Magnetic Particle Imaging tracks the long-term fate of in vivo neural cell implants with high image contrast.

We demonstrate that Magnetic Particle Imaging (MPI) enables monitoring of cellular grafts with high contrast, sensitivity, and quantitativeness. MPI directly detects the intense magnetization of iron-oxide tracers using low-frequency magnetic fields. MPI is safe, noninvasive and offers superb sensitivity, with great promise for clinical translation and quantitative single-cell tracking. Here we report the first MPI cell tracking study, showing 200-cell detection in vitro and in vivo monitoring of human neural graft clearance over 87 days in rat brain

hESC-secreted proteins can be enriched for multiple regenerative therapies by heparin-binding.

This work builds upon our findings that proteins secreted by hESCs exhibit pro-regenerative activity, and determines that hESC-conditioned medium robustly enhances the proliferation of both muscle and neural progenitor cells. Importantly, this work establishes that it is the proteins that bind heparin which are responsible for the pro-myogenic effects of hESC-conditioned medium, and indicates that this strategy is suitable for enriching the potentially therapeutic factors. Additionally, this work shows that hESC-secreted proteins act independently of the mitogen FGF-2, and suggests that FGF-2 is unlikely to be a pro-aging molecule in the physiological decline of old muscle repair. Moreover, hESC-secreted factors improve the viability of human cortical neurons in an Alzheimers disease (AD) model, suggesting that these factors can enhance the maintenance and regeneration of multiple tissues in the aging body

Directed Evolution of Adeno-associated Virus for Enhanced Gene Delivery and Gene Targeting in Human Pluripotent Stem Cells

Efficient approaches for the precise genetic engineering of human pluripotent stem cells (hPSCs) can enhance both basic and applied stem cell research. Adenoassociated virus (AAV) vectors are of particular interest for their capacity to mediate efficient gene delivery to and gene targeting in various cells. However, natural AAV serotypes offer only modest transduction of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), which limits their utility for efficiently manipulating the hPSC genome. Directed evolution is a powerful means to generate viral vectors with novel capabilities, and we have applied this approach to create a novel AAV variant with high gene delivery efficiencies (~50%) to hPSCs, which are importantly accompanied by a considerable increase in gene-targeting frequencies, up to 0.12%. While this level is likely sufficient for numerous applications, we also show that the gene-targeting efficiency mediated by an evolved AAV variant can be further enhanced (\u3e1%) in the presence of targeted double stranded breaks (DSBs) generated by the co-delivery of artificial zinc finger nucleases (ZFNs). Thus, this study demonstrates that under appropriate selective pressures, AAV vectors can be created to mediate efficient gene targeting in hPSCs, alone or in the presence of ZFNmediated double-stranded DNA breaks

High-Density Microwell Chip for Culture and Analysis of Stem Cells

With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)-differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field

Gene Expression Profile of Neuronal Progenitor Cells Derived from hESCs: Activation of Chromosome 11p15.5 and Comparison to Human Dopaminergic Neurons

BACKGROUND: We initiated differentiation of human embryonic stem cells (hESCs) into dopamine neurons, obtained a purified population of neuronal precursor cells by cell sorting, and determined patterns of gene transcription. METHODOLOGY: Dopaminergic differentiation of hESCs was initiated by culturing hESCs with a feeder layer of PA6 cells. Differentiating cells were then sorted to obtain a pure population of PSA-NCAM-expressing neuronal precursors, which were then analyzed for gene expression using Massive Parallel Signature Sequencing (MPSS). Individual genes as well as regions of the genome which were activated were determined. PRINCIPAL FINDINGS: A number of genes known to be involved in the specification of dopaminergic neurons, including MSX1, CDKN1C, Pitx1 and Pitx2, as well as several novel genes not previously associated with dopaminergic differentiation, were expressed. Notably, we found that a specific region of the genome located on chromosome 11p15.5 was highly activated. This region contains several genes which have previously been associated with the function of dopaminergic neurons, including the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, IGF2, and CDKN1C, which cooperates with Nurr1 in directing the differentiation of dopaminergic neurons. Other genes in this region not previously recognized as being involved in the functions of dopaminergic neurons were also activated, including H19, TSSC4, and HBG2. IGF2 and CDKN1C were also found to be highly expressed in mature human TH-positive dopamine neurons isolated from human brain samples by laser capture. CONCLUSIONS: The present data suggest that the H19-IGF2 imprinting region on chromosome 11p15.5 is involved in the process through which undifferentiated cells are specified to become neuronal precursors and/or dopaminergic neurons

Generation of Dopaminergic Neurons from Human Embryonic Stem Cells

Since the first successful derivation of human embryonic stem cells (hESC), rapid progress has been attained in the development of strategies in differentiation of these cells into various neural lineages, with the fundamental objective of using these cells for replacement and repair of damaged neuronal circuits in the central nervous system (CNS). Of particular interest are midbrain dopaminergic (mDA) neurons, which play a central role in regulation of voluntary movement. Degeneration or loss of function of mDA neurons in the nigrostriatal pathway is associated with Parkinson disease (PD). Stromal-Derived Inducing Activity (SDIA) is recognized as one of the most efficient methods in restricting ESC differentiation to a dopaminergic lineage, and refers to the property of mouse stromal cell lines such as PA6 or MS5 to cause ESC to differentiate to DA neurons. Although this strategy has been extensively used to generate mDA neurons from hESC, the biochemical nature of SDIA is yet unknown.  In the present study mDA neurons were generated from the BG01V2 hESC line by SDIA. To examine whether SDIA exerts its effect directly on hESC and is responsible for early dopaminergic induction, neural progenitor cells (NPC) were enyzmatically isolated from the co-cultures and allowed to differentiate in feeder-free conditions. The isolated cells were committed to a mesencephalic neural lineage, and were capable of maintaining their phenotype and developing into postmitotic mDA neurons in feeder-free conditions. The mDA neurons showed neuronal excitability and dopamine transporter function. The in vitro proliferation and differentiation of the NPC was also investigated by a BrDU incorporation assay. Next, the maintenance of cellular memory and capacity for proliferation of the mesencephalic NPC was assessed. The NPC could be expanded in vitro by five-fold as neurospheres for up to two weeks while retaining their DA differentiation potential, but did not retain a stable phenotype over extended periods of time. Preliminary transplantation experiments of neurospheres in striatal lesioned animals indicated, however, that these cells could survive and conserve their phenotype in vivo. To gain additional insight into the biochemical role of SDIA in early dopaminergic induction of hESC, the separate contributions of cell surface activity and secreted factors were examined. The data revealed that the PA6 cell surface activity promoted cell survival and was mainly responsible for enhanced neurogenesis of hESC, whereas secreted factors provided DA lineage-specific instructions. In order to identify the soluble factors responsible for the DA phenotype-inducing component of SDIA, the gene expression profile of PA6 cells was compared to that of cell lines lacking the DA-inducing property. A number of soluble factors known to be associated with CNS development that were highly expressed in PA6 cells were identified as potential DA differentiation-inducing candidates. These differentially-expressed genes included stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). When these factors, termed SPIE, were applied to the hESC, they induced dopaminergic neuronal differentiation of hESC line, BG01V2 and other karyotypically normal hESC lines in vitro. Thus, it appears that SPIE comprises the DA phenotype-inducing property of SDIA. This may provide a simple and direct means of differentiating hESC to form DA neurons in a single step, without a requirement for co-culture, animal cell lines, or animal products.QC 2010091

A novel combination of factors, termed SPIE, which promotes dopaminergic neuron differentiation from human embryonic stem cells.

BACKGROUND:Stromal-Derived Inducing Activity (SDIA) is one of the most efficient methods of generating dopaminergic (DA) neurons from embryonic stem cells (ESC). DA neuron induction can be achieved by co-culturing ESC with the mouse stromal cell lines PA6 or MS5. The molecular nature of this effect, which has been termed "SDIA" is so far unknown. Recently, we found that factors secreted by PA6 cells provided lineage-specific instructions to induce DA differentiation of human ESC (hESC). METHODOLOGY/PRINCIPAL FINDINGS:In the present study, we compared PA6 cells to various cell lines lacking the SDIA effect, and employed genome expression analysis to identify differentially-expressed signaling molecules. Among the factors highly expressed by PA6 cells, and known to be associated with CNS development, were stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). When these four factors, the combination of which was termed SPIE, were applied to hESC, they induced differentiation to TH-positive neurons in vitro. RT-PCR and western blot analysis confirmed the expression of midbrain specific markers, including engrailed 1, Nurr1, Pitx3, and dopamine transporter (DAT) in cultures influenced by these four molecules. Electrophysiological recordings showed that treatment of hESC with SPIE induced differentiation of neurons that were capable of generating action potentials and forming functional synaptic connections. CONCLUSIONS/SIGNIFICANCE:The combination of SDF-1, PTN, IGF2, and EFNB1 mimics the DA phenotype-inducing property of SDIA and was sufficient to promote differentiation of hESC to functional midbrain DA neurons. These findings provide a method for differentiating hESC to form DA neurons, without a requirement for the use of animal-derived cell lines or products

hESC-secreted proteins can be enriched for multiple regenerative therapies by heparin-binding.

This work builds upon our findings that proteins secreted by hESCs exhibit pro-regenerative activity, and determines that hESC-conditioned medium robustly enhances the proliferation of both muscle and neural progenitor cells. Importantly, this work establishes that it is the proteins that bind heparin which are responsible for the pro-myogenic effects of hESC-conditioned medium, and indicates that this strategy is suitable for enriching the potentially therapeutic factors. Additionally, this work shows that hESC-secreted proteins act independently of the mitogen FGF-2, and suggests that FGF-2 is unlikely to be a pro-aging molecule in the physiological decline of old muscle repair. Moreover, hESC-secreted factors improve the viability of human cortical neurons in an Alzheimer's disease (AD) model, suggesting that these factors can enhance the maintenance and regeneration of multiple tissues in the aging body