Kathleen M. Klueber

Adult human olfactory stem cells.

The location of stem cells within the adult CNS makes them impractical for surgical removal and autologous transplantation. Their limited availability and histocompatibility issues further restrict their use. In contrast, olfactory neuroepithelium (ONe) located in the nasal passageways has a continuous regenerative capability and can be biopsied readily. To investigate the potential of human ONe to provide viable populations of pluripotent cells, ONe was harvested from cadavers 6-18 h postmortem, dissociated, plated and fed every 3-4 days. Heterogeneous populations of neurons, glia, and epithelia were identified with lineage-specific markers. After several weeks, 5-10% of the cultures produced a population of rapidly dividing cells, which in turn, produced neurospheres containing at least two subpopulations based on neuronal and glial specific antigens. Most contained one or more neuronal markers; a few were positive for A2B5 and/or GFAP. To determine if growth modulators would affect the neurosphere forming cells, they were exposed to dibutyryl-cAMP. The nucleotide reduced cell division and increased process formation. Although the cells had been passaged more than 70 times, their viability remained constant as shown by the MTT viability index. Donor age or sex were not limiting factors, because neurospheres have been established from cadavers of both sexes from 50 to 95 years old at time of death. The ex vivo expansion of these cells will provide a patient-specific population of cells for immunological, genetic and pharmacological evaluation. Our long-term goal is to determine the utility of these cells to facilitate CNS repair.

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.

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.

Endoscopic biopsy of human olfactory epithelium as a source of progenitor cells.

Background The adult central nervous system contains progenitor cells; however, invasive surgery is required for their harvest. Olfactory neuroepithelium (ONe) has attracted attention because it is extracranial and contains progenitor cells that account for its regenerative capacity. Olfactory progenitor cells have been cultured from postmortem ONe. Our aim was to determine if olfactory progenitors could be obtained via biopsy from patients in a feasible, effective, and safe manner. Methods Endoscopic biopsy was performed on individuals undergoing sinus surgery (n = 42). Olfactory function was assessed pre- and postoperatively. Specimens were cultured under conditions for olfactory progenitor cell development. Results Progenitor cells emerged in cultures from 50% of our patients. The superior turbinate, biopsied with cutting punch forceps, gave the highest yield. No adverse impact on olfaction or complications with the biopsy were observed. Conclusion Endoscopic biopsy of ONe for obtaining olfactory progenitor cells from living donors is feasible, effective, and safe.

Clonal analysis of adult human olfactory neurosphere forming cells

Olfactory neuroepithelium (ONe) is unique because it contains progenitor cells capable of mitotic division that replace damaged or lost neurons throughout life. We isolated populations of ONe progenitors from adult cadavers and patients undergoing nasal sinus surgery that were heterogeneous and consisted of neuronal and glial progenitors. Progenitor lines have been obtained from these cultures that continue to divide and form nestin positive neurospheres. In the present study, we used clonal and population analyses to probe the self-renewal and multipotency of the neurosphere forming cells (NSFCs). NSFCs plated at the single cell level produced additional neurospheres; dissociation of these spheres resulted in mitotically active cells that continued to divide and produce spheres as long as they were subcultured. The mitotic activity of clonal NSFCs was assessed using bromodeoxyuridine (BrdU) incorporation. Lineage restriction of the clonal cultures was determined using a variety of antibodies that were characteristic of different levels of neuronal commitment: β-tubulin isotype III, neural cell adhesion molecule (NCAM) and microtubule associated protein (MAP2), or glial restriction: astrocytes, glial fibrillary acidic protein (GFAP); and oligodendrocytes, galactocerebroside (GalC). Furthermore, nestin expression, a marker indicative of progenitor nature, decreased in defined medium compared to serum-containing medium. Therefore, adult human ONe-derived neural progenitors retain their capacity for self-renewal, can be clonally expanded, and offer multipotent lineage restriction. Therefore, they are a unique source of progenitors for future cell replacement strategies in the treatment of neurotrauma and neurodegenerative diseases.

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.

Role of Transcription Factors in Motoneuron Differentiation of Adult Human Olfactory Neuroepithelial‐Derived Progenitors

Neurosphereforming cell (NSFC) lines have been established from cultures of human adult olfactory neuroepithelium. Few of these cells ever express mature neuronal or glial markers in minimal essential medium supplemented with 10% fetal bovine serum or defined medium. However, these neural progenitors have the potential to differentiate along glial or neuronal lineages. To evaluate the potential of NSFCs to form motoneurons, transcription factors Olig2, Ngn2, and HB9 were introduced into NSFCs to determine if their expression is sufficient for motoneuron specification and differentiation, as has been shown in the early development of the avian and murine central nervous systems in vivo. NSFCs transfected with Olig2, Ngn2, and HB9 alone exhibited no phenotypic lineage restriction. In contrast, simultaneous transfection of Ngn2 and HB9 cDNA increased the expression of Isl1/2, a motoneuron marker, when the cells were maintained in medium supplemented with retinoic acid, forskolin, and sonic hedgehog. Furthermore, a population of Olig2‐expressing NSFCs also expressed Ngn2. Cotransfection of NSFCs with Olig2 and HB9, but not Olig2 and Ngn2, increased Isl1/2 expression. Coculture of NSFCs trans‐fected with Ngn2‐HB92 or Olig2 and HB9 with purified chicken skeletal muscle demonstrated frequent contacts that resembled neuromuscular junctions. These studies demonstrate that transcription factors governing the early development of chick and mouse motoneuron formation are able to drive human adult olfactory neuroepithelial progenitors to differentiate into motoneurons in vitro. Our long‐term goal is to develop cell populations for future studies of the therapeutic utility of these olfactory‐derived NSFCs for autologous cell replacement strategies for central nervous system trauma and neurodegenerative diseases.

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.

Identification and culture of olfactory neural progenitors from GFP mice

The olfactory epithelium (OE) is one of the best sources for obtaining adult stem cells from the nervous system, because it contains neural progenitors that regenerate continuously throughout life. The OE is accessible through the nasal cavity, which facilitates stem cell harvest for examination and transplantation. The mitotic activity of OE progenitors can be stimulated by intranasal irrigation with zinc sulfate (ZnSO4). In the study reported here, we focused on OE from a transgenic mouse line transfected with green fluorescent protein (GFP). Histological examination demonstrated the site of highest yield of OE in the transgenic and wild type littermates. Cultures were established from that site four days in vitro following ZnSO4 exposure. The GFP-derived primary cultures contained a heterogeneous population of fluorescent cells. After 10-12 days, a population of round, mitotically active cells emerged that formed fluorescent neurospheres. The neurosphere forming cells (NSFCs) were collected and subcultured up to four times. The NSFCs were primarily neuronal with only a few cells of glial lineage. Furthermore, the NSFCs were nestin positive and keratin negative, suggesting that they were neural progenitors. The endogenous GFP fluorescence of these cells provides a readily identifiable label that will facilitate their identification following transplantation into nontransfected hosts. They should provide a useful model for evaluating the potential therapeutic utility of OE progenitors in neurodegenerative diseases and neurotrauma repair.