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.     

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: ss-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.     

Immunomagnetic separation of adult human olfactory neural progenitors

Olfactory neuroepithelium (ONe) has lifelong regenerative capacity owing to the presence of mitotically active progenitors. The accessibility of ONe makes it a unique source of progenitors for cell replacement strategies in the CNS. We have established lines of neurosphere forming cells (NSFCs) from adult postmortem ONe and patients undergoing nasal sinus surgery by endoscopic biopsy. These heterogeneous lines are composed primarily of an immature neuronally restricted and a small glial restricted subpopulation. More homogeneous subpopulations of the NSFCs are essential for detailed study of factors influencing their lineage restriction. Immunomagnetic bead separation using an antibody against tyrosine kinase (Trk) receptors (Trk-pan, which recognizes Trk-A, B, C) resulted in viable, enriched positive and negative subpopulations that could be analyzed immunocytochemically. The positive cells remained positive for the first week after which the number of Trk-pan expressing cells decreased. The negative subpopulation began to express Trk-pan immunoreactivity after five days in vitro. Both subpopulations reverted to the heterogeneous composition after two weeks. Furthermore, most NSFCs were positive for Trk-B, a few for Trk-A, while no reactivity was observed for Trk-C. Because NSFCs produce brain derived neurotrophic factor (BDNF) and express Trk B, the specific receptor for BDNF, it is likely that population dynamics are under a paracrine and/or autocrine regulatory mechanism. Lineage restriction analysis demonstrated that the isolated subpopulation had a restriction potential equivalent to the original heterogeneous population. These studies characterize further the NSFCs and support the future potential therapeutic use of ONe-derived progenitors for CNS injury and neurodegenerative disorders.     

Differentiation of human olfactory system-derived stem cells into dopaminergic neuron-like cells: A comparison between olfactory bulb and mucosa as two sources of stem cells

Cell transplantation has become a possible therapeutic approach in the treatment of neurodegenerative diseases of the nervous system by replacing lost cells. The current study aimed to make a comparison between the differentiation capacity of the olfactory bulb neural stem cells (OB-NSCs) and olfactory ectomesenchymal stem cells (OE-MSCs) into dopaminergic-like neurons under the inductive effect of transforming growth factor β (TGF-β). After culturing and treating with TGF-β, the differentiation capacities of both types of stem cells into dopaminergic neuron-like cells were evaluated. Quantitative real-time polymerase chain reaction analysis 3 weeks after induction demonstrated that the mRNA expression of the dopaminergic activity markers tyrosine hydroxylase (TH), dopamine transporter (DAT), paired box gene 2 (PAX2), and PAX5 in the neuron-like cells derived from OB-NSCs was significantly higher than those derived from OE-MSCs. These findings were further supported by the immunocytochemistry staining showing that the expression of the tyrosine hydroxylase, DAT, PAX2, and paired like homeodomain 3 seemed to be slightly higher in OB-NSCs compared with OE-MSCs. Despite the lower differentiation capacity of OE-MSCs, other considerations such as a noninvasive and easier harvesting process, faster proliferation attributes, longer life span, autologous transplantability, and also the easier and inexpensive cultural process of the OE-MSCs, cumulatively make these cells the more appropriate alternative in the case of autologous transplantation during the treatment process of neurodegenerative disorders like Parkinson's disease.     

Clonal growth of normal human uroepithelial cells

Summary We report the development of culture conditions which routinely support clonal growth of normal human uroepithelial cells (HUC). Secondary cultures seeded at clonal densities and grown under conditions described herein have a colony-forming efficiency (CFE) and colony size that will be useful for in vitro experiments. Primary cultures were dispersed to single cells and seeded in a supplemented Ham's F12 medium containing 1% fetal bovine serum together with 3×10⁵ lethally irradiated Swiss 3T3 feeder cells on plastic substrates preequilibrated with F12 medium containing 5 or 10% serum. Using these conditions, the average CFE was 16.1±2.5%. A cloning efficiency of 4.9±1.5% was obtained under the same conditions in serum-free F12+ when supplemented with a mixture of trace elements or 0.1 mM ethanolamine. The epithelial nature of the cloned cells was confirmed by morphology and by positive immunofluorescent staining for human epithelial keratin proteins. To make this system useful for mutagenesis experiments, a clone of Swiss 3T3 feeder cells resistant to 5 μg/ml 6-thioguanine (6TG) was derived from the parental cell line. This 6-TG-resistant Swiss 3T3 clone supports HUC clonal growth with a CFE of 17.9±2.0% CFE. We also report clonal growth of HUC without feeder cells using supplemented MCDB 170 medium containing 70 μg/ml bovine pituitary extract. The average cloning efficiency using these conditions was 5.7±1.7%. This work was supported by NIH grant 29525 to C. A. R. L. J. L. is a recipient of National Science Foundation predoctoral fellowship.     

Clonal growth characteristics of adult human prostatic epithelial cells

Summary The ability of human epithelial cells derived from adult prostatic tissues to undergo clonal growth in culture was examined. In a previously described serum-free culture system, such cells exhibited a density-dependent growth requirement. It was found that raising the level of one of the constituents of the culture medium, bovine pituitary extract, to 100 μg/ml permitted excellent clonal growth when as few as 100 cells were inoculated/60-mm² dish. Raising the levels of supplements other than pituitary extract (cholera toxin, epidermal growth factor, hydrocortisone, or insulin) did not produce this result. The average colony-forming efficiency of cells derived from primary or early passage cultures was approximately 25%. When single cell suspensions were prepared from tissue isolates and directly analyzed for clonal growth, colony-forming efficiencies were approximately 5%, perhaps indicating the proportion of stem cells with proliferative potential in the original isolates. The colony-forming efficiency of a cell population derived from cancer tissue was not significantly different from those of populations derived from normal tissues.     

Y1 receptors are critical for the proliferation of adult mouse precursor cells in the olfactory neuroepithelium

While the regenerative capacity of the olfactory neuroepithelium has been well studied less is known about the molecular events controlling precursor cell activity. Neuropeptide Y (NPY) is expressed at high levels in the olfactory system, and NPY has been shown to play a role in neuroregeneration of the brain. In this study, we show that the numbers of olfactory neurospheres derived from NPY, NPY/peptide YY, and Y1 receptor knockout mice are decreased compared with wild type (WT) controls. Furthermore, flow cytometric analysis of isolated horizontal basal cells, globose basal cells, and glandular cells showed that only glandular cells derived from WT mice, but not from NPY and Y1 receptor knockout mice, formed secondary neurospheres suggesting a critical role for NPY signaling in this process. Interestingly, olfactory function tests revealed that olfaction in Y1 knockout mice is impaired compared with those of WT mice, probably because of the reduced number of olfactory neurons formed. Together these results indicate that NPY and the Y1 receptor are required for the normal proliferation of adult olfactory precursors and olfactory function.     

Strengths and limitations of the neurosphere culture system

After the initial reports of free-floating cultures of neural stem cells termed neurospheres (1,2), a wide array of studies using this promising culture system emerged. In theory, this was a nearperfect system for large-scale production of neural cells for use in cell replacement therapies and to assay for and characterize neural stem cells. More than a decade later, after rigorous scrutiny and ample experimental testing of the neurosphere culture system, it has become apparent that the culture system suffers from several disadvantages, and its usefulness is limited for several applications. Nevertheless, the bulk of high-quality research produced over the last decade has also shown that under the right circumstances and for the appropriate purposes, neurospheres hold up to their initial promise. This article discusses the pros and cons of the neurosphere culture system regarding its three major applications: as an assay for neural stem cells, as a model system for neurogenesis and neural development, and for expansion of neural stem cells for transplantation purposes.     

Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.     

Inducible lineage tracing of Pax7‐descendant cells reveals embryonic origin of adult satellite cells

Descendants of E9.5 Pax7‐expressing (in red, β‐gal+) central dermomyotome cells contribute to myofibers (in green, MF‐20+, a myosin heavy chain protein) in the E16.5 mouse embryo. Nuclei are blue (DAPI). The β‐gal+/MF‐20_ cells that are intermingled with myofibers are likely myogenic progenitors. See the article by Lepper and Fan in this issue.     

A novel cell line isolated from the murine olfactory mucosa

Summary A clonal cell line (designated MOE CL1) was isolated from the olfactory mucosa of the mouse. A number of growth and biochemical parameters were studied. Our experiments suggest that MOE CL1 cells are olfactory basal cells (undifferentiated neuroblasts). A portion of this research was supported by USPHS Research Grant AM-18932.     

基于小分子筛选的嗅上皮类器官培养体系的建立

嗅上皮接收和传导气味信号是嗅觉系统的重要组成部分。嗅上皮的损伤在通常情况下可自发恢复,但特定疾病或衰老造成的嗅上皮损伤会引起嗅觉功能减退和嗅觉障碍。嗅上皮主要由基底细胞、支持细胞以及嗅感觉神经元组成。为了在体外建立包含多种细胞类型的嗅上皮类器官,本研究采用3D细胞培养技术,通过筛选小分子药物,构建了包含多种细胞类型的嗅上皮类器官模型,包含水平基底样细胞、球形基底样细胞、支持样细胞和嗅感觉神经元样细胞多种细胞类型。类器官培养体系中多种生长因子和小分子化合物在细胞增殖速度、细胞组成以及不同细胞类型标志基因的表达水平等方面对类器官产生影响。Wnt信号通路激活剂CHIR-99021能够提高嗅上皮类器官的成克隆率和增殖速度且有利于提高嗅上皮类器官中嗅感觉神经元样细胞标志基因的表达水平;培养体系的任一因子均能提高类器官中cKit阳性的球形基底样细胞克隆比例;表皮生长因子(epidermal growth factor,EGF)和维生素C均有利于类器官中水平基底样细胞标志基因的表达。本研究建立的嗅上皮类器官系统模拟了嗅上皮干细胞分化产生多种嗅上皮细胞类型的过程,为研究嗅上皮组织损伤再生、嗅觉障碍病理...     

A new culturing strategy improves functional neuronal development of human neural progenitor cells

Cell replacement therapies that rely on in vitro differentiation of human neural progenitor cells are a promising strategy to compensate the progressive cell loss in neurodegenerative disorders like Parkinson's disease. We and others observed, that the functional differentiation of progenitors in standard differentiation medium remains limited. The aim of the present study was to optimize neuronal in vitro differentiation by mimicking the physiological shift from depolarizing to hyperpolarizing conditions that occurs during early brain development. Differentiation was initiated using a depolarizing high potassium- and low sodium-containing medium. Subsequently, the high potassium-containing medium was replaced by a hyperpolarizing medium containing low potassium and high sodium concentrations. This two-phase strategy significantly promoted the expression of neuronal markers, enhanced neurite growth, enlarged sodium inward currents, and increased action potential firing. Thus, depolarizing followed by hyperpolarizing culture conditions enable developing human neural progenitor cells to adopt more mature functional qualities.     

Clonal expansion of human pluripotent stem cells on gelatin-coated surface

The research of human pluripotent stem cells is important for providing the molecular basis for their future application to regenerative medicine. To date, they are usually cultured on feeder cells and passaged by partial dissociation with either enzymatic or mechanical methods, which are problematic for the research using them in the convenience and reproducibility. Here we established a new culture system that allows handling as easily as culturing feeder-free mouse ES cells. This newly developed culture system is based on the combinatorial use of ROCK inhibitor and soluble fibronectin, which enables us to expand human pluripotent stem cells from single cell dissociation on gelatin-coated surface without any feeder cells. In this new culture system, these human pluripotent stem cells can stably grow, even if in clonal density with keeping expression of stem cell markers. These cells also have abilities to differentiate into three germ layers in vivo and in vitro. Furthermore, no chromosomal abnormalities are found even after sequential passage. Therefore this system will dramatically simplify genetic engineering of these human pluripotent stem cells or defining process of their signal pathway.     

Efficient non-viral transfection of adult neural stem/progenitor cells, without affecting viability, proliferation or differentiation

BACKGROUND: Neurogenesis occurs in defined areas of the adult mammalian brain, including the dentate gyrus of the hippocampus. Rat neural stem/progenitor cells isolated from this region retain their multipotency in vitro and in vivo after grafting into the adult brain. Molecular signalling and lineage selection in these cells may be examined using genetic manipulation. However, valid analysis requires that this manipulation should not affect cellular viability, proliferation or differentiation. METHODS: We screened several transfection protocols to develop a method which met these criteria. We then tested the effects of transfection on viability, proliferation and differentiation into the three neural lineages: neurons, astrocytes and oligodendrocytes. RESULTS: In initial testing, ExGen500 and FuGene6 efficiently transfected adult neural stem/progenitor cells, in vitro. After optimisation, these agents transfected 16% and 11% of cells, respectively. FuGene6-treated cells did not differ from untransfected cells in their viability or rate of proliferation, whereas these characteristics were significantly reduced following ExGen500 transfection. Importantly, neither agent affected the pattern of differentiation following transfection. Both agents could be used to genetically label cells, and track their differentiation into the three neural lineages, after grafting onto ex vivo organotypic hippocampal slice cultures. CONCLUSIONS: These data demonstrate that non-viral transfection may be used to genetically manipulate neural stem/progenitor cells, without adversely affecting their growth or perturbing lineage selection. Such a method is valuable for examining the molecular mechanisms of cell fate determination in vitro. Furthermore, this protocol may be exploited in the development of cell-based gene therapy strategies.     

Clonal analysis of the proliferation potential of human bone marrow mesenchymal stem cells as a function of potency

Human mesenchymal stem cells (MSCs) from bone marrow are a heterogeneous ensemble of progenitors and lineage-committed cells, with a broad range of regenerative properties. Ex vivo expansion to produce sufficient quantities of MSCs is essential for most therapeutic applications. The present study resolves the relationship between proliferation potential of MSCs and their potency. Clonal analysis generated single-cell derived colonies of MSCs that were classified according to their trilineage potential to exhibit adipo- (A), chondro- (C), and osteogenesis (O) as a measure of potency. Multipotent OAC clones were highly proliferative with colony-forming efficiencies that ranged from 35% to 90%; whereas, O clones formed colonies with an efficiency of 5% or less (P < 0.01). Similar trends were evident during ex vivo expansion: for example, the median specific growth rate was 0.8 day(-1) (20 h doubling time) for cultures inoculated with OAC clones and was 5-fold less for inocula of O clones (P < 0.01). OA and OC clones had similar proliferation potentials. More than 75% of cells in subconfluent cultures inoculated with O clones stained positive for senescence-associated β-galactosidase activity vs. less than 10% for OAC clones (P < 0.001). Apoptotic cells were in the minority for all potency groups. Preliminary data generated during clonal analysis suggest that osteogenic potential of MSCs to produce mineralized matrix is a function of potency, as well. These results are discussed in the context of the preparation of efficacious MSC therapies by ex vivo expansion.