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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.

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.

Ishemija mozga dovodi do teških posljedica na moždano tkivo, a moždani udar je jedan od najvećih problema moderne medicine. Budući da matične stanice imaju veliki potencijal u liječenju raznih bolesti, njihova trasplantacija je predložena kao moguća metoda u smanjivanju štetnih posljedica ishemije na moždano tkivo. Ipak, vrlo se malo zna o utjecaju ishemije na same matične stanice. Cilj ovog istraživanja je bio utvrditi utjecaj ishemije na ponašanje i diferencijaciju živčanih matičnih stanica uzgajanih u kulturi. Nakon izolacije matičnih stanica iz mišjih zametaka, napravljena je analiza kretanja stanica, njihovog rasta i diferencijacije uz pomoć visokorezolucijskog mikroskopa EVOS te njegovog poluautomatskog softvera Cell Tracker. Nakon što su stanice izložene ishemiji, analiziran je niz parametara (ukupna prijeđena udaljenost svih stanica, maksimalna dosegnuta brzina, maksimalna prijeđena udaljenost po jednoj stanici i udio naglih skretanja po stanici), a statistički značajna razlika je nađena u ukupnoj udaljenosti koje pređu sve stanice u kulturi. Ovim istraživanjem smo uspostavili nov način analize utjecaja različitih čimbenika na matične stanice, te smo pokazali da ishemija mijenja njihovo ponašanje., Brain ischemia leads to serious brain damage and stroke is one of the major problems of modern medicine. Since stem cells have great potential in treating various diseases, their transplantation is suggested as a possible method of reducing the damaging consequences of ischemia on brain tissue. Still, it is not much known about the effect of ischemia on the stem cells. The aim of this study was to determine the effect of ischemia on the behavior and differentiation of neural stem cells grown in the culture. After isolation of stem cells from mouse embryos, analysis of cell movement, their growth and differentiation was made with the help of a high-resolution EVOS microscope and its semi-automatic Cell Tracker software. After the cells were exposed to ischemia, a number of parameters were analyzed (the length of the total traveled distance of all cells, the maximum speed, the maximum distance reached per cell and the rate of sudden rotation per cell). The statistically significant difference was found in the length of the total traveled distance of all cells. This research has established a new way of analyzing the effects of various factors on stem cells, and we have shown that ischemia changes their behavior.