Improved method of producing human neural progenitor cells of high purity and in large quantities from pluripotent stem cells for transplantation studies.

Transplantation of human neural progenitor cells (hNPCs) is a promising therapeutic approach for various diseases of the central nervous system (CNS). Reliable testing of hNPC transplantation in animal models of neurological diseases requires that these cells can be produced in sufficient amounts, show consistent homogeneity as a neural cell population, and be reliably labeled for in vivo tracking. In addition, the cells should be characterized as being at the optimal state of differentiation favoring successful engraftment. Here, we show that high numbers of purified hNPCs can be produced from human embryonic stem cells (hESCs) by manually selecting specifically sized and shaped spheres followed by fluorescence-activated cell sorting based on the relative cell size. In addition, we report that labeling of hNPCs with ultra-small superparamagnetic iron oxide (USPIO) particles does not affect the cellular morphology or growth. More importantly, we show that the transduction with lentiviral vector encoding green fluorescent protein (GFP) decreases the neurality of the cell population. We conclude that our cost-effective protocol of generating hNPCs is widely applicable for preclinical studies on CNS disorders. This improved method of producing large quantities of high-purity hNPCs maybe useful also when generating hNPCs from human induced pluripotent stem (hiPS) cell lines. However, caution should be used when lenti-GFP transduction is applied for hNPC labeling.

Multiple cellular and molecular mechanisms are involved in human Aß clearance by transplanted adult astrocytes.

Astrocytes and microglia are able to degrade potentially neurotoxic ß-amyloid (Aß) deposits typical for Alzheimer's disease (AD) pathology. Contrary to microglia, astrocytes degrade human Aß from tissue sections in vitro without any additional stimulation, but it has remained unclear whether transplanted astrocytes are able to clear deposited human Aß in vivo. We transplanted adult mouse astrocytes into the hippocampi of transgenic mice mimicking AD and observed their fate, effects on microglial responses, and Aß clearance. After 2-months follow-up time, we discovered a significant reduction in Aß burden compared with AD mice infused with PBS only. The remaining Aß deposits were fragmented and most of the Aß immunoreactivity was seen within the transplanted astrocytes. Concomitant to Aß reduction, both CD68 and CD45 immunoreactivities were significantly upregulated but phagocytic microglia were often surrounding and engulfing Aß burdened, TUNEL-positive astrocytes rather than co-localizing with Aß alone. Astrocytes are known to degrade Aß also by secreting proteases involved in Aß catabolism. To study the contribution of neprilysin (NEP), angiotensin-converting enzyme-1 (ACE-1), and endothelin-converting enzyme-2 (ECE-2) in human Aß clearance, we utilized an ex vivo assay to demonstrate that adult astrocytes respond to human Aß by upregulating NEP expression. Further, incubation of adult astrocytes with known inhibitors of NEP, ACE-1, or ECE-2 significantly inhibited the removal of human Aß from the tissue suggesting an important role for these proteases in Aß clearance by adult astrocytes ex vivo.