Ex Vivo Expansion of Human Mesenchymal Stem Cells in Defined Serum-Free Media

Human mesenchymal stem cells (hMSCs) are presently being evaluated for their therapeutic potential in clinical studies to treat various diseases, disorders, and injuries. To date, early-phase studies have indicated that the use of both autologous and allogeneic hMSCs appear to be safe; however, efficacy has not been demonstrated in recent late-stage clinical trials. Optimized cell bioprocessing protocols may enhance the efficacy as well as safety of hMSC therapeutics. Classical media used for generating hMSCs are typically supplemented with ill-defined supplements such as fetal bovine serum (FBS) or human-sourced alternatives. Ideally, culture media are desired to have well-defined serum-free formulations that support the efficient production of hMSCs while maintaining their therapeutic and differentiation capacity. Towards this objective, we review here current cell culture media for hMSCs and discuss medium development strategies

Extremely low frequency magnetic field induces human neuronal differentiation through NMDA receptor activation

Magnetic fields with different frequency and intensity parameters exhibit a wide range of effects on different biological models. Extremely low frequency magnetic field (ELF MF) exposure is known to augment or even initiate neuronal differentiation in several in vitro and in vivo models. This effect holds potential for clinical translation into treatment of neurodegenerative conditions such as autism, Parkinson's disease and dementia by promoting neurogenesis, non-invasively. However, the lack of information on underlying mechanisms hinders further investigation into this phenomenon. Here, we examine involvement of glutamatergic Ca2+ channel, N-methyl-D-aspartate (NMDA) receptors in the process of human neuronal differentiation under ELF MF exposure. We show that human neural progenitor cells (hNPCs) differentiate more efficiently under ELF MF exposure in vitro, as demonstrated by the abundance of neuronal markers. Furthermore, they exhibit higher intracellular Ca2+ levels as evidenced by c-fos expression and more elongated mature neurites. We were able to neutralize these effects by blocking NMDA receptors with memantine. As a result, we hypothesize that the effects of ELF MF exposure on neuronal differentiation originate from the effects on NMDA receptors, which sequentially triggers Ca2+-dependent cascades that lead to differentiation. Our findings identify NMDA receptors as a new key player in this field that will aid further research in the pursuit of effect mechanisms of ELF MFs.European Molecular Biology Organization Short Term Fellowships, ASTF 7502. This work was partially supported by TUBITAK Projects under Grant No. 117Z864, Bogazici University Research Fund by Grant Number 6701. A.J. Salgado and A. Marote acknowledge the financial support from: Prémios Santa Casa Neurociências–Prize Melo e Castro for Spinal Cord Injury Research (MC-04/17); Portuguese Foundation for Science and Technology Pre–Doctoral fellowship to A. Marote PDE/BDE/113598/2015 and IF Development Grant to A. J. Salgado. This work is funded by national funds through FCT under the scope of grant reference TUBITAK/0007/2014. This article has been developed under the scope of the projects NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). This work has been funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038 and POCI-01-0145-FEDER-02920

Secretome of Mesenchymal Progenitors from the Umbilical Cord Acts as Modulator of Neural/Glial Proliferation and Differentiation

It was recently shown that the conditioned media (CM) of Human Umbilical Cord Perivascular Cells (HUCPVCs), a mesenchymal progenitor population residing within the Wharton Jelly of the umbilical cord, was able to modulate in vitro the survival and viability of different neuronal and glial cells populations. In the present work, we aimed to assess if the secretome of HUCPVCs is able to 1) induce the differentiation of human telencephalon neural precursor cells (htNPCs) in vitro, and 2) modulate neural/glial proliferation, differentiation and survival in the dentate gyrus (DG) of adult rat hippocampus. For this purpose, two separate experimental setups were performed: 1) htNPCs were incubated with HUCPVCs-CM for 5 days after which neuronal differentiation was assessed and, 2) HUCPVCs, or their respective CM, were injected into the DG of young adult rats and their effects assessed 7 days later. Results revealed that the secretome of HUCPVCs was able to increase neuronal cell differentiation in vitro; indeed, higher densities of immature (DCX+ cells) and mature neurons (MAP-2(+) cells) were observed when htNPCs were incubated with the HUCPVCs-CM. Additionally, when HUCPVCs and their CM were injected in the DG, results revealed that both cells or CM were able to increase the endogenous proliferation (BrdU(+) cells) 7 days after injection. It was also possible to observe an increased number of newborn neurons (DCX+ cells), upon injection of HUCPVCs or their respective CM. Finally western blot analysis revealed that after CM or HUCPVCs transplantation, there was an increase of fibroblast growth factor-2 (FGF-2) and, to a lesser extent, of nerve growth factor (NGF) in the DG tissue. Concluding, our results have shown that the transplantation of HUCPVCs or the administration of their secretome were able to potentiate neuronal survival and differentiation in vitro and in vivo.Foundation Calouste Gulbenkian for funds under the scope of the Gulbenkian Programme to Support Cutting Edge Research in Life Sciences; Portuguese Foundation for Science and Technology (FCT) for Ciência 2007 program and IF Development Grant (A.J. Salgado), and pre-doctoral fellowship to F.G. Teixeira (SFRH / BD / 69637 / 2010); John E. Davies for kindly providing the HUCPVCs used in this workinfo:eu-repo/semantics/publishedVersio

Exploiting the impact of the secretome of MSCs isolated from different tissue sources on neuronal differentiation and axonal growth

Cell transplantation using Mesenchymal stem cell (MSC) secretome have recently been presented as a possible free-based therapy for CNS related disorders. MSC secretome is rich in several bio-factors that act synergically towards the repair of damaged tissues, thus making it an ideal candidate for regenerative applications. Great effort is currently being made to map the molecules that compose the MSC secretome. Previous proteomic characterization of the secretome (in the form of conditioned media - CM) of MSCs derived from adipose tissue (ASC), bone-marrow (BMSC) and umbilical cord (HUCPVC) was performed by our group, where proteins relevant for neuroprotection, neurogenic, neurodifferentiation, axon guidance and growth functions were identified. Moreover, we have found significant differences among the expression of several molecules, which may indicate that their therapeutic outcome might be distinct. Having this in mind, in the present study, the neuroregulatory potential of ASC, BMSC and HUCPVC CM in promoting neurodifferentiation and axonal outgrowth was tested in vitro, using human telencephalon neuroprogenitor cells and dorsal root ganglion explants, respectively. The CM from the three MSC populations induced neuronal differentiation from human neural progenitor cells, as well as neurite outgrowth from dorsal root ganglion explants. Moreover, all the MSC populations promoted the same extent of neurodifferentiation, while ASC CM demonstrated higher potential in promoting axonal growth.The authors acknowledge the financial support by Premios Santa Casa Neurociencias - Prize Melo e Castro for Spinal Cord ^ Injury Research (MC-17-2013 and MC-04-2017); Portuguese Foundation for Science and Technology (Doctoral fellowships PDE/ BDE/113596/2015 and SFRH/BD/120124/2016 to R.C Assunçao Silva ~ and B. Mendes-Pinheiro, respectively; Post-doctoral fellowhip to F.G. Teixeira and Patrícia Patrício - SFRH/BPD/118408/2016 and SFRH/BPD/116249/2016; IF Starting Grant to L. Pinto and IF Development Grant to A. J. Salgado); Canada Research Chair in Biomedical Engineering (LAB). This work is funded by national funds through FCT under the scope of grante reference TUBITAK/0007/ 2014. This article has been developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). This work has been funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology, under the scope of the project POCI-01-0145-FEDER-007038. HUCPVCs and ASCs were kindly provided by Prof. John E. Davies (University of Toronto, Canada) and Prof. Jeff Gimble (LaCell Inc, USA).info:eu-repo/semantics/publishedVersio

Influence of passage number on the impact of the secretome of adipose tissue stem cells on neural survival, neurodifferentiation and axonal growth

Mesenchymal stem cells (MSCs), and within them adipose tissue derived stem cells (ASCs), have been shown to have therapeutic effects on central nervous system (CNS) cell populations. Such effects have been mostly attributed to soluble factors, as well as vesicles, present in their secretome. Yet, little is known about the impact that MSC passaging might have in the secretion therapeutic profile. Our aim was to show how human ASCs (hASCs) passage number influences the effect of their secretome in neuronal survival, differentiation and axonal growth. For this purpose, post-natal rat hippocampal primary cultures, human neural progenitor cell (hNPCs) cultures and dorsal root ganglia (DRGs) explants were incubated with secretome, collected as conditioned media (CM), obtained from hASCs in P3, P6, P9 and P12. Results showed no differences when comparing percentages of MAP-2 positive cells (a mature neuronal marker) in neuronal cultures or hNPCs, after incubation with hASCs secretome from different passages. The same was observed regarding DRG neurite outgrowth. In order to characterize the secretomes obtained from different passages, a proteomic analysis was performed, revealing that its composition did not vary significantly with passage number P3 to P12. Results allowed us to identify several key proteins, such as pigment epithelium derived factor (PEDF), DJ-1, interleucin-6 (IL-6) and galectin, all of which have already proven to play neuroprotective and neurodifferentiating roles. Proteins that promote neurite outgrowth were also found present, such as semaphorin 7A and glypican-1. We conclude that cellular passaging does not influence significantly hASCs's secretome properties especially their ability to support post-natal neuronal survival, induce neurodifferentiation and promote axonal growth.Prémios Santa Casa Neurociências - Prize Melo e Castro for Spinal Cord Injury Research (MC-17-2013 and MC-04-2017), Canada Research Chair in Biomedical Engineering (LAB), Northern Portugal Regional Operational Programme (NORTE 2020),, European Regional Development Fund (FEDER), Competitiveness Factors Operational Programme (COMPETE), National Mass Spectrometry Network (RNEM)info:eu-repo/semantics/publishedVersio