Kmenové buňky pro své jedinečné vlastnosti – plasticitu, slibují všestranné využití, zejména možnostireparace většiny orgánů a tkání lidského těla – buněčná terapie. Autoři ve svémsdělení prezentují svojeexperimentální výsledky na modelu neurálních kmenových buněk (NSCs). Hlavním cílem je průkazzachování hemopoetické identity NSCs. Subletálně celotělově ozářené myši dávkou LD 8,5 Gy bylyzachráněné transplantací značených (X-Gal+) NSCs. Průkaz zvýšeného záchytu NSCs ve slezině násvedlo k myšlence hledání vztahu hemopoetického mikroprostředí a hemopoetické identity. Průkazzachování hemopoetické identity byl proveden pomocí kultivace CFU-GM z kostní dřeně – kde bylyidentifikovány buňky vykazující X-Gal pozitivitu (stanovení těchto buněk bylo provedeno pomocíhistochemického testu). Naše experimenty poukazují na různé možnosti, jak transplantace NSCs můžeovlivnit poškozenou hematopoézu.
Stem cells for their unique property - plasticity promise a universal utilization,mainly, in the reparationof most organs and tissues in the human body. In this communication, the authors presents theirexperimental results in a model of neural stem cells (NCSc) where the main goal was to preserve theNSCs hemopoietic identity. Mice given whole body sublethal irradiation with a dose of LD 8.5 Gy weresaved by transplantation of labelled (X-Gal+) NSCs. The demonstration of increased uptake of NSCs inthe spleen led us to study the relationship of hemopoietic microenvironment and hemopoietic identity.The proof of hemopoeitic identity was performed by cultivation of CFU-GM from the bone marrow -where cells exhibiting X-Gal positivity were identified by histochemistry. Our experiments showdifferent ways how NSCs transplantation may influence the damaged hematopoiesis.
- MeSH
- Radiation Dosage MeSH
- Research Support as Topic MeSH
- Hematopoiesis MeSH
- Stem Cells physiology MeSH
- Bone Marrow MeSH
- Cells, Cultured MeSH
- Models, Animal MeSH
- Mice MeSH
- Bone Marrow Transplantation MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Review MeSH
- Comparative Study MeSH
The isolation of neural stem cells from fetal and adult mammalian CNS and the demonstration of functional neurogenesis in adult CNS have offered perspectives for treatment of many devastating hereditary and acquired neurological diseases. Due to this enormous potential, neural stem cells are a subject of extensive molecular profiling studies with a search for new markers and regulatory pathways governing their self-renewal as opposed to differentiation. Several in-depth proteomic studies have been conducted on primary or immortalized cultures of neural stem cells and neural progenitor cells, and yet more remains to be done. Additionally, neurons and glial cells have been obtained from embryonic stem cells and mesenchymal stem cells, and proteins associated with the differentiation process have been characterized to a certain degree with a view to further investigations. This review summarizes recent findings relevant to the proteomics of neural stem cells and discusses major proteins significantly regulated during neural stem cell differentiation with a view to their future use in cell-based regenerative and reparative therapy.
- MeSH
- Cell Differentiation MeSH
- Financing, Organized MeSH
- Stem Cells cytology chemistry MeSH
- Humans MeSH
- Neurons cytology chemistry MeSH
- Proteins physiology genetics MeSH
- Proteomics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
Among various strategies employed for spinal cord injury, stem cell therapy is a potential treatment. So far, a variety of stem cells have been evaluated in animal models and humans with spinal cord injury, and epidermal neural crest stem cells represent one of the attractive types in this area. Although these multipotent stem cells have been assessed in several spinal cord injury models by independent laboratories, extensive work remains to be done to ascertain whether these cells can safely improve the outcome following human spinal cord injury. Among the models that closely mimic human spinal cord injury, the in vitro model of injury in organotypic spinal cord slice culture has been identified as one of the faithful platforms for injury-related investigations. In this study, green fluorescent protein-expressing stem cells were grafted into injured organotypic spinal cord slice culture and their survival was examined by confocal microscope seven days after transplantation. Data obtained from this preliminary study showed that these stem cells can survive on top of the surface of injured slices, as observed on day seven following their transplantation. This result revealed that this in vitro model of injury can be considered as a suitable context for further evaluation of epidermal neural crest stem cells before their application in large animals.
- MeSH
- Models, Biological MeSH
- Cell Death MeSH
- Neural Crest cytology MeSH
- Epidermal Cells MeSH
- Epidermis MeSH
- Stem Cells cytology MeSH
- Cells, Cultured MeSH
- Spinal Cord cytology MeSH
- Rats, Wistar MeSH
- Stem Cell Transplantation MeSH
- Cell Shape MeSH
- Cell Survival MeSH
- Green Fluorescent Proteins metabolism MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
Damaged neural tissue is regenerated by neural stem cells (NSCs), which represent a rare and difficult-to-culture cell population. Therefore, alternative sources of stem cells are being tested to replace a shortage of NSCs. Here we show that mouse adipose tissue-derived mesenchymal stem cells (MSCs) can be effectively differentiated into cells expressing neuronal cell markers. The differentiation protocol, simulating the inflammatory site of neural injury, involved brain tissue extract, fibroblast growth factor, epidermal growth factor, supernatant from activated splenocytes and electrical stimulation under physiological conditions. MSCs differentiated using this protocol displayed neuronal cell morphology and expressed genes for neuronal cell markers, such as neurofilament light (Nf-L), medium (Nf-M) and heavy (Nf-H) polypeptides, synaptophysin (SYP), neural cell adhesion molecule (NCAM), glutamic acid decarboxylase (GAD), neuron-specific nuclear protein (NeuN), βIII-tubulin (Tubb3) and microtubule-associated protein 2 (Mtap2), which are absent (Nf-L, Nf-H, SYP, GAD, NeuN and Mtap2) or only slightly expressed (NCAM, Tubb3 and Nf-M) in undifferentiated cells. The differentiation was further enhanced when the cells were cultured on nanofibre scaffolds. The neural differentiation of MSCs, which was detected at the level of gene expression, was confirmed by positive immunostaining for Nf-L protein. The results thus show that the simulation of conditions in an injured neural tissue and inflammatory environment, supplemented with electrical stimulation under physiological conditions and cultivation of cells on a three-dimensional (3D) nanofibre scaffold, form an effective protocol for the differentiation of MSCs into cells with neuronal markers. Copyright © 2015 John Wiley & Sons, Ltd.
- MeSH
- Cell Differentiation * MeSH
- Antigens, Differentiation biosynthesis MeSH
- Mesenchymal Stem Cells metabolism pathology MeSH
- Mice, Inbred BALB C MeSH
- Mice MeSH
- Nerve Tissue metabolism pathology MeSH
- Neural Stem Cells metabolism pathology MeSH
- Inflammation metabolism pathology MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: A well-characterized method has not yet been established to reproducibly, efficiently, and safely isolate large numbers of clinical-grade multipotent human neural stem cells (hNSCs) from embryonic stem cells (hESCs). Consequently, the transplantation of neurogenic/gliogenic precursors into the CNS for the purpose of cell replacement or neuroprotection in humans with injury or disease has not achieved widespread testing and implementation. METHODS: Here, we establish an approach for the in vitro isolation of a highly expandable population of hNSCs using the manual selection of neural precursors based on their colony morphology (CoMo-NSC). The purity and NSC properties of established and extensively expanded CoMo-NSC were validated by expression of NSC markers (flow cytometry, mRNA sequencing), lack of pluripotent markers and by their tumorigenic/differentiation profile after in vivo spinal grafting in three different animal models, including (i) immunodeficient rats, (ii) immunosuppressed ALS rats (SOD1G93A), or (iii) spinally injured immunosuppressed minipigs. RESULTS: In vitro analysis of established CoMo-NSCs showed a consistent expression of NSC markers (Sox1, Sox2, Nestin, CD24) with lack of pluripotent markers (Nanog) and stable karyotype for more than 15 passages. Gene profiling and histology revealed that spinally grafted CoMo-NSCs differentiate into neurons, astrocytes, and oligodendrocytes over a 2-6-month period in vivo without forming neoplastic derivatives or abnormal structures. Moreover, transplanted CoMo-NSCs formed neurons with synaptic contacts and glia in a variety of host environments including immunodeficient rats, immunosuppressed ALS rats (SOD1G93A), or spinally injured minipigs, indicating these cells have favorable safety and differentiation characteristics. CONCLUSIONS: These data demonstrate that manually selected CoMo-NSCs represent a safe and expandable NSC population which can effectively be used in prospective human clinical cell replacement trials for the treatment of a variety of neurodegenerative disorders, including ALS, stroke, spinal traumatic, or spinal ischemic injury.
Neural crest cells (NCCs) derive early in vertebrate ontogenesis from neural tube as a population of migratory cells with exquisite differentiation potential. Abnormalities in NCC behaviour are cause of debilitating diseases including cancers and a spectrum of neurocristopathies. Thanks to their multilineage differentiation capacity NCCs offer a cell source for regenerative medicine. Both these aspects make NCC biology an important issue to study, which can currently be addressed using methodologies based on pluripotent stem cells. Here we contributed to understanding the biology of human NCCs by refining the protocol for differentiation/propagation of NCClike cells from human embryonic stem cells and by characterizing the molecular and functional phenotype of such cells. Most importantly, we improved formulation of media for NCC culture, we found that poly-L-ornithine combined with fibronectin provide good support for NCC growth, we unravelled the tendency of cultured NCCs to maintain heterogeneity of CD271 expression, and we showed that NCCs derived here possess the capacity to react to BMP4 signals by dramatically up-regulating MSX1, which is linked to odontogenesis.
- MeSH
- Adapalene MeSH
- Biomarkers metabolism MeSH
- Cell Differentiation * drug effects MeSH
- Neural Crest cytology drug effects metabolism MeSH
- Embryonic Stem Cells cytology drug effects metabolism MeSH
- Phenotype MeSH
- Bone Morphogenetic Protein 4 pharmacology MeSH
- Humans MeSH
- Naphthalenes metabolism MeSH
- Polymerase Chain Reaction MeSH
- Flow Cytometry MeSH
- MSX1 Transcription Factor metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Neural crest (NC) is a transient embryonic tissue, whose cells are motile and multipotent until they reach their destination and differentiate according to microenvironmental cues into a variety of cell types. However, a subpopulation of these cells remains multipotent. They were found, among other locations, in a bulge of adult murine whisker follicle and were designated epidermal neural crest stem cells (EPI-NCSCs). The aim of this work is to ascertain whether the EPI-NCSCs could be isolated from human hair follicles as well. Due to their exceptional properties, they could represent potential candidates for stem cell therapy. The presented work focuses on the isolation and characterization of EPI-NCSCs from human skin. We obtained a population of cells that expressed markers of NC, NC progeny and general stem cell markers. After prolonged cultivation, the subpopulation of cells spontaneously differentiated into some of NC derivatives, i.e. neurons, smooth muscle cells and Schwann cell progenitors. Targeted differentiation with neuregulin 1 highly increased the number of Schwann cells in the culture. Human EPI-NCSCs could also grow under non-adherent conditions and form 3-dimensional spheres. Microarray analysis was performed and gene profile of human EPI-NCSCs was compared with the list of key genes of murine EPI-NCSCs and the list of genes up-regulated in newly induced NC cells. This revealed 94% and 88% similarity, respectively. All presented results strongly support the NCSC identity and multipotency of isolated human cells. These cells could thus be used in regenerative medicine, especially because of the easy accessibility of donor tissue.
- MeSH
- Cell Differentiation MeSH
- Neural Crest cytology metabolism MeSH
- Financing, Organized MeSH
- Immunohistochemistry MeSH
- Stem Cells cytology metabolism MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Multipotent Stem Cells cytology metabolism MeSH
- Mice MeSH
- Neurons MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Schwann Cells MeSH
- Oligonucleotide Array Sequence Analysis MeSH
- Gene Expression Profiling MeSH
- Stem Cell Transplantation MeSH
- Vibrissae MeSH
- Hair Follicle cytology MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
Embryonic neural stem cells (NSCs), comprising neuroepithelial and radial glial cells, are indispensable precursors of neurons and glia in the mammalian developing brain. Since the process of neurogenesis occurs in a hypoxic environment, the question arises of how NSCs deal with low oxygen tension and whether it affects their stemness. Genes from the hypoxia-inducible factors (HIF) family are well known factors governing cellular response to hypoxic conditions. In this study, we have discovered that the endogenous stabilization of hypoxia-inducible factor 1α (Hif1α) during neural induction is critical for the normal development of the NSCs pool by preventing its premature depletion and differentiation. The knock-out of the Hif1α gene in mESC-derived neurospheres led to a decrease in self-renewal of NSCs, paralleled by an increase in neuronal differentiation. Similarly, neuroepithelial cells differentiated in hypoxia exhibited accelerated neurogenesis soon after Hif1α knock-down. In both models, the loss of Hif1α was accompanied by an immediate drop in neural repressor Hes1 levels while changes in Notch signaling were not observed. We found that active Hif1α/Arnt1 transcription complex bound to the evolutionarily conserved site in Hes1 gene promoter in both neuroepithelial cells and neural tissue of E8.5 - 9.5 embryos. Taken together, these results emphasize the novel role of Hif1α in the regulation of early NSCs population through the activation of neural repressor Hes1, independently of Notch signaling.
- MeSH
- Cell Differentiation MeSH
- Cell Line MeSH
- Hypoxia MeSH
- Neural Stem Cells * MeSH
- Neurogenesis MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Engineered tumor-homing neural stem cells (NSCs) have shown promise in treating cancer. Recently, we transdifferentiated skin fibroblasts into human-induced NSCs (hiNSC) as personalized NSC drug carriers. Here, using a SOX2 and spheroidal culture-based reprogramming strategy, we generated a new hiNSC variant, hiNeuroS, that was genetically distinct from fibroblasts and first-generation hiNSCs and had significantly enhanced tumor-homing and antitumor properties. In vitro, hiNeuroSs demonstrated superior migration to human triple-negative breast cancer (TNBC) cells and in vivo rapidly homed to TNBC tumor foci following intracerebroventricular (ICV) infusion. In TNBC parenchymal metastasis models, ICV infusion of hiNeuroSs secreting the proapoptotic agent TRAIL (hiNeuroS-TRAIL) significantly reduced tumor burden and extended median survival. In models of TNBC leptomeningeal carcinomatosis, ICV dosing of hiNeuroS-TRAIL therapy significantly delayed the onset of tumor formation and extended survival when administered as a prophylactic treatment, as well as reduced tumor volume while prolonging survival when delivered as established tumor therapy.
- MeSH
- Fibroblasts MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Neoplastic Stem Cells pathology MeSH
- Neural Stem Cells * pathology MeSH
- Triple Negative Breast Neoplasms * pathology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
