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.

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.

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.

Dynamic imaging of the growth plate cartilage reveals multiple contributors to skeletal morphogenesis.

The diverse morphology of vertebrate skeletal system is genetically controlled, yet the means by which cells shape the skeleton remains to be fully illuminated. Here we perform quantitative analyses of cell behaviours in the growth plate cartilage, the template for long bone formation, to gain insights into this process. Using a robust avian embryonic organ culture, we employ time-lapse two-photon laser scanning microscopy to observe proliferative cells' behaviours during cartilage growth, resulting in cellular trajectories with a spreading displacement mainly along the tissue elongation axis. We build a novel software toolkit of quantitative methods to segregate the contributions of various cellular processes to the cellular trajectories. We find that convergent-extension, mitotic cell division, and daughter cell rearrangement do not contribute significantly to the observed growth process; instead, extracellular matrix deposition and cell volume enlargement are the key contributors to embryonic cartilage elongation.

Synergic silencing of costimulatory molecules prevents cardiac allograft rejection.

BACKGROUND: While substantial progress has been made in blocking acute transplant rejection with the advent of immune suppressive drugs, chronic rejection, mediated primarily by recipient antigen presentation, remains a formidable problem in clinical transplantation. We hypothesized that blocking co-stimulatory pathways in the recipient by induction of RNA interference using small interference RNA (siRNA) expression vectors can prolong allogeneic heart graft survival. METHOD: Vectors expressing siRNA specifically targeting CD40 and CD80 were prepared. Recipients (BALB/c mice) were treated with CD40 and/or CD80 siRNA expression vectors via hydrodynamic injection. Control groups were injected with a scrambled siRNA vector and sham treatment (PBS). After treatment, a fully MHC-mismatched (BALB/c to C57/BL6) heart transplantation was performed. RESULT: Allogeneic heart graft survival (>100 days) was approximately 70% in the mice treated simultaneously with CD40 and CD80 siRNA expression vectors with overall reduction in lymphocyte interstitium infiltration, vascular obstruction, and edema. Hearts transplanted into CD40 or CD80 siRNA vector-treated recipients had an increased graft survival time compared to negative control groups, but did not survive longer than 40 days. In contrast, allogenic hearts transplanted into recipients treated with scrambled siRNA vector and PBS stopped beating within 10-16 days. Real-time PCR (RT-PCR) and flow cytometric analysis showed an upregulation of FoxP3 expression in spleen lymphocytes and a concurrent downregulation of CD40 and CD80 expression in splenic dendritic cells of siRNA-treated mice. Functional suppressive activity of splenic dendritic cells (DCs) isolated from tolerant recipients was demonstrated in a mixed lymphocyte reaction (MLR). Furthermore, DCs isolated from CD40- and CD80-treated recipients promoted CD4+CD25+FoxP3+ regulatory T cell differentiation in vitro. CONCLUSION: This study demonstrates that the simultaneous silencing of CD40 and CD80 genes has synergistic effects in preventing allograft rejection, and may therefore have therapeutic potential in clinical transplantation.

Exogenous endothelial cells as accelerators of hematopoietic reconstitution.

Despite the successes of recombinant hematopoietic-stimulatory factors at accelerating bone marrow reconstitution and shortening the neutropenic period post-transplantation, significant challenges remain such as cost, inability to reconstitute thrombocytic lineages, and lack of efficacy in conditions such as aplastic anemia. A possible means of accelerating hematopoietic reconstitution would be administration of cells capable of secreting hematopoietic growth factors. Advantages of this approach would include: a) ability to regulate secretion of cytokines based on biological need; b) long term, localized production of growth factors, alleviating need for systemic administration of factors that possess unintended adverse effects; and c) potential to actively repair the hematopoietic stem cell niche. Here we overview the field of hematopoietic growth factors, discuss previous experiences with mesenchymal stem cells (MSC) in accelerating hematopoiesis, and conclude by putting forth the rationale of utilizing exogenous endothelial cells as a novel cellular therapy for acceleration of hematopoietic recovery.

Electrophysiological characterization of Grueneberg ganglion olfactory neurons: spontaneous firing, sodium conductance, and hyperpolarization-activated currents.

Mammals rely on their acute olfactory sense for their survival. The most anterior olfactory subsystem in the nose, the Grueneberg ganglion (GG), plays a role in detecting alarm pheromone, cold, and urinary compounds. GG neurons respond homogeneously to these stimuli with increases in intracellular [Ca(2+)] or transcription of immediate-early genes. In this electrophysiological study, we used patch-clamp techniques to characterize the membrane properties of GG neurons. Our results offer evidence of functional heterogeneity in the GG. GG neurons fire spontaneously and independently in several stable patterns, including phasic and repetitive single-spike modes of discharge. Whole cell recordings demonstrated two distinct voltage-gated fast-inactivating Na(+) currents with different steady-state voltage dependencies and different sensitivities to tetrodotoxin. Hodgkin-Huxley simulations showed that these Na(+) currents confer dual mechanisms of action potential generation and contribute to different firing patterns. Additionally, GG neurons exhibited hyperpolarization-activated inward currents that modulated spontaneous firing in vitro. Thus, in GG neurons, the heterogeneity of firing patterns is linked to the unusual repertoire of ionic currents. The membrane properties described here will aid the interpretation of chemosensory function in the GG.

Reduction of tumorigenicity by placental extracts.

The influence of adult stem cells on tumor growth is paradoxical. On one hand, angiogenic factors secreted by stem cells are known to be essential for tumor vascularization. On the other hand, stem cell-derived factors can reportedly induce tumor differentiation or direct death of tumor cells. Both the placenta and umbilical cord are rich sources of stem cells with immune modulatory and tissue-healing properties; however, the effects of placental components on cancer cells have not been fully defined. Here we demonstrate that extracts of placental lysates reduce the malignancy of a variety of human tumor cell lines in a species-unrestricted manner. Using a standard model of leukemia cell differentiation, we demonstrated that addition of placental extracts to tumor cells, or co-culture of tumor cells with the CD34(+) cells from umbilical cord blood, induced tumor cell differentiation. Inhibition of tumor growth and metastasis in vivo was also observed following administration of placental extracts. These data support the concept of non-toxic biological therapy of cancer using stem cell derivatives, possibly through the induction of tumor cell differentiation.

Differential phase-contrast, swept-source optical coherence tomography at 1060 nm for in vivo human retinal and choroidal vasculature visualization.

Human retinal and choroidal vasculature was visualized by a differential phase-contrast (DPC) method using high-speed, swept-source optical coherence tomography (SS-OCT) at 1060 nm. The vasculature was recognized as regions of motion by creating differential phase-variance (DPV) tomograms: multiple B-scans of individual slices through the retina were collected and the variance of the phase differences was calculated. DPV captured the small vessels and the meshwork of capillaries associated with the inner retina in en-face images over 4 mm(2). The swept-source laser at 1060 nm offered the needed phase sensitivity to perform DPV and generated en-face images that capture motion in the inner choroidal layer exceeding the capabilities of previous spectrometer-based instruments. In comparison with the power Doppler phase-shift method, DPV provided better visualization of the foveal avascular zone in en-face images.

Gene silencing of IL-12 in dendritic cells inhibits autoimmune arthritis.

BACKGROUND: We have previously demonstrated that immune modulation can be accomplished by administration of gene silenced dendritic cells (DC) using siRNA. In this study, we demonstrate the therapeutic utilization of shRNA-modified DC as an antigen-specific tolerogenic vaccine strategy for autoimmune arthritis. METHODS: A shRNA that specifically targets IL-12 p35 was designed and cloned into a plasmid vectors (IL-12 shRNA). Bone marrow-derived DC from DBA/1 mice were transfected with the IL-12 shRNA construct in vitro. Mice with collagen II (CII)-induced arthritis (CIA) were treated with the modified DCs expressing the shRNA. Recall response and disease progression were assessed. RESULTS: After gene silencing of IL-12 in DC, DC were shown to selectively inhibit T cell proliferation on recall responses and in an MLR. In murine CIA, we demonstrated that administration of IL-12 shRNA-expressing DC that were pulsed with CII inhibited progression of arthritis. The therapeutic effects were evidenced by decreased clinical scores, inhibition of inflammatory cell infiltration in the joint, and suppression of T cell and B cell responses to CII. CONCLUSION: We demonstrate a novel tolerance-inducing protocol for the treatment of autoimmune inflammatory joint disease in which the target antigen is known, utilizing DNA-directed RNA interference.

Deep and fast live imaging with two-photon scanned light-sheet microscopy.

We implemented two-photon scanned light-sheet microscopy, combining nonlinear excitation with orthogonal illumination of light-sheet microscopy, and showed its excellent performance for in vivo, cellular-resolution, three-dimensional imaging of large biological samples. Live imaging of fruit fly and zebrafish embryos confirmed that the technique can be used to image up to twice deeper than with one-photon light-sheet microscopy and more than ten times faster than with point-scanning two-photon microscopy without compromising normal biology.

Tumor vaccines in 2010: need for integration.

Induction of tumor-specific immunity is an attractive approach to cancer therapy, however to date every major pivotal trial has resulted in failure. While the phenomena of tumor-mediated immune suppression has been known for decades, only recently have specific molecular pathways been elucidated, and for the first time, rationale means of intervening and observing results of intervention have been developed. In this review we describe major advances in our understanding of tumor escape from immunological pressure and provide some possible therapeutic scenarios for enhancement of efficacy in future cancer vaccine trials.

Lasers, stem cells, and COPD.

The medical use of low level laser (LLL) irradiation has been occurring for decades, primarily in the area of tissue healing and inflammatory conditions. Despite little mechanistic knowledge, the concept of a non-invasive, non-thermal intervention that has the potential to modulate regenerative processes is worthy of attention when searching for novel methods of augmenting stem cell-based therapies. Here we discuss the use of LLL irradiation as a "photoceutical" for enhancing production of stem cell growth/chemoattractant factors, stimulation of angiogenesis, and directly augmenting proliferation of stem cells. The combination of LLL together with allogeneic and autologous stem cells, as well as post-mobilization directing of stem cells will be discussed.

Grueneberg ganglion olfactory subsystem employs a cGMP signaling pathway.

The mammalian olfactory sense employs several olfactory subsystems situated at characteristic locations in the nasal cavity to detect and report on different classes of odors. These olfactory subsystems use different neuronal signal transduction pathways, receptor expression repertoires, and axonal projection targets. The Grueneberg ganglion (GG) is a newly appreciated olfactory subsystem with receptor neurons located just inside of the nostrils that project axons to a unique domain of interconnected glomeruli in the caudal olfactory bulb. It is not well understood how the GG relates to other olfactory subsystems in contributing to the olfactory sense. Furthermore, the range of chemoreceptors and the signal transduction cascade utilized by the GG have remained mysterious. To resolve these unknowns, we explored the molecular relationship between the GG and the GC-D neurons, another olfactory subsystem that innervates similarly interconnected glomeruli in the same bulbar region. We found that mouse GG neurons express the cGMP-associated signaling proteins phosphodiesterase 2a, cGMP-dependent kinase II, and cyclic nucleotide gated channel subunit A3 coupled to a chemoreceptor repertoire of cilia-localized particulate guanylyl cyclases (pGC-G and pGC-A). The primary cGMP signaling pathway of the GG is shared with the GC-D neurons, unifying their target glomeruli as a unique center of olfactory cGMP signal transduction. However, the distinct chemoreceptor repertoire in the GG suggests that the GG is an independent olfactory subsystem. This subsystem is well suited to detect a unique set of odors and to mediate behaviors that remained intact in previous olfactory perturbations.

The Grueneberg ganglion projects to the olfactory bulb.

The Grueneberg ganglion is a compact cluster of neurons in the rostral nasal vestibule once thought to be a component of the terminal nerve, a non-sensory nerve that does not innervate the olfactory bulb. Its strong expression of olfactory marker protein, a pan-olfactory marker, in mice led us to re-examine this conclusion. Here, we demonstrate that the Grueneberg ganglion projects axons from the nasal vestibule, along the septum, through the cribriform plate and onto the olfactory necklace domain of the olfactory bulbs where it forms glomeruli. Its expression of olfactory marker protein, combined with its direct wiring to the olfactory bulb, strongly suggest that the Grueneberg ganglion is a component of the olfactory pathway.

The elongation factors Pandora/Spt6 and Foggy/Spt5 promote transcription in the zebrafish embryo.

Precise temporal and spatial control of transcription is a fundamental component of embryonic development. Regulation of transcription elongation can act as a rate-limiting step during mRNA synthesis. The mechanisms of stimulation and repression of transcription elongation during development are not yet understood. We have identified a class of zebrafish mutations (pandora, sk8 and s30) that cause multiple developmental defects, including discrete problems with pigmentation, tail outgrowth, ear formation and cardiac differentiation. We demonstrate that the pandora gene encodes a protein similar to Spt6, a proposed transcription elongation factor. Additionally, the sk8 and s30 mutations are null alleles of the foggy/spt5 locus, which encodes another transcription elongation factor. Through real-time RT-PCR analysis, we demonstrate that Spt6 and Spt5 are both required for efficient kinetics of hsp70 transcription in vivo. Altogether, our results suggest that Spt6 and Spt5 play essential roles of comparable importance for promoting transcription during embryogenesis. This study provides the first genetic evidence for parallel functions of Spt6 and Spt5 in metazoans and establishes a system for the future analysis of transcription elongation during development.