Treating Parkinson's Disease with Human Bone Marrow Mesenchymal Stem Cell Secretome: A Translational Investigation Using Human Brain Organoids and Different Routes of In Vivo Administration.

Parkinson's disease (PD) is the most common movement disorder, characterized by the progressive loss of dopaminergic neurons from the nigrostriatal system. Currently, there is no treatment that retards disease progression or reverses damage prior to the time of clinical diagnosis. Mesenchymal stem cells (MSCs) are one of the most extensively studied cell sources for regenerative medicine applications, particularly due to the release of soluble factors and vesicles, known as secretome. The main goal of this work was to address the therapeutic potential of the secretome collected from bone-marrow-derived MSCs (BM-MSCs) using different models of the disease. Firstly, we took advantage of an optimized human midbrain-specific organoid system to model PD in vitro using a neurotoxin-induced model through 6-hydroxydopamine (6-OHDA) exposure. In vivo, we evaluated the effects of BM-MSC secretome comparing two different routes of secretome administration: intracerebral injections (a two-site single administration) against multiple systemic administration. The secretome of BM-MSCs was able to protect from dopaminergic neuronal loss, these effects being more evident in vivo. The BM-MSC secretome led to motor function recovery and dopaminergic loss protection; however, multiple systemic administrations resulted in larger therapeutic effects, making this result extremely relevant for potential future clinical applications.

Cerebral Malaria Model Applying Human Brain Organoids.

Neural injuries in cerebral malaria patients are a significant cause of morbidity and mortality. Nevertheless, a comprehensive research approach to study this issue is lacking, so herein we propose an in vitro system to study human cerebral malaria using cellular approaches. Our first goal was to establish a cellular system to identify the molecular alterations in human brain vasculature cells that resemble the blood-brain barrier (BBB) in cerebral malaria (CM). Through transcriptomic analysis, we characterized specific gene expression profiles in human brain microvascular endothelial cells (HBMEC) activated by the Plasmodium falciparum parasites. We also suggest potential new genes related to parasitic activation. Then, we studied its impact at brain level after Plasmodium falciparum endothelial activation to gain a deeper understanding of the physiological mechanisms underlying CM. For that, the impact of HBMEC-P. falciparum-activated secretomes was evaluated in human brain organoids. Our results support the reliability of in vitro cellular models developed to mimic CM in several aspects. These systems can be of extreme importance to investigate the factors (parasitological and host) influencing CM, contributing to a molecular understanding of pathogenesis, brain injury, and dysfunction.

Enhanced neuronal differentiation by dynamic piezoelectric stimulation.

Electroactive smart materials play an important role for tissue regenerative applications. Poly(vinylidene fluoride) (PVDF) is a specific subtype of piezoelectric electroactive material that generates electrical potential upon mechanical stimulation. This work focuses on the application of piezoelectric PVDF films for neural differentiation. Human neural precursor cells (hNPCs) are cultured on piezoelectric poled and non-poled ß-PVDF films with or without a pre-coating step of poly-d-lysine and laminin (PDL/L). Subsequently, hNPCs differentiation into the neuronal lineage is assessed (MAP2+ and DCX+ ) under static or dynamic (piezoelectric stimulation) culture conditions. The results demonstrate that poled and coated ß-PVDF films induce neuronal differentiation under static culture conditions which is further enhanced with mechanical stimulation. In silico calculations of the electrostatic potential of different domains of laminin, highlight the high polarity of those domains, which shows a clear preference to interact with the varying surface electric field of the piezoelectric material under mechanical stimulation. These interactions might explain the higher neuronal differentiation induced by poled ß-PVDF films pre-coated with PDL/L under dynamic conditions. Our results suggest that electromechanical stimuli, such as the ones induced by piezoelectric ß-PVDF films, are suitable to promote neuronal differentiation and hold great promise for the development of neuroregenerative therapies.

Cellular Aging Secretes: a Comparison of Bone-Marrow-Derived and Induced Mesenchymal Stem Cells and Their Secretome Over Long-Term Culture.

Mesenchymal stem cells (MSCs) hold promising therapeutic potential in several clinical applications, mainly due to their paracrine activity. The implementation of future secretome-based therapeutic strategies requires the use of easily accessible MSCs sources that provide high numbers of cells with homogenous characteristics. MSCs obtained from induced pluripotent stem cells (iMSCs) have been put forward as an advantageous alternative to the gold-standard tissue sources, such as bone marrow (BM-MSCs). In this study, we aimed at comparing the secretome of BM-MSCs and iMSCs over long-term culture. For that, we performed a broad characterization of both sources regarding their identity, proteomic secretome analysis, as well as replicative senescence and associated phenotypes, including its effects on MSCs secretome composition and immunomodulatory action. Our results evidence a rejuvenated phenotype of iMSCs, which is translated into a superior proliferative capacity before the induction of replicative senescence. Despite this significant difference between iMSCs and BM-MSCs proliferation, both untargeted and targeted proteomic analysis revealed a similar secretome composition for both sources in pre-senescent and senescent states. These results suggest that shifting from the use of BM-MSCs to a more advantageous source, like iMSCs, may yield similar therapeutic effects as identified over the past years for this gold-standard MSC source.

Cerebral Organoids to Study Central Mechanisms of Pain: The Effect of Stem Cell Secretome on Opioid Receptors and Neuroplasticity.

Over 90% of chronic pain (CP) patients receive opioids-based treatments, which led to a public health crisis with lasting impacts on social and economic wellbeing based on opioid addiction. Opioids act through activation of µ (MOR), δ (DOR), and κ (KOR) opioid receptors, which are broadly and differentially distributed throughout the brain. Chronic opioid consumption leads to brain changes such as alterations on neurotransmission, dendritic branching, and spine density, as well as an increase in apoptosis. To overcome opioid-related issues, extensive efforts have been made to search for an alternative treatment. Bioactive molecules secreted by stem cells, collectively known as secretome, have shown a positive impact in different pain models. However, there is a lack of studies on the role of secretome in modulating opioid receptors. By using cerebral organoids (CeO), a self-organized, functional, and multicellular 3D structure that resemble the brain, we were able to identify MOR, DOR, and KOR at different stages of maturation. Treatment with secretome increased MOR expression highlighting a possible role in pain signaling mechanisms. Opioid treatments did not impact the expression of neuronal maturation markers but together with secretome, they increased astrogliogenesis. Opioid-treated organoids presented higher dopamine secretion recapitulating an important physiological event after opioid exposure. This work demonstrates that CeO is an important model system for the study of opioid signaling with potential implications to the understanding of basic mechanisms related to pain physiology.

Unilateral Intrastriatal 6-Hydroxydopamine Lesion in Mice: A Closer Look into Non-Motor Phenotype and Glial Response.

Parkinson's disease (PD) is a prevalent movement disorder characterized by the progressive loss of dopaminergic neurons in substantia nigra pars compacta (SNpc). The 6-hydroxydopamine (6-OHDA) lesion is still one of the most widely used techniques for modeling Parkinson's disease (PD) in rodents. Despite commonly used in rats, it can be challenging to reproduce a similar lesion in mice. Moreover, there is a lack of characterization of the extent of behavioral deficits and of the neuronal loss/neurotransmitter system in unilateral lesion mouse models. In this study, we present an extensive behavioral and histological characterization of a unilateral intrastriatal 6-OHDA mouse model. Our results indicate significant alterations in balance and fine motor coordination, voluntary locomotion, and in the asymmetry's degree of forelimb use in 6-OHDA lesioned animals, accompanied by a decrease in self-care and motivational behavior, common features of depressive-like symptomatology. These results were accompanied by a decrease in tyrosine hydroxylase (TH)-labelling and dopamine levels within the nigrostriatal pathway. Additionally, we also identify a marked astrocytic reaction, as well as proliferative and reactive microglia in lesioned areas. These results confirm the use of unilateral intrastriatal 6-OHDA mice for the generation of a mild model of nigrostriatal degeneration and further evidences the recapitulation of key aspects of PD, thereby being suitable for future studies beholding new therapeutical interventions for this disease.

Fractionating stem cells secretome for Parkinson's disease modeling: Is it the whole better than the sum of its parts?

Human mesenchymal stem cells (hMSCs) secretome has been have been at the forefront of a new wave of possible therapeutic strategies for central nervous system neurodegenerative disorders, as Parkinson's disease (PD). While within its protein fraction, several promising proteins were already identified with therapeutic properties on PD, the potential of hMSCs-secretome vesicular fraction remains to be elucidated. Such highlighting is important, since hMSCs secretome-derived vesicles can act as biological nanoparticles with beneficial effects in different pathological contexts. Therefore, in this work, we have isolated hMSCs secretome vesicular fraction, and assessed their impact on neuronal survival, and differentiation on human neural progenitors' cells (hNPCs), and in a 6-hydroxydopamine (6-OHDA) rat model of PD when compared to hMSCs secretome (as a whole) and its protein derived fraction. From the results, we have found hMSCs vesicular fraction as polydispersity source of vesicles, which when applied in vitro was able to induce hNPCs differentiation at the same levels as the whole secretome, while the protein separated fraction was not able to induce such effect. In the context of PD, although distinct effects were observed, hMSCs secretome and its derived fractions displayed a positive impact on animals' motor and histological performance, thereby indicating that hMSCs secretome and its different fractions may impact different mechanisms and pathways. Overall, we concluded that the use of the secretome collected from hMSCs and its different fractions might be active modulators of different neuroregeneration mechanisms, which could open new therapeutical opportunities for their future use as a treatment for PD.

Impact of Aging on the 6-OHDA-Induced Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder. The neurodegeneration leading to incapacitating motor abnormalities mainly occurs in the nigrostriatal pathway due to the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Several animal models have been developed not only to better understand the mechanisms underlying neurodegeneration but also to test the potential of emerging disease-modifying therapies. However, despite aging being the main risk factor for developing idiopathic PD, most of the studies do not use aged animals. Therefore, this study aimed at assessing the effect of aging in the unilateral 6-hydroxydopamine (6-OHDA)-induced animal model of PD. For this, female young adult and aged rats received a unilateral injection of 6-OHDA into the medial forebrain bundle. Subsequently, the impact of aging on 6-OHDA-induced effects on animal welfare, motor performance, and nigrostriatal integrity were assessed. The results showed that aging had a negative impact on animal welfare after surgery. Furthermore, 6-OHDA-induced impairments on skilled motor function were significantly higher in aged rats when compared with their younger counterparts. Nigrostriatal histological analysis further revealed an increased 6-OHDA-induced dopaminergic cell loss in the SNpc of aged animals when compared to young animals. Overall, our results demonstrate a higher susceptibility of aged animals to 6-OHDA toxic insult.

Bone Marrow Mesenchymal Stem Cells' Secretome Exerts Neuroprotective Effects in a Parkinson's Disease Rat Model.

Parkinson's disease (PD) is characterized by a selective loss of dopamine (DA) neurons in the human midbrain causing motor dysfunctions. The exact mechanism behind dopaminergic cell death is still not completely understood and, so far, no cure or neuroprotective treatment for PD is available. Recent studies have brought attention to the variety of bioactive molecules produced by mesenchymal stem cells (MSCs), generally referred to as the secretome. Herein, we evaluated whether human MSCs-bone marrow derived (hBMSCs) secretome would be beneficial in a PD pre-clinical model, when compared directly with cell transplantation of hBMSCs alone. We used a 6-hydroxydpomanie (6-OHDA) rat PD model, and motor behavior was evaluated at different time points after treatments (1, 4, and 7 weeks). The impact of the treatments in the recovery of DA neurons was estimated by determining TH-positive neuronal densities in the substantia nigra and fibers in the striatum, respectively, at the end of the behavioral characterization. Furthermore, we determined the effect of the hBMSCs secretome on the neuronal survival of human neural progenitors in vitro, and characterized the secretome through proteomic-based approaches. This work demonstrates that the injection of hBMSCs secretome led to the rescue of DA neurons, when compared to transplantation of hBMSCs themselves, which can explain the recovery of secretome-injected animals' behavioral performance in the staircase test. Moreover, we observed that hBMSCs secretome induces higher levels of in vitro neuronal differentiation. Finally, the proteomic analysis revealed that hBMSCs secrete important exosome-related molecules, such as those related with the ubiquitin-proteasome and histone systems. Overall, this work provided important insights on the potential use of hBMSCs secretome as a therapeutic tool for PD, and further confirms the importance of the secreted molecules rather than the transplantation of hBMSCs for the observed positive effects. These could be likely through normalization of defective processes in PD, namely proteostasis or altered gene transcription, which lately can lead to neuroprotective effects.

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.

Filling the Gap: Neural Stem Cells as A Promising Therapy for Spinal Cord Injury.

Spinal cord injury (SCI) can lead to severe motor, sensory and social impairments having a huge impact on patients' lives. The complex and time-dependent SCI pathophysiology has been hampering the development of novel and effective therapies. Current treatment options include surgical interventions, to stabilize and decompress the spinal cord, and rehabilitative care, without providing a cure for these patients. Novel therapies have been developed targeting different stages during trauma. Among them, cell-based therapies hold great potential for tissue regeneration after injury. Neural stem cells (NSCs), which are multipotent cells with inherent differentiation capabilities committed to the neuronal lineage, are especially relevant to promote and reestablish the damaged neuronal spinal tracts. Several studies demonstrate the regenerative effects of NSCs in SCI after transplantation by providing neurotrophic support and restoring synaptic connectivity. Therefore, human clinical trials have already been launched to assess safety in SCI patients. Here, we review NSC-based experimental studies in a SCI context and how are they currently being translated into human clinical trials.

Capacitive technologies for highly controlled and personalized electrical stimulation by implantable biomedical systems.

Cosurface electrode architectures are able to deliver personalized electric stimuli to target tissues. As such, this technology holds potential for a variety of innovative biomedical devices. However, to date, no detailed analyses have been conducted to evaluate the impact of stimulator architecture and geometry on stimuli features. This work characterizes, for the first time, the electric stimuli delivered to bone cellular tissues during in vitro experiments, when using three capacitive architectures: stripped, interdigitated and circular patterns. Computational models are presented that predict the influence of cell confluence, cosurface architecture, electrodes geometry, gap size between electrodes and power excitation on the stimuli delivered to cellular layers. The results demonstrate that these stimulators are able to deliver osteoconductive stimuli. Significant differences in stimuli distributions were observed for different stimulator designs and different external excitations. The thickness specification was found to be of utmost importance. In vitro experiments using an osteoblastic cell line highlight that cosurface stimulation at a low frequency can enhance osteoconductive responses, with some electrode-specific differences being found. A major feature of this type of work is that it enables future detailed analyses of stimuli distribution throughout more complex biological structures, such as tissues and organs, towards sophisticated biodevice personalization.

Generation of an induced pluripotent stem cell line (CSC-46) from a patient with Parkinson's disease carrying a novel p.R301C mutation in the GBA gene.

Mutations in the glucocerebrosidase (GBA) gene have been associated with the development of Parkinson's disease (PD). An induced pluripotent stem cell (iPSC) line was generated from a 60-year old patient diagnosed with PD and carrying a new mutation variant p.R301C in GBA. Using non-integrating Sendai virus-based technology, we utilized OCT3/4, SOX2, c-MYC and KLF4 transcription factors to reprogram skin fibroblasts into iPSCs. The generated iPSC line retained the mutation, displayed expression of common pluripotency markers, differentiated into the three germ layers, and exhibited normal karyotype. The iPSC line can be further used for studying PD pathogenesis.

Cell secretome based approaches in Parkinson's disease regenerative medicine.

INTRODUCTION: The available therapeutic strategies for Parkinson's disease (PD) rely only on the amelioration of the symptomatology of the disease, lacking neuroprotection or neuroregeneration capacities. Therefore, the development of disease modifying strategies is extremely important for the management of PD in the long term. AREAS COVERED: In this review, the authors provide an overview of the current therapeutic approaches for PD and the emerging use of stem cell transplantation as an alternative. Particularly, the use of the secretome from mesenchymal stem cells (MSCs), as well as some methodologies used for the modulation of their paracrine signaling, will be discussed. Indeed, there is a growing body of literature highlighting the use of paracrine factors and vesicles secreted from different cell populations, for this purpose. EXPERT OPINION: Secretome from MSCs has shown its potential as a therapy for PD. Nevertheless, in the coming years, research should focus in several key aspects to enable the translation of this strategy from the bench to the bedside.

Electrically polarized PLLA nanofibers as neural tissue engineering scaffolds with improved neuritogenesis.

Tissue engineering is evolving towards the production of smart platforms exhibiting stimulatory cues to guide tissue regeneration. This work explores the benefits of electrical polarization to produce more efficient neural tissue engineering platforms. Poly (l-lactic) acid (PLLA)-based scaffolds were prepared as solvent cast films and electrospun aligned nanofibers, and electrically polarized by an in-lab built corona poling device. The characterization of the platforms by thermally stimulated depolarization currents reveals a polarization of 60 × 10-10C cm-2 that is stable on poled electrospun nanofibers for up to 6 months. Further in vitro studies using neuroblastoma cells reveals that platforms' polarization potentiates Retinoic Acid-induced neuronal differentiation. Additionally, in differentiating embryonic cortical neurons, poled aligned nanofibers further increased neurite outgrowth by 30% (+70 µm) over non-poled aligned nanofibers, and by 50% (+100 µm) over control conditions. Therefore, the synergy of topographical cues and electrical polarization of poled aligned nanofibers places them as promising biocompatible and bioactive platforms for neural tissue regeneration. Given their long lasting induced polarization, these PLLA poled nanofibrous scaffolds can be envisaged as therapeutic devices of long shelf life for neural repair applications.

Generation of an induced pluripotent stem cell line (CSC-41) from a Parkinson's disease patient carrying a p.G2019S mutation in the LRRK2 gene.

The leucine-rich repeat kinase 2 (LRRK2) p.G2019S mutation is the most common genetic cause of Parkinson's disease (PD). An induced pluripotent stem cell (iPSC) line CSC-41 was generated from a 75-year old patient diagnosed with PD caused by a p.G2019S mutation in LRRK2. Skin fibroblasts were reprogrammed using a non-integrating Sendai virus-based technology to deliver OCT3/4, SOX2, c-MYC and KLF4 transcription factors. The generated iPSC line exhibits expression of common pluripotency markers, differentiates into the three germ layers and has a normal karyotype. The iPSC line can be used to explore the association between LRRK2 mutation and PD.

Generation of a human induced pluripotent stem cell line (CSC-42) from a patient with sporadic form of Parkinson's disease.

Skin fibroblasts were collected from a 44-year-old patient with sporadic case of Parkinson's disease (PD). The non-integrating Sendai virus vector encoding OCT3/4, SOX2, c-MYC and KLF4 was used to reprogram fibroblasts into induced pluripotent stem cells (iPSCs). Generated iPSCs had normal karyotypes, expressed common stem cell markers, and were capable of differentiating into all three germ layers. Generated line could be used for PD modeling to understand the mechanisms that influence the disorder.

Generation of an integration-free induced pluripotent stem cell line (CSC-43) from a patient with sporadic Parkinson's disease.

An induced pluripotent stem cell (iPSC) line was generated from a 36-year-old patient with sporadic Parkinson's disease (PD). Skin fibroblasts were reprogrammed using the non-integrating Sendai virus technology to deliver OCT3/4, SOX2, c-MYC and KLF4 factors. The generated cell line (CSC-43) exhibits expression of common pluripotency markers, in vitro differentiation into three germ layers and normal karyotype. This iPSC line can be used to study the mechanisms underlying the development of PD.

Generation of a human induced pluripotent stem cell line (CSC-40) from a Parkinson's disease patient with a PINK1 p.Q456X mutation.

Parkinson's disease (PD) is a neurodegenerative disease with unknown etiology. Here we show the generation of an induced pluripotent stem cell (iPSC) line, named CSC-40, from dermal fibroblasts obtained from a 59-year-old male patient with a homozygous p.Q456X mutation in the PTEN-induced putative kinase 1 (PINK/PARK6) gene and a confirmed diagnosis of PD, which could be used to model familial PD. A non-integrating Sendai virus-based delivery of the reprogramming factors OCT3/4, SOX2, c-MYC and KLF4 was employed. The CSC-40 cell line showed normal karyotyping and fingerprinting following transduction as well as sustained expression of several pluripotency markers and the ability to differentiate into all three germ layers.

Generation of an induced pluripotent stem cell line (CSC-44) from a Parkinson's disease patient carrying a compound heterozygous mutation (c.823C>T and EX6 del) in the PARK2 gene.

Mutations in the PARK2 gene, which encodes PARKIN, are the most frequent cause of autosomal recessive Parkinson's disease (PD). We report the generation of an induced pluripotent stem cell (iPSC) line from a 78-year-old patient carrying a compound heterozygous mutation (c.823C>T and EX6del) in the PARK2 gene. Skin fibroblasts were reprogrammed using the non-integrating Sendai virus technology to deliver OCT3/4, SOX2, c-MYC and KLF4 factors. The generated cell line CSC-44 exhibits expression of common pluripotency markers, in vitro differentiation into the three germ layers and normal karyotype. This iPSC line can be used to explore the association between PARK2 mutations and PD.