Different Neurogenic Potential in the Subnuclei of the Postnatal Rat Cochlear Nucleus

In patients suffering from hearing loss, the reduced or absent neural input induces morphological changes in the cochlear nucleus (CN). Neural stem cells have recently been identified in this first auditory relay. Afferent nerve signals and their impact on the immanent neural stem and progenitor cells already impinge upon the survival of early postnatal cells within the CN. This auditory brainstem nucleus consists of three different subnuclei: the anteroventral cochlear nucleus (AVCN), the posteroventral cochlear nucleus (PVCN), and the dorsal cochlear nucleus (DCN). Since these subdivisions differ ontogenetically and physiologically, the question arose whether regional differences exist in the neurogenic niche. CN from postnatal day nine Sprague-Dawley rats were microscopically dissected into their subnuclei and cultivated in vitro as free-floating cell cultures and as whole-mount organ cultures. In addition to cell quantifications, immunocytological and immunohistological studies of the propagated cells and organ preparations were performed. The PVCN part showed the highest mitotic potential, while the AVCN and DCN had comparable activity. Specific stem cell markers and the ability to differentiate into cells of the neural lineage were detected in all three compartments. The present study shows that in all subnuclei of rat CN, there is a postnatal neural stem cell niche, which, however, differs significantly in its potential. The results can be explained by the origin from different regions in the rhombic lip, the species, and the various analysis techniques applied. In conclusion, the presented results provide further insight into the neurogenic potential of the CN, which may prove beneficial for the development of new regenerative strategies for hearing loss.

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Long range channelrhodopsin-assisted circuit mapping of inferior colliculus neurons with blue and red-shifted channelrhodopsins

10.1101/756957 ◽

2019 ◽

Author(s):

David Goyer ◽

Michael T. Roberts

Keyword(s):

Electrical Stimulation ◽

Patch Clamp ◽

Inferior Colliculus ◽

Long Range ◽

Cochlear Nucleus ◽

Auditory Brainstem ◽

Dorsal Cochlear Nucleus ◽

Multiple Sources ◽

Patch Clamp Recording

ABSTRACTWhen investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, making it difficult to isolate inputs from individual sources with electrical stimulation. However, by using channelrhodopsin assisted circuit mapping (CRACM) this limitation can now be overcome. Here, we report a method to use CRACM to map ascending inputs from lower auditory brainstem nuclei and commissural inputs to an identified class of neurons in the inferior colliculus (IC), the midbrain nucleus of the auditory system. In the IC, local, commissural, ascending, and descending axons are heavily intertwined and therefore indistinguishable with electrical stimulation. By injecting a viral construct to drive expression of a channelrhodopsin in a presynaptic nucleus, followed by patch clamp recording to characterize the presence and physiology of channelrhodopsin-expressing synaptic inputs, projections from a specific source to a specific population of IC neurons can be mapped with cell type-specific accuracy. We show that this approach works with both Chronos, a blue light-activated channelrhodopsin, and ChrimsonR, a red-shifted channelrhodopsin. In contrast to previous reports from the forebrain, we find that ChrimsonR is robustly trafficked down the axons of dorsal cochlear nucleus principal neurons, indicating that ChrimsonR may be a useful tool for CRACM experiments in the brainstem. The protocol presented here includes detailed descriptions of the intracranial virus injection surgery, including stereotaxic coordinates for targeting injections to the dorsal cochlear nucleus and IC of mice, and how to combine whole cell patch clamp recording with channelrhodopsin activation to investigate long-range projections to IC neurons. Although this protocol is tailored to characterizing auditory inputs to the IC, it can be easily adapted to investigate other long-range projections in the auditory brainstem and beyond.SUMMARYChannelrhodopsin-assisted circuit mapping (CRACM) is a precision technique for functional mapping of long-range neuronal projections between anatomically and/or genetically identified groups of neurons. Here, we describe how to utilize CRACM to map auditory brainstem connections, including the use of a red-shifted opsin, ChrimsonR.

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Characterization of Human Chondrocytes from Less- vs. Severely-Affected Osteoarthritic Cartilage and Evaluation of their Ability to Develop into In Vitro 3D Models

10.20944/preprints201810.0269.v1 ◽

2018 ◽

Author(s):

Nazatul Nurzazlin Binti Zakariah ◽

Shamsul Bin Sulaiman ◽

Nor Hamdan Bin Yahaya ◽

Rizal Abdul Rani ◽

Ruszymah Binti Haji Idrus ◽

...

Keyword(s):

Stem Cell ◽

Morphological Changes ◽

Cartilage Degeneration ◽

3D Models ◽

Joint Disease ◽

Stem Cell Markers ◽

Osteoarthritic Cartilage ◽

Cell Markers ◽

Inflammatory Conditions

Osteoarthritis (OA) is a joint disease involving cartilage degeneration. This study aimed to compare properties of chondrocytes from less-affected (LA-Cartilage) and severely-affected (SA-Cartilage) of human OA articular cartilage. Based on Dougados classification, OA cartilage was classified into two groups; less-affected (Grade 0–1) and severely-affected (Grade 2–3). Chondrocytes from each group were cultured until passage (P) 4. Growth, migration, stem cell properties and chondrogenic properties under normal and inflammatory conditions, and the formation of in vitro 3D cartilage tissues were compared between groups. The growth and migratory properties of LA-chondrocytes and SA-chondrocytes were similar, except that the migration rate of SA-chondrocytes was significantly higher at P0 compared to LA-chondrocytes. Both LA-chondrocytes and SA-chondrocytes expressed mesenchymal stem cell markers and tri-lineage differentiation, but the expression of stem cell markers decreased significantly with increasing passage number. Exposure to inflammatory conditions induced distinct morphological changes and significant increases in expression of SOX9 at P4 and MMP3 at P1 for LA-chondrocytes. LA-chondrocytes and SA-chondrocytes able to develop into in vitro 3D constructs, but SA-chondrocytes exhibited superior cartilage-like properties. Chondrocytes from both less- and severely-affected regions are suitable to be used in clinical applications, however, chondrocytes from severely-affected regions could be a more favorable cell source.

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Isolation and Characterization of Human Epidermal Stem Cells

Clinical Science ◽

10.1042/cs0910141 ◽

1996 ◽

Vol 91 (2) ◽

pp. 141-146 ◽

Cited By ~ 24

Author(s):

P. H. Jones

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Matrix Proteins ◽

Stem Cell Markers ◽

Epidermal Stem Cells ◽

Cell Behaviour ◽

Isolation And Characterization ◽

Integrin Family

1. The keratinocytes in human epidermis are constantly turned over and replaced by a population of stem cells located in the basal epidermal layer. Until recently there were no markers allowing the isolation of viable epidermal stem cells. However, it has now been shown that epidermal stem cells can be isolated both in vitro and direct from the epidermis as they express high levels of functional β1 integrin family receptors for extracellular matrix proteins. 2. The evidence for integrins as stem cell markers and the insights that have been gained into stem cell behaviour are reviewed.

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Development of hyperactivity after hearing loss in a computational model of the dorsal cochlear nucleus depends on neuron response type

Hearing Research ◽

10.1016/j.heares.2008.02.006 ◽

2008 ◽

Vol 240 (1-2) ◽

pp. 57-72 ◽

Cited By ~ 31

Author(s):

Roland Schaette ◽

Richard Kempter

Keyword(s):

Hearing Loss ◽

Computational Model ◽

Cochlear Nucleus ◽

Dorsal Cochlear Nucleus ◽

Response Type ◽

Neuron Response

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Differentiation of human pluripotent teratocarcinoma stem cells induced by bone morphogenetic protein-2

Reproduction Fertility and Development ◽

10.1071/rd98097 ◽

1998 ◽

Vol 10 (8) ◽

pp. 551 ◽

Cited By ~ 27

Author(s):

Martin F. Pera ◽

Daniella Herszfeld

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Retinoic Acid ◽

Cell Line ◽

Bone Morphogenetic Protein ◽

Morphological Changes ◽

Bone Morphogenetic Protein 2 ◽

Stem Cell Markers ◽

Potent Inducer ◽

Morphogenetic Protein

Pluripotent human teratocarcinoma stem cells cultured in vitro provide a resource for the study of early embryonic development in man, as well as a means for discovery of novel factors controlling cell differentiation and commitment. We previously reported that the human teratocarcinoma stem cell line GCT 27X-1 could be induced to differentiate into an endodermal progenitor cell by treatment with high doses of retinoic acid. A search for polypeptide inducers of differentiation in this system has identified bone morphogenetic protein-2 (BMP-2) as a potent inducer of differentiation. In cell line GCT 27X-1, treatment with BMP-2 reduces proliferation, induces morphological changes similar to obtained following treatment with retinoic acid, and causes a decrease in the expression of transcripts for the stem cell markers CD30 and Oct-4. Preliminary immunochemical studies indicate that the differentiated cells produced by BMP-2 are endodermal precursors with a pattern of marker expression similar to that found in retinoic acid treated cells. Models of endoderm differentiation in humans will be useful for identifying the molecules which mediate cell interactions in development, and in achieving directed differentiation of cells for use in transplantation.

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5-Azacytidine-Induced Cardiomyocyte Differentiation of Very Small Embryonic-Like Stem Cells

Stem Cells International ◽

10.1155/2020/5162350 ◽

2020 ◽

Vol 2020 ◽

pp. 1-8

Author(s):

XiaoLin Sun ◽

HongXiao Li ◽

Ye Zhu ◽

Pei Xu ◽

QiSheng Zuo ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Ethical Issues ◽

Troponin T ◽

Embryonic Stem ◽

Stem Cell Population ◽

Pacemaker Activity ◽

Stem Cell Markers ◽

Seed Cells

The use of stem cells in generating cell-based pacemaker therapies for bradyarrhythmia is currently being considered. Due to the propensity of stem cells to form tumors, as well as ethical issues surrounding their use, the seed cells used in cardiac biological pacemakers have limitations. Very small embryonic-like stem cells (VSELs) are a unique and rare adult stem cell population, which have the same structural, genetic, biochemical, and functional characteristics as embryonic stem cells without the ethical controversy. In this study, we investigated the ability of rat bone marrow- (BM-) derived VSELs to differentiate in vitro into cardiomyocytes by 5-Azacytidine (5-AzaC) treatment. The morphology of VSELs treated with 10 μM 5-AzaC increased in volume and gradually changed to cardiomyocyte-like morphology without massive cell death. Additionally, mRNA expression of the cardiomyocyte markers cardiac troponin-T (cTnT) and α-sarcomeric actin (α-actin) was significantly upregulated after 5-AzaC treatment. Conversely, stem cell markers such as Nanog, Oct-4, and Sox2 were continuously downregulated posttreatment. On day 14 post-5-AzaC treatment, the positive expression rates of cTnT and α-actin were 18.41±1.51% and 19.43±0.51%, respectively. Taken together, our results showed that rat BM-VSELs have the ability to differentiate into cardiomyocytes in vitro. These findings suggest that VSELs would be useful as seed cells in exploring the mechanism of biological pacemaker activity.

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Focal Adhesion Kinase Inhibition Contributes to Tumor Cell Survival and Motility in Neuroblastoma Patient-Derived Xenografts

Scientific Reports ◽

10.1038/s41598-019-49853-z ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Laura L. Stafman ◽

Adele P. Williams ◽

Raoud Marayati ◽

Jamie M. Aye ◽

Hooper R. Markert ◽

...

Keyword(s):

Stem Cell ◽

Tumor Cell ◽

Cell Survival ◽

Cell Lines ◽

Human Condition ◽

Migration And Invasion ◽

Stem Cell Markers ◽

Cell Markers ◽

Cycle Arrest

Abstract Patient-derived xenografts (PDXs) provide an opportunity to evaluate the effects of therapies in an environment that more closely resembles the human condition than that seen with long-term passage cell lines. In the current studies, we investigated the effects of FAK inhibition on two neuroblastoma PDXs in vitro. Cells were treated with two small molecule inhibitors of FAK, PF-573,228 (PF) and 1,2,4,5-benzentetraamine tetrahydrochloride (Y15). Following FAK inhibition, cell survival and proliferation decreased significantly and cell cycle arrest was seen in both cell lines. Migration and invasion assays were used to determine the effect of FAK inhibition on cell motility, which decreased significantly in both cell lines in the presence of either inhibitor. Finally, tumor cell stemness following FAK inhibition was evaluated with extreme limiting dilution assays as well as with immunoblotting and quantitative real-time PCR for the expression of stem cell markers. FAK inhibition decreased formation of tumorspheres and resulted in a corresponding decrease in established stem cell markers. FAK inhibition decreased many characteristics of the malignant phenotype, including cancer stem cell like features in neuroblastoma PDXs, making FAK a candidate for further investigation as a potential target for neuroblastoma therapy.

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Improving the immunosuppressive potential of articular chondroprogenitors in a three-dimensional culture setting

Scientific Reports ◽

10.1038/s41598-020-73188-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Guillermo Bauza ◽

Anna Pasto ◽

Patrick Mcculloch ◽

David Lintner ◽

Ava Brozovich ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Articular Cartilage ◽

Cartilage Repair ◽

Adipogenic Differentiation ◽

3D Culture ◽

Cell Populations ◽

Stem Cell Markers ◽

3D Scaffold

Abstract Cartilage repair in osteoarthritic patients remains a challenge. Identifying resident or donor stem/progenitor cell populations is crucial for augmenting the low intrinsic repair potential of hyaline cartilage. Furthermore, mediating the interaction between these cells and the local immunogenic environment is thought to be critical for long term repair and regeneration. In this study we propose articular cartilage progenitor/stem cells (CPSC) as a valid alternative to bone marrow-derived mesenchymal stem cells (BMMSC) for cartilage repair strategies after trauma. Similar to BMMSC, CPSC isolated from osteoarthritic patients express stem cell markers and have chondrogenic, osteogenic, and adipogenic differentiation ability. In an in vitro 2D setting, CPSC show higher expression of SPP1 and LEP, markers of osteogenic and adipogenic differentiation, respectively. CPSC also display a higher commitment toward chondrogenesis as demonstrated by a higher expression of ACAN. BMMSC and CPSC were cultured in vitro using a previously established collagen-chondroitin sulfate 3D scaffold. The scaffold mimics the cartilage niche, allowing both cell populations to maintain their stem cell features and improve their immunosuppressive potential, demonstrated by the inhibition of activated PBMC proliferation in a co-culture setting. As a result, this study suggests articular cartilage derived-CPSC can be used as a novel tool for cellular and acellular regenerative medicine approaches for osteoarthritis (OA). In addition, the benefit of utilizing a biomimetic acellular scaffold as an advanced 3D culture system to more accurately mimic the physiological environment is demonstrated.

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