Adult Human Neural Stem Cells Research Articles

EPO regulates neuronal differentiation of adult human neural-crest derived stem cells in a sex-specific manner

BackgroundSexual differences in the biology of human stem cells are increasingly recognized to influence their proliferation, differentiation and maturation. Especially in neurodegenerative diseases such as Alzheimers disease (AD), Parkinson’s disease (PD) or ischemic stroke, sex is a key player for disease progression and recovery of damaged tissue. Recently, the glycoprotein hormone erythropoietin (EPO) has been implicated as a regulator of neuronal differentiation and maturation in female rats.MethodsIn this study, we used adult human neural crest-derived stem cells (NCSCs) as a model system for exploring potential sex specific effects of EPO on human neuronal differentiation. We started with expression validation of the specific EPO receptor (EPOR) by performing PCR analysis in the NCSCs. Next, EPO mediated activation of nuclear factor-κB (NF-κB) via Immunocytochemistry (ICC) was performed, followed by investigating the sex-specific effects of EPO on neuronal differentiation by determining morphological changes in axonal growth and neurite formation accompanied by ICC.ResultsUndifferentiated male and female NCSCs showed a ubiquitous expression of the EPO receptor (EPOR). EPO treatment resulted in a statistically profound (male p = 0.0022, female p = 0.0012) nuclear translocation of NF-κB RELA in undifferentiated NCSCs of both sexes. But after one week of neuronal differentiation, we could show a highly significant (p = 0,0079) increase of nuclear NF-κB RELA in females only. In contrast, we observed a strong decrease (p = 0,0022) of RELA activation in male neuronal progenitors. Extending the view on the role of sex during human neuronal differentiation, here we demonstrate a significant increase of axon lengths in female NCSCs-derived neurons upon EPO-treatment (+ EPO: 167,73 (SD = 41,66) µm, w/o EPO: 77,68 (SD = 18,31) µm) compared to their male counterparts (+ EPO: 68,37 (SD = 11,97) µm, w/o EPO: 70,23 (SD = 12,89) µm).ConclusionOur present findings therefore show for the first time an EPO-driven sexual dimorphism in neuronal differentiation of human neural-crest derived stem cells and emphasize sex-specific variability as a crucial parameter in stem cell biology and for treating neurodegenerative diseases.

JAK2/STAT3 pathway mediates neuroprotective and pro-angiogenic treatment effects of adult human neural stem cells in middle cerebral artery occlusion stroke animal models.

Mismatches between pre-clinical and clinical results of stem cell therapeutics for ischemic stroke limit their clinical applicability. To overcome these discrepancies, precise planning of pre-clinical experiments that can be translated to clinical trials and the scientific elucidation of treatment mechanisms is important. In this study, adult human neural stem cells (ahNSCs) derived from temporal lobe surgical samples were used (to avoid ethical and safety issues), and their therapeutic effects on ischemic stroke were examined using middle cerebral artery occlusion animal models. 5 × 105 ahNSCs was directly injected into the lateral ventricle of contralateral brain hemispheres of immune suppressed rat stroke models at the subacute phase of stroke. Compared with the mock-treated group, ahNSCs reduced brain tissue atrophy and neurological sensorimotor and memory functional loss. Tissue analysis demonstrated that the significant therapeutic effects were mediated by the neuroprotective and pro-angiogenic activities of ahNSCs, which preserved neurons in ischemic brain areas and decreased reactive astrogliosis and microglial activation. The neuroprotective and pro-angiogenic effects of ahNSCs were validated in in vitro stroke models and were induced by paracrine factors excreted by ahNSCs. When the JAK2/STAT3 signaling pathway was inhibited by a specific inhibitor, AG490, the paracrine neuroprotective and pro-angiogenic effects of ahNSCs were reversed. This pre-clinical study that closely simulated clinical settings and provided treatment mechanisms of ahNSCs for ischemic stroke may aid the development of protocols for subsequent clinical trials of ahNSCs and the realization of clinically available stem cell therapeutics for ischemic stroke.

Significant Therapeutic Effects of Adult Human Neural Stem Cells for Spinal Cord Injury Are Mediated by Monocyte Chemoattractant Protein-1 (MCP-1).

The limited capability of regeneration in the human central nervous system leads to severe and permanent disabilities following spinal cord injury (SCI) while patients suffer from no viable treatment option. Adult human neural stem cells (ahNSCs) are unique cells derived from the adult human brain, which have the essential characteristics of NSCs. The objective of this study was to characterize the therapeutic effects of ahNSCs isolated from the temporal lobes of focal cortical dysplasia type IIIa for SCI and to elucidate their treatment mechanisms. Results showed that the recovery of motor functions was significantly improved in groups transplanted with ahNSCs, where, in damaged regions of spinal cords, the numbers of both spread and regenerated nerve fibers were observed to be higher than the vehicle group. In addition, the distance between neuronal nuclei in damaged spinal cord tissue was significantly closer in treatment groups than the vehicle group. Based on an immunohistochemistry analysis, those neuroprotective effects of ahNSCs in SCI were found to be mediated by inhibiting apoptosis of spinal cord neurons. Moreover, the analysis of the conditioned medium (CM) of ahNSCs revealed that such neuroprotective effects were mediated by paracrine effects with various types of cytokines released from ahNSCs, where monocyte chemoattractant protein-1 (MCP-1, also known as CCL2) was identified as a key paracrine mediator. These results of ahNSCs could be utilized further in the preclinical and clinical development of effective and safe cell therapeutics for SCI, with no available therapeutic options at present.

Transcriptome Analysis Reveals High Similarities between Adult Human Cardiac Stem Cells and Neural Crest-Derived Stem Cells.

Simple SummaryThe regeneration of nearly all organs of the human body mainly depends on the functionality of adult stem cell populations that reside in their respective niches and can be activated upon injuries or other damages. These stem cell populations greatly differ in their expression profile of molecular markers, which greatly influences their potential use in regenerative medicine. Neural crest-derived stem cells are a prominent subpopulation of adult stem cells and are known for their high regenerative potential. Within this study, we compared two adult human stem cell populations, namely neural crest-derived inferior turbinate stem cells from the nasal cavity and human cardiac stem cells from the heart, using global gene expression profiling. Here, we found differences that correspond to the tissue sources of origin but also similarities in the expression of markers that are associated with the neural crest. Further classifying nasal stem cells and cardiac stem cells in a broader context, we identified clear similarities between both populations and other adherent stem cell populations compared to non-adherent progenitor cells of the blood system. The analyses provided here might help to understand the differences and similarities between different adult human stem cell populations.For the identification of a stem cell population, the comparison of transcriptome data enables the simultaneous analysis of tens of thousands of molecular markers and thus enables the precise distinction of even closely related populations. Here, we utilized global gene expression profiling to compare two adult human stem cell populations, namely neural crest-derived inferior turbinate stem cells (ITSCs) of the nasal cavity and human cardiac stem cells (hCSCs) from the heart auricle. We detected high similarities between the transcriptomes of both stem cell populations, particularly including a range of neural crest-associated genes. However, global gene expression likewise reflected differences between the stem cell populations with regard to their niches of origin. In a broader analysis, we further identified clear similarities between ITSCs, hCSCs and other adherent stem cell populations compared to non-adherent hematopoietic progenitor cells. In summary, our observations reveal high similarities between adult human cardiac stem cells and neural crest-derived stem cells from the nasal cavity, which include a shared relation to the neural crest. The analyses provided here may help to understand underlying molecular regulators determining differences between adult human stem cell populations.

Ectopic expression of L1CAM ectodomain alters differentiation and motility, but not proliferation, of human neural progenitor cells

Adult human neural progenitor and stem cells have been implicated as a potential source of brain cancer causing cells, but specific events that might cause cells to progress towards a transformed phenotype remain unclear. The L1CAM (L1) cell adhesion/recognition molecule is expressed abnormally by human glioma cancer cells and is released as a large extracellular ectodomain fragment, which stimulates cell motility and proliferation. This study investigates the effects of ectopic overexpression of the L1 long ectodomain (L1LE; ˜180 kDa) on the motility, proliferation, and differentiation of human neural progenitor cells (HNPs). L1LE was ectopically expressed in HNPs using a lentiviral vector. Surprisingly, overexpression of L1LE resulted in reduced HNP motility in vitro, in stark contrast to the effects on glioma and other cancer cell types. L1LE overexpression resulted in a variable degree of maintenance of HNP proliferation in media without added growth factors but did not increase proliferation. In monolayer culture, HNPs expressed a variety of differentiation markers. L1LE overexpression resulted in loss of glutamine synthetase (GS) and β3-tubulin expression in normal HNP media, and reduced vimentin and increased GS expression in the absence of added growth factors. When co-cultured with chick embryonic brain cell aggregates, HNPs show increased differentiation potential. Some HNPs expressed p-neurofilaments and oligodendrocytic O4, indicating differentiation beyond that in monolayer culture. Most HNP-L1LE cells lost their vimentin and GFAP (glial fibrillary acidic protein) staining, and many cells were positive for astrocytic GS. However, these cells rarely were positive for neuronal markers β3-tubulin or p-neurofilaments, and few HNP oligodendrocyte progenitors were found. These results suggest that unlike for glioma cells, L1LE does not increase HNP cell motility, but rather decreases motility and influences the differentiation of normal brain progenitor cells. Therefore, the effect of L1LE on increasing motility and proliferation appears to be limited to already transformed cells.

Identification of a Novel High Yielding Source of Multipotent Adult Human Neural Crest-Derived Stem Cells.

Due to their extraordinarily broad differentiation potential and persistence during adulthood, adult neural crest-derived stem cells (NCSCs) are highly promising candidates for clinical applications, particularly when facing the challenging treatment of neurodegenerative diseases or complex craniofacial injuries. Successful application of human NCSCs in regenerative medicine and pharmaceutical research mainly relies on the availability of sufficient amounts of tissue for cell isolation procedures. Facing this challenge, we here describe for the first time a novel population of NCSCs within the middle turbinate of the human nasal cavity. From a surgical point of view, high amounts of tissue are routinely and easily removed during nasal biopsies. Investigating the presence of putative stem cells in obtained middle turbinate tissue by immunohistochemistry, we observed Nestin+/p75NTR+/S100+/α smooth muscle actin (αSMA)- cells, which we successfully isolated and cultivated in vitro. Cultivated middle turbinate stem cells (MTSCs) kept their expression of neural crest and stemness markers Nestin, p75 NTR and S100 and showed the capability of sphere formation and clonal growth, indicating their stem cell character. Application of directed in vitro differentiation assays resulted in successful differentiation of MTSCs into osteogenic and neuronal cell types. Regarding the high amount of tissue obtained during surgery as well as their broad differentiation capability, MTSCs seem to be a highly promising novel neural crest stem cell population for applications in cell replacement therapy and pharmacological research.

Regenerative potential of primary adult human neural stem cells on micropatterned bio-implants boosts motor recovery

BackgroundThe adult brain is unable to regenerate itself sufficiently after large injuries. Therefore, hopes rely on therapies using neural stem cell or biomaterial transplantation to sustain brain reconstruction. The aim of the present study was to evaluate the improvement in sensorimotor recovery brought about by human primary adult neural stem cells (hNSCs) in combination with bio-implants.MethodshNSCs were pre-seeded on implants micropatterned for neurite guidance and inserted intracerebrally 2 weeks after a primary motor cortex lesion in rats. Long-term behaviour was significantly improved after hNSC implants versus cell engraftment in the grip strength test. MRI and immunohistological studies were conducted to elucidate the underlying mechanisms of neuro-implant integration.ResultshNSC implants promoted tissue reconstruction and limited hemispheric atrophy and glial scar expansion. After 3 months, grafted hNSCs were detected on implants and expressed mature neuronal markers (NeuN, MAP2, SMI312). They also migrated over a short distance to the reconstructed tissues and to the peri-lesional tissues, where 26% integrated as mature neurons. Newly formed host neural progenitors (nestin, DCX) colonized the implants, notably in the presence of hNSCs, and participated in tissue reconstruction. The microstructured bio-implants sustained the guided maturation of both grafted hNSCs and endogenous progenitors.ConclusionsThese immunohistological results are coherent with and could explain the late improvement observed in sensorimotor recovery. These findings provide novel insights into the regenerative potential of primary adult hNSCs combined with microstructured implants.

Therapeutic potential of human olfactory bulb neural stem cells for spinal cord injury in rats.

Adult human olfactory bulb neural stem cells (OBNSCs) were isolated from human patients undergoing craniotomy for tumor resection. They were genetically engineered to overexpresses green fluorescent protein (GFP) to help trace them following engraftment. Spinal cord injury (SCI) was induced in rats using standard laminectomy protocol, and GFP-OBNSC were engrafted into rat model of SCI at day 7 post injury. Three rat groups were used: (i) Control group, (ii) Sham group (injected with cerebrospinal fluid) and treated group (engrafted with OBNSCs). Tissues from different groups were collected weekly up to 2 months. The collected tissues were fixed in 4% paraformaldehyde, processed for paraffin sectioning, immunohistochemically stained for different neuronal and glial markers and examined with bright-field fluorescent microscopy. Restoration of sensory motor functions we assessed on a weekly bases using the BBB score. To assess the therapeutic potential of OBNSCs-GFP and their ability to survive, proliferate, differentiate and to restore lost sensory motor functions following their engraftment in spinal cord injury (SCI). GFP-OBNSC were engrafted into a rat model of SCI at day 7 post injury and were followed-up to 8 weeks using behavioral and histochemical methods. All transplanted animals exhibited successful engraftment. The survival rate was about 30% of initially transplanted cells. Twenty-seven percent of the engrafted cells differentiated along the NG2 and O4-positive oligodendrocyte lineage, 16% into MAP2 and β-tubulin-positive neurons, and 56% into GFAP-positive astrocytes. GFP-OBNSCs had survived for >8 weeks after engraftment and were differentiated into neurons, astrocytes and oligodendrocytes, The engrafted cells were distributed throughout gray and white matter of the cord with no evidence of abnormal morphology or any mass formation indicative of tumorigenesis. However, the engrafted cells failed to restore lost sensory and motor functions as evident from behavioral analysis using the BBB score test.

Survival, differentiation, and neuroprotective mechanisms of human stem cells complexed with neurotrophin-3-releasing pharmacologically active microcarriers in an ex vivo model of Parkinson's disease.

Stem cell-based regenerative therapies hold great potential for the treatment of degenerative disorders such as Parkinson's disease (PD). We recently reported the repair and functional recovery after treatment with human marrow-isolated adult multilineage inducible (MIAMI) cells adhered to neurotrophin-3 (NT3) releasing pharmacologically active microcarriers (PAMs) in hemiparkinsonian rats. In order to comprehend this effect, the goal of the present work was to elucidate the survival, differentiation, and neuroprotective mechanisms of MIAMI cells and human neural stem cells (NSCs), both adhering to NT3-releasing PAMs in an ex vivo organotypic model of nigrostriatal degeneration made from brain sagittal slices. It was shown that PAMs led to a marked increase in MIAMI cell survival and neuronal differentiation when releasing NT3. A significant neuroprotective effect of MIAMI cells adhering to PAMs was also demonstrated. NSCs barely had a neuroprotective effect and differentiated mostly into dopaminergic neuronal cells when adhering to PAM-NT3. Moreover, those cells were able to release dopamine in a sufficient amount to induce a return to baseline levels. Reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay analyses identified vascular endothelial growth factor (VEGF) and stanniocalcin-1 as potential mediators of the neuroprotective effect of MIAMI cells and NSCs, respectively. It was also shown that VEGF locally stimulated tissue vascularization, which might improve graft survival, without excluding a direct neuroprotective effect of VEGF on dopaminergic neurons. These results indicate a prospective interest of human NSC/PAM and MIAMI cell/PAM complexes in tissue engineering for PD. Stem cell-based regenerative therapies hold great potential for the treatment of degenerative disorders such as Parkinson's disease (PD). The present work elucidates and compares the survival, differentiation, and neuroprotective mechanisms of marrow-isolated adult multilineage inducible cells and human neural stem cells both adhered to neurotrophin-3-releasing pharmacologically active microcarriers in an ex vivo organotypic model of PD made from brain sagittal slices.

Identification of Nucleoside Analogs as Inducers of Neuronal Differentiation in a Human Reporter Cell Line and Adult Stem Cells

Nucleoside analogs (NSAs) were among the first chemotherapeutic agents and could also be useful for the manipulation of cell fate. To investigate the potential of NSAs for the induction of neuronal differentiation, we developed a novel phenotypic assay based on a human neuron-committed teratocarcinoma cell line (NT2) as amodel for neuronal progenitors and constructed a NT2-based reporter cell line that expressed eGFP under the control of a neuron-specific promoter. We tested 38 structurally related NSAs and determined their activity to induce neuronal differentiation by immunocytochemistry of neuronal marker proteins, live cell imaging, fluorometric detection and immunoblot analysis. We identified twelve NSAs, which induced neuronal differentiation to different extents. NSAs with highest activity carried a halogen substituent at their pyrimidine nucleobase and an unmodified or 2'-O-methyl substituted 2-deoxy-β-D-ribofuranosyl residue as glyconic moiety. Cladribine, a purine nucleoside with similar structural features and in use to treat leukemia and multiple sclerosis, induced also differentiation of adult human neural crest-derived stem cells. Our results suggest that NSAs could be useful for the manipulation of neuronal cell fate in cell replacement therapy or treatment of neurodegenerative disorders. The data on the structure and function relationship will help to design compounds with increased activity and low toxicity.

Intrastriatal transplantation of adult human neural crest-derived stem cells improves functional outcome in parkinsonian rats.

Parkinson's disease (PD) is considered the second most frequent and one of the most severe neurodegenerative diseases, with dysfunctions of the motor system and with nonmotor symptoms such as depression and dementia. Compensation for the progressive loss of dopaminergic (DA) neurons during PD using current pharmacological treatment strategies is limited and remains challenging. Pluripotent stem cell-based regenerative medicine may offer a promising therapeutic alternative, although the medical application of human embryonic tissue and pluripotent stem cells is still a matter of ethical and practical debate. Addressing these challenges, the present study investigated the potential of adult human neural crest-derived stem cells derived from the inferior turbinate (ITSCs) transplanted into a parkinsonian rat model. Emphasizing their capability to give rise to nervous tissue, ITSCs isolated from the adult human nose efficiently differentiated into functional mature neurons in vitro. Additional successful dopaminergic differentiation of ITSCs was subsequently followed by their transplantation into a unilaterally lesioned 6-hydroxydopamine rat PD model. Transplantation of predifferentiated or undifferentiated ITSCs led to robust restoration of rotational behavior, accompanied by significant recovery of DA neurons within the substantia nigra. ITSCs were further shown to migrate extensively in loose streams primarily toward the posterior direction as far as to the midbrain region, at which point they were able to differentiate into DA neurons within the locus ceruleus. We demonstrate, for the first time, that adult human ITSCs are capable of functionally recovering a PD rat model.

Interaction of adult human neural crest-derived stem cells with a nanoporous titanium surface is sufficient to induce their osteogenic differentiation

Osteogenic differentiation of various adult stem cell populations such as neural crest-derived stem cells is of great interest in the context of bone regeneration. Ideally, exogenous differentiation should mimic an endogenous differentiation process, which is partly mediated by topological cues. To elucidate the osteoinductive potential of porous substrates with different pore diameters (30nm, 100nm), human neural crest-derived stem cells isolated from the inferior nasal turbinate were cultivated on the surface of nanoporous titanium covered membranes without additional chemical or biological osteoinductive cues. As controls, flat titanium without any topological features and osteogenic medium was used. Cultivation of human neural crest-derived stem cells on 30nm pores resulted in osteogenic differentiation as demonstrated by alkaline phosphatase activity after seven days as well as by calcium deposition after 3weeks of cultivation. In contrast, cultivation on flat titanium and on membranes equipped with 100nm pores was not sufficient to induce osteogenic differentiation. Moreover, we demonstrate an increase of osteogenic transcripts including Osterix, Osteocalcin and up-regulation of Integrin β1 and α2 in the 30nm pore approach only. Thus, transplantation of stem cells pre-cultivated on nanostructured implants might improve the clinical outcome by support of the graft adherence and acceleration of the regeneration process.

Culture bag systems for clinical applications of adult human neural crest-derived stem cells

IntroductionFacing the challenging treatment of neurodegenerative diseases as well as complex craniofacial injuries such as those common after cancer therapy, the field of regenerative medicine increasingly relies on stem cell transplantation strategies. Here, neural crest-derived stem cells (NCSCs) offer many promising applications, although scale up of clinical-grade processes prior to potential transplantations is currently limiting. In this study, we aimed to establish a clinical-grade, cost-reducing cultivation system for NCSCs isolated from the adult human nose using cGMP-grade Afc-FEP bags.MethodsWe cultivated human neural crest-derived stem cells from inferior turbinate (ITSCs) in a cell culture bag system using Afc-FEP bags in human blood plasma-supplemented medium. Investigations of viability, proliferation and expression profile of bag-cultured ITSCs were followed by DNA-content and telomerase activity determination. Cultivated ITSCs were introduced to directed in vitro differentiation assays to assess their potential for mesodermal and ectodermal differentiation. Mesodermal differentiation was determined using an enzyme activity assay (alkaline phosphatase, ALP), respective stainings (Alizarin Red S, Von Kossa and Oil Red O), and RT-PCR, while immunocytochemistry and synaptic vesicle recycling were applied to assay neuroectodermal differentiation of ITSCs.ResultsWhen cultivated within Afc-FEP bags, ITSCs grew three-dimensionally in a human blood plasma-derived matrix, thereby showing unchanged morphology, proliferation capability, viability and expression profile in comparison to three dimensionally-cultured ITSCs growing in standard cell culture plastics. Genetic stability of bag-cultured ITSCs was further accompanied by unchanged telomerase activity. Importantly, ITSCs retained their potential to differentiate into mesodermal cell types, particularly including ALP-active, Alizarin Red S-, and Von Kossa-positive osteogenic cell types, as well as adipocytes positive in Oil Red O assays. Bag culture further did not affect the potential of ITSCs to undergo differentiation into neuroectodermal cell types coexpressing β-III-tubulin and MAP2 and exhibiting the capability for synaptic vesicle recycling.ConclusionsHere, we report for the first time the successful cultivation of human NCSCs within cGMP-grade Afc-FEP bags using a human blood plasma-supplemented medium. Our findings particularly demonstrate the unchanged differentiation capability and genetic stability of the cultivated NCSCs, suggesting the great potential of this culture system for future medical applications in the field of regenerative medicine.

Generation of Neuronal Progenitor Cells in Response to Tumors in the Human Brain

Data from transgenic mouse models show that neuronal progenitor cells (NPCs) migrate toward experimental brain tumors and modulate the course of pathology. However, the pathways whereby NPCs are attracted to CNS neoplasms are not fully understood and it is unexplored if NPCs migrate toward brain tumors (high-grade astrocytomas) in humans. We analyzed the tumor-parenchyma interface of neurosurgical resections for the presence of (NPCs) and distinguished these physiological cells from the tumor mass. We observed that polysialic acid neural cell adhesion molecule-positive NPCs accumulate at the border of high-grade astrocytomas and display a marker profile consistent with immature migratory NPCs. Importantly, these high-grade astrocytoma-associated NPCs did not carry genetic aberrations that are indicative of the tumor. Additionally, we observed NPCs accumulating in CNS metastases. These metastatic tumors are distinguished from neural cells by defined sets of markers. Transplanting murine glioma cells embedded in a cell-impermeable hollow fiber capsule into the brains of nestin-gfp reporter mice showed that diffusible factors are sufficient to induce a neurogenic reaction. In vitro, vascular endothelial growth factor (VEGF) secreted from glioma cells increases the migratory and proliferative behavior of adult human brain-derived neural stem and progenitor cells via stimulation of VEGF receptor-2 (VEGFR-2). In vivo, inhibiting VEGFR-2 signaling with a function-blocking antibody led to a reduction in NPC migration toward tumors. Overall, our data reveal a mechanism by which NPCs are attracted to CNS tumors and suggest that NPCs accumulate in human high-grade astrocytomas.

Over-expression of hNGF in adult human olfactory bulb neural stem cells promotes cell growth and oligodendrocytic differentiation.

The adult human olfactory bulb neural stem/progenitor cells (OBNC/PC) are promising candidate for cell-based therapy for traumatic and neurodegenerative insults. Exogenous application of NGF was suggested as a promising therapeutic strategy for traumatic and neurodegenerative diseases, however effective delivery of NGF into the CNS parenchyma is still challenging due mainly to its limited ability to cross the blood–brain barrier, and intolerable side effects if administered into the brain ventricular system. An effective method to ensure delivery of NGF into the parenchyma of CNS is the genetic modification of NSC to overexpress NGF gene. Overexpression of NGF in adult human OBNSC is expected to alter their proliferation and differentiation nature, and thus might enhance their therapeutic potential. In this study, we genetically modified adult human OBNS/PC to overexpress human NGF (hNGF) and green fluorescent protein (GFP) genes to provide insight about the effects of hNGF and GFP genes overexpression in adult human OBNS/PC on their in vitro multipotentiality using DNA microarray, immunophenotyping, and Western blot (WB) protocols. Our analysis revealed that OBNS/PC-GFP and OBNS/PC-GFP-hNGF differentiation is a multifaceted process involving changes in major biological processes as reflected in alteration of the gene expression levels of crucial markers such as cell cycle and survival markers, stemness markers, and differentiation markers. The differentiation of both cell classes was also associated with modulations of key signaling pathways such MAPK signaling pathway, ErbB signaling pathway, and neuroactive ligand-receptor interaction pathway for OBNS/PC-GFP, and axon guidance, calcium channel, voltage-dependent, gamma subunit 7 for OBNS/PC-GFP-hNGF as revealed by GO and KEGG. Differentiated OBNS/PC-GFP-hNGF displayed extensively branched cytoplasmic processes, a significant faster growth rate and up modulated the expression of oligodendroglia precursor cells markers (PDGFRα, NG2 and CNPase) respect to OBNS/PC-GFP counterparts. These findings suggest an enhanced proliferation and oligodendrocytic differentiation potential for OBNS/PC-GFP-hNGF as compared to OBNS/PC-GFP.

Comparison of glioma stem cells to neural stem cells from the adult human brain identifies dysregulated Wnt- signaling and a fingerprint associated with clinical outcome

Glioblastoma is the most common brain tumor. Median survival in unselected patients is <10 months. The tumor harbors stem-like cells that self-renew and propagate upon serial transplantation in mice, although the clinical relevance of these cells has not been well documented. We have performed the first genome-wide analysis that directly relates the gene expression profile of nine enriched populations of glioblastoma stem cells (GSCs) to five identically isolated and cultivated populations of stem cells from the normal adult human brain. Although the two cell types share common stem- and lineage-related markers, GSCs show a more heterogeneous gene expression. We identified a number of pathways that are dysregulated in GSCs. A subset of these pathways has previously been identified in leukemic stem cells, suggesting that cancer stem cells of different origin may have common features. Genes upregulated in GSCs were also highly expressed in embryonic and induced pluripotent stem cells. We found that canonical Wnt-signaling plays an important role in GSCs, but not in adult human neural stem cells. As well we identified a 30-gene signature highly overexpressed in GSCs. The expression of these signature genes correlates with clinical outcome and demonstrates the clinical relevance of GSCs.

Reprogramming of adult human neural stem cells into induced pluripotent stem cells

Background Since an effective method for generating induced pluripotent stem cells (iPSCs) from human neural stem cells (hNSCs) can offer us a promising tool for studying brain diseases, here we reported direct reprogramming of adult hNSCs into iPSCs by retroviral transduction of four defined factors. Methods NSCs were successfully isolated and cultured from the hippocampus tissue of epilepsy patients. When combined with four factors (OCT3/4, SOX2, KLF4, and c-MYC), iPSCs colonies were successfully obtained. Results Morphological characterization and specific genetic expression confirmed that these hNSCs-derived iPSCs showed embryonic stem cells-like properties, which include the ability to differentiate into all three germ layers both in vitro and in vivo. Conclusion Our method would be useful for generating human iPSCs from NSCs and provide an important tool for studying neurological diseases.

Micro/nano-ingénierie pour le contrôle de la croissance de cellules neuronales et ingénierie tissulaire appliquée au système nerveux central

Central nervous system pathologies are often characterized by the loss of cell populations. A promising therapy now being developed consists in using bioactive materials, associating grafted cells to biopolymers which provide a scaffold for the in vitro building of new tissues, to be implanted in vivo. In the present article, the state of the art of this field, at crossroads between microtechnology and neuroscience, is described in detail; thereafter our own approach and results about interactions between adult human neural stem cells and microstructured polymers are summarized and discussed. In a second part, some central nervous system repair strategies, based on cerebral tissue engineering, are presented. We will report the main results of our studies to work out and characterize in vivo a cerebral bioprosthesis.

Experimental and clinical factors influencing long-term stable in vitro expansion of multipotent neural cells from human adult temporal lobes

Autologous adult human neural stem cells may be used for regenerative cell therapies bypass potential ethical problems. However, stable in vitro expansion protocols and experimental/clinical factors influencing primary cultures need to be further elucidated for clinically applicable techniques. To address these issues, we obtained biopsy specimens from 23 temporal lobe epilepsy patients and adult human multipotent neural cells (ahMNCs) were primarily cultured in a defined attachment culture condition. When the success of primary cultures was defined as stable expansion of cells (>ten in vitro passages) and expression of NSC markers, success rate of the primary culture was 39% (nine of 23 temporal lobes). During the long-term expansion, expressions of NSC markers and differentiation potentials into astrocytes and neurons were maintained. After the 18th sub-culture, spontaneous senescence and differentiation were observed, and the cultivated ahMNCs ceased their proliferation. The culture results were not affected by seizure characteristics; however, an older age (>40years) and a smaller sample volume (<2ml) were found to exert negative influences on the primary culture results. Furthermore therapeutic effects of ahMNCs against stroke were analyzed in an animal model. Transplantation of ahMNCs cells reduced infarction volumes and enhanced motor activity, significantly. The results here would provide promising experimental and clinical strategy of using patient-specific autologous ahMNCs in regenerative medicine in the future.

Transcription factors expressed in embryonic and adult olfactory bulb neural stem cells reveal distinct proliferation, differentiation and epigenetic control

TF genomic markers associated with neurogenesis, proliferation, differentiation, and epigenetic control in human embryonic neural stem cells (hENSC(, and adult human olfactory bulb neural stem cells (OBNSC) were studied by immunohistochemistry (IHC) and DNA microarray. The biological impact of TF gene changes in the examined cell types was estimated using DAVID to specify a different GO class and signaling pathway based on KEGG database. Eleven, and twenty eight TF genes were up-regulated (fold change≤2–39) in OBNSC, and hENSC respectively. KEGG pathway analysis for the up-regulated TF genes revealed significant enrichments for the basal transcription factor pathway, and Notch signaling pathway in OBNSCs, and hENSCs, respectively. Immunofluorescence analysis revealed a significantly greater number of β-tubulin III (TUBB3), MAP, glial fibrillary acidic protein (GFAP), and O4 in hENSC when compared to those in OBNSC. Furthermore, the expression of epigenetic-related TF-genes SMARCC1, TAF12, and UHRF1 increased significantly in OBNSC when compared with hENSC.