A method for rapid derivation and propagation of neural progenitors from human embryonic stem cells.

Neuronal loss is a common feature of many neurological disorders, including stroke, Parkinson's disease, Alzheimer's disease and traumatic brain injury. Human embryonic stem cell (hESC)-derived neural progenitors (NPs) may provide new ways of treatment for several diseases and injuries in the brain, as well as enhance our understanding of early human development. Here we report a method for rapid generation of proliferating NPs from feeder free cultures of undifferentiated hESCs. In this rapid and simple protocol, NPs are derived by seeding undifferentiated hESC on adherent surfaces of laminin or gelatine with normal hESC culturing medium and with the addition of basic fibroblast growth factor. After the first passage, adherent monolayer progenitors are derived that express early neuroectodermal and progenitor markers, such as Nestin, Sox1, Sox2, Sox3, Internexin, Musashi-1, NCAM, and Pax6. This novel protocol renders hESCs suitable for large scale progenitor production and long-term propagation, and the progenitors have the capacity to differentiate in vitro into all three neural lineages (neurons, astrocytes and oligodendrocytes). This method allows rapid, cost-efficient production of expandable progenitors that may be a source of cells for the restoration of cellular and functional loss after neurodegeneration and/or provide a useful source of progenitor cells for studying early brain development.

Electrospun polyurethane scaffolds for proliferation and neuronal differentiation of human embryonic stem cells.

Adult central nervous system (CNS) tissue has a limited capacity to recover after trauma or disease. Hence, tissue engineering scaffolds intended for CNS repair and rehabilitation have been subject to intense research effort. Electrospun porous scaffolds, mimicking the natural three-dimensional environment of the in vivo extracellular matrix (ECM) and providing physical support, have been identified as promising candidates for CNS tissue engineering. The present study demonstrates in vitro culturing and neuronal differentiation of human embryonic stem cells (hESCs) on electrospun fibrous polyurethane scaffolds. Electrospun scaffolds composed of biocompatible polyurethane resin (Desmopan 9370A, Bayer MaterialScience AG) were prepared with a vertical electrospinning setup. Resulting scaffolds, with a thickness of approximately 150 microm, exhibited high porosity (84%) and a bimodal pore size distribution with peaks at 5-6 and 1 microm. The mean fiber diameter was measured to approximately 360 nm with a standard deviation of 80 nm. The undifferentiated hESC line SA002 (Cellartis AB, Göteborg, Sweden) was seeded and cultured on the produced scaffolds and allowed propagation and then differentiation for up to 47 days. Cultivation of hESC on electrospun fibrous scaffolds proved successful and neuronal differentiation was observed via standard immunocytochemistry. The results indicate that predominantly dopaminergic tyrosine hydroxylase (TH) positive neurons are derived in co-culture with fibrous scaffolds, in comparison to reference cultures under the same differentiation conditions displaying large proportions of GFAP positive cell types. Scanning electron micrographs confirm neurite outgrowth and connection to adjacent cells, as well as cell attachment to individual fibers of the fibrous scaffold. Consequently, electrospun polyurethane scaffolds have been proven feasible as a substrate for hESC propagation and neuronal differentiation. The physical interaction between cells and the fibrous scaffold indicates that these scaffolds provide a three-dimensional physical structure; a potential candidate for neural tissue engineering repair and rehabilitation.

Human embryonic stem cell-derived mesenchymal progenitors--potential in regenerative medicine.

Tissue engineering and cell therapy require large-scale production of homogeneous populations of lineage-restricted progenitor cells that easily can be induced to differentiate into a specific tissue. We have developed straightforward protocols for the establishment of human embryonic stem (hES) cell-derived mesenchymal progenitor (hES-MP) cell lines. The reproducibility was proven by derivation of multiple hES-MP cell lines from 10 different hES cell lines. To illustrate clinical applicability, a xeno-free hES-MP cell line was also derived. None of the markers characteristic for undifferentiated hES cells were detected in the hES-MP cells. Instead, these cells were highly similar to mesenchymal stem cells with regard to morphology and expression of markers. The safety of hES-MP cells following transplantation was studied in severely combined immunodeficient (SCID) mice. The implanted hES-MP cells gave rise to homogeneous, well-differentiated tissues exclusively of mesenchymal origin and no teratoma formation was observed. These cells further have the potential to differentiate toward the osteogenic, adipogenic, and chondrogenic lineages in vitro. The possibility of easily and reproducibly generating highly expandable hES-MP cell lines from well-characterized hES cell lines with differentiation potential into several mesodermal tissues entails an enormous potential for the field of regenerative medicine.

Human neuroblasts migrate to the olfactory bulb via a lateral ventricular extension.

The rostral migratory stream (RMS) is the main pathway by which newly born subventricular zone cells reach the olfactory bulb (OB) in rodents. However, the RMS in the adult human brain has been elusive. We demonstrate the presence of a human RMS, which is unexpectedly organized around a lateral ventricular extension reaching the OB, and illustrate the neuroblasts in it. The RMS ensheathing the lateral olfactory ventricular extension, as seen by magnetic resonance imaging, cell-specific markers, and electron microscopy, contains progenitor cells with migratory characteristics and cells that incorporate 5-bromo-2'-deoxyuridine and become mature neurons in the OB.