Janine Müller

Isolation of Novel Multipotent Neural Crest-Derived Stem Cells from Adult Human Inferior Turbinate

Adult human neural crest-derived stem cells (NCSCs) are of extraordinary high plasticity and promising candidates for the use in regenerative medicine. Here we describe for the first time a novel neural crest-derived stem cell population within the respiratory epithelium of human adult inferior turbinate. In contrast to superior and middle turbinates, high amounts of source material could be isolated from human inferior turbinates. Using minimally-invasive surgery methods isolation is efficient even in older patients. Within their endogenous niche, inferior turbinate stem cells (ITSCs) expressed high levels of nestin, p75(NTR), and S100. Immunoelectron microscopy using anti-p75 antibodies displayed that ITSCs are of glial origin and closely related to nonmyelinating Schwann cells. Cultivated ITSCs were positive for nestin and S100 and the neural crest markers Slug and SOX10. Whole genome microarray analysis showed pronounced differences to human ES cells in respect to pluripotency markers OCT4, SOX2, LIN28, and NANOG, whereas expression of WDR5, KLF4, and c-MYC was nearly similar. ITSCs were able to differentiate into cells with neuro-ectodermal and mesodermal phenotype. Additionally ITSCs are able to survive and perform neural crest typical chain migration in vivo when transplanted into chicken embryos. However ITSCs do not form teratomas in severe combined immunodeficient mice. Finally, we developed a separation strategy based on magnetic cell sorting of p75(NTR) positive ITSCs that formed larger neurospheres and proliferated faster than p75(NTR) negative ITSCs. Taken together our study describes a novel, readily accessible source of multipotent human NCSCs for potential cell-replacement therapy.

1,8-Cineol inhibits nuclear translocation of NF-κB p65 and NF-κB-dependent transcriptional activity

Natural plant-derived products are commonly applied to treat a broad range of human diseases, including cancer as well as chronic and acute airway inflammation. In this regard, the monoterpene oxide 1,8-cineol, the active ingredient of the clinically approved drug Soledum®, is well-established for the therapy of airway diseases, such as chronic sinusitis and bronchitis, chronic obstructive pulmonary disease and bronchial asthma. Although clinical trials underline the beneficial effects of 1,8-cineol in treating inflammatory diseases, the molecular mode of action still remains unclear. Here, we demonstrate for the first time a 1,8-cineol-depending reduction of NF-κB-activity in human cell lines U373 and HeLa upon stimulation using lipopolysaccharides (LPS). Immunocytochemistry further revealed a reduced nuclear translocation of NF-κB p65, while qPCR and western blot analyses showed strongly attenuated expression of NF-κB target genes. Treatment with 1,8-cineol further led to increased protein levels of IκBα in an IKK-independent matter, while FRET-analyses showed restoring of LPS-associated loss of interaction between NF-κB p65 and IκBα. We likewise observed reduced amounts of phosphorylated c-Jun N-terminal kinase 1/2 protein in U373 cells after exposure to 1,8-cineol. In addition, 1,8-cineol led to decreased amount of nuclear NF-κB p65 and reduction of its target gene IκBα at protein level in human peripheral blood mononuclear cells. Our findings suggest a novel mode of action of 1,8-cineol through inhibition of nuclear NF-κB p65 translocation via IκBα resulting in decreased levels of proinflammatory NF-κB target genes and may therefore broaden the field of clinical application of this natural drug for treating inflammatory diseases.

Intrastriatal Transplantation of Adult Human Neural Crest-Derived Stem Cells Improves Functional Outcome in Parkinsonian Rats

Parkinsons 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.

1,8-Cineol Reduces Mucus-Production in a Novel Human Ex Vivo Model of Late Rhinosinusitis

Inflammatory diseases of the respiratory system such as rhinosinusitis, chronic obstructive pulmonary disease, or bronchial asthma are strongly associated with overproduction and hypersecretion of mucus lining the epithelial airway surface. 1,8-cineol, the active ingredient of the pharmaceutical drug Soledum, is commonly applied for treating such inflammatory airway diseases. However, its potential effects on mucus overproduction still remain unclear.In the present study, we successfully established ex vivo cultures of human nasal turbinate slices to investigate the effects of 1,8-cineol on mucus hypersecretion in experimentally induced rhinosinusitis. The presence of acetyl-α-tubulin-positive cilia confirmed the integrity of the ex vivo cultured epithelium. Mucin-filled goblet cells were also detectable in nasal slice cultures, as revealed by Alcian Blue and Periodic acid-Schiff stainings. Treatment of nasal slice cultures with lipopolysaccharides mimicking bacterial infection as observed during late rhinosinusitis led to a significantly increased number of mucin-filled goblet cells. Notably, the number of mucin-filled goblet cells was found to be significantly decreased after co-treatment with 1,8-cineol. On a molecular level, real time PCR-analysis further showed 1,8-cineol to significantly reduce the expression levels of the mucin genes MUC2 and MUC19 in close association with significantly attenuated NF-κB-activity. In conclusion, we demonstrate for the first time a 1,8-cineol-dependent reduction of mucin-filled goblet cells and MUC2-gene expression associated with an attenuated NF-κB-activity in human nasal slice cultures. Our findings suggest that these effects partially account for the clinical benefits of 1,8-cineol-based therapy during rhinosinusitis. Therefore, topical application of 1,8-cineol may offer a novel therapeutic approach to reduce bacteria-induced mucus hypersecretion.

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.

Hsc70 Is a Novel Interactor of NF-kappaB p65 in Living Hippocampal Neurons

Signaling via NF-κB in neurons depends on complex formation with interactors such as dynein/dynactin motor complex and can be triggered by synaptic activation. However, so far a detailed interaction map for the neuronal NF-κB is missing. In this study we used mass spectrometry to identify novel interactors of NF-κB p65 within the brain. Hsc70 was identified as a novel neuronal interactor of NF-κB p65. In HEK293 cells, a direct physical interaction was shown by co-immunoprecipitation and verified via in situ proximity ligation in healthy rat neurons. Pharmacological blockade of Hsc70 by deoxyspergualin (DSG) strongly decreased nuclear translocation of NF-κB p65 and transcriptional activity shown by reporter gene assays in neurons after stimulation with glutamate. In addition, knock down of Hsc70 via siRNA significantly reduced neuronal NF-κB activity. Taken together these data provide evidence for Hsc70 as a novel neuronal interactor of NF-κB p65.

Prolonged cultivation of hippocampal neural precursor cells shifts their differentiation potential and selects for aneuploid cells

Abstract Neural precursor cells (NPCs) are lineage-restricted neural stem cells with limited self-renewal, giving rise to a broad range of neural cell types such as neurons, astrocytes, and oligodendrocytes. Despite this developmental potential, the differentiation capacity of NPCs has been controversially discussed concerning the trespassing lineage boundaries, for instance resulting in hematopoietic competence. Assessing their in vitro plasticity, we isolated nestin+/Sox2+, NPCs from the adult murine hippocampus. In vitro-expanded adult NPCs were able to form neurospheres, self-renew, and differentiate into neuronal, astrocytic, and oligodendrocytic cells. Although NPCs cultivated in early passage efficiently gave rise to neuronal cells in a directed differentiation assay, extensively cultivated NPCs revealed reduced potential for ectodermal differentiation. We further observed successful differentiation of long-term cultured NPCs into osteogenic and adipogenic cell types, suggesting that NPCs underwent a fate switch during culture. NPCs cultivated for more than 12 passages were aneuploid (abnormal chromosome numbers such as 70 chromosomes). Furthermore, they showed growth factor-independent proliferation, a hallmark of tumorigenic transformation. In conclusion, our findings substantiate the lineage restriction of NPCs from adult mammalian hippocampus. Prolonged cultivation results, however, in enhanced differentiation potential, which may be attributed to transformation events leading to aneuploid cells.

An Investigation of the Specificity of Research Antibodies against NF-κB-subunit p65

The transcription factor nuclear factor kappa B (NF-κB) plays a pivotal role in a variety of biological processes in different mammalian tissues and organs, including the immune and central nervous systems (Hayden and Ghosh 2008). Many of the studies on the NF-κB signaling in different cellular systems were, at least partly, based on immunological analyses of the distribution of the NF-κB subunit p65. Recently, different p50 and p65 antibodies were analyzed and demonstrated that many of the tested antibodies were not fully specific for their antigen (Herkenham et al. 2011). Such improper epitope recognitions may lead to misinterpretations of the distribution, localization and activity of NF-κB. Further elucidating the specificity of commercially available antibodies against p65, we tested six different antibodies using immunocytochemistry (ICC) and western blots. Murine embryonic stem cells (mESCs) protein samples were used as a stringent negative control, as these cells are known to be p65-negative at the protein level (Luningschror et al. 2012), whereas protein from GFP-p65 knock-in mice (De Lorenzi et al. 2009) served as positive controls. We demonstrate inappropriate cross-reactivity of several commercially available p65 antibodies. We treated murine embryonic fibroblast (MEFs) with the cytokine tumor necrosis factor alpha (TNF-α) to trigger NF-κB signaling for the detection of nuclear immunoreactivity of the p65 antibodies using ICC. In all approaches, such TNF-α–dependent nuclear signals were observable (Fig. 1A). Non-stimulated MEFs showed no expected cytosolic signals for NF-κB p65, except when using antibodies sc-8008, sc-372 and E498 (Fig. 1A). All antibodies are summarized in Table 1 and all data in Table 2. Using western blots from cell lysates, the observations from ICC could partly be confirmed (Fig. 1B). Interestingly, sc-7151 marked a single band at the size of p65 after having shown inappropriate cross-reactivity in ICC. On the contrary, E498 did not mark a band at all. Apparently, this antibody is highly specific for p65 but only in its native form and not after a denaturing SDS-PAGE; although, according to the manufacturer, this antibody is suitable for western blotting. It should be noted, however, that this antibody is no longer available for purchase. As a stringent negative control, we tested the antibodies on mESCs in western blots as well as in ICC. In western blots, all antibodies, except sc-372, did not mark a band (Fig. 1B) and also demonstrated no immunoreactivity in ICC. One representative staining using MAB3026 is shown in Figure 1C. The sc-372 antibody demonstrated strong cytosolic immunoreactivity in repeated approaches (Fig. 1C), confirming the result from the western blot, where it marked one single band at a size comparable with p65 (Fig. 1B). Figure 1. Antibodies against NF-κB p65 show cross-reactivity in mouse embryonic fibroblasts (MEFs) and mouse embryonic stem cells (mESCs). (A) Immunocytochemical staining of TNF-α–treated MEFs using six different commercially available antibodies ... Table 1. List of NF-κB p65 Antibodies Used in this Study. Table 2. Summary of All Observations Regarding p65 Antibody Specificity. Additionally, we studied the potential co-localization of GFP-expression in a GFP-p65 knock-in mouse line. These mice express a GFP-p65 fusion protein from the endogenous p65 locus that functionally substitutes p65 (De Lorenzi et al. 2009). Therefore, MEFs derived from this mouse line—hereafter referred to as GFP-p65 MEFs—are highly suitable for performing co-localization studies to gain further insight into the specificity of the p65 antibodies. For sc-8008 and MAB3026 antibodies, co-immunostaining was performed with ab290 anti-GFP antibody (Abcam; Cambridge, MA). All antibodies demonstrated nuclear co-localization with the GFP signals in TNF-α–treated cells. A representative immunostaining of TNF-α–treated cells using the sc-8008 antibody is shown in Figure 2A. In non-stimulated GFP-p65 MEFs, only signals from sc-372 and sc-8008 antibodies co-localized with the GFP signal (Fig. 2A, ​,B).B). To summarize the presented data, only the sc-8008 antibody showed the expected immunoreactivity in all approaches of our test models. Interestingly, this is in contrast to the results by Herkenham et al. (2011), wherein presumed non-specificity of sc-8008 was indicated by the presentation of bands of variable sizes in western blots of different tissues from TNF receptor 1/p65 double-knockout mice (Herkenham et al. 2011). Additionally, they presumed that the sc-372 antibody was very specific for p65, whereas we demonstrate its inappropriate cross-reactivity in mESCs. Remarkably, the above presented contrary results regarding sc-372 and sc-8008 might be due to batch fluctuations. It is unlikely that the same batches were used in our analyses and in the 2011 study by Herkenham and colleagues. Figure 2. Immunocytochemistry of GFP-p65 murine embryonic fibroblasts (MEFs) for potential co-localization of GFP with anti-p65 antibodies. (A) Immunostaining using the ab290 anti-GFP antibody and the sc-8008. ICC was performed as described for wildtype MEFs in ... In the case of MAB3026, we demonstrated non-specificity of binding, which was analogous to the findings of Herkenham et al (2011). With the western blot results being not completely unambiguous regarding MAB3026 (see Table 2), the ICC staining revealed non-specific nuclear immunoreactivity independent of TNF-α treatment. MAB3026, originally created in our laboratory, is intended to mark “active p65”, as its epitope includes the nuclear localization signal, which is only exposed when p65 is activated. Then named alpha-p65M, it passed stringent tests for validity, marking exclusively p65 in p65-overexpressing HEK 293 cells in western blot and immunostaining (Kaltschmidt et al. 1995). It was transferred to Boehringer-Mannheim as Clone 12H11, resold to Roche and finally bought by Chemicon, and it is now sold as MAB3026. Being generated in the early ‘90s, transferred from one company to the next, we presume that there may be a mutation in Clone 12H11. With the NLS being rather conserved, a mutation that changes the recognition of the epitope slightly might lead to the recognition of other NLS-containing proteins. This hypothesis would be in agreement with our observations of TNF-α–independent nuclear immunoreactivity. The antibodies sc-109 and sc-7151 both demonstrated inappropriate cross-reactivity and usage of these antibodies, especially in ICC, is therefore not recommended. Our results indicate that not all of the commonly used antibodies against p65 exclusively bind to p65, at least in the batches that we have tested. Even antibodies that mark specifically p65 in western blotting do not necessarily show specific immunoreactivity in ICC. Low amounts of p65 in cells require higher concentrations of the antibody, which increases the risk of non-specific binding. Therefore, the usage of these antibodies should be conducted with awareness of the limitations of each antibody, and great care should be taken to exclude false-positive results, which can lead to misinterpretations of the localization and activity of p65. To avoid this, rigorous testing of every new batch of antibody prior to its application is highly recommended.

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 a model 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.

Methods for the modulation and analysis of NF-κB-dependent adult neurogenesis.

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-κB plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-κB activity can be used. In this study, methods are described for the analysis of NF-κB-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.