Juvenile skeletogenesis in anciently diverged sea urchin clades.

Mechanistic understanding of evolutionary divergence in animal body plans devolves from analysis of those developmental processes that, in forms descendant from a common ancestor, are responsible for their morphological differences. The last common ancestor of the two extant subclasses of sea urchins, i.e., euechinoids and cidaroids, existed well before the Permian/Triassic extinction (252 mya). Subsequent evolutionary divergence of these clades offers in principle a rare opportunity to solve the developmental regulatory events underlying a defined evolutionary divergence process. Thus (i) there is an excellent and fairly dense (if yet incompletely analyzed) fossil record; (ii) cladistically confined features of the skeletal structures of modern euechinoid and cidaroid sea urchins are preserved in fossils of ancestral forms; (iii) euechinoids and cidaroids are among current laboratory model systems in molecular developmental biology (here Strongylocentrotus purpuratus [Sp] and Eucidaris tribuloides [Et]); (iv) skeletogenic specification in sea urchins is uncommonly well understood at the causal level of interactions of regulatory genes with one another, and with known skeletogenic effector genes, providing a ready arsenal of available molecular tools. Here we focus on differences in test and perignathic girdle skeletal morphology that distinguish all modern euechinoid from all modern cidaroid sea urchins. We demonstrate distinct canonical test and girdle morphologies in juveniles of both species by use of SEM and X-ray microtomography. Among the sharply distinct morphological features of these clades are the internal skeletal structures of the perignathic girdle to which attach homologous muscles utilized for retraction and protraction of Aristotles׳ lantern and its teeth. We demonstrate that these structures develop de novo between one and four weeks after metamorphosis. In order to study the underlying developmental processes, a method of section whole mount in situ hybridization was adapted. This method displays current gene expression in the developing test and perignathic girdle skeletal elements of both Sp and Et juveniles. Active, specific expression of the sm37 biomineralization gene in these muscle attachment structures accompanies morphogenetic development of these clade-specific features in juveniles of both species. Skeletogenesis at these clade-specific muscle attachment structures displays molecular earmarks of the well understood embryonic skeletogenic GRN: thus the upstream regulatory gene alx1 and the gene encoding the vegfR signaling receptor are both expressed at the sites where they are formed. This work opens the way to analysis of the alternative spatial specification processes that were installed at the evolutionary divergence of the two extant subclasses of sea urchins.

An allograft mouse model for the study of hearing loss secondary to vestibular schwannoma growth.

Vestibular schwannoma is a benign neoplasm arising from the Schwann cell sheath of the auditory-vestibular nerve. It most commonly affects both sides in the genetic condition Neurofibromatosis type 2, causing progressive high frequency sensorineural hearing loss. Here, we describe a microsurgical technique and stereotactic coordinates for schwannoma cell grafting in the vestibular nerve region that recapitulates local tumor growth in the cerebellopontine angle and inner auditory canal with resulting hearing loss. Tumor growth was monitored by bioluminescence and MRI in vivo imaging, and hearing assessed by auditory brainstem responses. These techniques, by potentially enabling orthotopic grafting of a variety of cell lines will allow studies on the pathogenesis of tumor-related hearing loss and preclinical drug evaluation, including hearing endpoints, for NF2-related and sporadic schwannomas.

Quantitative noninvasive measurement of cerebrospinal fluid flow in shunted hydrocephalus.

OBJECTIVE: Standard MRI protocols lack a quantitative sequence that can be used to evaluate shunt-treated patients with a history of hydrocephalus. The objective of this study was to investigate the use of phase-contrast MRI (PC-MRI), a quantitative MR sequence, to measure CSF flow through the shunt and demonstrate PC-MRI as a useful adjunct in the clinical monitoring of shunt-treated patients. METHODS: The rapid (96 seconds) PC-MRI sequence was calibrated using a flow phantom with known flow rates ranging from 0 to 24 mL/hr. Following phantom calibration, 21 patients were scanned with the PC-MRI sequence. Multiple, successive proximal and distal measurements were gathered in 5 patients to test for measurement error in different portions of the shunt system and to determine intrapatient CSF flow variability. The study also includes the first in vivo validations of PC-MRI for CSF shunt flow by comparing phase-contrast-measured flow rate with CSF accumulation in a collection burette obtained in patients with externalized distal shunts. RESULTS: The PC-MRI sequence successfully measured CSF flow rates ranging from 6 to 54 mL/hr in 21 consecutive pediatric patients. Comparison of PC-MRI flow measurement and CSF volume collected in a bedside burette showed good agreement in a patient with an externalized distal shunt. Notably, the distal portion of the shunt demonstrated lower measurement error when compared with PC-MRI measurements acquired in the proximal catheter. CONCLUSIONS: The PC-MRI sequence provided accurate and reliable clinical measurements of CSF flow in shunt-treated patients. This work provides the necessary framework to include PC-MRI as an immediate addition to the clinical setting in the noninvasive evaluation of shunt function and in future clinical investigations of CSF physiology.

Decorating metal oxide surfaces with fluorescent chlorosulfonated corroles.

We have prepared 2,17-bis(chlorosulfonyl)-5,10,15-tris(pentafluorophenyl)corrole (1), 2,17-bis(chlorosulfonyl)-5,10,15-tris(pentafluorophenyl)corrolatoaluminum(III) (1-Al), and 2,17-bis(chlorosulfonyl)-5,10,15-tris(pentafluorophenyl)corrolatogallium(III) (1-Ga). The metal complexes 1-Al and 1-Ga were isolated and characterized by electronic absorption and NMR spectroscopies, as well as by mass spectrometry. Relative emission quantum yields for 1, 1-Al, and 1-Ga, determined in toluene, are 0.094, 0.127, and 0.099, respectively. Reactions between 1, 1-Al, and 1-Ga and TiO2 nanoparticles (NPs) result in corrole-TiO2 NP conjugates. The functionalized NP surfaces were investigated by solid-state Fourier transform infrared and X-ray photoelectron spectroscopies and by confocal fluorescence imaging. The fluorescence images for 1-Al-TiO2 and 1-Ga-TiO2 suggest a promising application of these NP conjugates as contrast agents for noninvasive optical imaging.

Development of an ultrafast brain MR neuronavigation protocol for ventricular shunt placement.

OBJECTIVE: Advancements in MRI technology have provided improved ways to acquire imaging data and to more seamlessly incorporate MRI into modern pediatric surgical practice. One such situation is image-guided navigation for pediatric neurosurgical procedures, including intracranial catheter placement. Image-guided surgery (IGS) requires acquisition of CT or MR images, but the former carries the risk of ionizing radiation and the latter is associated with long scan times and often requires pediatric patients to be sedated. The objective of this project was to circumvent the use of CT and standard-sequence MRI in ventricular neuronavigation by investigating the use of fast MR sequences on the basis of 3 criteria: scan duration comparable to that of CT acquisition, visualization of ventricular morphology, and image registration with surface renderings comparable to standard of care. The aim of this work was to report image development, implementation, and results of registration accuracy testing in healthy subjects. METHODS: The authors formulated 11 candidate MR sequences on the basis of the standard IGS protocol, and various scan parameters were modified, such as k-space readout direction, partial k-space acquisition, sparse sampling of k-space (i.e., compressed sensing), in-plane spatial resolution, and slice thickness. To evaluate registration accuracy, the authors calculated target registration error (TRE). A candidate sequence was selected for further evaluation in 10 healthy subjects. RESULTS: The authors identified a candidate imaging protocol, termed presurgical imaging with compressed sensing for time optimization (PICO). Acquisition of the PICO protocol takes 25 seconds. The authors demonstrated noninferior TRE for PICO (3.00 ± 0.19 mm) in comparison with the default MRI neuronavigation protocol (3.35 ± 0.20 mm, p = 0.20). CONCLUSIONS: The developed and tested sequence of this work allowed accurate intraoperative image registration and provided sufficient parenchymal contrast for visualization of ventricular anatomy. Further investigations will evaluate use of the PICO protocol as a substitute for CT and conventional MRI protocols in ventricular neuronavigation.

Episodic live imaging of cone photoreceptor maturation in GNAT2-EGFP retinal organoids.

Fluorescent reporter pluripotent stem cell (PSC) derived retinal organoids are powerful tools to investigate cell type-specific development and disease phenotypes. When combined with live imaging, they enable direct and repeated observation of cell behaviors within a developing retinal tissue. Here, we generated a human cone photoreceptor reporter line by CRISPR/Cas9 genome editing of WTC11-mTagRFPT-LMNB1 human induced pluripotent stem cells (iPSCs) by inserting enhanced green fluorescent protein (EGFP) coding sequences and a 2A self-cleaving peptide at the N-terminus of Guanine Nucleotide-Binding Protein Subunit Alpha Transducin 2 (GNAT2). In retinal organoids generated from these iPSCs, the GNAT2-EGFP allele robustly and exclusively labeled both immature and mature cones starting at culture day 34. Episodic confocal live imaging of hydrogel immobilized retinal organoids allowed tracking of morphological maturation of individual cones for >18 weeks and revealed inner segment accumulation of mitochondria and growth at 12.2 cubic microns per day from day 126 to day 153. Immobilized GNAT2-EGFP cone reporter organoids provide a valuable tool for investigating human cone development and disease.

Episodic live imaging of cone photoreceptor maturation in GNAT2-EGFP retinal organoids.

Fluorescent reporter pluripotent stem cell-derived retinal organoids are powerful tools to investigate cell type-specific development and disease phenotypes. When combined with live imaging, they enable direct and repeated observation of cell behaviors within a developing retinal tissue. Here, we generated a human cone photoreceptor reporter line by CRISPR/Cas9 genome editing of WTC11-mTagRFPT-LMNB1 human induced pluripotent stem cells (iPSCs) by inserting enhanced green fluorescent protein (EGFP) coding sequences and a 2A self-cleaving peptide at the N-terminus of guanine nucleotide-binding protein subunit alpha transducin 2 (GNAT2). In retinal organoids generated from these iPSCs, the GNAT2-EGFP alleles robustly and exclusively labeled immature and mature cones. Episodic confocal live imaging of hydrogel immobilized retinal organoids allowed tracking of the morphological maturation of individual cones for >18 weeks and revealed inner segment accumulation of mitochondria and growth at 12.2 µm3 per day from day 126 to day 153. Immobilized GNAT2-EGFP cone reporter organoids provide a valuable tool for investigating human cone development and disease.

Novel cell lines established from pediatric brain tumors.

The paucity of cell culture models for childhood brain tumors prompted us to establish pediatric cell lines for use in biological experiments and preclinical developmental therapeutic studies. Three cell lines were established, CHLA-200 (GBM), CHLA-259 (anaplastic medulloblastoma) and CHLA-266 (atypical teratoid rhabdoid tumor, AT/RT). Consistent with an AT/RT origin, CHLA-266 lacked INI1 expression and had monosomy 22. All lines had unique DNA short tandem repeat "fingerprints" matching that of the patient's tumor tissue and were adherent on tissue culture plastic, but differed in morphology and doubling times. CHLA-200 had a silent mutation in TP53. CHLA-259 and CHLA-266 had wild-type TP53. All three lines were relatively resistant to multiple drugs when compared to the DAOY medulloblastoma cell line, using the DIMSCAN fluorescence digital image microscopy cytotoxicity assay. RNA expression of MYC and MYCN were quantified using RT-PCR (Taqman). CHLA-200 expressed MYC, DAOY and CHLA-259 expressed MYCN, and CHLA-266 expressed both MYCN and MYC. CHLA-200 was only tumorigenic subcutaneously, but CHLA-259 and CHLA-266 were tumorigenic both subcutaneously and in brains of NOD/SCID mice. Immunohistochemistry of the xenografts revealed GFAP staining in CHLA-200 and PGP 9.5 staining in CHLA-259 and CHLA-266 tumors. As expected, INI1 expression was lacking in CHLA-266 (AT/RT). These three new cell lines will provide useful models for research of pediatric brain tumors.

Imaging Pulmonary Blood Vessels and Ventilation-Perfusion Mismatch in COVID-19.

COVID-19 hypoxemic patients although sharing a same etiology (SARS-CoV-2 infection) present themselves quite differently from one another. Patients also respond differently to prescribed medicine and to prone Vs supine bed positions. A severe pulmonary ventilation-perfusion mismatch usually triggers moderate to severe COVID-19 cases. Imaging can aid the physician in assessing severity of COVID-19. Although useful for their portability X-ray and ultrasound serving on the frontline to evaluate lung parenchymal abnormalities are unable to provide information about pulmonary vasculature and blood flow redistribution which is a consequence of hypoxemia in COVID-19. Advanced imaging modalities such as computed tomography, single-photon emission tomography, and electrical impedance tomography use a sharp algorithm visualizing pulmonary ventilation-perfusion mismatch in the abnormal and in the apparently normal parenchyma. Imaging helps to access the severity of infection, lung performance, ventilation-perfusion mismatch, and informs strategies for medical treatment. This review summarizes the capacity of these imaging modalities to assess ventilation-perfusion mismatch in COVID-19. Despite having limitations, these modalities provide vital information on blood volume distribution, pulmonary embolism, pulmonary vasculature and are useful to assess severity of lung disease and effectiveness of treatment in COVID-19 patients.

Automatic abdominal fat assessment in obese mice using a segmental shape model.

PURPOSE: To develop a computerized image analysis method to assess the quantity and distribution of abdominal fat tissues in an obese (ob/ob) mouse model relevant to 7 T magnetic resonance imaging (MRI). MATERIALS AND METHODS: A novel segmental shape model is presented that separates visceral adipose tissue (VAT) from subcutaneous adipose tissue (SAT). With shape and distance constraints, it deforms a contour inwards from the skin to the muscle wall and separates the connecting adipose tissues in an ob/ob mouse. The fat tissues are segmented by the adaptive fuzzy C means method to compensate for intensity variation in adipose images. The results were obtained by logical operations applied on the extracted fat images and the separated adipose masks. RESULTS: The method was validated by manual segmentations on 109 axial slice images from 7 ob/ob mice. The average correlation coefficients of measured sizes between the automatic and manual results for total adipose tissue (TAT) is 0.907; SAT is 0.944; VAT is 0. 950. The average Dice coefficient of their positions for TAT is 0.941, SAT is 0.935, and VAT is 0.920. CONCLUSION: The automated results correlate well with manual segmentations and the method can be used to increase laboratory automation.

Adipose segmentation in small animals at 7T: a preliminary study.

BACKGROUND: Small animal MRI at 7 Tesla (T) provides a useful tool for adiposity research. For adiposity researchers, separation of fat from surrounding tissues and its subsequent quantitative or semi- quantitative analysis is a key task. This is a relatively new field and a priori it cannot be known which specific biological questions related to fat deposition will be relevant in a specific study. Thus it is impossible to predict what accuracy and what spatial resolution will be required in all cases and even difficult what accuracy and resolution will be useful in most cases. However the pragmatic time constraints and the practical resolution ranges are known for small animal imaging at 7T. Thus we have used known practical constraints to develop a method for fat volume analysis based on an optimized image acquisition and image post processing pair. METHODS: We designed a fat segmentation method based on optimizing a variety of factors relevant to small animal imaging at 7T. In contrast to most previously described MRI methods based on signal intensity of T1 weighted image alone, we chose to use parametric images based on Multi-spin multi-echo (MSME) Bruker pulse sequence which has proven to be particularly robust in our laboratory over the last several years. The sequence was optimized on a T1 basis to emphasize the signal. T2 relaxation times can be calculated from the multi echo data and we have done so on a pixel by pixel basis for the initial step in the post processing methodology. The post processing consists of parallel paths. On one hand, the weighted image is precisely divided into different regions with optimized smoothing and segmentation methods; and on the other hand, a confidence image is deduced from the parametric image according to the distribution of relaxation time relationship of typical adipose. With the assistance of the confidence image, a useful software feature was implemented to which enhances the data and in the end results in a more reliable and flexible method for adipose evaluation. RESULTS: In this paper, we describe how we arrived at our recommended procedures and key aspects of the post-processing steps. The feasibility of the proposed method is tested on both simulated and real data in this preliminary research. A research tool was created to help researchers segment out fat even when the anatomical information is of low quality making it difficult to distinguish between fat and non-fat. In addition, tool is designed to allow the operator to make adjustments to many of the key steps for comparison purposes and to quantitatively assess the difference these changes make. Ultimately our flexible software lets the researcher define key aspects of the fat segmentation and quantification. CONCLUSIONS: Combining the full T2 parametric information with the optimized first echo image information, the research tool enhances the reliability of the results while providing more flexible operations than previous methods. The innovation in the method is to pair an optimized and very specific image acquisition technique to a flexible but tuned image post processing method. The separation of the fat is aided by the confidence distribution of regions produced on a scale relevant to and dictated by practical aspects of MRI at 7T.

Distribution of 14C-Latanoprost Following a Single Intracameral Administration Versus Repeated Topical Administration.

PURPOSE: To qualitatively evaluate the ocular and periocular distribution of 14C-latanoprost following a single intracameral administration or repeated topical ocular administration in beagle dogs and cynomolgus monkeys. METHODS: In the dog study, three animals received an intracameral dose of 14C-latanoprost bilaterally and were euthanized at 1, 2, and 4 h post dose; three control animals received topical 14C-latanoprost bilaterally once daily for 5 days and were euthanized at 1, 4, and 24 h post final dose. Sagittal 40-µm sections of eyes with surrounding tissues were collected and processed for autoradiography. Methods in the monkey study were similar; two animals received a unilateral intracameral dose of 14C-latanoprost. RESULTS: After intracameral dosing in dogs, radioactivity was concentrated in the cornea, iris, ciliary body, and anterior chamber with no radioactivity detected in the eyelids or other periorbital tissues. After topical dosing, radioactivity was distributed in the bulbar conjunctiva, cornea, anterior chamber, iris, ciliary body, upper and lower eyelids, and periorbital tissues (fat/muscle). After intracameral dosing in monkeys, radioactivity was concentrated in the anterior chamber, cornea, iris, ciliary body, and posteriorly along the uveoscleral outflow pathway; there was no radioactivity in the eyelids or periorbital tissues aside from signal in the nasolacrimal duct, likely from reflux of 14C-latanoprost into the tear film. CONCLUSIONS: Intracameral delivery resulted in more selective target tissue drug exposure. Intracameral drug delivery has potential to reduce ocular surface and periocular adverse effects associated with topical administration of prostaglandin analogues, such as eyelash growth and periorbital fat atrophy.

The activity of zoledronic Acid on neuroblastoma bone metastasis involves inhibition of osteoclasts and tumor cell survival and proliferation.

Metastasis to the bone is seen in 56% of patients with neuroblastoma and contributes to morbidity and mortality. Using a murine model of bone invasion, we have reported previously that neuroblastoma cells invade the bone by activating osteoclasts. Here, we investigated the antitumoral and antiosteolytic activities of zoledronic acid, a bisphosphonate inhibitor of osteoclasts, in combination with cytotoxic chemotherapy in our model. We first show that zoledronic acid given at the same time (early prevention) or 2 weeks after tumor cell injection (late prevention) significantly prevented the formation of severe osteolytic lesions. It also prevented formation of these lesions when given 4 weeks after tumor cell injection (intervention) when combined with chemotherapy including cyclophosphamide and topotecan. The combination of zoledronic acid + cyclophosphamide/topotecan also significantly improved survival (P < 0.001). In mice treated with zoledronic acid, we observed a marked inhibition of osteoclasts inside the bone associated with a decrease in tumor cell proliferation and increase in tumor cell apoptosis. In vitro, zoledronic acid inhibited neuroblastoma cell proliferation and induced apoptosis, and these effects were significantly enhanced by the addition of 4-hydroxyperoxycyclophosphamide (4-HC). The proapoptotic effect of zoledronic acid and zoledronic acid in combination with 4-HC on tumor cells was associated with an increase in caspase-3 activity and a decrease in phosphorylated Bcl-2, Bcl-2, and Bcl-X(L) expression. Zoledronic acid inhibited the association of Ras with the plasma membrane and activation of c-Raf, Akt, and extracellular signal-regulated kinase 1/2. The data indicate that zoledronic acid, in addition to inhibiting osteoclasts, is active against tumor cells and suggest that zoledronic acid in combination with cytotoxic chemotherapy may be effective in children with neuroblastoma that has metastasized to the bone.

Continuous and bolus intraventricular topotecan prolong survival in a mouse model of leptomeningeal medulloblastoma.

Leptomeningeal metastasis remains a difficult clinical challenge. Some success has been achieved by direct administration of therapeutics into the cerebrospinal fluid (CSF) circumventing limitations imposed by the blood brain barrier. Here we investigated continuous infusion versus bolus injection of therapy into the CSF in a preclinical model of human Group 3 medulloblastoma, the molecular subgroup with the highest incidence of leptomeningeal disease. Initial tests of selected Group 3 human medulloblastoma cell lines in culture showed that D283 Med and D425 Med were resistant to cytosine arabinoside and methotrexate. D283 Med cells were also resistant to topotecan, whereas 1 µM topotecan killed over 99% of D425 Med cells. We therefore introduced D425 Med cells, modified to express firefly luciferase, into the CSF of immunodeficient mice. Mice were then treated with topotecan or saline in five groups: continuous intraventricular (IVT) topotecan via osmotic pump (5.28 µg/day), daily bolus IVT topotecan injections with a similar daily dose (6 µg/day), systemic intraperitoneal injections of a higher daily dose of topotecan (15 µg/day), daily IVT pumped saline and daily intraperitoneal injections of saline. Bioluminescence analyses revealed that both IVT topotecan treatments effectively slowed leptomeningeal tumor growth in the brains. Histological analysis showed that they were associated with localized brain necrosis, possibly due to backtracking of topotecan around the catheter. In the spines, bolus IVT topotecan showed a trend towards slower tumor growth compared to continuous (pump) IVT topotecan, as measured by bioluminescence. Both continuous and bolus topotecan IVT showed longer survival compared to other groups. Thus, both direct IVT topotecan CSF delivery methods produced better anti-medulloblastoma effect compared to systemic therapy at the dosages used here.

PID1 increases chemotherapy-induced apoptosis in medulloblastoma and glioblastoma cells in a manner that involves NFκB.

Phosphotyrosine Interaction Domain containing 1 (PID1; NYGGF4) inhibits growth of medulloblastoma, glioblastoma and atypical teratoid rhabdoid tumor cell lines. PID1 tumor mRNA levels are highly correlated with longer survival in medulloblastoma and glioma patients, suggesting their tumors may have been more sensitive to therapy. We hypothesized that PID1 sensitizes brain tumors to therapy. We found that PID1 increased the apoptosis induced by cisplatin and etoposide in medulloblastoma and glioblastoma cell lines. PID1 siRNA diminished cisplatin-induced apoptosis, suggesting that PID1 is required for cisplatin-induced apoptosis. Etoposide and cisplatin increased NFκB promoter reporter activity and etoposide induced nuclear translocation of NFκB. Etoposide also increased PID1 promoter reporter activity, PID1 mRNA, and PID1 protein, which were diminished by NFκB inhibitors JSH-23 and Bay117082. However, while cisplatin increased PID1 mRNA, it decreased PID1 protein. This decrease in PID1 protein was mitigated by the proteasome inhibitor, bortezomib, suggesting that cisplatin induced proteasome dependent degradation of PID1. These data demonstrate for the first time that etoposide- and cisplatin-induced apoptosis in medulloblastoma and glioblastoma cell lines is mediated in part by PID1, involves NFκB, and may be regulated by proteasomal degradation. This suggests that PID1 may contribute to responsiveness to chemotherapy.

Optimization of a Neural Stem-Cell-Mediated Carboxylesterase/Irinotecan Gene Therapy for Metastatic Neuroblastoma.

Despite improved survival for children with newly diagnosed neuroblastoma (NB), recurrent disease is a significant problem, with treatment options limited by anti-tumor efficacy, patient drug tolerance, and cumulative toxicity. We previously demonstrated that neural stem cells (NSCs) expressing a modified rabbit carboxylesterase (rCE) can distribute to metastatic NB tumor foci in multiple organs in mice and convert the prodrug irinotecan (CPT-11) to the 1,000-fold more toxic topoisomerase-1 inhibitor SN-38, resulting in significant therapeutic efficacy. We sought to extend these studies by using a clinically relevant NSC line expressing a modified human CE (hCE1m6-NSCs) to establish proof of concept and identify an intravenous dose and treatment schedule that gave maximal efficacy. Human-derived NB cell lines were significantly more sensitive to treatment with hCE1m6-NSCs and irinotecan as compared with drug alone. This was supported by pharmacokinetic studies in subcutaneous NB mouse models demonstrating tumor-specific conversion of irinotecan to SN-38. Furthermore, NB-bearing mice that received repeat treatment with intravenous hCE1m6-NSCs and irinotecan showed significantly lower tumor burden (1.4-fold, p = 0.0093) and increased long-term survival compared with mice treated with drug alone. These studies support the continued development of NSC-mediated gene therapy for improved clinical outcome in NB patients.

Structural and Functional Characterization of Human Stem-Cell-Derived Retinal Organoids by Live Imaging.

Purpose: Human pluripotent stem cell (hPSC)-derived retinal organoids are a platform for investigating retinal development, pathophysiology, and cellular therapies. In contrast to histologic analysis in which multiple specimens fixed at different times are used to reconstruct developmental processes, repeated analysis of the same living organoids provides a more direct means to characterize changes. New live imaging modalities can provide insights into retinal organoid structure and metabolic function during in vitro growth. This study employed live tissue imaging to characterize retinal organoid development, including metabolic changes accompanying photoreceptor differentiation. Methods: Live hPSC-derived retinal organoids at different developmental stages were examined for microanatomic organization and metabolic function by phase contrast microscopy, optical coherence tomography (OCT), fluorescence lifetime imaging microscopy (FLIM), and hyperspectral imaging (HSpec). Features were compared to those revealed by histologic staining, immunostaining, and microcomputed tomography (micro-CT) of fixed organoid tissue. Results: We used FLIM and HSpec to detect changes in metabolic activity as organoids differentiated into organized lamellae. FLIM detected increased glycolytic activity and HSpec detected retinol and retinoic acid accumulation in the organoid outer layer, coinciding with photoreceptor genesis. OCT enabled imaging of lamellae formed during organoid maturation. Micro-CT revealed three-dimensional structure, but failed to detect lamellae. Conclusions: Live imaging modalities facilitate real-time and nondestructive imaging of retinal organoids as they organize into lamellar structures. FLIM and HSpec enable rapid detection of lamellar structure and photoreceptor metabolism. Live imaging techniques may aid in the continuous evaluation of retinal organoid development in diverse experimental and cell therapy settings.

Intestinal adaptation in proximal and distal segments: Two epithelial responses diverge after intestinal separation.

BACKGROUND: In short bowel syndrome, luminal factors influence adaptation in which the truncated intestine increases villus lengths and crypt depths to increase nutrient absorption. No study has evaluated the effect of adaptation within the distal intestine after intestinal separation. We evaluated multiple conditions, including Igf1r inhibition, in proximal and distal segments after intestinal resection to evaluate the epithelial effects of the absence of mechanoluminal stimulation. METHODS: Short bowel syndrome was created in adult male zebrafish by performing a proximal stoma with ligation of the distal intestine. These zebrafish with short bowel syndrome were compared to sham-operated zebrafish. Groups were treated with the Igf1r inhibitor NVP-AEW541, DMSO, a vehicle control, or water for 2 weeks. Proximal and distal intestine were analyzed by hematoxylin and eosin for villus epithelial circumference, inner epithelial perimeter, and circumference. We evaluated BrdU+ cells, including costaining for ß-catenin, and the microbiome was evaluated for changes. Reverse transcription quantitative polymerase chain reaction was performed for ß-catenin, CyclinD1, Sox9a, Sox9b, and c-Myc. RESULTS: Proximal intestine demonstrated significantly increased adaptation compared to sham-operated proximal intestine, whereas the distal intestine showed no adaptation in the absence of luminal flow. Addition of the Igf1r inhibitor resulted in decreased adaption in the distal intestine but an increase in distal proliferative cells and proximal ß-catenin expression. While some proximal proliferative cells in short bowel syndrome colocalized ß-catenin and BrdU, the distal proliferative cells did not co-stain for ß-catenin. Sox9a increased in the distal limb after division but not after inhibition with the Igf1r inhibitor. There was no difference in alpha diversity or species richness of the microbiome between all groups. CONCLUSION: Luminal flow in conjunction with short bowel syndrome significantly increases intestinal adaption within the proximal intestine in which proliferative cells contain ß-catenin. Addition of an Igf1r inhibitor decreases adaptation in both proximal and distal limbs while increasing distal proliferative cells that do not colocalize ß-catenin. Igf1r inhibition abrogates the increase in distal Sox9a expression that otherwise occurs in short bowel syndrome. Mechanoluminal flow is an important stimulus for intestinal adaptation.

Human Neural Stem Cell Biodistribution and Predicted Tumor Coverage by a Diffusible Therapeutic in a Mouse Glioma Model.

Engineered neural stem cells (NSCs) intrinsically migrating to brain tumors offer a promising mechanism for local therapeutic delivery. However, difficulties in quantitative assessments of NSC migration and in estimates of tumor coverage by diffusible therapeutics have impeded development and refinement of NSC-based therapies. To address this need, we developed techniques by which conventional serial-sectioned formalin-fixed paraffin-embedded (FFPE) brains can be analyzed in their entirety across multiple test animals. We considered a conventional human glioblastoma model: U251 glioma cells orthotopically engrafted in immunodeficient mice receiving intracerebral (i.c.) or intravenous (i.v.) administrations of NSCs expressing a diffusible enzyme to locally catalyze chemotherapeutic formation. NSC migration to tumor sites was dose-dependent, reaching 50%-60% of total administered NSCs for the i.c route and 1.5% for the i.v. route. Curiously, the most efficient NSC homing was seen with smaller NSC doses, implying existence of rate-limiting process active during administration and/or migration. Predicted tumor exposure to a diffusing therapeutic (assuming a 50 µm radius of action) could reach greater than 50% of the entire tumor volume for i.c. and 25% for i.v. administration. Within individual sections, coverage of tumor area could be as high as 100% for i.c. and 70% for i.v. routes. Greater estimated therapeutic coverage was observed for larger tumors and for larger tumor regions in individual sections. Overall, we have demonstrated a framework within which investigators may rationally evaluate NSC migration to, and integration into, brain tumors, and therefore enhance understanding of mechanisms that both promote and limit this therapeutic modality. Stem Cells Translational Medicine 2017;6:1522-1532.

Longitudinal microPET imaging of brain tumor growth with F-18-labeled RGD peptide.

PURPOSE: EMD 121974, a potent cyclic RGD peptide inhibitor of alphav-integrins, demonstrated effectiveness in suppressing brain tumor growth in both preclinical models and phases I/II clinical trials. The ability to non-invasively evaluate alphav-integrin expression provides a novel and unique way to better understand brain tumor angiogenesis in relationship to alphav-integrin expression, and allow for direct assessment of anti-integrin treatment efficacy. PROCEDURES: We developed a F-18-labeled RGD peptide [F-18]FB-RGD and performed serial microPET imaging scans to follow brain tumor growth and angiogenesis as a function of time in an orthotopic U87MG glioblastoma xenograft model in athymic nude mice. RESULTS: The tumor was barely visible on microPET at the size of