Tandis Vazin

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. Adeno- associated 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 (>1%) 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 ZFN- mediated double-stranded DNA breaks.

Human embryonic stem cells: Derivation, culture, and differentiation: A review

The greatest therapeutic promise of human embryonic stem cells (hESC) is to generate specialized cells to replace damaged tissue in patients suffering from various degenerative diseases. However, the signaling mechanisms involved in lineage restriction of ESC to adopt various cellular phenotypes are still under investigation. Furthermore, for progression of hESC-based therapies towards clinical applications, appropriate culture conditions must be developed to generate genetically stable homogenous populations of cells, to hinder possible adverse effects following transplantation. Other critical challenges that must be addressed for successful cell implantation include problems related to survival and functional efficacy of the grafted cells. This review initially describes the derivation of hESC and focuses on recent advances in generation, characterization, and maintenance of these cells. We also give an overview of original and emerging differentiation strategies used to convert hESC to different cell types. Finally, we will discuss transplantation studies of hESC-derived cells with respect to safety and functional recovery.

Multivalent ligands control stem cell behaviour in vitro and in vivo

There is broad interest in designing nanostructured materials that can interact with cells and regulate key downstream functions1–7. In particular, materials with nanoscale features may enable control over multivalent interactions, which involve the simultaneous binding of multiple ligands on one entity to multiple receptors on another and are ubiquitous throughout biology8–10. Cellular signal transduction of growth factor and morphogen cues that play critical roles in regulating cell function and fate often begins with such multivalent binding of ligands, either secreted or cell-surface tethered, to target cell receptors, leading to receptor clustering11–18. Cellular mechanisms that orchestrate ligand-receptor oligomerisation are complex, however, and the capacity to control multivalent interactions and thereby modulate key signaling events within living systems is therefore currently very limited. Here we demonstrate the design of potent multivalent conjugates that can organise stem cell receptors into nanoscale clusters and control stem cell behaviour in vitro and in vivo. The ectodomain of ephrin-B2, normally an integral membrane protein ligand, was conjugated to a soluble biopolymer to yield multivalent nanoscale conjugates that potently induced signaling in neural stem cells and promoted their neuronal differentiation both in culture and within the brain. Super-resolution microscopy analysis yielded insights into the organisation of receptor-ligand clusters at the nanoscale. We also found that synthetic multivalent conjugates of ephrin-B1 strongly enhanced human embryonic and induced pluripotent stem cell differentiation into functional dopaminergic neurons. Multivalent bioconjugates thus represent powerful tools and potential nanoscale therapeutics for controlling the behaviour of target stem cells in vitro and in vivo.

Quantitative Magnetic Particle Imaging Monitors the Transplantation, Biodistribution, and Clearance of Stem Cells In Vivo

Stem cell therapies have enormous potential for treating many debilitating diseases, including heart failure, stroke and traumatic brain injury. For maximal efficacy, these therapies require targeted cell delivery to specific tissues followed by successful cell engraftment. However, targeted delivery remains an open challenge. As one example, it is common for intravenous deliveries of mesenchymal stem cells (MSCs) to become entrapped in lung microvasculature instead of the target tissue. Hence, a robust, quantitative imaging method would be essential for developing efficacious cell therapies. Here we show that Magnetic Particle Imaging (MPI), a novel technique that directly images iron-oxide nanoparticle-tagged cells, can longitudinally monitor and quantify MSC administration in vivo. MPI offers near-ideal image contrast, depth penetration, and robustness; these properties make MPI both ultra-sensitive and linearly quantitative. Here, we imaged, for the first time, the dynamic trafficking of intravenous MSC administrations using MPI. Our results indicate that labeled MSC injections are immediately entrapped in lung tissue and then clear to the liver within one day, whereas standard iron oxide particle (Resovist) injections are immediately taken up by liver and spleen. Longitudinal MPI-CT imaging also indicated a clearance half-life of MSC iron oxide labels in the liver at 4.6 days. Finally, our ex vivo MPI biodistribution measurements of iron in liver, spleen, heart, and lungs after injection showed excellent agreement (R2 = 0.943) with measurements from induction coupled plasma spectrometry. These results demonstrate that MPI offers strong utility for noninvasively imaging and quantifying the systemic distribution of cell therapies and other therapeutic agents.

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.

The effect of multivalent Sonic hedgehog on differentiation of human embryonic stem cells into dopaminergic and GABAergic neurons

Stem cell differentiation is regulated by complex repertoires of signaling ligands which often use multivalent interactions, where multiple ligands tethered to one entity interact with multiple cellular receptors to yield oligomeric complexes. One such ligand is Sonic hedgehog (Shh), whose posttranslational lipid modifications and assembly into multimers enhance its biological potency, potentially through receptor clustering. Investigations of Shh typically utilize recombinant, monomeric protein, and thus the impact of multivalency on ligand potency is unexplored. Among its many activities, Shh is required for ventralization of the midbrain and forebrain and is therefore critical for the development of midbrain dopaminergic (mDA) and forebrain gamma-aminobutyric acid (GABA) inhibitory neurons. We have designed multivalent biomaterials presenting Shh in defined spatial arrangements and investigated the role of Shh valency in ventral specification of human embryonic stem cells (hESCs) into these therapeutically relevant cell types. Multivalent Shh conjugates with optimal valencies, compared to the monomeric Shh, increased the percentages of neurons belonging to mDA or forebrain GABAergic fates from 33% to 60% or 52% to 86%, respectively. Thus, multivalent Shh bioconjugates can enhance neuronal lineage commitment of pluripotent stem cells and thereby facilitate efficient derivation of neurons that could be used to treat Parkinsons and epilepsy patients.

Quantitative stem cell imaging with magnetic particle imaging

MPI stem cell imaging - sensitivity and linearity: 9 pelletized, labeled hESC-derived cell populations (Fig. 2A) with varying cell numbers were imaged successively in the FFL imager. Fig. 2B shows the maximum image intensity from each reconstructed cell pellet image as a function of cell number. When a linear fit was applied, we found a strong linear correlation between MPI signal intensity and cell number, with R2 > 0.99. Additionally, the detection sensitivity of the projection MPI system was found to be slightly over 104 cells. We found no detectable MPI signal from unlabeled cell populations. Mice imaging: A MPI image of postmortem cell injections containing 4×105 and 6×105 is shown in Fig. 2C. The MPI image shows excellent contrast with minimal tissue effects. Additionally, the ratio of total MPI signal between the 6×105 and 4×105 injection regions was found to be 1.5, as expected.

In vivo magnetic nanoparticle cytometer for stem cells in small animals

Parkinsons Disease (PD) is a degenerative disorder that causes the malfunction and death of neurons in the substantia nigra and afflicts hundreds of thousands of Americans. There is no known cure for PD. However, stem cell transplant therapy holds promise for reversing the effects of PD. Preclinically, the mouse model of stem cell therapy for PD is used to assess stem cell viability and analyze neuron regeneration in the substantia nigra. However, tracking stem cell viability in these experiments remains difficult at best because conventional imaging methods may inaccurately quantify stem cell number in vivo due to penetration depth or other effects. Here, we propose a new magnetic nanoparticle cytometer for small animals to quantify cells implanted in vivo labeled with superparamagnetic iron oxide (SPIO) nanoparticles. This cytometer, which uses MPI principles, is well-suited for determining stem cell viability as it provides linear signals for accurate quantification of cell number in vivo without suffering from depth or tissue attenuation or relying on ionizing radiation or radioactive materials with short half-lives.