Lothar Just

Expansion and differentiation of neural progenitors derived from the human adult enteric nervous system.

BACKGROUND & AIMS Neural stem and progenitor cells from the enteric nervous system have been proposed for use in cell-based therapies against specific neurogastrointestinal disorders. Recently, enteric neural progenitors were generated from human neonatal and early postnatal (until 5 years after birth) gastrointestinal tract tissues. We investigated the proliferation and differentiation of enteric nervous system progenitors isolated from human adult gastrointestinal tract. METHODS Human enteric spheroids were generated from adult small and large intestine tissues and then expanded and differentiated, depending on the applied cell culture conditions. For implantation studies, spheres were grafted into fetal slice cultures and embryonic aganglionic hindgut explants from mice. Differentiating enteric neural progenitors were characterized by 5-bromo-2-deoxyuridine labeling, in situ hybridization, immunocytochemistry, quantitative real-time polymerase chain reaction, and electrophysiological studies. RESULTS The yield of human neurosphere-like bodies was increased by culture in conditional medium derived from fetal mouse enteric progenitors. We were able to generate proliferating enterospheres from adult human small or large intestine tissues; these enterospheres could be subcultured and maintained for several weeks in vitro. Spheroid-derived cells could be differentiated into a variety of neuronal subtypes and glial cells with characteristics of the enteric nervous system. Experiments involving implantation into organotypic intestinal cultures showed the differentiation capacity of neural progenitors in a 3-dimensional environment. CONCLUSIONS It is feasible to isolate and expand enteric progenitor cells from human adult tissue. These findings offer new strategies for enteric stem cell research and future cell-based therapies.

Generation of pluripotent stem cells from adult human testis

Human primordial germ cells and mouse neonatal and adult germline stem cells are pluripotent and show similar properties to embryonic stem cells. Here we report the successful establishment of human adult germline stem cells derived from spermatogonial cells of adult human testis. Cellular and molecular characterization of these cells revealed many similarities to human embryonic stem cells, and the germline stem cells produced teratomas after transplantation into immunodeficient mice. The human adult germline stem cells differentiated into various types of somatic cells of all three germ layers when grown under conditions used to induce the differentiation of human embryonic stem cells. We conclude that the generation of human adult germline stem cells from testicular biopsies may provide simple and non-controversial access to individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells.

Dopaminergic properties and function after grafting of attached neural precursor cultures

Generation of dopaminergic (DA) neurons from multipotent embryonic progenitors represents a promising therapeutical strategy for Parkinsons disease (PD). Aim of the present study was the establishment of enhanced cell culture conditions, which optimize the use of midbrain progenitor cells in animal models of PD. In addition, the progenitor cells were characterized during expansion and differentiation according to morphological and electrophysiological criteria and compared to primary tissue. Here, we report that CNS precursors can be expanded in vitro up to 40-fold and afterwards be efficiently differentiated into DA neurons. After 4-5 days under differentiation conditions, more than 70% of the neurons were TH+, equivalent to 30% of the total cell population. Calcium imaging revealed the presence of calcium-permeable AMPA receptors in the differentiated precursors which are capable to contribute to many developmental processes. The overall survival rate, degree of reinnervation and the behavioral performance after transplantation of 4 days in-vitro-differentiated cells were similar to results after direct grafting of E14 ventral mesencephalic cells, whereas after shorter or longer differentiation periods, respectively, less effects were achieved. Compared to the amount of in-vitro-generated DA neurons, the survival rate was only 0.8%, indicating that these cells are very vulnerable. Our results suggest that expanded and differentiated DA precursors from attached cultures can survive microtransplantation and integrate within the striatum in terms of behavioral recovery. However, there is only a short time window during in vitro differentiation, in which enough cells are already differentiated towards a DA phenotype and simultaneously not too mature for implantation. However, additional factors and/or genetical manipulation of these expanded progenitors will be required to increase their in vivo survival in order to improve both the ethical and the technical outlook for the use of fetal tissue in clinical transplantation.

Glucocorticoid adrenal steroids and glucocorticoid-inducible kinase isoforms in the regulation of GluR6 expression

Generation of memory is enhanced during stress, an effect attributed to stimulation of neuronal learning by adrenal glucocorticoids. The glucocorticoid‐dependent genes include the serum‐ and glucocorticoid‐inducible kinase SGK1. SGK1 is activated through the phosphatidylinositol 3 kinase (PI3‐kinase) pathway by growth factors such as insulin‐like growth factor‐1 (IGF1) or tumour growth factor β (TGF‐β). Previously, a fourfold higher expression of SGK1 has been observed in fast‐learning rats as compared with slow‐learning rats. The mechanisms linking glucocorticoids or SGK1 with neuronal function have, however, remained elusive. We show here that treatment of mice with the glucocorticoid dexamethasone (238 μg day−1 for 8–20 days) enhances hippocampal expression of GluR6. Immunohistochemistry reveals significantly enhanced GluR6 protein abundance at neurones but not at astrocytes in mice. Immunohistochemistry and patch clamp on hippocampal neurones in primary culture reveal upregulation of GluR6 protein abundance and kainate‐induced currents following treatment with dexamethasone (1 μm) and TGF‐β (1 μm). In Xenopus oocytes expressing rat GluR6, coexpression of SGK1 strongly increases glutamate‐induced current at least partially by increasing the abundance of GluR6 protein in the plasma membrane. The related kinases SGK2 and SGK3 similarly stimulate GluR6, but are less effective than SGK1. The observations point to a novel mechanism regulating GluR6 which contributes to the regulation of neuronal function by glucocorticoids.

Regulation of GluR1 abundance in murine hippocampal neurones by serum‐ and glucocorticoid‐inducible kinase 3

Phosphatidylinositol 3 kinase (PI3‐kinase) is activated during and is required for hippocampal glutamate receptor‐dependent long‐term potentiation. It mediates the delivery of AMPA receptors to the neuronal surface. Among the downstream targets of PI3‐kinase are three members of the serum‐ and glucocorticoid‐inducible kinase family, SGK1, SGK2 and SGK3. In Xenopus oocytes expressing the AMPA subunit GluR1, we show that SGK3, and to a lesser extent SGK2, but not SGK1, increase glutamate‐induced currents by increasing the abundance of GluR1 protein in the cell membrane. We further show Sgk3 mRNA expression in the hippocampus by RT‐PCR and in situ hybridization. According to Western blotting, the hippocampal abundance of GluR1 is significantly lower in gene‐targeted mice lacking SGK3 (Sgk3−/−) than in their wild‐type littermates (Sgk3+/+). The present observations disclose a novel mechanism in the regulation of GluR1.

Identification of human cells in brain xenografts and in neural co-cultures of rat by in situ hybridisation with Alu probe

Transplantation of human cells into animal models of neurodegenerative disorders is an important scientific application to analyse the survival and developmental capacity of grafted human cells under in vivo conditions. It is critical, therefore, to have a reliable method to distinguish between human and animal cells. In the present study, we describe a combined in situ hybridisation and immunocytochemistry method for the identification of human cells in cultured rat brain cells and xenografts. The specific Alu probe we utilised, which corresponds to the consensus sequence of human Alu repeats was evaluated by southern blot hybridisation of zoo blot and by in situ hybridisation of primary and neoplastic cells from man, rat, mouse, and hamster. This method allows a definite identification of human cells in neural xenografts and, in combination with additional in situ techniques, a further detection of grafted cells.

Human SK-Mel 28 melanoma cells resume neural crest cell migration after transplantation into the chick embryo.

Melanocytes are derived from the neural crest. We questioned whether the migratory mechanism during the invasive growth of melanoma cells is the same as that in neural crest cell migration. We transplanted human SK-Mel 28 melanoma cells into the neural tube of the chick embryo stage 11–13 and, after up to 6 days of total incubation, traced the cells by immunohistochemistry in serial paraffin sections. SK-Mel 28 cells were integrated into the host neural crest and were found in the roof plate of the neural tube, along the medial neural crest cell pathway, in the sclerotome and, finally, in developing sympathetic ganglia. At stage 21, massive segmental emigration between myotome and disintegrating dermatome was observed at the level of the upper limb bud. The melanoma cells, in contrast with the chick neural crest cells, were HNK-1-negative. They retained the premelanosome epitope HMB-45. For definite identification and exclusion of fusion with chick embryo cells, in situ hybridization with the human-specific Alu sequence was performed. The results showed that human SK-Mel 28 melanoma cells were capable of resuming neural crest cell migration in the embryo.

Decreased intestinal glucose transport in the sgk3-knockout mouse

Xenopus oocyte coexpression experiments revealed the capacity of the serum- and glucocorticoid-inducible kinase isoform 3 (SGK3) to up-regulate a variety of transport systems including the sodium-dependent glucose transporter SGLT1. The present study explored the functional significance of SGK3-dependent regulation of intestinal transport. To this end, experiments were performed in gene targeted mice lacking functional sgk3 (sgk3−/−) and their wild type littermates (sgk3+/+). Oral food intake and fecal dry weight were significantly larger in sgk3−/− than in sgk3+/+ mice. Glucose-induced current (Ig) in Ussing chamber as a measure of Na+ coupled glucose transport was significantly smaller in sgk3−/− than in sgk3+/+ mouse jejunal segments. Fasting plasma glucose concentrations were significantly lower in sgk3−/− than in sgk3+/+ mice. Intestinal electrogenic transport of phenylalanine, cysteine, glutamine and proline were not significantly different between sgk3−/− and sgk3+/+ mice. In conclusion, SGK3 is required for adequate intestinal Na+ coupled glucose transport and impaired glucose absorption may contribute to delayed growth and decreased plasma glucose concentrations of SGK3 deficient mice. The hypoglycemia might lead to enhanced food intake to compensate for impaired intestinal absorption.

Isolation and Culture of Ventral Mesencephalic Precursor Cells and Dopaminergic Neurons from Rodent Brains

The ability to isolate ventral midbrain (VM) precursor cells and neurons provides a powerful means to characterize their differentiation properties and to study their potential for restoring dopamine (DA) neurons degenerated in Parkinsons disease (PD). Preparation and maintenance of DA VM in primary culture involves a number of critical steps to yield healthy cells and appropriate data. Here, we offer a detailed description of protocols to consistently prepare VM DA cultures from rat and mouse embryonic fetal-stage midbrain. We also present methods for organotypic culture of midbrain tissue, for differentiation as aggregate cultures, and for adherent culture systems of DA differentiation and maturation, followed by a synopsis of relevant analytical read-out options. Isolation and culture of rodent VM precursor cells and DA neurons can be exploited for studies of DA lineage development, of neuroprotection, and of cell therapeutic approaches in animal models of PD.

In vivo biocompatibility and biodegradation of a novel thin and mechanically stable collagen scaffold.

The demand for scaffolds comprised of natural materials such as collagen has increased in recent years. However, many scaffolds rely on chemical or physical modifications in order to comply with the necessary requirements for biomedical engineering. We evaluated the in vivo biocompatibility and biodegradation of a novel, thin, mechanically stable, and chemically non-crosslinked collagen cell carrier (CCC). CCC was implanted subcutaneously into 25 adult Lewis rats and biopsies were taken on days 7, 14, 21, 42, and 84 after surgery. For histological analysis, paraffin sections of implanted skin were immunolabeled for CD68 and stained by hematoxylin-eosin and Masson-Goldners trichrome method. Macroscopic analysis of skin surface during wound healing process showed a normal physiological reaction. Biodegradation of CCC was completed 42 days after subcutaneous implantation. Histological evaluation revealed no evidence of encapsulation, scar formation, or long-term vascularization and inflammation. The collagen type I based biomaterial demonstrated a high in vivo biocompatibility, low irritability, complete resorption, and replacement by autologous tissue. The in vivo biocompatibility and degradation behavior encourage for further evaluation of CCC in surgical applications and regenerative medicine.