Olfactory Neuroepithelial Neural Progenitor Cells from Subjects with Bipolar I Disorder.

BACKGROUND: Research into the pathophysiology of bipolar disorder (BD) is limited by the inability to examine brain cellular processes in subjects with the illness. METHODS: Endoscopic biopsy was performed in subjects with bipolar I disorder to establish olfactory neural progenitor (ONP) cell lines. Olfactory function was assessed prebiopsy and postbiopsy using the University of Pennsylvania Smell Identification Test (UPSIT). Cells were characterized to determine their lineage. RESULTS: There were no significant complications associated with the biopsy procedure, including olfaction. Outpatient olfactory neuroepithelial biopsy yielded ONP cells in three out of 13 biopsy attempts (23.1%). ONPs were positive for neuron-specific proteins (ß-tubulin III, nestin, hexaribonucleotide binding protein-3, and peripherin) and glia-specific proteins (glial fibrillary acidic protein and myelin basic protein). CONCLUSIONS: ONP cells can be obtained safely from awake outpatients and are potentially useful for pathophysiological studies of bipolar illness and perhaps other neuropsychiatric conditions. Such cells allow for the investigation of potential pathological cellular processes without the confounding factors of genetic manipulation, which is required for induced pluripotent cells.

Adult human olfactory epithelial-derived progenitors: a potential autologous source for cell-based treatment for Parkinson's disease.

Human adult olfactory epithelial-derived neural progenitors (hONPs) can differentiate along several neural lineages in response to morphogenic signals in vitro. A previous study optimized the transfection paradigm for the differentiation of hONPs to dopaminergic neurons. This study engrafted cells modified by the most efficient transfection paradigm for dopaminergic neural restriction and pretransfected controls into a unilateral neurotoxin, 6-hydroxydopamine-induced parkinsonian rat model. Approximately 35% of the animals engrafted with hONPs had improved behavioral recovery as demonstrated by the amphetamine-induced rotation test, as well as a corner preference and cylinder paw preference, over a period of 24 weeks. The pre- and post-transfected groups produced equivalent responses, indicating that the toxic host environment supported hONP dopaminergic differentiation in situ. Human fibroblasts used as a cellular control did not diminish the parkinsonian rotational deficits at any point during the study. Increased numbers of tyrosine hydroxylase (TH)-positive cells were detected in the engrafted brains compared with the fibroblast-implanted and medium-only controls. Engrafted TH-positive hONPs were detected for a minimum of 6 months in vivo; they were multipolar, had long processes, and migrated beyond their initial injection sites. Higher dopamine levels were detected in the striatum of behaviorally improved animals than in equivalent regions of their nonrecovered counterparts. Throughout these experiments, no evidence of tumorigenicity was observed. These results support our hypothesis that human adult olfactory epithelial-derived progenitors represent a unique autologous cell type with promising potential for future use in a cell-based therapy for patients with Parkinson's disease.

Human adult olfactory neural progenitors promote axotomized rubrospinal tract axonal reinnervation and locomotor recovery.

We investigated the effects of engrafted human adult olfactory neuroepithelial neurosphere forming cells (NSFCs) on regeneration and reinnervation of rubrospinal tract (RST) axons and locomotor recovery following partial cervical hemisection that completely ablated the RST. Weekly behavioral testing showed greater functional recovery of forelimb use during the 12 weeks after NSFCs engraftment than in the control rats. Anterograde tracing with biotinylated dextran amine (BDA) confirmed the presence of RST axons within the white matter 4-8 segments caudal to the grafts. Both immunofluorescence and immunoelectron microscopy revealed the BDA-labeled RST axonal terminals reestablished synaptic connections with motoneurons in the ventral horn of the distal cervical spinal cord. Further study of forelimb functional recovery in NSFCs-engrafted subgroups considered the effects of a second dorsolateral funiculotomy, irreversibly destroying the recovery, and the injection of muscimol, blocking RST neuronal activity reversibly. These results highlight the unique potential of human olfactory neuroepithelial-derived progenitors as an autologous cell source for spinal cord repair.

Human adult olfactory neural progenitors rescue axotomized rodent rubrospinal neurons and promote functional recovery.

Previously, our lab reported the isolation of patient-specific neurosphere-forming progenitor lines from human adult olfactory epithelium from cadavers as well as patients undergoing nasal sinus surgery. RT-PCR and ELISA demonstrated that the neurosphere-forming cells (NSFCs) produced BDNF. Since rubrospinal tract (RST) neurons have been shown to respond to exogenous BNDF, it was hypothesized that if the NSFCs remained viable following engraftment into traumatized spinal cord, they would rescue axotomized RS neurons from retrograde cell atrophy and promote functional recovery. One week after a partial cervical hemisection, GFP-labeled NSFCs suspended in Matrigel matrix or Matrigel matrix alone was injected into the lesion site. GFP-labeled cells survived up to 12 weeks in the lesion cavity or migrated within the ipsilateral white matter; the apparent number and mean somal area of fluorogold (FG)-labeled axotomized RST neurons were greater in the NSFC-engrafted rats than in lesion controls. Twelve weeks after engraftment, retrograde tracing with FG revealed that some RST neurons regenerated axons 4-5 segments caudal to the engraftment site; anterograde tracing with biotinylated dextran amine confirmed regeneration of RST axons through the transplants within the white matter for 3-6 segments caudal to the grafts. A few RST axons terminated in gray matter close to motoneurons. Matrix alone did not elicit regeneration. Behavioral analysis revealed that NSFC-engrafted rats displayed better performance during spontaneous vertical exploration and horizontal rope walking than lesion Matrigel only controls 11 weeks post transplantation. These results emphasize the unique potential of human olfactory neuroepithelial-derived progenitors as an autologous source of stem cells for spinal cord repair.

Human adult olfactory neuroepithelial derived progenitors retain telomerase activity and lack apoptotic activity.

Olfactory epithelium (OE) contains a population of progenitors responsible for its life-long regenerative capacity. Procedures for the isolation of these progenitors have been established [F.J. Roisen, K.M. Klueber, C.L. Lu, L.M. Hatcher, A. Dozier, C.B. Shields, Adult human olfactory stem cells, Brain Res., 890 (2001) 11-12.] and over 40 patient-specific cell lines from adult postmortem OE and endoscopic biopsy from patients undergoing nasal sinus surgery have been obtained. As these cells emerged in primary cultures, they formed neurospheres (NSFCs). The purpose of the present study was to further characterize these adult human olfactory-derived progenitors. Subcultures of the NSFCs have been passaged nearly 200 times, with a mitotic cycle of 18-20 h. Telomerase activity remains in stem cells; therefore, ELISA was employed to determine the telomerase activity of different lines and passages. Since progenitors undergo low levels of apoptosis, the levels of apoptosis were also examined in these populations. The levels of telomerase and apoptotic activity in 12 NSFC lines remained relatively constant irrespective of donor age, culture duration, or sex. To further study the apoptotic characteristics of the NSFCs, nine different caspases (cysteine proteases) known to be critical in apoptosis were evaluated using gene-microarrays comparing cells from a single line at passages 14, 88, and 183. No increases were found in caspase activity in all passages studied. ELISA confirmed the absence of caspase activity over the entire range of passages. This study further suggests that NSFCs can be obtained and used from patients, irrespective of age, sex, or time in culture without altered viability expanding the potential utility of these cells for autologous transplantation and possible diagnostic testing.

Induction of oligodendrocytes from adult human olfactory epithelial-derived progenitors by transcription factors.

Neurosphere-forming cell (NSFC) lines have been derived from cultures of adult olfactory neuroepithelium obtained from patients and cadavers. These progenitors remain undifferentiated when maintained in minimal essential medium with 10% fetal bovine serum, but have the potential to differentiate along glial or neuronal lineages. However, few of these cells ever express mature neuronal or glial markers in defined medium. To evaluate the potential of NSFCs to form oligodendrocytes, two transcription factors, Olig2 and Nkx2.2, were introduced into NSFCs to determine whether their expression is sufficient for oligodendrocyte differentiation, as has been shown in the embryonic avian and murine central nervous systems in vivo. NSFCs transfected with Olig2 or Nkx2.2 alone exhibited no phenotypic lineage restriction. In contrast, simultaneous transfection of Olig2 and Nkx2.2 cDNA produced characteristic oligodendrocyte morphology and antigenicity, including myelin basic protein (MBP). Furthermore, a population of Olig2-expressing NSFCs also expressed Sox10. Cotransfection of NSFCs with Nkx2.2 and Sox10, but not Olig2 and Sox10, produced a MBP(+) oligodendrocytic phenotype. Coculture of NSFCs transfected with Olig2 and Nkx2.2 or Nkx2.2 and Sox10 with purified sensory neurons, demonstrated frequent contacts between NSFC processes and axons, including the early stages of ensheathment. These studies demonstrate transcription factors governing early development of chick and mouse oligodendrocyte formation, also apply to human progenitors isolated from adult olfactory neuroepithelium. Our long-term goal is to develop cell populations for future studies used to determine the therapeutic utility of these olfactory-derived NSFCs for autologous transplantation into donors with central nervous system trauma or neurodegenerative diseases.

Adult human olfactory neural progenitors cultured in defined medium.

Neurosphere-forming cells (NSFCs) derived from primary cultures of adult human olfactory epithelium were established in minimum essential medium (MEM) with Hanks balanced salts and 10% heat-inactivated fetal bovine serum (FBS). A totally defined medium (DM) was employed to examine their proliferation, lineage restriction and differentiation. DMEM/F12 (DF) was found to support NSFCs and served as the base medium for this study. NSFCs were adapted to the DM through serial serum reductions at successive feedings. NSFCs in DF supplemented with N2, B27 or insulin attained saturation density and formed extensive processes. Immunolocalization of lineage specific markers [i.e., nestin, beta-tubulin III, peripherin, neural cell adhesion molecule, A2B5, O4, microtubule-associated-protein-2 (MAP2) and glial fibrillary acidic protein], as well as 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and ornithine decarboxylase assays were employed to characterize the NSFCs. The effects of trophic factors including epidermal growth factor (EGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophic factors (NT-3), and basic fibroblast growth factor (bFGF) were evaluated. With the reduction of serum and the addition N2, B27, and other nutrients, there was a change in lineage restriction including an increase the expression of A2B5 and other glial markers as well as the expression of mature neuronal markers with a simultaneous reduction of nestin reactivity. NSFCs proliferated and maintained their pluripotency for over a year in the DM. Further studies will determine the utility of NSFCs for cell replacement therapy.

Neurotrophins regulate proliferation and survival of two microglial cell lines in vitro.

Microglia are thought to play a key role in the development and regeneration of the central nervous system although the mechanisms regulating their presence and activity are not fully understood. Substantial evidence suggests that members of the neurotrophin family such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 and -4 (NT-3/4) have a dramatic effect on both neurons and perineuronal cells. This study employed two murine microglial lines, BV-2 and N9, to examine the action of these neurotrophins on the mitotic activity and survival of microglia in vitro. Neurotrophins were incorporated into the media at the time of plating and cell number and levels of mitochondrial dehydrogenase activity (MTT) were determined at various time points in vitro. NGF increased cell number and MTT levels of both cell lines in a dose-dependent manner. BV-2 was more sensitive to NGF than N9. Similar responses were elicited by BDNF, although the sensitivity of each cell line was different than that found for NGF. NT-3 and NT-4 had no effect on cell proliferation. However, NT-4 had an effect on the survival of BV-2 and N9 cells. The response of these cells to neurotrophins was blocked by K252a, a tyrosine kinase inhibitor, suggesting that actions of neurotrophins were mediated by high-affinity tyrosine kinase receptors (Trk). Immunolocalization studies revealed positive Trk (pan) reactivity in the above cell lines and in primary microglia, but an absence of the low-affinity p75 neurotrophin receptor. Western blot analysis supported the above observations. These studies suggest that in addition to their neurotrophic actions, NGF and BDNF may also regulate microglial dynamics, thereby influencing the surrounding milieu during neuronal regeneration.