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Objectives: To describe neuronal differentiation from the disomic (D21) and trisomic (T21) isogenic human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) derived from persons with partial and full trisomy 21, and mosaic Down syndrome (DS). Methods: NSC were generated form the isogenic iPSCs following published protocol (1) and expanded in our laboratory. NSCs, between passage 5 and 10 were seeded on the commercially available mouse astrocytes and differentiated to the mature neurons 100 days in vitro (DIV) (2). Moreover, cerebral organoids were generated following published protocol (3) with our modification (4) and fixed at 30, 50, 70 and 100 DIV and analysed by immunohistochemistry. Results: In our previous work we published neuronal differentiation from EBs D3 and 47-1 (5). Here we find that Tubb3 positive cells show a specific neuronal phenotype. Disomic cells showed normal neuronal morphology, on the other hand trisomic cells showed a very specific cellular dysmorphology: neurons were smaller, shorter with thicker processes, and abnormal branching patterns. The same pattern we have observed in neurons derived form iPSCs in monolayer as well as in cerebral organoids. Both, neurons in monolayer, as well as in cerebral organoids expressed all neuronal markers: pan neuronal markers (Dcx, MAP2. Tubb3, SMI, 3R-Tau) and cortical layer specific markers (Reelen, Brn2, Ctip2, SATB2, TBR1 and TBR2). Conclusion: Trisomic cells could differentiate to mature, synaptic active neurons but morphology of trisomic cells shows a unique and specific dysmorphology pattern.

Human, mammalian, brain development is a very complex process and starts shortly after gastrulation. Human in vitro models for brain development as well as pathological changes were published in last few years, eliminating the ethical concerns that accompany the study of aborted foetal material. One of the recent approaches is the development of induced pluripotent stem cell (iPSC) technology and different types of organoids, which can be applied to any species. In our previous work we developed iPSCs from an adult individual with constitutional mosaicism for Down Syndrome (DS) (1). From those iPSCs, cerebral organoids were grown and corticogenesis and Alzheimer-like pathology in vitro were studied (2). Mixed cerebral (2), as well as midbrain (unpublished) organoids were grown in vitro from the isogenic, trisomic and disomic iPSCs. Organoids were analysed histologically by IMARIS software. Foetal human brains were stained with the same combinations of antibodies and the data were compared. Anonymized post-mortem brain samples were obtained from the Brain Bank of the Croatian Institute for Brain Research (CIBR). Isogenic cerebral organoids expressed the markers of all six cortical layers (Reelin, FOXG1, SATB2, TBR1, Ctip2 and Brn2) after 100 days differentiation in vitro (DIV). At the early time point of DIV30 the majority of cells were Nestin, SOX2, PAX6 positive. At that time point neurogenesis had already started, so we observed TRBR2, DCX, MAP2 and TUBB3 positive cells in disomic and trisomic organoids. Following further neuronal differentiation of cerebral organoids we observed decreased number of neural stem cells and increased number of neuronal markers specific for all six layers. On the other hand, midbrain organoids expressed markers specific for midbrain. At the early time points midbrain organoids expressed neuronal stem cells markers, similar to cerebral organoids, but with organoid maturation we found midbrain specific cells such as NURR1 and OTX2 specific for the early stages, and later Dopamine β Hydroxylase, NET and TH. Finally, all those markers were compared with expression in foetal human brain from individuals with Down Syndrome as well as euploid controls age, sex and brain region matched. Our data showed that isogenic cerebral organoids grown from iPSCs are good tool for multidisciplinary studies. In our group we study corticogenesis, synaptogenesis, pathology and possible drug treatments.

Background: Specific cell types from people with Down syndrome (DS) show faster accumulation of DNA damage and epigenetic senescence marks. Causative mechanisms remain un-proven ranging from amplified chromosomal instability to actions of chromosome 21 genes. Plasma immunoglobulin G (IgG) glycosylation profiles are established as a reliable predictor of biological and chronological ageing. Methods: We performed IgG glycan profiling of n=246 individuals with DS (208 adults and 38 children) clinically characterised for co- morbidities, from three European countries, and compared these to age-, sex- and demography- matched general populations. Results: We uncovered very significantly increased IgG glycosylation ageing marks associated with DS, beginning in early childhood. Average levels of IgG glycans without galactose (G0) and those with two galactoses (G2) as a function of age in persons with DS corresponded to levels detected in 19 years older euploid individuals. Sub-cohorts of DS without any known co-morbidities from different countries showed a similar result, eliminating DS- linked autoimmune diseases, frequent infections, or Alzheimer’s dementia as indirect causes of accelerated biological ageing. Importantly, no difference in slope of the curve relating IgG- glycan-age to chronological age was observed for any of the 3 analysed populations, discouraging the amplified-chromosomal-instability explanation. Remarkably, IgG-glycan profiles of a child with DS caused by a short segmental duplication of only 31 genes on chromosome-21 (DS-critical-region) had values like age-matched whole-chromosome-trisomy- 21 children, pointing to a causative contribution of one or more of the 31 genes. Conclusion: This shifts the paradigm from an (essentially untreateable) “amplified instability” mechanism, to a specific gene-overdose-driven cause of a progeria-like syndrome, opening possibilities for therapeutic amelioration strategies. We generated an induced pluripotent stem cell (iPSC) model of this child with partial trisomy 21, and editing experiments using CRISPR-Cas9 are underway in order to dissect the causative overdosed genes and mechanisms responsible for the accelerated ageing in DS.

The goal was to explore the difference in N- glycosylation between euploid disomic (D21) and trisomic (T21) isogenic human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) derived from person with mosaic Down syndrome (DS) trisomy of 21st chromosome.....

Toll-like receptor 2 (TLR2) deficient mice model is widely used for investigation of microglial role in regulating inflammatory response following ischemic brain lesion (1). Additional functions and broader cell-type expression of TLRs have been evidenced in brain tissue which makes TLRs knock- out mice (such as TLR2-KO) a promising model for investigating neuron-microglia interactions, and microglial regulation of plasticity and excitability (2). Plasma membrane Ca2+ ATPases (PMCAs) participate significantly in ion homeostasis maintenance and signal transduction in brain tissue, thus we aimed to examine PMCAs protein expression and catalytic activity across brain regions in TLR2-KO mice. Results acquired by Western blot analysis and spectrophotometric enzyme activity measurements exhibit strikingly different protein expression and enzyme activity in brain tissue samples derived from TLR2-KO when compared with wild-type (WT) mice. Observed abundance of PMCA expression across cortex, hippocampus and cerebellum suggests that ubiquitous isoforms, PMCA 1 and 4 are less affected by TLR2 deficiency than neuron specific isoforms, PMCA 2 and 3. Notably, higher expression of PMCA2 in hippocampus and lower in cerebellum of TLR2-KO coincide with catalytic PMCA activity variations, higher activity is detected in hippocampus and lower in cerebellum of TLR2-KO compared with WT controls. Here collected evidence suggests that TLR2 deficiency is accompanied by a disturbance of neuronal Ca2+ signaling system. Further investigation should clarify in more details a potential impact of TLR2 on establishing proper neuron-microglia interactions and regulation of neurotransmission. [1.] Gorup D, Škokić S, Kriz J, Gajović S. Tlr2 Deficiency is Associated with Enhanced Elements of Neuronal Repair and Caspase 3 Activation Following Brain Ischemia. Sci Rep. 2019 ; 9(1):1–10. [2.] Yamamoto M, Kim M, Imai H, Itakura Y, Ohtsuki G. Microglia- Triggered Plasticity of Intrinsic Excitability Modulates Psychomotor Behaviors in Acute Cerebellar Inflammation. Cell Rep [Internet]. 2019 ; 28(11):2923-2938.e8. Available from: https://doi.org/10.1016/j.celrep.2019.07.078 [3.] Hajieva P, Baeken MW, Moosmann B. The role of Plasma Membrane Calcium ATPases (PMCAs) in neurodegenerative disorders. Neurosci Lett. 2018 ; 663(September 8. Available from: https://doi.org/10.1016/j.neulet.2017.09.033 Funding: Croatian Science Foundation (IP-2016-06- 8638), European Social Fund (ESF), and Centre of Excellence for basic, clinical and translational neuroscience.

Transmembrane proteins, neuroplastin (Np), and Toll-like receptor 2 (TLR2) are implicated in tuning the neuro-architecture. Np isoforms (Np55 and Np65) are related to synaptic plasticity and neuronal ion homeostasis by interactions with plasma membrane Ca2+ ATPases (PMCA). TLR2 is mainly expressed on microglia, but much broader cellular localization of TLR2 has been established, particularly during neurodevelopment. To test the hypothesis that TLR2 deficiency affects membrane dynamics, we analysed membrane protein phenotype in the brains of TLR2 knock-out (KO) mice. Cortical, cerebellar, and hippocampal tissue derived from male TLR2-KO and age-matched control (C) mice (N=36+36) underwent systematic biochemical profiling encompassing: analysis of Np55, Np65, PMCA, Na+/K+-ATPase (NKA) expression by Western blot (WB) ; transcriptional variations of selected genes by qPCR ; synaptic proteome by mass spectrometry (MS) ; activity of NKA and PMCA by spectrophotometry. MS analysis revealed astonishing differences in the synaptic proteome. Increased Np55, Np65, and NKA expression in cortex, cerebellum, and hippocampus was determined by WB in TLR2 vs. C. These findings were confirmed at a transcriptional level using qPCR. WB analysis of 4 PMCA isoforms showed that lack of TLR2 is associated with decreased expression of PMCA2 in cortex and cerebellum and increased in hippocampus. Changes in NKA and PMCA activity were also observed in TLR2 KO vs. C. Multiple-level analysis revealed that TLR2 deficiency leads to altered expression of proteins associated with synaptic plasticity and ion homeostasis. Observed proteomic changes are related to glutamatergic transmission, neuronal cytoskeleton organization, and mitochondrion energy metabolism. Further investigation may clarify previously undescribed roles of TLR2 in neuron-microglia interactions, synaptic connections arrangement, and neurotransmission.

Aim of the study was to determine the immunoreactivity pattern and unravel potential changes of two isoforms of synaptic transmembrane glycoprotein neuroplastin (Np55 and Np65) in brains of Toll-like receptor 2 deficient (TLR2D) mice in comparison with wild-type (WT) animals. For the purposes of immunohistochemical analysis by confocal microscopy, mouse brain sections derived from TLR2D and WT mice (n=10) were subjected to immunofluorescence labeling using antibodies against Np55, Np65, TLR2, and specific cell-type markers for neurons and different glial cells. Our preliminary Western blot analysis of cortical, hippocampal and cerebellar membrane fractions revealed changed expression levels of two Np isoforms in TLR2D vs WT mice. Here presented immunohistochemical data indicate higher Np55 and Np65 signal intensity in cortical and hippocampal area of TLR2D vs WT mice, and lower Np55 signal intensity in cerebellum of TLR2D mice, which corresponds to our previous Western blot results. In addition, Np was found to be expressed only in neuronal cells in both TLR2D and WT mice. TLR2D mice are a widely used model for studying neuroinflammatory component of different neuropathological disorders and Np is known to be involved in synaptic organization and affected in neurodegenerative disorders. It may be expected that deficiency of TLR2 has impact on cell-cell communication due to changed molecular signatures of neurons and surrounding glial cells. Having in mind evidenced role of TLR2D in activated microglia, deciphering biochemical phenotype of TLR2D mice could as well contribute to understanding potential involvement of neuroplastin in neuroinflammation.

Toll-like receptors (TLRs) are widely expressed in mammalian brain. Involvement in neurodegeneration and hypoxia has been evidenced for several microglial TLRs. In order to investigate interplay of specific microglial and neuronal transmembrane proteins and its potential role in neurodegeneration, we use TLR2 knock-out (KO ) mice (C57BL/6 TLR2-/-).Our preliminary findings indicate different expression of synaptic transmembrane protein neuroplastin (Np) in brain tissue of TLR2 KO mice in comparison with wild- type littermates (WT).Neuroplastin, a cell- adhesion molecule found in two isoforms (Np65 and Np55) is an essential auxiliary subunit of plasma membrane Ca2+ ATPase (PMCA ). In addition, Np is influenced by glycosphingolipid environment of the membrane, which is also the case for another ion pumping enzyme, Na+/K+-ATPase (NKA). Since Np, PMCA and NKA all seem to favor specific lipid milieu in order to function optimally, and at least some of them are confirmed to interact with one another, we investigated the expression of these proteins in TLR2 KO compared to WT across different brain regions.We isolated membrane fractions from cortical, hippocampal and cerebellar tissue of 5 WT and 5 KO animals, and analyzed the expression of Np, NKA and PMCA by Western blotting. Data revealed increased expression level of Np isoforms and PMCA in cortex and hippocampus in TLR2 KO, and decreased expression of Np55 and PMCA in the cerebellum. NKA exhibited the same expression pattern in TLR2 KO vs WT, and increased expression in all regions analyzed (cortex, hippocampus, cerebellum). Furthermore, we investigated the cortical NKA activity which is lower in TLR2 KO mice.This data together with ongoing immunohistochemistry and transcriptional analysis will enable more detailed neurobiochemical characterization of TLR2 KO model, and give insight into functional implications of differential regional distribution of here analyzed proteins involved in synaptic plasticity and membrane ion transport.

The aim of this study was to investigate the expression of two isoforms of synaptic glycoprotein neuroplastin (Np), Np55 and Np65, in the brain cortex and cerebellum of mouse lacking Toll-like receptor 2 (TLR2). Since TLRs are widely expressed in mammalian brain and involved in neurodegeneration and hypoxic stress, TLR2 knock-out (KO) mouse model (B6.129-Tlr2tm1Kir/J) is especially intriguing to analyze Np expression due to following evidence: 1) there is a threefold increase in Np65 in rat forebrain following transient ischemia suggesting a role of Np65 in recovery from ischemic insult ; 2) Np contributes to neuronal energy metabolism by acting as chaperone of monocarboxylate transporter MCT2 ; 3) Np65 KO mice have been found to be more susceptible to ischemic brain lesions. In TLR2 KO mice, attenuated neurodegeneration upon hypoxic stimuli has been described due to reduced microglial activation. To fill in the gaps in biochemical description of this model we investigated expression of Np55 and Np65 isoforms in brain tissue derived from wt and TLR2 KO mice. Membrane fractions were isolated from cortical and cerebellar tissue samples dissected from 22 animals, in which expression of two Np isoforms was determined by Western blotting. Results showed statistically significant increase of both Np55 and Np65 in cortex and decrease of Np55 in cerebellum in male KO vs wt mice. Obtained data are introduction for a larger study which will address potential interplay of Np and TLR2 as well as influence of hypoxia on Np expression in different brain regions.

Spinal root avulsion results in paralysis and sensory loss, and is commonly associated with chronic pain. In addition to the failure of avulsed dorsal root axons to regenerate into the spinal cord, avulsion injury leads to extensive neuroinflammation and degeneration of second-order neurons in the dorsal horn. The ultimate objective in the treatment of this condition is to counteract degeneration of spinal cord neurons and to achieve functionally useful regeneration/reconnection of sensory neurons with spinal cord neurons. Here we compare survival and migration of murine boundary cap neural crest stem cells (bNCSCs) and embryonic stem cells (ESCs)- derived, predifferentiated neuron precursors after their implantation acutely at the junction between avulsed dorsal roots L3-L6 and the spinal cord. Both types of cells survived transplantation, but showed distinctly different modes of migration. Thus, bNCSCs migrated into the spinal cord, expressed glial markers and formed elongated tubes in the peripheral nervous system (PNS) compartment of the avulsed dorsal root transitional zone (DRTZ) area. In contrast, the ESC transplants remained at the site of implantation and differentiated to motor neurons and interneurons. These data show that both stem cell types successfully survived implantation to the acutely injured spinal cord and maintained their differentiation and migration potential. These data suggest that, depending on the source of neural stem cells, they can play different beneficial roles for recovery after dorsal root avulsion.

Introduction: This study was based on the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J which under the control of Thy1 gene promoter expresses yellow fluorescent protein (YFP) in all parts of neurons. In this study, we have determined and compared expression pattern of: 1) Thy1 - YFP positive cells during in vitro differentiation of neural stem cells (NSC), 2) during embryonic development of Thy1 – YFP mouse embryo and 3) after transplantation of Thy1 – YFP cells into the stroke-affected mouse brain. Materials and Methods: Neural stem cells were isolated from the forebrain of 14.5 old mouse embryos and cultured as neurospheres. Immunocytochemistry and RT-PCR were performed after cells differentiation. Embryonic slices were prepared for immunohistochemistry. In third part of this study, PKH26 labeled NSC were transplanted into the stroke-affected mouse brain obtained by MCAO method. Results: THY1 - YFP expression was described during differentiation of NSC and observed in neural progenitors as well as in mature neurons. Neural progenitors were nestin positive while mature cell were not. THY1 – YFP positive cells were positive for mature neuronal markers (MAP2, β3-tubulin and NeuN) during the whole differentiation period, while astrocytes which were GFAP positive were not expressing YFP. During embryonic development THY1 – YFP positive cells were observed from E12.5 and the expression steadily increased to the end of pregnancy. Positive cells were present in both, central and peripheral nervous system. After transplantation, cells survived and incorporated into the stroke-affected brain. Conclusion: Our results showed that mature neurons as well as neuronal progenitor cells do express THY1 – YFP construct in in vitro conditions, during embryonic development and after transplantation into the stroke-affected mouse brain. This suggests that in addition to analyses of neuronal differentiation in B6.Cg- Tg(Thy1-YFP)16Jrs/J mouse, NSCs isolated from this strain can be successfully used in studies where tracing of cells with neuronal fate is needed.

INTRODUCTION: Biocompatibility, safety and risk assessments of superparamagnetic iron oxide nanoparticles (SPIONs) are of the highest priority in testing their potential applications in biomedicine. Improvement of the biological properties of SPIONs may be achieved by different functionalization and surface modifications. AIMS: This study investigated how different surface functionalizations of SPIONs – uncoated, coated with D-mannose or coated with poly-L-lysine - affected biocompatibility. By using murine neural stem cells (NSCs) as important model system for the regenerative medicine we had a goal to reveal the possible mechanism of toxicity of SPIONs on NSCs, mitochondrial membrane potential, cell membrane potential and DNA damage. RESULTS: We observed hyperpolarization of mitochondrial membrane, dissipated cell membrane potential and increased DNA damages in NSCs, irrespective of the surface coating applied for SPION stabilization. Although surface coating should prevent the toxic effects of SPIONs, our results showed that all of the tested SPION types affected the NSCs similarly indicating that mitochondrial homeostasis is their major cellular target. CONCLUSIONS: Despite the claimed biomedical benefits of SPIONs, the refined determination of their effects on various cellular functions presented in this work highlights the need for further safety evaluations. This investigation helps to fill the knowledge gaps on the criteria that should be considered in evaluating the biocompatibility and safety of novel nanoparticles. ACKNOWLEDGMENTS: This study was supported by EU FP7 grant GlowBrain (REGPOT-2012-CT2012-316120), by EU ESF project MladiMozak (030201-0180) and by the Czech Science Foundation (project no. 16-01128J).

Objective In order to analyze morphologic processes occurring during development of mammalian CNS, several specific mouse strains have been constructed. This study was based on the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J which under the control of Thy1 gene promoter expresses yellow fluorescent protein (YFP) in neurons. In this study, we have determined and compared pattern of Thy1 - YFP expression during embryonic development as well as during differentiation of neural stem cells culture. Material and methods Pregnant females, at different stages of gestation were used for embryo isolation. The embryos were fixed and cryosectioned on 20µm thick slices. Neural stem cells were isolated from the forebrain of 14.5 old murine embryos and cultured as neurospheres. After dissociation, cells were plated on cover slips. The cells were fixed 1, 3, 5 and 7 and prepared to the immunocytochemistry. Results First signs of expression were observed at embryonic day 12.5 in ventral horn of the spinal cord. During development of the embryo in the cranio-caudal direction. In accordance with the process of neurogenesis which primarily occurs in mid-gestation, expression of YFP in neurons after E12.5 became much more pronounced. With embryo maturation, expression became stronger in the brain and by E17.5 expression was observed in the optic nerve and retina. Furthermore, expression of YFP was determined during in vitro NSC differentiation. YFP expression was observed in neural progenitors as well as in mature neurons. YFP positive cells stained positive for Map2 (mature neuronal marker), while astrocytes which were GFAP positive were not expressing YFP. Conclusion Our results showed that, in addition to mature neurons, neuronal progenitor cells express this construct both in in vivo and in vitro conditions. This suggests that in addition to analyses of neuronal differentiation in B6.Cg- Tg(Thy1-YFP)16Jrs/J mouse, NSCs isolated from this strain can be successfully used in studies where tracing of cells with neuronal fate is needed.

In order to analyze morphologic processes occurring during development of mammalian nervous system, several specific mouse strains have been constructed. This study was based on the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J which under the control of Thy1 gene promoter expresses yellow fluorescent protein (YFP) in all parts of neurons. This allows to precisely describe development of otherwise less visible parts of nervous cells, including axon and dendrites. In order to use these cells for transplantation, we had to determine pattern of Thy1 - YFP expression during embryonic development. During the first 10 days of embryonic development expression of YFP was not observed. First signs of expression were observed at embryonic day 12.5 (E12.5) in ventral horn of the spinal cord. During development of the embryo in the cranio-caudal direction, a stronger expression of YFP was observed in the cranial part of the spinal cord. Moreover, expression was significantly stronger in the spinal cord than in the brain. In accordance with the process of neurogenesis which primarily occurs in mid-gestation, expression of YFP in neurons after E12.5 became much more pronounced. With embryo maturation, expression became stronger in the brain and by E17.5 expression was observed in the optic nerve and retina. In the newborn mouse P0 expression was observed in the olfactory bulb. Our preliminary results showed that, except neurons, progenitor cells also express this construct. Therefore, stem cells isolated from Thy1 - YFP at E14.5 can be successfully traced after transplantation in the brain and their processes arising during cell differentiation can be recognized using a bright fluorescence of YFP.

In order to analyze morphologic processes occurring during development of mammalian nervous system, several specific mouse strains have been constructed. This study was based on the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J which under the control of Thy1 gene promoter expresses yellow fluorescent protein (YFP) in all parts of neurons. This allows to precisely describing development of otherwise less visible parts of nervous cells, including axon and dendrites. During the first 10 days of embryonic development expression of YFP was not observed. First signs of expression were observed at embryonic day 12.5 (E12.5) in ventral horn of the spinal cord. During development of the embryo in the cranio-caudal direction, a stronger expression of YFP was observed in the cranial part of the spinal cord. Moreover, expression was significantly stronger in the spinal cord than in the brain. In accordance with the process of neurogenesis which primarily occurs in mid-gestation, expression of YFP in nerve cells after E12.5 become much more pronounced. Thus expression of YFP was observed in the brain, especially at the basis of the brain by E13.5. With embryo maturation, expression became stronger in the brain and by E17.5 expression was observed in the optic nerve and retina. In the newborn mouse P0 expression was observed in the olfactory bulb. Analyses of expression of Thy-YFP construct in B6.Cg-Tg(Thy1-YFP)16Jrs/J strain revealed a high rate of development of nervous system cells starting at E12.5 and increasing during later stages of gestation and reaching the first days of postnatal life.

Neuronal nuclear localization of STAM2 protein, a member of the endosome-associated complex ESCRT-0

Stem cell research raises hopes for incurable neurodegenerative diseases. In amyotrophic lateral sclerosis (ALS), affecting the motoneurones of the central nervous system (CNS), stem cell-based therapy aims to replace dying host motoneurones by transplantation of cells in disease-affected regions. Moreover, transplanted stem cells can serve as a source of trophic factors providing neuroprotection, slowing down neuronal degeneration and disease progression. Aim: To determine the profile of seven trophic factors expressed by mesenchymal stem cells (MSC) and neural stem cells (NSC) upon stimulation with CNS protein extracts from SOD1-linked ALS rat model. Methods: Culture of rat MSC, NSC and fibroblasts were incubated with brain and spinal cord extracts from SOD1(G93A) transgenic rats and mRNA expression of seven growth factors was measured by quantitative PCR. Results: MSC, NSC and fibroblasts exhibited different expression patterns. Nerve growth factor and brain-derived neurotropic factor were significantly upregulated in both NSC and MSC cultures upon stimulation with SOD1(G93A) CNS extracts. Fibroblast growth factor 2, insulin-like growth factor and glial-derived neurotropic factor were upregulated in NSC, while the same factors were downregulated in MSC. Vascular endothelial growth factor A upregulation was restricted to MSC and fibroblasts. Surprisingly, SOD1(G93A) spinal cord, but not the brain extract, upregulated brain-derived neurotropic factor in MSC and glial-derived neurotropic factor in NSC. Conclusions: These results suggest that inherent characteristics of different stem cell populations define their healing potential and raise the concept of ALS environment in stem cell transplantation.