques were employed. Results: Quality attributes of a uniquely processed UCB-allograft (UCBr) were enumerated based on identity (cell viability, immunophenotyping, proteomic profiling, and quantification of relevant cytokines); safety (bioburden and microbiological screening), purity (endotoxin levels) and potency (effect of UCBr on chondrocytes and mesenchymal stem cells derived exosomes). These attributes were stable up to 24 months in cryopreserved UCBr. Conclusion: We identified a comprehensive panel of tests to establish the clinical efficacy and quality control attributes of a UCB-sourced allograft.

Cytokines in umbilical cord blood-derived cellular product: a mechanistic insight into bone repair.

AIM: Umbilical cord blood (UCB) finds frequent applications in regenerative medicine. We evaluated the role of cytokines present in a uniquely processed, UCB-derived cellular allograft product (UCBp). MATERIALS & METHODS: Luminex multiplex assay and standard cell biology methods were employed. RESULTS: Study with allografts from 33 donors identified 44 quantifiable cytokines in the UCBp derived conditioned media (CM). The UCBp-CM elevated proliferation and migration rates of mesenchymal stem cells (MSCs) and bone marrow stromal cells. Moreover, UCBp-CM induced secretion of VEGF-A and osteoprotegerin, which promoted angiogenesis of endothelial cells and positively influenced the osteogenic differentiation of MSCs, respectively. CONCLUSION: Cytokines in UCBp stimulate cellular processes important for bone regeneration, making UCBp an excellent candidate for potential applications in orthopedic procedures like bone non-union and spinal fusion.

Biochemical characterization of pure dehydrated binate amniotic membrane: role of cytokines in the spotlight.

AIM: Placental allografts used for tissue regeneration differ in membrane compositions and processing techniques. A uniquely folded dehydrated binate amniotic membrane (DBAM) was biochemically characterized to evaluate its potential role in wound healing. METHODS: Histology, Luminex-based immunoassay and standard in vitro cell biology techniques were employed. RESULTS: Histological staining confirmed that the DBAM was chorion free with epithelial cell layer of the respective amnion membranes facing outward. DBAM had quantifiable levels of relevant cytokines that induced proliferation and migration while bolstering secretory activity of the cells. DBAM retained biological efficacy at a broad range of temperatures. CONCLUSION: Cytokines in DBAM stimulate bone marrow stromal and stem cells that may lead to tissue regeneration and wound healing in a clinical setup.