Long-term pre-clinical evaluation of an injectable chitosan nanocellulose hydrogel with encapsulated adipose-derived stem cells in an ovine model for IVD regeneration.

The potential therapeutic benefit of adipose-derived stem cells (ASCs) encapsulated in an injectable hydrogel for stimulating intervertebral disc (IVD) regeneration has been assessed by a number of translational and preclinical studies. However, previous work has been primarily limited to small animal models and short-term outcomes of only a few weeks. Long-term studies in representative large animal models are crucial for translation into clinical success, especially for permanent stabilization of major defects such as disc herniation. An injectable chitosan carboxymethyl cellulose hydrogel scaffold loaded with ASCs was evaluated regarding its intraoperative handling, crosslinking kinetics, cell viability, fully-crosslinked viscoelasticity, and long-term therapeutic effects in an ovine model. Three IVDs per animal were damaged in 10 sheep. Subcutaneous adipose tissue was the source for autologous ASCs. Six weeks after IVD damage, two of the damaged IVDs were treated via ASC-loaded hydrogel injection. After 12 months following the implantation, IVD disc height and histological and cellular changes were assessed. This system was reliable and easy to handle intraoperatively. Over 12 months, IVD height was stabilized and degeneration progression significantly mitigated compared to untreated, damaged IVDs. Here we show for the first time in a large animal model that an injectable chitosan carboxymethyl cellulose hydrogel system with encapsulated ASCs is able to affect long-term stabilization of an injured IVD and significantly decrease degeneration processes as compared to controls.

Intervertebral Disc Regeneration Injection of a Cell-Loaded Collagen Hydrogel in a Sheep Model.

Degenerated intervertebral discs (IVDs) were treated with autologous adipose-derived stem cells (ASC) loaded into an injectable collagen scaffold in a sheep model to investigate the implant's therapeutic potential regarding the progression of degeneration of previously damaged discs. In this study, 18 merino sheep were subjected to a 3-step minimally invasive injury and treatment model, which consisted of surgically induced disc degeneration, treatment of IVDs with an ASC-loaded collagen hydrogel 6 weeks post-operatively, and assessment of the implant's influence on degenerative tissue changes after 6 and 12 months of grazing. Autologous ASCs were extracted from subcutaneous adipose tissue and cultivated in vitro. At the end of the experiment, disc heights were determined by µ-CT measurements and morphological tissue changes were histologically examined.Histological investigations show that, after treatment with the ASC-loaded collagen hydrogel implant, degeneration-specific features were observed less frequently. Quantitative studies of the degree of degeneration did not demonstrate a significant influence on potential tissue regeneration with treatment. Regarding disc height analysis, at both 6 and 12 months after treatment with the ASC-loaded collagen hydrogel implant a stabilization of the disc height can be seen. A complete restoration of the intervertebral disc heights however could not be achieved.The reported injection procedure describes in a preclinical model a translational therapeutic approach for degenerative disc diseases based on adipose-derived stem cells in a collagen hydrogel scaffold. Further investigations are planned with the use of a different injectable scaffold material using the same test model.

Fluorescent protein-expressing neural progenitor cells as a tool for transplantation studies.

The purpose of this study was to generate quadruple fluorescent protein (QFP) transgenic mice as a source for QFP-expressing neural stem and progenitor cells (NSCs/NPCs) that could be utilized as a tool for transplantation research. When undifferentiated, these NSCs only express cyan fluorescent protein (CFP); however, upon neuronal differentiation, the cells express yellow fluorescent protein (YFP). During astrocytic differentiation, the cells express green fluorescent protein (GFP), and during oligodendrocytic differentiation, the cells express red fluorescent protein (DsRed). Using immunocytochemistry, immunoblotting, flow cytometry and electrophysiology, quadruple transgenic NPCs (Q-NPCs) and GFP-sorted NPCs were comprehensively characterized in vitro. Overall, the various transgenes did not significantly affect proliferation and differentiation of transgenic NPCs in comparison to wild-type NPCs. In contrast to a strong CFP and GFP expression in vitro, NPCs did not express YFP and dsRed either during proliferation or after differentiation in vitro. GFP-positive sorted NPCs, expressing GFP under the control of the human GFAP promoter, demonstrated a significant improvement in astroglial differentiation in comparison to GFP-negative sorted NPCs. In contrast to non-sorted and GFP-positive sorted NPCs, GFP-negative sorted NPCs demonstrated a high proportion of neuronal differentiation and proved to be functional in vitro. At 6 weeks after the intracerebroventricular transplantation of Q-NPCs into neonatal wild-type mice, CFP/DCX (doublecortin) double-positive transplanted cells were observed. The Q-NPCs did not express any other fluorescent proteins and did not mature into neuronal or glial cells. Although this model failed to visualize NPC differentiation in vivo, we determined that activation of the NPC glial fibrillary acid protein (GFAP) promoter, as indicated by GFP expression, can be used to separate neuronal and glial progenitors as a valuable tool for transplantation studies.

The influence of immunosuppressive drugs on neural stem/progenitor cell fate in vitro.

In allogenic and xenogenic transplantation, adequate immunosuppression plays a major role in graft survival, especially over the long term. The effect of immunosuppressive drugs on neural stem/progenitor cell fate has not been sufficiently explored. The focus of this study is to systematically investigate the effects of the following four different immunotherapeutic strategies on human neural progenitor cell survival/death, proliferation, metabolic activity, differentiation and migration in vitro: (1) cyclosporine A (CsA), a calcineurin inhibitor; (2) everolimus (RAD001), an mTOR-inhibitor; (3) mycophenolic acid (MPA, mycophenolate), an inhibitor of inosine monophosphate dehydrogenase and (4) prednisolone, a steroid. At the minimum effective concentration (MEC), we found a prominent decrease in hNPCs' proliferative capacity (BrdU incorporation), especially for CsA and MPA, and an alteration of the NAD(P)H-dependent metabolic activity. Cell death rate, neurogenesis, gliogenesis and cell migration remained mostly unaffected under these conditions for all four immunosuppressants, except for apoptotic cell death, which was significantly increased by MPA treatment.

Erythropoietin enhances cell proliferation and survival of human fetal neuronal progenitors in normoxia.

Extensive data reporting the neurogenerative, neuroprotective and neuroregenerative potential of erythropoietin (EPO), mainly on RNA level, can be found in the literature. However, there is still a poor knowledge on the response of neuronal progenitor cells (NPC) upon stimulation with EPO in terms of the protein species involved. Herein, the effect of EPO on the proliferation of human mesencephalic NPC (hmNPC) under normoxia is monitored using cellular assays and proteomic analysis (two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry). The administration of EPO increased the proliferation of hmNPC within 4 days after application. It positively influenced the cell-cycle progression by affecting the G2 phase of the cell cycle. A proteomic analysis of the protein expression in hmNPC cultures 4 days after EPO treatment identified 8 proteins differentially expressed in EPO-treated cultures. It is likely that one or more of the identified proteins are involved in cellular pathways that promote cell proliferation and differentiation of hmNPC under normoxia. Their further characterization could provide cellular targets for the development of new therapeutic agents to treat CNS injury. Moreover, as EPO signaling is hypoxia-inducible, our findings may also indicate the beneficial effect of EPO to mimic hypoxia, while bypassing its negative effects, to culture human fetal midbrain-derived progenitor cells.

Neuroprotective effects of the beta-catenin stabilization in an oxygen- and glucose-deprived human neural progenitor cell culture system.

ß-Catenin stabilization achieved either via GSK-3ß specific inhibition or involving canonical Wnt signalling pathway, contributes to neuroprotection in an oxygen-glucose deprivation (4h OGD) in vitro hypoxia model performed on human cortical neural progenitor cells previously differentiated into neurons and glia. Neuroprotection mechanisms include both acquiring tolerance to injury throughout preconditioning (72 h prior to OGD) or being pro-survival during 24h reoxygenation after the insult. Four hours of OGD induced apoptotic cell death elevation to 73 ± 1% vs. 12% measured in control and the LDH level, indicative of necrotic cell injury, elevation by 67 ± 7% (set to 100%). A significant reduction in apoptosis occurred at 24h reoxygenation with indirubin supplement which was 49 ± 6% at 2.5 µM BIO while LDH level was only 47 ± 5% of OGD. Kenpaullone was efficient in reducing both cell deaths at 5 µM (apoptosis 38 ± 1% and necrosis 33 ± 3% less than in OGD). Wnt agonist reduced apoptosis to 45 ± 3% at 0.01 µM, while LDH value was decreased to a level of 53 ± 5% of control. Our findings suggest that GSK-3beta inhibitors/ß-catenin stabilizers may ultimately be useful drugs in neuroprotection and neuroregeneration therapies in vivo.

Genome-wide expression profiling and functional network analysis upon neuroectodermal conversion of human mesenchymal stem cells suggest HIF-1 and miR-124a as important regulators.

Tissue-specific stem cells, such as bone-marrow-derived human mesenchymal stem cells (hMSCs), are thought to be lineage restricted and therefore, could only be differentiated into cell types of the tissue of origin. Several recent studies however have suggested that these types of stem cells might be able to break barriers of germ layer commitment and differentiate in vitro into cells with neuroectodermal properties. We reported earlier about efficient conversion of adult hMSCs into a neural stem cell (NSC)-like population (hmNSCs, for human marrow-derived NSC-like cells) with all major properties of NSCs including functional neuronal differentiation capacity. Here we compared the transcriptomes from hMSCs and hmNSCs using a novel strategy by combining classic Affymetrix oligonucleotide microarray profiling with regulatory and protein interaction network analyses to shed light on regulatory protein networks involved in this neuroectodermal conversion process. We found differential regulation of extracellular matrix protein transcripts, up-regulation of distinct neuroectodermal and NSCs marker genes and local chromosomal transcriptional up-regulation at chromosome 4q13.3. In comparison to hMSCs and primary adult hippocampal NSCs, the transcriptome of hmNSCs displayed minor overlap with both other cell populations. Advanced bioinformatics of regulated genes upon neuroectodermal conversion identified transcription factor networks with HIF-1 and microRNA miR-124a as potential major regulators. Together, transgerminal neuroectodermal conversion of hMSCs into NSC-like cells is accompanied by extensive changes of their global gene expression profile, which might be controlled in part by transcription factor networks related to HIF-1 and miR-124a.

Rat embryonic liver cell expansion and differentiation on NH3 plasma-grafted PEEK-WC-PU membranes.

Biomaterials can potentially influence stem and progenitor cell proliferation and differentiation in both a positive and a negative way. Herein, we report on the expansion and differentiation of rat embryonic (E17) liver (RLC-18) cells on new bioactive membrane made of PEEK-WC-PU, whose surface was grafted with nitrogen functionalities by means of NH(3) glow discharges. The performance of the developed membrane was evaluated by analyzing the expression of the liver specific functions of cells cultured in a 6-well gas-permeable bioreactor. It was found that native and NH(3) plasma-grafted PEEK-WC-PU membranes enabled expansion of liver cells in the bioreactor. Liver embryonic cells on the membranes exhibited higher functional activities compared to those cultured on conventional culture dishes as demonstrated by higher albumin and urea production. They showed gene expression of alpha-fetoprotein and albumin in a time-dependent manner of the hepatic differentiation process. LDH assay and SEM analyses revealed that a high number of viable liver stem cells attached to the membranes. Unexpectedly, liver progenitors cultured on membranes had higher telomerase activity than ones in the plates, preventing cell senescence. Thus, membranes are able to sustain in vitro the same in vivo liver functions and to allow the expansion of progenitor cells.

Emerging role of LRRK2 in human neural progenitor cell cycle progression, survival and differentiation.

Despite a comprehensive mapping of the Parkinson's disease (PD)-related mRNA and protein leucine-rich repeat kinase 2 (LRRK2) in the mammalian brain, its physiological function in healthy individuals remains enigmatic. Based on its structural features and kinase properties, LRRK2 may interact with other proteins involved in signalling pathways. Here, we show a widespread LRRK2 mRNA and/or protein expression in expanded or differentiated human mesencephalic neural progenitor cells (hmNPCs) and in post-mortem substantia nigra PD patients. Using small interfering RNA duplexes targeting LRRK2 in hmNPCs following their differentiation into glia and neurons, we observed a reduced number of dopaminergic neurons due to apoptosis in LRRK2 knockdown samples. LRRK2-deficient hmNPCs exhibited elevated cell cycle- and cell death-related markers. In conclusion, a reduction of LRRK2 expression in hmNPCs severely impaired dopaminergic differentiation and/or survival of dopaminergic neurons most likely via preserving or reactivating the cell cycle.

Increase of intracellular Ca2+ by adenine and uracil nucleotides in human midbrain-derived neuronal progenitor cells.

Nucleotides play an important role in brain development and may exert their action via ligand-gated cationic channels or G protein-coupled receptors. Patch-clamp measurements indicated that in contrast to AMPA, ATP did not induce membrane currents in human midbrain derived neuronal progenitor cells (hmNPCs). Various nucleotide agonists concentration-dependently increased [Ca(2+)](i) as measured by the Fura-2 method, with the rank order of potency ATP>ADP>UTP>UDP. A Ca(2+)-free external medium moderately decreased, whereas a depletion of the intracellular Ca(2+) storage sites by cyclopiazonic acid markedly depressed the [Ca(2+)](i) transients induced by either ATP or UTP. Further, the P2Y(1) receptor antagonistic PPADS and MRS 2179, as well as the nucleotide catalyzing enzyme apyrase, allmost abolished the effects of these two nucleotides. However, the P2Y(1,2,12) antagonistic suramin only slightly blocked the action of ATP, but strongly inhibited that of UTP. In agreement with this finding, UTP evoked the release of ATP from hmNPCs in a suramin-, but not PPADS-sensitive manner. Immunocytochemistry indicated the co-localization of P2Y(1,2,4)-immunoreactivities (IR) with nestin-IR at these cells. In conclusion, UTP may induce the release of ATP from hmNPCs via P2Y(2) receptor-activation and thereby causes [Ca(2+)](i) transients by stimulating a P2Y(1)-like receptor.

Non-hypoxic stabilization of hypoxia-inducible factor alpha (HIF-alpha): relevance in neural progenitor/stem cells.

Hypoxia-inducible factor-1 (HIF-1) plays an important role in neural progenitor cell (NPC) propagation and dopaminergic differentiation. In the presence of oxygen and iron, hypoxia-inducible factor 1 alpha (HIF-1alpha) is rapidly degraded via the prolyl hydroxylase (PHD)/VHL pathway. In addition to hypoxia, various non-hypoxic stimuli can stabilize HIF-1alpha in NPCs and influence the transcription of HIF-regulated genes. Here, we investigate various hypoxia mimetics: deferoxamine (DFO), ciclopirox olamine (CPX), dimethyloxallyl glycine (DMOG), a novel HIF-PHD inhibitor (FG-4497) and cobalt chloride (CoCl(2)) with respect to their ability to enhance in vitro proliferation, neurogenesis and dopaminergic differentiation of human fetal mesencephalic NPCs (hmNPCs) in ambient oxygen (21%). Although able to stabilize HIF-1alpha, iron chelators (DFO and CPX) and DMOG were toxic to hmNPCs. CoCl(2) was beneficial only towards neuronal and dopaminergic differentiation, while FG-4497 enhanced proliferation, neurogenesis and dopaminergic differentiation of hmNPCs. Both CoCl(2) and FG-4497 were protective to human dopaminergic neurons. Finally, exposure to hyperbaric oxygen (HBO) also stabilized HIF-1alpha in hmNPCs and induced neurogenesis in vitro. These findings suggest that several HIF stabilizing agents or conditions can rescue impaired neurons and promote neurogenesis in vitro.

Non-viral gene transfer by nucleofection allows stable gene expression in human neural progenitor cells.

Human neural progenitor cells (hNPCs) are a promising source to treat various neurodegenerative diseases. Potential applications are to use such cells for reprogramming to induce pluripotent stem cells or for secretion of proteins into the brain. These applications usually involve expression of heterologously expressed genes which is difficult to achieve in hNPCs. We tested several protocols for non-viral gene transfer and different promoters. Nucleofection and the cytomegalovirus enhancer/chicken beta-actin promoter allowed expression of foreign genes in hNPCs for up to 6 months. Treatment with the antibiotic G418 enabled us to select stably transfected cells which were subcloned and continued to express the NPC marker nestin. Differentiation of stably nucleofected hNPCs revealed that multipotency was maintained following long-term expansion of subcloned hNPCs. After differentiation for 3 weeks in vitro or in vivo following striatal transplantations transfected hNPCs expressed voltage-gated sodium channels suggesting the development of functional properties during neuronal maturation. In conclusion, stably nucleofected hNPCs can be isolated, subcloned, and expanded for up to 6 months without loss of their differentiation potential. These data provide a basis for future studies using hNPCs to investigate the neuronal differentiation in vivo after transplantation, the feasibility as a vector for gene (protein) therapy, and the induction of pluripotent stem cells.

Labeling of human neural precursor cells using ferromagnetic nanoparticles.

Fetal human neural precursor cells (NPCs) are unique with respect to their capacity to proliferate and to preserve their potential to differentiate into neurons and glia. Human mesencephalic neural precursor cells (hmNPCs) provide a source for dopaminergic neurons. Preclinical and clinical research will benefit from reliable in vivo tracking of transplanted cells. Here, we investigate the potency of very small superparamagnetic iron oxide particles (VSOPs) to label hmNPCs, the effect of VSOPs on survival, proliferation, and differentiation of hmNPCs, and the sensitivity of 1.5T magnetic resonance imaging (MRI) to detect labeled cells in living rats following transplantation. When incubated with VSOPs at 1.5 mM, >95% of hmNPCs incorporated VSOPs without detectable impact on cell viability (>90%) or proliferative capacity, as measured by the expression of proliferating cell nuclear antigen (PCNA) and cell cycle distribution. Labeled hmNPCs differentiate into neurons (>30%) and glia with no detectable difference compared to nonlabeled cells. Following transplantation into rat striata, marked paramagnetic signal changes were detected for as long as three months postsurgery using MRI, corresponding to the histologically-identified graft. Our data indicate that hmNPCs can be labeled with VSOPs without impairment of viability, proliferation, or multipotency. Labeled, transplanted cells are detectable in vivo using 1.5T MRI.

Dopamine D2/D3 receptor stimulation fails to promote dopaminergic neurogenesis of murine and human midbrain-derived neural precursor cells in vitro.

The potential application of neural precursor cells (NPCs) in brain repair of neurodegenerative diseases has placed the factors capable of stimulating neurogenesis under increasing attention. Among these factors are dopamine (DA) D2/D3 receptor agonists, like 7-hydroxy-dipropylaminotetralin (7-OH-DPAT). The purpose of this investigation was to explore proliferating and neurostimulating effects of this drug in murine and human NPCs derived from the fetal midbrain. In both cell types, dopamine D2 and D3 receptors were detected by microarray data analysis and quantitative RT-PCR. Despite D2/D3 receptors expression, treatment with 7-OH-DPAT did not affect proliferation, survival, or neurogenesis of murine and human NPCs. Our data question the relevance of neuroregenerative effects of dopamine agonists for human predopaminergic cells as well as patients with Parkinson's disease.

Transcription profiling of adult and fetal human neuroprogenitors identifies divergent paths to maintain the neuroprogenitor cell state.

Global gene expression profiling was performed using RNA from adult human hippocampus-derived neuroprogenitor cells (NPCs) and multipotent frontal cortical fetal NPCs compared with adult human mesenchymal stem cells (hMSCs) as a multipotent adult stem cell control, and adult human hippocampal tissue, to define a gene expression pattern that is specific for human NPCs. The results were compared with data from various databases. Hierarchical cluster analysis of all neuroectodermal cell/tissue types revealed a strong relationship of adult hippocampal NPCs with various white matter tissues, whereas fetal NPCs strongly correlate with fetal brain tissue. However, adult and fetal NPCs share the expression of a variety of genes known to be related to signal transduction, cell metabolism and neuroectodermal tissue. In contrast, adult NPCs and hMSCs overlap in the expression of genes mainly involved in extracellular matrix biology. We present for the first time a detailed transcriptome analysis of human adult NPCs suggesting a relationship between hippocampal NPCs and white matter-derived precursor cells. We further provide a framework for standardized comparative gene expression analysis of human brain-derived NPCs with other stem cell populations or differentiated tissues. Disclosure of potential conflicts of interest is found at the end of this article.

Lack of hypoxia-inducible factor-1 alpha impairs midbrain neural precursor cells involving vascular endothelial growth factor signaling.

Oxygen tension is critical for proliferation of human and murine midbrain-derived neural precursor cells (mNPCs). Here, we conditionally inactivated the hypoxia-responsive transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha) in murine NPCs to determine its role in proliferation, survival, and dopaminergic differentiation in vitro as well as survival of murine dopaminergic neurons in vivo. HIF-1alpha conditional knock-out (HIF-1alpha CKO) mNPCs showed midbrain-specific impairment of survival and proliferation. Dopaminergic differentiation of HIF-1alpha CKO mNPCs in vitro was markedly reduced. Expression of vascular endothelial growth factor (VEGF) mRNA was reduced in HIF-1alpha CKO mNPCs, whereas erythropoietin signaling was not affected. Treatment of HIF-1alpha CKO mNPCs with 50 ng/ml VEGF partially recovered proliferation and dopaminergic differentiation in vitro. In substantia nigra (SN) of adult HIF-1alpha CKO mice, protein levels of dopaminergic marker molecules such as tyrosine hydroxylase (TH) and aldehyde dehydrogenase were reduced by 41 and 61%, respectively. The cell survival marker Bcl-2 was reduced by 58% while caspase-3 was activated. Nonbiased stereological cell counts of TH-positive neurons in SN of young adult HIF-1alpha CKO mice revealed a reduction of 31% compared with cre/wt mice (in which the wild-type Hif1a allele is expressed in parallel with the Cre recombinase allele). However, we found no impairment of striatal dopamine concentrations or locomotor behavior. In conclusion, HIF-1alpha seems to be a transcription factor relevant to the development and survival of substantia nigra dopaminergic neurons involving VEGF signaling.

Uracil nucleotides stimulate human neural precursor cell proliferation and dopaminergic differentiation: involvement of MEK/ERK signalling.

Isolation and propagation of neural stem cells derived from human brain tissue uniquely enables the study of human neurogenesis in vitro. In addition, ex vivo-expanded human neural stem/precursor cells (NPCs) may offer novel therapeutic strategies. We investigated the effects of extracellular nucleotides on the proliferation and differentiation of human mesencephalic neural stem/precursor cells (hmNPCs). When combined with the mitogens epidermal growth factor and fibroblast growth factor 2, UTP (1 microm) boosted proliferation of hmNPCs as shown by increased expression of the proliferation marker proliferating cell nuclear antigen (330%). UTP-induced proliferation was abrogated by the preferential P2Y receptor blocker pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). UTP also stimulated dopaminergic differentiation. Treatment with UTP (100 microm) increased the number of tyrosine hydroxylase (TH)-positive cells and TH protein by 267 and 319% respectively. UTP-stimulated dopaminergic differentiation of hmNPCs was blocked by the P2 receptor antagonists suramin (10 microm) and PPADS (100 microm). In addition, UDP (1 microm) enhanced TH protein expression by 194%. During differentiation, treatment with UTP stimulated the extracellular signal-regulated kinase (ERK) pathway. Both ERK1/2 phosphorylation and dopaminergic differentiation were inhibited by U0126, a selective ERK kinase inhibitor, as well as by suramin. When other P2 receptor agonists (ATP, ADP and adenosine 5'-O-(2-thiophosphate) (ADPbetaS); all 100 microm) were applied, both proliferation and dopaminergic differentiation of NPCs were compromised. We conclude that uracil nucleotides exert specific P2 receptor-mediated effects on midbrain-derived human NPCs, and may be used to enhance both proliferation and dopaminergic differentiation.

Low extracellular calcium is sufficient for survival and proliferation of murine mesencephalic neural precursor cells.

Various media and Ca2+ concentrations are employed to culture neural progenitor cells (NPCs). We have therefore explored the effects of extracellular calcium concentrations on the survival, proliferation, spontaneous apoptosis and self-renewal capacity of mesencephalic NPCs grown adherently and as free-floating neurospheres. We employed EMEM supplemented with various concentrations of extracellular CaCl2 (0.1-1 mM). Raising the calcium concentration from 0.1 mM to 0.6 mM resulted in an increased number of NPCs growing as a monolayer and increased the protein yield of cells growing in neurospheres (24+/-3 microg total proteins in 0.1 mM Ca2+ medium vs. 316+/-34 microg proteins in 1 mM Ca2+ medium). Concentrations more than 0.6 mM did not result in a further improvement of proliferation or survival. Elimination of calcium from our control medium by 1 mM EGTA resulted in a decrease in cell number from 82+/-2 x 10(4) NPCs/ml observed in control medium to 62+/-2 x 10(4) NPCs/ml observed in calcium-free media. Protein yield dropped significantly in calcium-free media, accompanied by the decreased expression of the proliferation marker PCNA and the pro-survival marker Bcl-2. Two weeks of expansion as neurospheres caused spontaneous cell death in more than 90% of NPCs grown in 0.1 mM CaCl2 EMEM compared with 42% in 1 mM CaCl2 EMEM. Although the action of Ca2+ on NPCs appears to be complex, the presented data strongly suggest that extracellular calcium plays a crucial role in the maintenance of NPCs in a healthy and proliferating state; physiological concentrations (>1.0 mM) are not required, a concentration of 0.5 mM being adequate for cell maintenance.

Cryopreservation does not affect proliferation and multipotency of murine neural precursor cells.

Stem cell research offers unique opportunities for developing new medical therapies for devastating diseases and a new way to explore fundamental questions of biology. Establishing an efficient freezing protocol for neural precursor cells (NPCs) is of great importance for advances in cell-based therapies. We used fluorescence-activated cell sorter-based cell death/survival analysis and Western blot analysis of proliferation markers (proliferating cell nuclear antigen) and prosurvival proteins (Bcl-2) to study the effect of a variety of cryoprotective agents on fetal mouse forebrain NPCs. Neurospheres frozen at -70 degrees C or in liquid nitrogen in a rate-controlled manner and thawed after 5 days retained viability of 60%-70% measured 24 hours after thawing. However, 1 week after thawing, viability dropped to 50%-60%. Using a clonogenic sphere formation assay, we showed that recovery rate of frozen NPCs was approximately 26% and did not significantly differ between dimethyl sulfoxide (DMSO)- and glycerol-supplemented samples. Application of the caspase inhibitor zVAD-fmk during freezing or in the first week after thawing resulted in protection of cryopreserved neurospheres after thawing but not during the freezing process, indicating that apoptosis limits recovery of NPCs. Cell survival was not reduced in cells that were enzymatically separated before cryopreservation. Optimal protection of NPCs was achieved when 10% DMSO alone or in a combination with 10% fetal calf serum (FCS) was used. However, 10% glycerol alone was equally effective. Using these protocols, NPCs retained their multipotency and differentiated into both glial (GFAP-positive) and neuronal (Tuj1-positive) cells. Percentage of Tuj1-positive cells in 5% and 10% DMSO, in 10% DMSO + 10% FCS, and in 10% glycerol remained at the same level as before freezing and varied from 5%-7%. We conclude that cryopreservation (up to 1 month at -70 degrees C and up to 1 year in liquid nitrogen) does not markedly alter the rate of proliferation and multipotency of murine neural precursor cells.

Low atmospheric oxygen avoids maturation, senescence and cell death of murine mesencephalic neural precursors.

The efficient generation of specific brain cells in vitro may serve as a source of cells for brain repair in several devastating neurological diseases. Production of dopaminergic neurons from precursor cells for transplantation in Parkinson's disease has become a major research goal. We found that murine mesencephalic neurospheres were viable and proliferated, preserved telomerase activity, pluripotency and dopaminergic commitment for many weeks when cultured in 3% O2, whereas exposing these cells to 21% oxygen prohibited long-term expansion. Microarray data suggest that a variety of genes related to the cell cycle, cell maturation and apoptosis are differentially regulated in midbrain-derived precursors cultured in 3 versus 21% oxygen after 1-2 months. Taken together, we hypothesize that sustained high oxygen has deleterious effects on the self-renewal capacity of mesencephalic neural precursors, possibly accelerating maturation and senescence resulting in overall cell loss. Gene regulation governed by low oxygen tension may be relevant to the normal development and survival of midbrain neurons.