Neuroectodermal Cell Types Research Articles

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.

Enrichment and Characterization of Human Dermal Stem/Progenitor Cells by Intracellular Granularity

Adult stem cells from the dermis would be an attractive cell source for therapeutic purposes as well as studying the process of skin aging. Several studies have reported that human dermal stem/progenitor cells (hDSPCs) with multipotent properties exist within the dermis of adult human skin. However, these cells have not been well characterized, because methods for their isolation or enrichment have not yet been optimized. In the present study, we enriched high side scatter (SSC(high))-hDSPCs from normal human dermal fibroblasts using a structural characteristic, intracellular granularity, as a sorting parameter. The SSC(high)-hDSPCs had high in vitro proliferation properties and expressed high levels of SOX2 and S100B, similar to previously identified mouse SOX2+ hair follicle dermal stem cells. The SSC(high)-hDSPCs could differentiate into not only mesodermal cell types, for example, adipocytes, chondrocytes, and osteoblasts, but also neuroectodermal cell types, such as neural cells. In addition, the SSC(high)-hDSPCs exhibited no significant differences in the expression of nestin, vimentin, SNAI2, TWIST1, versican, and CORIN compared with non-hDSPCs. These cells are therefore different from the previously identified multipotent fibroblasts and skin-derived progenitors. In this study, we suggest that hDSPCs can be enriched by using characteristic of their high intracellular granularity, and these SSC(high)-hDSPCs exhibit high in vitro proliferation and differentiation potentials.

Epigenetic conversion of human adult bone mesodermal stromal cells into neuroectodermal cell types for replacement therapy of neurodegenerative disorders

Tissue-specific stem cells, such as bone marrow-derived mesodermal stromal cells (MSCs), are thought to be lineage restricted and, therefore, could only be differentiated into cell types of the tissue of origin. Several recent studies, however, suggest that these types of stem cells might be able to break barriers of germ layer commitment and differentiate in vitro and/or in vivo into cells of different tissues, such as neuroectodermal cell types. Recently, protocols for high-yield generation of undifferentiated neural stem cell (NSC)-like cells from MSCs of primate and human origin were reported. Undifferentiated NSCs are commonly used and are more suitable for neurotransplantation compared with fully differentiated neural cells, as differentiated neural cells are well known to poorly survive detachment and subsequent transplantation procedures. These human MSC-derived NSC-like cells (MSC-NSCs) grow in neurosphere-like structures and express high levels of early neuroectodermal markers, but lose characteristics of MSCs. In the presence of selected growth factors, human MSC-NSCs can be differentiated into the three main neural phenotypes: astroglia, oligodendroglia and neurons. Compared with direct differentiation of human MSCs into mature neural cells, the conversion step seems to be essential to generate mature functional neuroectodermal cells. This review describes the techniques for the conversion of human MSCs into NSCs and summarises the data on epigenetic conversion of human MSCs into immature neuroectodermal cells. These cells provide a powerful tool for investigating the molecular mechanisms of neural differentiation, and might serve as an autologous cell source to treat acute and chronic neurodegenerative diseases.

Comparative analysis of neuroectodermal differentiation capacity of human bone marrow stromal cells using various conversion protocols

Human adult bone marrow-derived mesodermal stromal cells (hMSCs) are able to differentiate into multiple mesodermal tissues, including bone and cartilage. There is evidence that these cells are able to break germ layer commitment and differentiate into cells expressing neuroectodermal properties. There is still debate about whether this results from cell fusion, aberrant marker gene expression or real neuroectodermal differentiation. Here we extend our work on neuroectodermal conversion of adult hMSCs in vitro by evaluating various epigenetic conversion protocols using quantitative RT-PCR and immunocytochemistry. Undifferentiated hMSCs expressed high levels of fibronectin as well as several neuroectodermal genes commonly used to characterize neural cell types, such as nestin, beta-tubulin III, and GFAP, suggesting that hMSCs retain the ability to differentiate into neuroectodermal cell types. Protocols using a direct differentiation of hMSCs into a neural phenotype failed to induce significant changes in morphology and/or expression of markers of early and mature glial/neuronal cells types. In contrast, a multistep protocol with conversion of hMSCs into a neural stem cell-like population and subsequent terminal differentiation in mature glia and neurons generated relevant morphological changes as well as significant increase of expression levels of marker genes for early and late neural cell types, such as nestin, neurogenin2, MBP, and MAP2ab, accompanied by a loss of their mesenchymal properties. Our data provide an impetus for differentiating hMSCs in vitro into mature neuroectodermal cells. Neuroectodermally converted hMSCs may therefore ultimately help in treating acute and chronic neurodegenerative diseases. Analysis of marker gene expression for characterization of neural cells derived from MSCs has to take into account that several early and late neuroectodermal genes are already expressed in undifferentiated MSCs.

Gene expression changes during step-wise differentiation of embryonic stem cells along the inner ear hair cell pathway

Conclusion. Our study outlines an alternative approach for the selection and investigation of genes involved in inner ear function. Objective. To gain understanding of the gene pathways involved in the development of the normal cochlea. Materials and methods. Microarray technology currently offers the most efficient approach to investigate gene expression and identify pathways involved in cell differentiation. Epidermal growth factor (EGF) induces cultures derived from the organ of Corti to proliferate and produce new hair cells. Since pluripotent embryonic stem (ES) cells have the capacity to generate all tissues, we induced murine ES cells to differentiate towards ectodermal and neuroectodermal cell types and from there investigated their commitment towards the hair cell lineage in the presence of EGF. Cells were collected at three points along the differentiation pathway and their expression profiles were determined using the Soares NMIE mouse inner ear cDNA library printed in microarray format. Results. Three genes up-regulated after addition of EGF (serine (or cysteine) proteinase inhibitor, clade H, member 1 (Serpinh1), solute carrier family 2 (facilitated glucose transporter), member 10 (Slc2a10) and secreted acidic cysteine-rich glycoprotein (Sparc)) were selected for further analysis and characterization. Of the three genes, Serpinh1 and Slc2a10 have never been implicated in the hearing process.

Cerebellar Involvement in Pick's Disease: Affliction of Mossy Fibers, Monodendritic Brush Cells, and Dentate Projection Neurons

Pick's disease chiefly is characterized by progressive degeneration of specific telencephalic cortical areas and associated subcortical nuclei. Components of the cerebellum also are affected. Immunoreactions for abnormally hyperphosphorylated tau protein, indicating the development of cytoskeletal anomalies in a few susceptible neuroectodermal cell types, permit visualization and identification of the pathology. Initially, accumulations of nonargyrophilic material appear in the perikarya and cellular processes of susceptible nerve cells. In some neuronal types, the abnormal deposits are transformed into more condensed inclusions, so-called Pick bodies in perikarya and Pick neurites in cellular processes, some of which become argyrophilic in the course of the disease. This study employs silver techniques and immunoreactions to draw attention to Pick's disease-associated lesions in the cerebellar cortex and cerebellar nuclei. Immunoreactive rosettes, which correspond to the terminal synaptic boutons of mossy fibers, frequently are encountered in the cerebellar granule cell layer. Some cases of Pick's disease also exhibit afflicted monodendritic brush cells in this layer. Single immunopositive Purkinje cells occasionally are seen as well. The brunt of the alterations is borne by cerebellar subdivisions receiving dense input from the telencephalic cortex through the pontocerebellar pathway (neocerebellum). The dentate nucleus shows immunoreactive axons with numerous varicose thickenings which remain confined to the reaches of this band-like nuclear gray and probably represent collaterals of altered mossy fibers. A large number of the dentate projection cells also contain the abnormal material in the perikarya, as well as in all of the neuronal processes. Many of these cells develop spherical nonargyrophilic condensations of this material. Output of the neocerebellum is conveyed to extended territories of the telencephalic cortex via the dentate nucleus and thalamus. Therefore, all of the cerebellar territories which receive major input from and generate output chiefly to the telencephalic cortex (pontocerebellum or neocerebellum) are notably afflicted in Pick's disease. Other subdivisions with preponderant input from the spinal cord and/or other noncortical sources remain intact or else are only minimally involved. It is concluded that the pattern of cerebellar involvement reflects Pick's disease-associated neocortical destruction.

Cell Type-Specific Regulation of Expression of Transcription Factor AP-2 in Neuroectodermal Cells

AP-2 is a cell type-specific DNA-binding transcription factor that regulates selected target genes in vertebrate organisms. Here we investigated cell type-specific expression and regulation of AP-2 in neuroectodermal cell lineages. During retinoic acid (RA)-mediated differentiation of P19 embryonal carcinoma cells into neuroectodermal cell types that include immunohistochemically defined neurons and astrocytes, we observed a strong induction of AP-2 transcripts and protein. In contrast, AP-2 mRNA was not induced in P19 cells which undergo mesoendodermal differentiation in response to 1% dimethylsulfoxide or low concentrations of RA, respectively. The potential of both neurons and astrocytes to express AP-2 was ascertained by using cerebellar neurons and astrocytes derived from newborn mice. Unlike these types of cells, microglial cells do not express AP-2. Dibutyryl cyclic AMP further enhanced levels of AP-2 transcripts in both P19 astrocytes and primary astrocytes which also respond to agents elevating intracellular cAMP (noradrenaline, isoproterenol, forskolin). The cAMP-dependent induction of AP-2 could be blocked by inhibitors of protein kinase A. In contrast to its action in P19 cells, RA had no effect on AP-2 mRNA levels in primary astrocytes. Our results indicate that AP-2 may play a role as a retinoic acid-sensitive regulator during differentiation of neurons and glia from an embryonic neural precursor. Furthermore, AP-2 may be involved in gene transcription in both mature neurons and astrocytes.

Inhibition of differentiation by leukemia inhibitory factor distinguishes two induction pathways in P19 embryonal carcinoma cells

Abstract The ability of leukemia inhibitory factor (LIF) to block differentiation of P19 embryonal carcinoma (EC) cells under a variety of induction conditions was determined. LIF inhibits differentiation under several conditions which lead to endodermal and mesodermal cell lineages including skeletal and cardiac muscle. In contrast, LIF does not block differentiation when cells are induced under conditions which lead to neuro-ecto-dermal cell types including neurons and astroglial cells. These studies demonstrate that P19 EC cell differentiation can be divided into LIF sensitive and insensitive pathways which correlate with differentiation of endo-dermal/mesodermal and neuro-ectodermal cell types, respectively. The effect of LIF on mRNA levels for several genes which have previously been implicated in mediating differentiation in P19 EC cells was determined. LIF has no effect on the mRNA levels for retinoic acid receptor (RAR) α, RAR β, RAR γ, jun A, jun D, c- fos, or fra-1. In contrast LIF stimulates jun B mRNA expression by a factor of four to six under all induction conditions.

Identification of the type-B receptor for platelet-derived growth factor in human embryonal carcinoma cells

The human embryonal carcinoma (EC) cell line Tera 2 clone 13 (T2/13) can be induced to differentiate in vitro into neuroectodermal cell types with retinoic acid. Undifferentiated cells are characterized by rapid proliferation, whereas differentiated cells show a prolonged generation time, have a limited life span, and possess new cell-surface markers. In the present study we establish that both differentiated and undifferentiated T2/13 cells express the type-B platelet-derived growth factor (PDGF) receptor mRNA and bind PDGF-BB with high affinity. Differentiation causes a three-fold increase in receptor number per cell and leaves the affinity of the receptors unaffected. These data are the first to describe expression of this receptor in EC cells. The biosynthesis and degradation of this receptor were studied in undifferentiated as well as in differentiated T2/13 cells using an anti-type-B receptor antibody. These experiments revealed that high concentrations of recombinant PDGF-AA did not accelerate receptor metabolism in both cell types. In contrast, human PDGF or recombinant PDGF-BB added to the culture dishes readily increased receptor degradation. These results demonstrate that T2/13 cells express functional type-B PDGF receptors and suggest that cells responsive to PDGF might be present during mammalian development before the onset of mesoderm formation.

Differentially regulated production of platelet-derived growth factor and of transforming growth factor beta by a human teratocarcinoma cell line

Abstract The human teratocarcinoma stem cell line Tera-2 clone 13 is induced by retinoic acid to differentiate in vitro into endodermal or neuroectodermal cell types. In the absence of externally added growth factors, Tera-2 clone 13 cells proliferated at the same rate as in the presence of serum growth factors. Analysis of serum-free medium conditioned by Tera-2 clone 13 cells showed the presence of a polypeptide immunologically and biochemically related to platelet-derived growth factor (PDGF). In addition transforming growth factor beta (TGF-β), but no TGF-α production could be detected. Tera-2 clone 13 cells specifically expressed high levels of the A-chain mRNA, but not the B-chain mRNA of PDGF. During retinoic acid induced differentiation the level of A-chain mRNA became markedly reduced. In contrast the TGF-β mRNA levels increased significantly upon differentiation. The implications of these findings are discussed in terms of regulation of growth and differentiation in early embryos as well in (human) teratocarcinomas.