Intra-individual variability in the neuroprotective and promyelinating properties of conditioned culture medium obtained from human adipose mesenchymal stromal cells.

Greater knowledge of mesenchymal stromal cell (MSC)-based therapies is driving the research into their secretome, identified as the main element responsible for their therapeutic effects. The aim of this study is to characterize the individual variability of the secretome of adipose tissue-derived MSCs (adMSCs) with regard to potential therapeutical applications in neurology. adMSCs were isolated from the intact adipose tissue of ten subjects undergoing abdominal plastic surgery or reduction mammoplasty. Two commercial lines were also included. We analyzed the expansion rate, production, and secretion of growth factors of interest for neurological applications (VEGF-A, BDNF, PDGF-AA and AA/BB, HGF, NGF, FGF-21, GDNF, IGF-I, IGF-II, EGF and FGF-2). To correlate these characteristics with the biological effects on the cellular targets, we used individual media conditioned with adMSCs from the various donors on primary cultures of neurons/astrocytes and oligodendrocyte precursor cells (OPCs) exposed to noxious stimuli (oxygen-glucose deprivation, OGD) to evaluate their protective and promyelinating properties, using MSC medium as a control group. The MSC secretome showed significant individual variability within the considered population with regard to PDGF-AA, PDGF-AB/BB, VEGF-A and BDNF. None of the MSC-derived supernatants affected neuron viability in normoxia, while substantial protection by high BDNF-containing conditioned MSC medium was observed in neuronal cultures exposed to OGD conditions. In OPC cultures, the MSC-derived supernatants protected cells from OGD-induced cell death, also increasing the differentiation in mature oligodendrocytes. Neuroprotection showed a positive correlation with VEGF-A, BDNF and PDGF-AA concentrations in the culture supernatants, and an inverse correlation with HGF, while OPC differentiation following OGD was positively correlated to PDGF-AA concentration. Despite the limited number of adMSC donors, this study showed significant individual variability in the biological properties of interest for neurological applications for adMSC secretome, an under-researched aspect which may represent an important step in the translation of MSC-derived acellular products to clinical practice. We also showed the potential protection capability of MSC conditioned medium on neuronal and oligodendroglial lineages exposed to oxygen-glucose deprivation. These effects are directly correlated to the concentration of specific growth factors, and indicate that the remyelination should be included as a primary target in MSC-based therapies.

Baldassarro VA ，Perut F ，Cescatti M ，Pinto V ，Fazio N ，Alastra G ，Parziale V ，Bassotti A ，Fernandez M ，Giardino L ，Baldini N ，Calzà L ... -  《Stem Cell Research & Therapy》

Neurotrophic and neuroprotective potential of human limbus-derived mesenchymal stromal cells.

The purpose of this study was to examine neurotrophic and neuroprotective effects of limbus stroma-derived mesenchymal stromal cells (L-MSCs) on cortical neurons in vitro and in vivo. Cultured L-MSCs were characterized by flow cytometry and immunofluorescence through the use of specific MSC marker antibodies. Conditioned media were collected from normoxia- and hypoxia-treated L-MSCs to assess neurotrophic effects. Neuroprotective potentials were evaluated through the use of in vitro hypoxic cortical neuron culture and in vivo rat focal cerebral ischemia models. Neuronal morphology was confirmed by immunofluorescence with the use of anti-MAP2 antibody. Post-ischemic infarct volume and motor behavior were assayed by means of triphenyltetrazolium chloride staining and open-field testing, respectively. Human growth antibody arrays and enzyme-linked immunoassays were used to analyze trophic/growth factors contained in conditioned media. Isolated human L-MSCs highly expressed CD29, CD90 and CD105 but not CD34 and CD45. Mesenchymal lineage cell surface expression pattern and differentiation capacity were identical to MSCs derived form human bone marrow and adipose tissue. The L-MSC normoxic and hypoxic conditioned media both promoted neurite outgrowth in cultured cortical neurons. Hypoxic conditioned medium showed superior neurotrophic function and neuroprotective potential with reduced ischemic brain injury and improved functional recovery in rat focal cerebral ischemia models. Human growth factor arrays and enzyme-linked immunoassays measurements showed neuroprotective and growth-associated cytokines (vascular endothelial growth factor [VEGF], VEGFR3, brain-derived neurotrophic factor, insulin-like growth factor -2 and hepatocyte growth factor) contained in conditioned media. Hypoxic exposure caused VEGF and brain-derived neurotrophic factor upregulation, possibly contributing to neurotrophic and neuroprotective effects. L-MSCs can secrete various neurotrophic factors stimulating neurite outgrowth and protecting neurons against brain ischemic injury through paracrine mechanism.

Liang CM ，Weng SJ ，Tsai TH ，Li IH ，Lu PH ，Ma KH ，Tai MC ，Chen JT ，Cheng CY ，Huang YS ... -  《-》

Paracrine-mediated neuroprotection and neuritogenesis of axotomised retinal ganglion cells by human dental pulp stem cells: comparison with human bone marrow and adipose-derived mesenchymal stem cells.

Mead B ，Logan A ，Berry M ，Leadbeater W ，Scheven BA ... -  《PLoS One》

Reduced neuroprotective potential of the mesenchymal stromal cell secretome with ex vivo expansion, age and progressive multiple sclerosis.

Clinical trials using ex vivo expansion of autologous mesenchymal stromal cells (MSCs) are in progress for several neurological diseases including multiple sclerosis (MS). Given that environment alters MSC function, we examined whether in vitro expansion, increasing donor age and progressive MS affect the neuroprotective properties of the MSC secretome. Comparative analyses of neuronal survival in the presence of MSC-conditioned medium (MSCcm) isolated from control subjects (C-MSCcm) and those with MS (MS-MSCcm) were performed following (1) trophic factor withdrawal and (2) nitric oxide-induced neurotoxicity. Reduced neuronal survival following trophic factor withdrawal was seen in association with increasing expansion of MSCs in vitro and MSC donor age. Controlling for these factors, there was an independent, negative effect of progressive MS. In nitric oxide neurotoxicity, MSCcm-mediated neuroprotection was reduced when C-MSCcm was isolated from higher-passage MSCs and was negatively associated with increasing MSC passage number and donor age. Furthermore, the neuroprotective effect of MSCcm was lost when MSCs were isolated from patients with MS. Our findings have significant implications for MSC-based therapy in neurodegenerative conditions, particularly for autologous MSC therapy in MS. Impaired neuroprotection mediated by the MSC secretome in progressive MS may reflect reduced reparative potential of autologous MSC-based therapy in MS and it is likely that the causes must be addressed before the full potential of MSC-based therapy is realized. Additionally, we anticipate that understanding the mechanisms responsible will contribute new insights into MS pathogenesis and may also be of wider relevance to other neurodegenerative conditions.

Sarkar P ，Redondo J ，Kemp K ，Ginty M ，Wilkins A ，Scolding NJ ，Rice CM ... -  《-》

Three-dimensional cell culture (3DCC) improves secretion of signaling molecules of mesenchymal stem cells (MSCs).

The secretome of mesenchymal stem cells (MSCs), also called MSC-conditioned media (MSC-CM), represents one of the promising strategies for cellular therapy and tissue repair and regeneration. MSC-CM contains growth factors and cytokines that control many cellular responses during development and regeneration. Traditional 2D cell culture (2DCC) has previously been used to generate MSC-CM while evidence has proved that the physiological and biological behaviors of cells in 2DCC are significantly different from those in 3D cell culture (3DCC). Therefore, the objective is to compare the content of MSC-CM generated from traditional 2DCC and 3DCC using a 3D scaffold. Adipose tissue-derived MSCs (AT-MSCs) were isolated from four donors (N = 4) and characterized according to the criteria stipulated by the International Society for Cell Therapy (ISCT). MSCs at passage 3 were grown in traditional 2DCC until 70% confluence and MSC-CM were collected at 24, 48, and 94 h. On the other hand, MSCs at passage 3 were grown on a polystyrene scaffold for 10 days to generate a 3D model of MSCs, and then MSC-CM was collected at 24, 48, and 94 h. MSC-CM from both 2DCC and 3DCC were analyzed for protein content using ELISA. Haematoxylin eosin (HE) staining and immunofluorescence (IF) were used to characterize the 3DCC of MSCs. MSCs from 2DCC were fibroblast like cells, and flow cytometry showed they were positive for CD73 and CD105 while being negative for CD14, CD19, and HLA-DR. They were also able to differentiate into adipocytes, osteoblasts, and chondrocytes. HE and IF showed that MSCs formed 3D model structures on the polystyrene scaffold. MSC-CM collected from both 2DCC and 3DCC contained growth factors, e.g., platelet derived growth factor (PDGF-AB), transforming growth factor-1 (TGF-1), hepatocyte growth factor (HGF), stromal derived factor-1 (SDF-1), interleukin 1 (IL-1), and interleukin 6 (IL-6). Concentrations of biomolecules secreted by MSCs in 3DCC were significantly higher than in 2DCC. It could be concluded that 3DCC of MSCs using a polystyrene scaffold is a novel approach to generate MSC secretome for therapeutic applications.

Al-Shaibani MBH 《-》