Creation of GMP-Compliant iPSCs From Banked Umbilical Cord Blood.

Many clinical trials are in progress using cells derived from induced pluripotent stem cells (iPSC) for immunotherapies and regenerative medicine. The success of these new therapies is underpinned by the quality of the cell population used to create the iPSC lines, along with the creation of iPSCs in a fully Good Manufacturing Practice (GMP)-compliant environment such that they can be used safely and effectively in the clinical setting. Umbilical cord blood (CB) from public cord blood banks is an excellent source of starting material for creation of iPSCs. All CB units are manufactured under GMP-conditions, have been screened for infectious diseases, with known family and medical history of the donor. Furthermore, the HLA tissue typing is known, thereby allowing identification of CB units with homozygous HLA haplotypes. CB cells are naïve with less exposure to environmental insults and iPSC can be generated with high efficiency. We describe a protocol that can be adopted by those seeking to create clinical-grade iPSC from banked CB. This protocol uses a small volume of thawed CB buffy to first undergo ex-vivo expansion towards erythroid progenitor cells, which are then used for reprogramming using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit. Resultant iPSC lines are tested to confirm pluripotency, genomic integrity, and stability. Cells are maintained in a feeder-free, xeno-free environment, using fully defined, commercially available reagents. Adoption of this protocol, with heed given to tips provided, allows efficient and robust creation of clinical-grade iPSC cell lines from small volumes of cryopreserved CB.

Human stem cells harboring a suicide gene improve the safety and standardisation of neural transplants in Parkinsonian rats.

Despite advancements in human pluripotent stem cells (hPSCs) differentiation protocols to generate appropriate neuronal progenitors suitable for transplantation in Parkinson's disease, resultant grafts contain low proportions of dopamine neurons. Added to this is the tumorigenic risk associated with the potential presence of incompletely patterned, proliferative cells within grafts. Here, we utilised a hPSC line carrying a FailSafeTM suicide gene (thymidine kinase linked to cyclinD1) to selectively ablate proliferative cells in order to improve safety and purity of neural transplantation in a Parkinsonian model. The engineered FailSafeTM hPSCs demonstrated robust ventral midbrain specification in vitro, capable of forming neural grafts upon transplantation. Activation of the suicide gene within weeks after transplantation, by ganciclovir administration, resulted in significantly smaller grafts without affecting the total yield of dopamine neurons, their capacity to innervate the host brain or reverse motor deficits at six months in a rat Parkinsonian model. Within ganciclovir-treated grafts, other neuronal, glial and non-neural populations (including proliferative cells), were significantly reduced-cell types that may pose adverse or unknown influences on graft and host function. These findings demonstrate the capacity of a suicide gene-based system to improve both the standardisation and safety of hPSC-derived grafts in a rat model of Parkinsonism.

Unprecedented Potential for Neural Drug Discovery Based on Self-Organizing hiPSC Platforms.

Human induced pluripotent stem cells (hiPSCs) have transformed conventional drug discovery pathways in recent years. In particular, recent advances in hiPSC biology, including organoid technologies, have highlighted a new potential for neural drug discovery with clear advantages over the use of primary tissues. This is important considering the financial and social burden of neurological health care worldwide, directly impacting the life expectancy of many populations. Patient-derived iPSCs-neurons are invaluable tools for novel drug-screening and precision medicine approaches directly aimed at reducing the burden imposed by the increasing prevalence of neurological disorders in an aging population. 3-Dimensional self-assembled or so-called 'organoid' hiPSCs cultures offer key advantages over traditional 2D ones and may well be gamechangers in the drug-discovery quest for neurological disorders in the coming years.

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

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 Parkinson's 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.

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.

Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros.

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34+ cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34+ cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34+ hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17+ vessels from which RUNX1C+ blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34+ hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

Pathophysiological insights into ALS with C9ORF72 expansions.

OBJECTIVE: Expansions of a hexanucleotide repeat in C9ORF72 are a common cause of familial amyotrophic lateral sclerosis (ALS) and a small proportion of sporadic ALS cases. We sought to examine clinical and neurophysiological features of familial and sporadic ALS with C9ORF72 expansions. METHODS: C9ORF72 was screened for expansions in familial and sporadic ALS. Clinical features of expansion positive cases are described. Cortical excitability studies used novel threshold tracking transcranal magnetic stimulation techniques with motor evoked responses recorded over the abductor pollicis brevis. RESULTS AND CONCLUSIONS: Analysis of large clinical cohorts identified C9ORF72 expansions in 38.5% (72/187) of ALS families and 3.5% (21/606) of sporadic ALS cases. Two expansion positive families were known to carry reported ANG mutations, possibly implicating an oligogenic model of ALS. 6% of familial ALS cases with C9ORF72 expansions were also diagnosed with dementia. The penetrance of ALS was 50% at age 58 years in male subjects and 63 years in female subjects. 100% penetrance of ALS was observed in male subjects by 86 years, while 6% of female subjects remained asymptomatic at age 82 years. Gender specific differences in age of onset were evident, with male subjects significantly more likely to develop ALS at a younger age. Importantly, features of cortical hyperexcitability were apparent in C9ORF72-linked familial ALS as demonstrated by significant reduction in short interval intracortical inhibition and cortical silent period duration along with an increase in intracortical facilitation and motor evoked potential amplitude, indicating that cortical hyperexcitability is an intrinsic process in C9ORF72-linked ALS.

FUS mutations in amyotrophic lateral sclerosis: clinical, pathological, neurophysiological and genetic analysis.

OBJECTIVE: FUS gene mutations were recently identified in familial amyotrophic lateral sclerosis (ALS). The present studies sought to define the clinical, post-mortem and neurophysiological phenotypes in ALS families with FUS mutations and to determine the frequency of FUS mutations in familial and sporadic ALS. METHODS: FUS was screened for mutations in familial and sporadic ALS cases. Clinical, post-mortem and neurophysiological features of large families with FUS mutations are described. RESULTS AND CONCLUSIONS: FUS mutations were evident in 3.2% (4/124) of familial ALS, representing the second most common gene abnormality to be described in familial ALS after SOD1. No mutations were present in 247 sporadic ALS cases. The clinical presentation in 49 affected patients was consistent with a predominantly lower motor neuron disorder, supported by post-mortem findings. Upper motor neuron involvement varied, with Wallerian degeneration of corticospinal tracts present in one post-mortem case but absent in a second case from the same family. Features of cortical hyperexcitability demonstrated upper motor neuron involvement consistent with other forms of familial and sporadic ALS. One case presented with frontotemporal dementia (FTD) indicating that this may be a rare presenting feature in families with FUS mutation. Ubiquitin-positive cytoplasmic skein-like inclusions were present in lower motor neurons, but in contrast to sporadic ALS, no TDP-43 pathology was evident. Mutation-specific clinical features were identified. Patients with a R521C mutation were significantly more likely to develop disease at a younger age, and dropped-head syndrome was a frequent feature. Reduced disease penetrance was evident among most affected families.

TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder characterized pathologically by ubiquitinated TAR DNA binding protein (TDP-43) inclusions. The function of TDP-43 in the nervous system is uncertain, and a mechanistic role in neurodegeneration remains speculative. We identified neighboring mutations in a highly conserved region of TARDBP in sporadic and familial ALS cases. TARDBPM337V segregated with disease within one kindred and a genome-wide scan confirmed that linkage was restricted to chromosome 1p36, which contains the TARDBP locus. Mutant forms of TDP-43 fragmented in vitro more readily than wild type and, in vivo, caused neural apoptosis and developmental delay in the chick embryo. Our evidence suggests a pathophysiological link between TDP-43 and ALS.

A novel locus for distal motor neuron degeneration maps to chromosome 7q34-q36.

The motor neuron diseases (MND) are a group of related neurodegenerative diseases that cause the relative selective progressive death of motor neurons. These diseases range from slowly progressive forms including hereditary motor neuropathy (HMN), to the rapidly progressive disorder amyotrophic lateral sclerosis (ALS). There is clinical and genetic overlap among these MNDs, implicating shared pathogenic mechanisms. We recruited a large family with a MND that was previously described as juvenile ALS and distal HMN. We identified a novel MND/HMN locus on chromosome 7q34-q36 following a genome-wide scan for linkage in this family. The disease causing mutation maps to a 26.2 cM (12.3 Mb) interval flanked by D7S2513 and D7S637 on chromosome 7q34-q36. Recombinant haplotype analysis including unaffected individuals suggests that the refined candidate interval spans 14.3 cM (6.3 Mb) flanked by D7S2511 and D7S798. One gene in the candidate interval, CDK5, was selected for immediate mutation analysis based upon its known association with an ALS-like phenotype in mice however, no mutations were identified. Identification of genes causing familial MND will lead to a greater understanding of the biological basis of both familial and sporadic motor neuron degeneration including ALS.