Jonathan C. Niclis

Human embryonic stem cell models of Huntington disease

Huntington disease (HD) is an incurable late-onset neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the HD gene (HTT). The major hallmark of disease pathology is neurodegeneration in the brain. Currently, there are no useful in-vitro human models of HD. Recently, two human embryonic stem cell (hESC) lines carrying partial (CAG(37)) and fully (CAG(51)) penetrant mutant alleles have been derived from affected IVF embryos identified following preimplantation genetic diagnosis (PGD). Fluorescence polymerase chain reaction (F-PCR) and Genescan analysis confirmed the original embryonic HD genotypes. Reverse transcription PCR (RT-PCR) analysis confirmed the expression of mutant transcripts and western blot analysis demonstrated expression of mutant huntingtin protein (HTT). After treatment with noggin, HD hESC formed neurospheres, which could be further differentiated into cells susceptible to neurodegeneration in HD, namely primary neurones and astrocytes. Small pool PCR analysis of neurosphere cells revealed instability of disease-length CAG repeats following differentiation. The presence of active HTT genes, neural differentiation capabilities and evidence of CAG repeat instability indicates these HD hESC lines may serve as valuable in-vitro human models of HD to better understand the mechanisms of neurodegeneration in patients, and for drug screening to identify new therapies for human clinical trials.

Human embryonic stem cell-derived oligodendrocytes: protocols and perspectives.

Oligodendrocytes play a fundamental supportive role in the mammalian central nervous system (CNS) as the myelinating-glial cells. Disruption of fast axonal transport mechanisms can occur as a consequence of mature oligodendrocyte loss following spinal cord injury, stroke, or due to neuroinflammatory conditions, such as multiple sclerosis. As a result of the limited remyelination ability in the CNS after injury or disease, human embryonic stem cells (hESCs) may prove to be a promising option for the generation and replacement of mature oligodendrocytes. Moreover, hESC-derived oligodendrocytes may be experimentally utilized to unravel fundamental questions of oligodendrocyte development, along with their therapeutic potential through growth factor support of axons and neurons. However, an intensive characterization and examination of hESC-derived oligodendrocytes prior to preclinical or clinical trials is required to facilitate greater success in their integration following cellular replacement therapy (CRT). Currently, the protocols utilized to derive oligodendrocytes from hESCs consist of significant variations in culture style, time-length of differentiation, and the provision of growth factors in culture. Further, these differing protocols also report disparate patterns in the expression of oligodendroglial markers by these derived oligodendrocytes, throughout their differentiation in culture. We have comprehensively reviewed the published protocols describing the derivation of oligodendrocytes from hESCs and the studies that examine their efficacy to remyelinate, along with the fundamental issues of their safety as a viable CRT. Additionally, this review will highlight particular issues of concern and suggestions for troubleshooting to provide investigators critical information for the future improvement of establishing in vitro hESC-derived oligodendrocytes.

Huntingtin Inclusions Trigger Cellular Quiescence, Deactivate Apoptosis, and Lead to Delayed Necrosis

Competing models exist in the literature for the relationship between mutant Huntingtin exon 1 (Httex1) inclusion formation and toxicity. In one, inclusions are adaptive by sequestering the proteotoxicity of soluble Httex1. In the other, inclusions compromise cellular activity as a result of proteome co-aggregation. Using a biosensor of Httex1 conformation in mammalian cell models, we discovered a mechanism that reconciles these competing models. Newly formed inclusions were composed of disordered Httex1 and ribonucleoproteins. As inclusions matured, Httex1 reconfigured into amyloid, and other glutamine-rich and prion domain-containing proteins were recruited. Soluble Httex1 caused a hyperpolarized mitochondrial membrane potential, increased reactive oxygen species, and promoted apoptosis. Inclusion formation triggered a collapsed mitochondrial potential, cellular quiescence, and deactivated apoptosis. We propose a revised model where sequestration of soluble Httex1 inclusions can remove the trigger for apoptosis but also co-aggregate other proteins, which curtails cellular metabolism and leads to a slow death by necrosis.

Characterization of forebrain neurons derived from late-onset Huntington's disease human embryonic stem cell lines.

Huntingtons disease (HD) is an incurable neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the Huntingtin (HTT) gene. Recently, induced pluripotent stem cell (iPSC) lines carrying atypical and aggressive (CAG60+) HD variants have been generated and exhibit disparate molecular pathologies. Here we investigate two human embryonic stem cell (hESC) lines carrying CAG37 and CAG51 typical late-onset repeat expansions in comparison to wildtype control lines during undifferentiated states and throughout forebrain neuronal differentiation. Pluripotent HD lines demonstrate growth, viability, pluripotent gene expression, mitochondrial activity and forebrain specification that is indistinguishable from control lines. Expression profiles of crucial genes known to be dysregulated in HD remain unperturbed in the presence of mutant protein and throughout differentiation; however, elevated glutamate-evoked responses were observed in HD CAG51 neurons. These findings suggest typical late-onset HD mutations do not alter pluripotent parameters or the capacity to generate forebrain neurons, but that such progeny may recapitulate hallmarks observed in established HD model systems. Such HD models will help further our understanding of the cascade of pathological events leading to disease onset and progression, while simultaneously facilitating the identification of candidate HD therapeutics.

Efficiently Specified Ventral Midbrain Dopamine Neurons from Human Pluripotent Stem Cells Under Xeno‐Free Conditions Restore Motor Deficits in Parkinsonian Rodents

Recent studies have shown evidence for the functional integration of human pluripotent stem cell (hPSC)‐derived ventral midbrain dopamine (vmDA) neurons in animal models of Parkinson’s disease. Although these cells present a sustainable alternative to fetal mesencephalic grafts, a number of hurdles require attention prior to clinical translation. These include the persistent use of xenogeneic reagents and challenges associated with scalability and storage of differentiated cells. In this study, we describe the first fully defined feeder‐ and xenogeneic‐free protocol for the generation of vmDA neurons from hPSCs and utilize two novel reporter knock‐in lines (LMX1A‐eGFP and PITX3‐eGFP) for in‐depth in vitro and in vivo tracking. Across multiple embryonic and induced hPSC lines, this “next generation” protocol consistently increases both the yield and proportion of vmDA neural progenitors (OTX2/FOXA2/LMX1A) and neurons (FOXA2/TH/PITX3) that display classical vmDA metabolic and electrophysiological properties. We identify the mechanism underlying these improvements and demonstrate clinical applicability with the first report of scalability and cryopreservation of bona fide vmDA progenitors at a time amenable to transplantation. Finally, transplantation of xeno‐free vmDA progenitors from LMX1A‐ and PITX3‐eGFP reporter lines into Parkinsonian rodents demonstrates improved engraftment outcomes and restoration of motor deficits. These findings provide important and necessary advancements for the translation of hPSC‐derived neurons into the clinic. Stem Cells Translational Medicine 2017;6:937–948Abstract Recent studies have shown evidence for the functional integration of human pluripotent stem cell (hPSC)‐derived ventral midbrain dopamine (vmDA) neurons in animal models of Parkinson’s disease. Although these cells present a sustainable alternative to fetal mesencephalic grafts, a number of hurdles require attention prior to clinical translation. These include the persistent use of xenogeneic reagents and challenges associated with scalability and storage of differentiated cells. In this study, we describe the first fully defined feeder‐ and xenogeneic‐free protocol for the generation of vmDA neurons from hPSCs and utilize two novel reporter knock‐in lines (LMX1A‐eGFP and PITX3‐eGFP) for in‐depth in vitro and in vivo tracking. Across multiple embryonic and induced hPSC lines, this “next generation” protocol consistently increases both the yield and proportion of vmDA neural progenitors (OTX2/FOXA2/LMX1A) and neurons (FOXA2/TH/PITX3) that display classical vmDA metabolic and electrophysiological properties. We identify the mechanism underlying these improvements and demonstrate clinical applicability with the first report of scalability and cryopreservation of bona fide vmDA progenitors at a time amenable to transplantation. Finally, transplantation of xeno‐free vmDA progenitors from LMX1A‐ and PITX3‐eGFP reporter lines into Parkinsonian rodents demonstrates improved engraftment outcomes and restoration of motor deficits. These findings provide important and necessary advancements for the translation of hPSC‐derived neurons into the clinic. Stem Cells Translational Medicine 2017;6:937–948

Long-distance axonal growth and protracted functional maturation of neurons derived from human induced pluripotent stem cells after intracerebral transplantation

The capacity for induced pluripotent stem (iPS) cells to be differentiated into a wide range of neural cell types makes them an attractive donor source for autologous neural transplantation therapies aimed at brain repair. Translation to the in vivo setting has been difficult, however, with mixed results in a wide variety of preclinical models of brain injury and limited information on the basic in vivo properties of neural grafts generated from human iPS cells. Here we have generated a human iPS cell line constitutively expressing green fluorescent protein as a basis to identify and characterize grafts resulting from transplantation of neural progenitors into the adult rat brain. The results show that the grafts contain a mix of neural cell types, at various stages of differentiation, including neurons that establish extensive patterns of axonal growth and progressively develop functional properties over the course of 1 year after implantation. These findings form an important basis for the design and interpretation of preclinical studies using human stem cells for functional circuit re‐construction in animal models of brain injury. Stem Cells Translational Medicine 2017;6:1547–1556

Three-dimensional imaging of human stem cells using soft X-ray tomography.

Three-dimensional imaging of human stem cells using transmission soft X-ray tomography (SXT) is presented for the first time. Major organelle types—nuclei, nucleoli, mitochondria, lysosomes and vesicles—were discriminated at approximately 50 nm spatial resolution without the use of contrast agents, on the basis of measured linear X-ray absorption coefficients and comparison of the size and shape of structures to transmission electron microscopy (TEM) images. In addition, SXT was used to visualize the distribution of a cell surface protein using gold-labelled antibody staining. We present the strengths of SXT, which include excellent spatial resolution (intermediate between that of TEM and light microscopy), the lack of the requirement for fixative or contrast agent that might perturb cellular morphology or produce imaging artefacts, and the ability to produce three-dimensional images of cells without microtome sectioning. Possible applications to studying the differentiation of human stem cells are discussed.

A PITX3-EGFP Reporter Line Reveals Connectivity of Dopamine and Non-dopamine Neuronal Subtypes in Grafts Generated from Human Embryonic Stem Cells

Summary Development of safe and effective stem cell-based therapies for brain repair requires an in-depth understanding of the in vivo properties of neural grafts generated from human stem cells. Replacing dopamine neurons in Parkinsons disease remains one of the most anticipated applications. Here, we have used a human PITX3-EGFP embryonic stem cell line to characterize the connectivity of stem cell-derived midbrain dopamine neurons in the dopamine-depleted host brain with an unprecedented level of specificity. The results show that the major A9 and A10 subclasses of implanted dopamine neurons innervate multiple, developmentally appropriate host targets but also that the majority of graft-derived connectivity is non-dopaminergic. These findings highlight the promise of stem cell-based procedures for anatomically correct reconstruction of specific neuronal pathways but also emphasize the scope for further refinement in order to limit the inclusion of uncharacterized and potentially unwanted cell types.

GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives

Summary The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that β-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives.

Shear Containment of BDNF within Molecular Hydrogels Promotes Human Stem Cell Engraftment and Postinfarction Remodeling in Stroke

Improved control over spatiotemporal delivery of growth factors is needed to enhance tissue repair. Current methods are limited–requiring invasive procedures, poor tissue targeting, and/or limited control over dosage and duration. Incorporation into implantable biomaterials enables stabilized delivery and avoids burst release/fluctuating doses. Here, the physical forces of fibrils formed by self‐assembly of epitope‐containing peptides are exploited. This biomimetic hydrogel is loaded with neurotrophic factor BDNF via a shear‐induced gel–solution transition, unique to noncovalent hydrogels. This results in a biomaterial with three desirable features: a nanofibrillar scaffold, presentation of a laminin epitope, and slow release of BDNF. In a stroke‐injury model, synergistic actions of this trimodal strategy on the integration of transplanted human neural progenitor cells, and protection of peri‐infarct tissue are identified. These BDNF‐functionalized hydrogels promote the integration of transplanted human embryonic stem cell–derived neural progenitors–resulting in larger grafts with greater cortical differentiation, appropriate for neuronal replacement. Furthermore, BDNF promotes the infiltration of host endothelial cells into the graft to augment vascularization of the graft, and adjacent penumbra tissue. These findings demonstrate the benefits of multifaceted tissue‐specific hydrogels to provide biomimetics of the host tissue, while sustain protein delivery, to promote endogenous and graft‐derived tissue repair.