Identification of cell barcodes from long-read single-cell RNA-seq with BLAZE.

Long-read single-cell RNA sequencing (scRNA-seq) enables the quantification of RNA isoforms in individual cells. However, long-read scRNA-seq using the Oxford Nanopore platform has largely relied upon matched short-read data to identify cell barcodes. We introduce BLAZE, which accurately and efficiently identifies 10x cell barcodes using only nanopore long-read scRNA-seq data. BLAZE outperforms the existing tools and provides an accurate representation of the cells present in long-read scRNA-seq when compared to matched short reads. BLAZE simplifies long-read scRNA-seq while improving the results, is compatible with downstream tools accepting a cell barcode file, and is available at https://github.com/shimlab/BLAZE .

Understanding and modeling regional specification of the human ganglionic eminence.

Inhibitory neurons originating from the ventral forebrain are associated with several neurological conditions. Distinct ventral forebrain subpopulations are generated from topographically defined zones; lateral-, medial- and caudal ganglionic eminences (LGE, MGE and CGE), yet key specification factors often span across developing zones contributing to difficulty in defining unique LGE, MGE or CGE profiles. Here we use human pluripotent stem cell (hPSC) reporter lines (NKX2.1-GFP and MEIS2-mCherry) and manipulation of morphogen gradients to gain greater insight into regional specification of these distinct zones. We identified Sonic hedgehog (SHH)-WNT crosstalk in regulating LGE and MGE fate and uncovered a role for retinoic acid signaling in CGE development. Unraveling the influence of these signaling pathways permitted development of fully defined protocols that favored generation of the three GE domains. These findings provide insight into the context-dependent role of morphogens in human GE specification and are of value for in vitro disease modeling and advancement of new therapies.

Understanding the Influence of Target Acquisition on Survival, Integration, and Phenotypic Maturation of Dopamine Neurons within Stem Cell-Derived Neural Grafts in a Parkinson's Disease Model.

Midbrain dopaminergic (DA) neurons include many subtypes characterized by their location, connectivity and function. Surprisingly, mechanisms underpinning the specification of A9 neurons [responsible for motor function, including within ventral midbrain (VM) grafts for treating Parkinson's disease (PD)] over adjacent A10, remains largely speculated. We assessed the impact of synaptic targeting on survival, integration, and phenotype acquisition of dopaminergic neurons within VM grafts generated from fetal tissue or human pluripotent stem cells (PSCs). VM progenitors were grafted into female mice with 6OHDA-lesions of host midbrain dopamine neurons, with some animals also receiving intrastriatal quinolinic acid (QA) injections to ablate medium spiny neurons (MSN), the A9 neuron primary target. While loss of MSNs variably affected graft survival, it significantly reduced striatal yet increased cortical innervation. Consequently, grafts showed reduced A9 and increased A10 specification, with more DA neurons failing to mature into either subtype. These findings highlight the importance of target acquisition on DA subtype specification during development and repair.SIGNIFICANCE STATEMENT Parish and colleagues highlight, in a rodent model of Parkinson's disease (PD), the importance of synaptic target acquisition in the survival, integration and phenotypic specification of grafted dopamine neurons derived from fetal tissue and human stem cells. Ablation of host striatal neurons resulted in reduced dopamine neuron survival within grafts, re-routing of dopamine fibers from striatal to alternate cortical targets and a consequential reduced specification of A9 dopamine neurons (the subpopulation critical for restoration of motor function) and increase in A10 DA neurons.

A combined cell and gene therapy approach for homotopic reconstruction of midbrain dopamine pathways using human pluripotent stem cells.

Midbrain dopamine (mDA) neurons can be replaced in patients with Parkinson's disease (PD) in order to provide long-term improvement in motor functions. The limited capacity for long-distance axonal growth in the adult brain means that cells are transplanted ectopically, into the striatal target. As a consequence, several mDA pathways are not re-instated, which may underlie the incomplete restoration of motor function in patients. Here, we show that viral delivery of GDNF to the striatum, in conjunction with homotopic transplantation of human pluripotent stem-cell-derived mDA neurons, recapitulates brain-wide mDA target innervation. The grafts provided re-instatement of striatal dopamine levels and correction of motor function and also connectivity with additional mDA target nuclei not well innervated by ectopic grafts. These results demonstrate the remarkable capacity for achieving functional and anatomically precise reconstruction of long-distance circuitry in the adult brain by matching appropriate growth-factor signaling to grafting of specific cell types.

Spontaneous Formation of ß-sheet Nano-barrels during the Early Aggregation of Alzheimer's Amyloid Beta.

Soluble low-molecular-weight oligomers formed during the early aggregation of amyloid peptides have been hypothesized as a major toxic species of amyloidogenesis. Herein, we performed the first synergic in silico, in vitro and in vivo validations of the structure, dynamics and toxicity of Aß42 oligomers. Aß peptides readily assembled into ß-rich oligomers comprised of extended ß-hairpins and ß-strands. Nanosized ß-barrels were observed with certainty with simulations, transmission electron microscopy and Fourier transform infrared spectroscopy, corroborated by immunohistochemistry, cell viability, apoptosis, inflammation, autophagy and animal behavior assays. Secondary and tertiary structural proprieties of these oligomers, such as the sequence regions with high ß-sheet propensities and inter-residue contact frequency patterns, were similar to the properties known for Aß fibrils. The unambiguous spontaneous formation of ß-barrels in the early aggregation of Aß42 supports their roles as the common toxic intermediates in Alzheimer's pathobiology and a target for Alzheimer's therapeutics.

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.

FGF-MAPK signaling regulates human deep-layer corticogenesis.

Despite heterogeneity across the six layers of the mammalian cortex, all excitatory neurons are generated from a single founder population of neuroepithelial stem cells. However, how these progenitors alter their layer competence over time remains unknown. Here, we used human embryonic stem cell-derived cortical progenitors to examine the role of fibroblast growth factor (FGF) and Notch signaling in influencing cell fate, assessing their impact on progenitor phenotype, cell-cycle kinetics, and layer specificity. Forced early cell-cycle exit, via Notch inhibition, caused rapid, near-exclusive generation of deep-layer VI neurons. In contrast, prolonged FGF2 promoted proliferation and maintained progenitor identity, delaying laminar progression via MAPK-dependent mechanisms. Inhibiting MAPK extended cell-cycle length and led to generation of layer-V CTIP2+ neurons by repressing alternative laminar fates. Taken together, FGF/MAPK regulates the proliferative/neurogenic balance in deep-layer corticogenesis and provides a resource for generating layer-specific neurons for studying development and disease.

Viral Delivery of GDNF Promotes Functional Integration of Human Stem Cell Grafts in Parkinson's Disease.

Dopaminergic neurons (DAns), generated from human pluripotent stem cells (hPSCs), are capable of functionally integrating following transplantation and have recently advanced to clinical trials for Parkinson's disease (PD). However, pre-clinical studies have highlighted the low proportion of DAns within hPSC-derived grafts and their inferior plasticity compared to fetal tissue. Here, we examined whether delivery of a developmentally critical protein, glial cell line-derived neurotrophic factor (GDNF), could improve graft outcomes. We tracked the response of DAns implanted into either a GDNF-rich environment or after a delay in exposure. Early GDNF promoted survival and plasticity of non-DAns, leading to enhanced motor recovery in PD rats. Delayed exposure to GDNF promoted functional recovery through increases in DAn specification, DAn plasticity, and DA metabolism. Transcriptional profiling revealed a role for mitogen-activated protein kinase (MAPK)-signaling downstream of GDNF. Collectively, these results demonstrate the potential of neurotrophic gene therapy strategies to improve hPSC graft outcomes.

Transcriptional Profiling of Xenogeneic Transplants: Examining Human Pluripotent Stem Cell-Derived Grafts in the Rodent Brain.

Human pluripotent stem cells are a valuable resource for transplantation, yet our ability to profile xenografts is largely limited to low-throughput immunohistochemical analysis by difficulties in readily isolating grafts for transcriptomic and/or proteomic profiling. Here, we present a simple methodology utilizing differences in the RNA sequence between species to discriminate xenograft from host gene expression (using qPCR or RNA sequencing [RNA-seq]). To demonstrate the approach, we assessed grafts of undifferentiated human stem cells and neural progenitors in the rodent brain. Xenograft-specific qPCR provided sensitive detection of proliferative cells, and identified germ layer markers and appropriate neural maturation genes across the graft types. Xenograft-specific RNA-seq enabled profiling of the complete transcriptome and an unbiased characterization of graft composition. Such xenograft-specific profiling will be crucial for pre-clinical characterization of grafts and batch-testing of therapeutic cell preparations to ensure safety and functional predictability prior to translation.

Isolation of LMX1a Ventral Midbrain Progenitors Improves the Safety and Predictability of Human Pluripotent Stem Cell-Derived Neural Transplants in Parkinsonian Disease.

Human pluripotent stem cells (hPSCs) are a promising resource for the replacement of degenerated ventral midbrain dopaminergic (vmDA) neurons in Parkinson's disease. Despite recent advances in protocols for the in vitro generation of vmDA neurons, the asynchronous and heterogeneous nature of the differentiations results in transplants of surprisingly low vmDA neuron purity. As the field advances toward the clinic, it will be optimal, if not essential, to remove poorly specified and potentially proliferative cells from donor preparations to ensure safety and predictable efficacy. Here, we use two novel hPSC knock-in reporter lines expressing GFP under the LMX1A and PITX3 promoters, to selectively isolate vm progenitors and DA precursors, respectively. For each cell line, unsorted, GFP+, and GFP- cells were transplanted into male or female Parkinsonian rodents. Only rats receiving unsorted cells, LMX1A-eGFP+, or PITX3-eGFP- cell grafts showed improved motor function over 6 months. Postmortem analysis revealed small grafts from PITX3-eGFP+ cells, suggesting that these DA precursors were not compatible with cell survival and integration. In contrast, LMX1A-eGFP+ grafts were highly enriched for vmDA neurons, and importantly excluded expansive proliferative populations and serotonergic neurons. These LMX1A-eGFP+ progenitor grafts accelerated behavioral recovery and innervated developmentally appropriate forebrain targets, whereas LMX1A-eGFP- cell grafts failed to restore motor deficits, supported by increased fiber growth into nondopaminergic target nuclei. This is the first study to use an hPSC-derived reporter line to purify vm progenitors, resulting in improved safety, predictability of the graft composition, and enhanced motor function.SIGNIFICANCE STATEMENT Clinical trials have shown functional integration of transplanted fetal-derived dopamine progenitors in Parkinson's disease. Human pluripotent stem cell (hPSC)-derived midbrain progenitors are now being tested as an alternative cell source; however, despite current differentiation protocols generating >80% correctly specified cells for implantation, resultant grafts contain a small fraction of dopamine neurons. Cell-sorting approaches, to select for correctly patterned cells before implantation, are being explored yet have been suboptimal to date. This study provides the first evidence of using 2 hPSC reporter lines (LMX1A-GFP and PITX3-GFP) to isolate correctly specified cells for transplantation. We show LMX1A-GFP+, but not PITX3-GFP+, cell grafts are more predictable, with smaller grafts, enriched in dopamine neurons, showing appropriate integration and accelerated functional recovery in Parkinsonian rats.

Acute or Delayed Systemic Administration of Human Amnion Epithelial Cells Improves Outcomes in Experimental Stroke.

BACKGROUND AND PURPOSE: Human amnion epithelial cells (hAECs) are nonimmunogenic, nontumorigenic, anti-inflammatory cells normally discarded with placental tissue. We reasoned that their profile of biological features, wide availability, and the lack of ethical barriers to their use could make these cells useful as a therapy in ischemic stroke. METHODS: We tested the efficacy of acute (1.5 hours) or delayed (1-3 days) poststroke intravenous injection of hAECs in 4 established animal models of cerebral ischemia. Animals included young (7-14 weeks) and aged mice (20-22 months) of both sexes, as well as adult marmosets of either sex. RESULTS: We found that hAECs administered 1.5 hours after stroke in mice migrated to the ischemic brain via a CXC chemokine receptor type 4-dependent mechanism and reduced brain inflammation, infarct development, and functional deficits. Furthermore, if hAECs administration was delayed until 1 or 3 days poststroke, long-term functional recovery was still augmented in young and aged mice of both sexes. We also showed proof-of-principle evidence in marmosets that acute intravenous injection of hAECs prevented infarct development from day 1 to day 10 after stroke. CONCLUSIONS: Systemic poststroke administration of hAECs elicits marked neuroprotection and facilitates mechanisms of repair and recovery.

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.

Human pluripotent stem cell derived midbrain PITX3(eGFP/w) neurons: a versatile tool for pharmacological screening and neurodegenerative modeling.

PITX3 expression is confined to adult midbrain dopaminergic (mDA) neurons. In this study we describe the generation and basic functional characteristics of mDA neurons derived from a human pluripotent stem cell (hPSC) line expressing eGFP under the control of the PITX3 promoter. Flow cytometry showed that eGFP was evident in 15% of the neuron population at day 12 of differentiation and this level was maintained until at least day 80. From days 20 to 80 of differentiation intracellular chloride decreased and throughout this period around ∼20% of PITX3(eGFP/w) neurons exhibited spontaneous Ca(2+) transients (from 3.3 ± 0.3 to 5.0 ± 0.1 min(-1), respectively). These neurons also responded to any of ATP, glutamate, acetylcholine, or noradrenaline with elevations of intracellular calcium. As neuronal cultures matured more dopamine was released and single PITX3(eGFP/w) neurons began to respond to more than one neurotransmitter. MPP(+) and tumor necrosis factor (TNF), but not prostaglandin E2, caused death of the ∼50% of PITX3(eGFP/w) neurons (day 80). Tracking eGFP using time lapse confocal microscopy over 24 h demonstrated significant TNF-mediated neurite retraction over time. This work now shows that these PITX3(eGFP/w) neurons are amenable to flow cytometry, release dopamine and respond to multiple neurotransmitters with elevations of intracellular calcium, we believe that they represent a versatile system for neuropharmacological and neurotoxicological studies.

Analysis of photovoltage decay transients in dye-sensitized solar cells.

It is shown that application of the so-called quasi-static approximation greatly simplifies the theoretical treatment of the open circuit photovoltage decay of dye-sensitized nanostructured solar cells (DSCs), since it removes the need to treat the kinetics of trapping and detrapping explicitly and leads to a straightforward analytical solution in the case of an exponential trap distribution. To identify the conditions under which the quasi-static approach is valid, transients calculated using the quasi-static approximation are compared with the results of numerical calculations that treat trapping and detrapping of electrons explicitly. The application of the quasi-static approach to derive the rate constant for the back-reaction of electrons from experimental photovoltage decay data is illustrated for an optimized DSC.

How does back-reaction at the conducting glass substrate influence the dynamic photovoltage response of nanocrystalline dye-sensitized solar cells?

In dye-sensitized nanocrystalline solar cells (DSC), the transfer of electrons from the conducting glass substrate to triiodide ions in solution is an important loss mechanism that can be suppressed by using thin compact blocking layers of TiO(2). Whereas back-reaction at the substrate is relatively unimportant under short circuit conditions, it must be taken into account at the maximum power point or at open circuit. The influence of the back-reaction on open circuit photovoltage decay measurements and on intensity modulated photovoltage (IMVS) measurements has been studied by model simulations and by experimental measurements. The simulations demonstrate that reliable information about DSC properties such as trapping distributions can only be derived from transient or periodic photovoltage responses if the back-reaction is suppressed by the use of suitable blocking layers.

Determination of the density and energetic distribution of electron traps in dye-sensitized nanocrystalline solar cells.

Electron transport and recombination in dye-sensitized nanocrystalline solar cells (DSCs) are strongly influenced by the presence of trapping states in the titanium dioxide particles, and collection of photoinjected electrons at the contact can require times ranging from milliseconds to seconds, depending on the illumination intensity. A direct method of determining the density and energetic distribution of the trapping states responsible for slowing electron transport has been developed. It involves extraction of trapped electrons by switching the cell from an open circuit to a short circuit after a period of illumination. An advantage of this charge extraction method is that it is less sensitive than other methods to shunting of the DSC by electron transfer at the conducting glass substrate. Results derived from charge extraction measurements on DSCs (with and without compact TiO(2) blocking layers) are compared with those obtained by analysis of the open circuit photovoltage decay.

Development of economic thresholds and monitoring systems for Helicoverpa armigera (Lepidoptera: Noctuidae) in tomatoes.

Field sampling methods and economic thresholds were developed to provide management recommendations for Helicoverpa armigera (Hübner) on processing tomatoes, based on a commercially acceptable damage level of 5% fruit damage. Population estimates from destructive sampling and a rapid 1-min plant scouting method were related to fruit damage, and a nominal economic threshold of one larva per plant was derived. The economic threshold was confirmed in a designed trial where it resulted in acceptable levels of fruit damage. The 1-min scouting method and economic threshold was validated in 17 commercial crops in the Gisborne, Poverty Bay region of New Zealand. Scouting in these fields was based on 10 plants in each of four quadrants proportionally representing the topography of each field. In unsprayed areas, egg and larval populations were usually below the economic threshold in early-planted crops but often exceeded thresholds in late-planted crops. In commercial demonstration trials where standard calendar spraying practice was compared with no spraying, calendar-based applications maintained fruit damage below 3.4%, but insecticide was applied unnecessarily to more than half the crops. Larval populations were a significant predictor of damage in these commercial crops. In 12 implementation trials, where spraying recommendations were based on the 1-min scouting threshold of one larva per plant, the worst fruit damage observed was 2.3%. The definition of an economic threshold, scouting methods, and establishment of parasitoids have reduced spray applications and contributed to the implementation of an integrated pest management program for processing tomatoes in New Zealand.

Prospective 153Sm-EDTMP therapy dosimetry by whole-body scintigraphy.

Samarium-153 ethylenediaminetetramethylene phosphonic acid (153Sm-EDTMP) effectively palliates painful bony metastases, but the standard recommended administered activity of 38 MBq.kg-1 may lead to significant myelotoxicity. Prospective individual dosimetry by urine collection and counting allow the bone marrow radiation dose to be limited to 2 Gy. Our novel whole-body scintigraphic method for prospective dosimetry was compared with the 5 h urine collection technique in 10 patients with bone metastases. Anterior and posterior whole-body images were obtained using identical acquisition parameters 10 min and 5 h after the intravenous injection of 740 MBq 153Sm-EDTMP. Total counts in each imaging study were corrected for background activity and time of injection and the bone activity at 5 h was determined. Bone activity was also calculated from a complete urine collection over 5 h, and these two values were compared. MIRD formulae were applied to calculate the radiation absorbed dose to the bone marrow from the injected activity. The total activity delivering a dose of 2 Gy to the bone marrow was then determined and constituted the amount given for therapy. Values for bone activity determined by imaging and by urine counting were concordant in all patients (correlation coefficient = 0.98). The total administered activity of 153Sm-EDTMP predicted on a 2 Gy bone marrow dose varied between 35 and 63% of the standard recommended regimen of 37 MBq.kg-1 and pain relief was experienced by eight of the ten patients. Administration of 153Sm-EDTMP according to the supplier's recommendations would have delivered bone marrow doses of 3.27-5.90 Gy in our patients, doses at which myelotoxicity would have been anticipated.

Stearoyl coenzyme A desaturase enzyme activity and mRNA levels are not different in subcutaneous adipose tissue from Angus and American Wagyu steers.

We proposed that greater stearoyl coenzyme A (CoA) desaturase enzyme activity caused the elevated monounsaturated fatty acids observed in American Wagyu adipose tissue. Stearoyl CoA desaturase mRNA concentrations and enzyme activities were measured in subcutaneous adipose samples from Angus (n = 5) and American Wagyu (n = 5), fed to the Japanese market end point. A rat liver stearoyl CoA desaturase cDNA clone was used to measure the relative amounts of stearoyl CoA desaturase mRNA. Enzyme activities and mRNA concentrations, as measured by laser densitometry of slot-blot autoradiograms, were not significantly different between the two breeds at this stage of growth. This investigation has demonstrated that, at this stage of maturity, differences in fatty acid composition between Angus and American Wagyu steers cannot be attributed to differences in stearoyl CoA desaturase enzyme activity.

166Ho-microsphere liver radiotherapy: a preclinical SPECT dosimetry study in the pig.

Liver metastases cause the majority of deaths from colorectal cancer and response to chemotherapy is poor. Intrahepatic arterial 90Y-microspheres may induce tumour regression but the beta-radiation dose is variable and cannot be determined in patients. The 81 keV gamma emission of holmium-166 (166Ho) was used to determine, by single photon emission computed tomographic (SPECT) imaging, the beta-radiation absorbed dose to normal liver in pigs following intrahepatic arterial administration of 166Ho-microspheres. The SPECT system was calibrated with anthropomorphic liver phantoms containing known activity concentrations of 166Ho-chloride. The relationship of SPECT counts to phantom activity concentration was linear with a correlation coefficient of r = 0.996. The SPECT pattern of liver distribution following successive administrations of tracer activities of 166Ho-microspheres was similar. The ratio of initial to total SPECT estimates of mean activity concentration in regions of interest, from which anatomically matched biopsy samples were later obtained and counted in an ionization chamber, showed good correlation (r = 0.924). Prospective SPECT dosimetry performed on a tracer activity of 166Ho-microspheres predicted the total administered activity required to deliver a prescribed radiation absorbed dose of 25 Gy to the liver within an error of +/- 8%. This study demonstrates the feasibility of prospective control of the absorbed radiation dose to the critical normal organ by SPECT dosimetry on a tracer dose of 166Ho-microspheres prior to administration of a therapy dose.