Nadja Zeltner

Large-scale screening using familial dysautonomia induced pluripotent stem cells identifies compounds that rescue IKBKAP expression.

Patient-specific induced pluripotent stem cells (iPSCs) represent a novel system for modeling human genetic disease and could develop into a key drug discovery platform. We recently reported disease-specific phenotypes in iPSCs from familial dysautonomia (FD) patients. FD is a rare but fatal genetic disorder affecting neural crest lineages. Here we demonstrate the feasibility of performing a primary screen in FD-iPSC derived neural crest precursors. Out of 6,912 compounds tested we characterized 8 hits that rescue expression of IKBKAP, the gene responsible for FD. One of those hits, SKF-86466, is shown to induce IKBKAP transcription via modulation of intracellular cAMP levels and PKA dependent CREB phosphorylation. SKF-86466 also rescues IKAP protein expression and the disease-specific loss of autonomic neuron marker expression. Our data implicate alpha-2 adrenergic receptor activity in regulating IKBKAP expression and demonstrate that small molecule discovery in an iPSC-based disease model can identify candidate drugs for potential therapeutic intervention.Patient-specific induced pluripotent stem cells (iPSCs) represent a novel system for modeling human genetic disease and could provide a source of cells for large-scale drug-discovery screens. Here we demonstrate the feasibility of performing a primary screen in neural crest precursors derived from iPSCs that were generated from individuals with familial dysautonomia (FD), a rare, fatal genetic disorder affecting neural crest lineages. We tested 6,912 small-molecule compounds and characterized eight that rescued expression of IKBKAP, the gene responsible for FD. One of the hits, SKF-86466, was found to induce IKBKAP transcription through modulation of intracellular cAMP levels and PKA-dependent CREB phosphorylation. SKF-86466 also rescued IKAP protein expression and the disease-specific loss of autonomic neuronal marker expression. Our data implicate alpha-2 adrenergic receptor activity in regulating IKBKAP expression and demonstrate that small-molecule discovery using an iPSC-based disease model can identify candidate drugs for potential therapeutic intervention.

Deriving human ENS lineages for cell therapy and drug discovery in Hirschsprung disease

The enteric nervous system (ENS) is the largest component of the autonomic nervous system, with neuron numbers surpassing those present in the spinal cord. The ENS has been called the ‘second brain’ given its autonomy, remarkable neurotransmitter diversity and complex cytoarchitecture. Defects in ENS development are responsible for many human disorders including Hirschsprung disease (HSCR). HSCR is caused by the developmental failure of ENS progenitors to migrate into the gastrointestinal tract, particularly the distal colon. Human ENS development remains poorly understood owing to the lack of an easily accessible model system. Here we demonstrate the efficient derivation and isolation of ENS progenitors from human pluripotent stem (PS) cells, and their further differentiation into functional enteric neurons. ENS precursors derived in vitro are capable of targeted migration in the developing chick embryo and extensive colonization of the adult mouse colon. The in vivo engraftment and migration of human PS-cell-derived ENS precursors rescue disease-related mortality in HSCR mice (Ednrbs-l/s-l), although the mechanism of action remains unclear. Finally, EDNRB-null mutant ENS precursors enable modelling of HSCR-related migration defects, and the identification of pepstatin A as a candidate therapeutic target. Our study establishes the first, to our knowledge, human PS-cell-based platform for the study of human ENS development, and presents cell- and drug-based strategies for the treatment of HSCR.

Site-specific integration of adeno-associated virus involves partial duplication of the target locus

A variety of viruses establish latency by integrating their genome into the host genome. The integration event generally occurs in a nonspecific manner, precluding the prediction of functional consequences from resulting disruptions of affected host genes. The nonpathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene. To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the target gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted target gene could potentially be preserved despite the resulting rearrangements. A latently infected, Mbs85-targeted mouse ES cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, maintained their multilineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a viral strategy for targeted genome addition with the apparent absence of functional consequences.

Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells

Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions to rapidly differentiate hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born cortical neurons. Combinatorial application of six pathway inhibitors induces post-mitotic cortical neurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 d of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole-brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders.

Near-perfect infectivity of wild-type AAV as benchmark for infectivity of recombinant AAV vectors

Viral vectors derived from adeno-associated viruses (AAVs) are widely used for gene transfer both in vitro and in vivo. The increasing use of AAV as a gene transfer vector, as well as recently shown immunological complications in clinical trials, highlight the necessity to define the specific activity of vector preparations beyond current standards. In this report, we determined the infectious, physical and genome-containing particle titers of several wild-type AAV type 2 (wtAAV2) and recombinant AAV type 2 (rAAV2) preparations that were produced and purified by standard methods. We found that the infectivity of wtAAV2 approaches a physical-to-infectious particle ratio of one. This near-perfect physical-to-infectious particle ratio defines a ‘ceiling’ for the theoretically achievable quality of recombinant AAV vectors. In comparison, for rAAV2, only approximately 50 out of 100 viral particles contained a genome and, more strikingly, only approximately 1 of the 100 viral particles was infectious. Our findings suggest that current strategies for rAAV vector design, production and/or purification should be amenable to improvements. Ultimately, this could result in the generation of near-perfect vector particles, a prospect with significant implications for gene therapy.

Pluripotent stem cells in neuropsychiatric disorders

Neuropsychiatric disorders place an enormous medical burden on patients across all social and economic ranks. The current understanding of the molecular and cellular causes of neuropsychiatric disease remains limited, which leads to a lack of targeted therapies. Human-induced pluripotent stem cell (iPSC) technology offers a novel platform for modeling the genetic contribution to mental disorders and yields access to patient-specific cells for drug discovery and personalized medicine. Here, we review recent progress in using iPSC technology to model and potentially treat neuropsychiatric disorders by focusing on the most prevalent conditions in psychiatry, including depression, anxiety disorders, bipolar disorder and schizophrenia.

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Capturing the biology of disease severity in a PSC-based model of familial dysautonomia

Familial dysautonomia (FD) is a debilitating disorder that affects derivatives of the neural crest (NC). For unknown reasons, people with FD show marked differences in disease severity despite carrying an identical, homozygous point mutation in IKBKAP, encoding IκB kinase complex–associated protein. Here we present disease-related phenotypes in human pluripotent stem cells (PSCs) that capture FD severity. Cells from individuals with severe but not mild disease show impaired specification of NC derivatives, including autonomic and sensory neurons. In contrast, cells from individuals with severe and mild FD show defects in peripheral neuron survival, indicating that neurodegeneration is the main culprit for cases of mild FD. Although genetic repair of the FD-associated mutation reversed early developmental NC defects, sensory neuron specification was not restored, indicating that other factors may contribute to disease severity. Whole-exome sequencing identified candidate modifier genes for individuals with severe FD. Our study demonstrates that PSC-based modeling is sensitive in recapitulating disease severity, which presents an important step toward personalized medicine.

20. Targeted Gene Delivery to Embryonic Stem Cells

To date, gene delivery relies on either extrachromosomal persistence or random integration of transgenes within the host nucleus. In addition, the need for safe and efficient gene targeting of ES cells has grown since recent developments in stem cell biology have focused considerable attention on the use of cell-based therapies for the treatment of complex diseases. The success of such an approach, however, will require the ability to genetically modify stem cells ex vivo. Adeno-associated virus has evolved the unique strategy to establish latency by integrating its genome site-specifically into a locus on human chromosome 19 at 19q13.4 (termed AAVS1). We have initiated a study aimed at adapting the underlying mechanism to the development of targeted transgene delivery strategies in ES cells. Differentiation assays of ES cells, both in vitro (embryoid body system) and in vivo (transgenic mice), offer the possibility to investigate whether AAVS1 represents a safe and suitable targeting site. We will discuss the infectivity of different recombinant AAV-based GFP viruses in ES cells. Furthermore, we will present evidence for targeted transgene delivery in these cells and address the safety as determined by differentiation of targeted ES cells into several different lineages. Taken together, our data suggest that a) AAV-mediated targeted gene delivery can be achieved into the mouse AAVS1 ortholog, b) targeted gene delivery to this locus is feasible in ES cells, c) as determined to date, disruption of AAVS1 does not interfere with multilineage in vitro differentiation of ES cells and d) that transgene expression is maintained throughout differentiation.