A Phase 2 Trial of Primary Tumor Stereotactic Body Radiation Therapy Boost Before Concurrent Chemoradiation for Locally Advanced Non-Small Cell Lung Cancer.

PURPOSE: Primary tumor failure is common in patients treated with chemoradiation (CRT) for locally advanced NSCLC (LA-NSCLC). Stereotactic body radiation therapy (SBRT) yields high rates of primary tumor control (PTC) in early-stage NSCLC. This trial tested an SBRT boost to the primary tumor before the start of CRT to improve PTC. METHODS AND MATERIALS: Patients with LA-NSCLC received an SBRT boost in 2 fractions (central location 12 Gy, peripheral location 16 Gy) to the primary tumor, followed by standard CRT (60 Gy in 30 fractions). The primary objective was PTC rate at 1 year, and the hypothesis was that the 1-year PTC rate would be ≥90%. Secondary objectives included objective response rate, regional and distant control, disease-free survival (DFS), and overall survival (OS). Correlative studies included functional magnetic resonance imaging and blood-based miRNA analysis. RESULTS: The study enrolled 21 patients (10 men and 11 women); the median age was 62 years (range, 52-78). The median pretreatment primary tumor size was 5.0 cm (range, 1.0-8.3). The most common nonhematologic toxicities were pneumonitis, fatigue, esophagitis/dysphagia, dyspnea, and cough. Only 1 treatment-related grade 4 nonhematologic toxicity occurred (respiratory failure/radiation pneumonitis), and no grade 5 toxicities occurred. The objective response rate at 3 and 6 months was 72.7% and 80.0%, respectively, and PTC at 1 and 2 years was 100% and 92.3%, respectively. The 2-year regional and distant control rates were 81.6% and 70.3%, respectively. Disease-free survival and overall survival at 2 years were 46.1% and 50.3%, respectively, and median survival was 37.8 months. Functional magnetic resonance imaging detected a mean relative decrease in blood oxygenation level-dependent signal of -87.1% (P = .05), and miR.142.3p was correlated with increased risk of grade ≥3 pulmonary toxicity (P = .01). CONCLUSIONS: Dose escalation to the primary tumor using upfront SBRT appears feasible and safe. PTC was high and other oncologic endpoints compared favorably to standard treatment. Functional magnetic resonance imaging suggested changes in oxygenation with the first SBRT boost dose, and miR.142.3p was correlated with pulmonary toxicity.

Comparison of epigenetic mediator expression and function in mouse and human embryonic blastomeres.

A map of human embryo development that combines imaging, molecular, genetic and epigenetic data for comparisons to other species and across pathologies would be greatly beneficial for basic science and clinical applications. Here, we compared mRNA and protein expression of key mediators of DNA methylation and histone modifications between mouse and human embryos, embryos from fertile/infertile couples, and following growth factor supplementation. We observed that individual mouse and human embryos are characterized by similarities and distinct differences in DNA methylation and histone modification patterns especially at the single-cell level. In particular, while mouse embryos first exhibited sub-compartmentalization of different histone modifications between blastomeres at the morula stage and cell sub-populations in blastocysts, differential histone modification expression was detected between blastomeres earlier in human embryos at the four- to eight-cell stage. Likewise, differences in epigenetic mediator expression were also observed between embryos from fertile and infertile couples, which were largely equalized in response to growth factor supplementation, suggesting that select growth factors might prevent alterations in epigenetic profiles during prolonged embryo culture. Finally, we determined that reduced expression via morpholino technologies of a single histone-modifying enzyme, Rps6ka4/Msk2, resulted in cleavage-stage arrest as assessed by time-lapse imaging and was associated with aneuploidy generation. Taken together, data document differences in epigenetic patterns between species with implications for fertility and suggest functional roles for individual epigenetic factors during pre-implantation development.

Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS.

Amyotrophic lateral sclerosis (ALS) causes motor neuron degeneration, paralysis, and death. Accurate disease modeling, identifying disease mechanisms, and developing therapeutics is urgently needed. We previously reported motor neuron toxicity through postmortem ALS spinal cord-derived astrocytes. However, these cells can only be harvested after death, and their expansion is limited. We now report a rapid, highly reproducible method to convert adult human fibroblasts from living ALS patients to induced neuronal progenitor cells and subsequent differentiation into astrocytes (i-astrocytes). Non-cell autonomous toxicity to motor neurons is found following coculture of i-astrocytes from familial ALS patients with mutation in superoxide dismutase or hexanucleotide expansion in C9orf72 (ORF 72 on chromosome 9) the two most frequent causes of ALS. Remarkably, i-astrocytes from sporadic ALS patients are as toxic as those with causative mutations, suggesting a common mechanism. Easy production and expansion of i-astrocytes now enables rapid disease modeling and high-throughput drug screening to alleviate astrocyte-derived toxicity.

Day 2 transfer in clinical ART.

Over the past 20 years, numerous techniques have enhanced assisted reproductive technology outcomes to help couples have >60,000 infants in the United States in 2008. Several different days for embryo transfers have been studied, but debate for the best timing of embryo transfer is still ongoing. With growing concern about multiple gestations and neonatal outcomes, early cleavage stage embryo transfer with novel embryo selection tools may be attractive to some patients and in vitro fertilization programs. In this review, we summarize clinical and basic studies relating to the timing of embryo transfer and highlight the possibilities of safe embryo transfer by combining advanced embryo screening tools with potentially high efficiency and low adverse effects on clinical outcome.

Transcriptomic signature of trophoblast differentiation in a human embryonic stem cell model.

Identification of genes involved in trophoblast differentiation is of great interest in understanding cellular and molecular mechanisms involved in placental development and is relevant clinically to fetal development, fertility, and maternal health. Herein, we investigated differentiation of human embryonic stem cells (hESCs) down the trophoblast lineage by culture with bone morphogenetic protein 4 (BMP4) over a 10-day period. Within 2 days, the stemness markers POU5F1 and NANOG were markedly down-regulated, followed temporally by up-regulation of the CDX2, KRT7, HLA-G, ID2, CGA, and CGB trophoblast markers. To understand, on a global scale, changes in the transcriptome during the differentiation of hESCs down the trophoblast lineage, a large-scale microarray analysis was performed. Through whole-genome analysis, more than 3800 genes displayed statistically significant and 2-fold or greater changes in expression during the time course. Of those genes that showed the largest increases, many were involved in processes associated with trophoblast biology; however, novel genes were also identified. Some of them are hypothesized to be associated mainly with extracellular matrix remodeling (e.g., NID2) and cell migration and invasion (e.g., RAB25). Using Ingenuity pathways analysis software to identify signaling pathways involved in trophoblast differentiation or function, we discovered that many genes are involved in WNT/beta-catenin, ERK/MAPK, NFKB, and calcium signaling pathways, suggesting potential roles for these families in trophoblast development. This work provides an in vitro functional genomic model with which to identify genes involved in trophoblast development.

Parthenogenic blastocysts derived from cumulus-free in vitro matured human oocytes.

BACKGROUND: Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis. METHODOLOGY/PRINCIPAL FINDING: Immature human oocytes were matured in vitro via supplementation with ovarian paracrine/autocrine factors that were selected based on expression of ligands in the cumulus cells and their corresponding receptors in oocytes. Matured oocytes were artificially activated to assess developmental competence. Gene expression profiles of parthenotes were compared to IVF/ICSI embryos at morula and blastocyst stages. Following incubation in medium supplemented with ovarian factors (BDNF, IGF-I, estradiol, GDNF, FGF2 and leptin), a greater percentage of oocytes demonstrated nuclear maturation and subsequently, underwent parthenogenesis relative to control. Similarly, cytoplasmic maturation was also improved as indicated by development to blastocyst stage. Parthenogenic blastocysts exhibited mRNA expression profiles similar to those of blastocysts obtained after IVF/ICSI with the exception for MKLP2 and PEG1. CONCLUSIONS/SIGNIFICANCE: Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer.

Developmental competence of immature and failed/abnormally fertilized human oocytes in nuclear transfer.

Somatic cell nuclear transfer holds great promise for basic studies of reprogramming human somatic cells and for the potential development of novel cell-based therapeutics. The aim of this study was to examine experimental aspects of human nuclear transfer via use of an abundant source of oocytes, those that are routinely discarded from assisted reproduction clinics. The results suggest and reinforce several findings based on the analysis of multiple parameters: first, it was observed that supplementation of commercial culture media with hormones promoted embryo development after parthenogenetic activation. Second, the use of the chemical activation reagent puromycin resulted in significant differences in cleavage rates in oocytes that were failed/abnormally fertilized after intracytoplasmic sperm injection relative to those from IVF (P < 0.05). Third, cycloheximide promoted cleavage rates >/=40% in both groups of oocytes; moreover, two blastocysts were produced following cycloheximide treatment. Finally, the use of a subset of oocytes for nuclear transfer resulted in cleaved embryos that expressed green fluorescent protein from a transgene in donor nuclei from human embryonic stem cells. In light of these results, it is suggested that the discarded oocytes can be used to investigate new human nuclear transfer protocols for embryonic stem cell derivation.

Noninvasive human nuclear transfer with embryonic stem cells.

INTRODUCTIONIn somatic cell nuclear transfer (SCNT), the nucleus of a somatic cell is transferred to an enucleated oocyte for reprogramming to an embryonic cell state through the use of the endogenous machinery. SCNT technology has been used to produce offspring, establish embryonic stem cells, and study epigenetic reprogramming, as mediated by oocytes, in several animal species. In humans, there are ethical and practical issues that limit availability of oocytes donated by women of reproductive age specifically for research. Thus, there is a need to more exhaustively explore alternatives, including oocyte sources and different SCNT protocols. Nuclear transfer (NT) techniques are important factors that impact development of NT embryos. The procedures of enucleation of oocyte genetic material and introduction of the donor nucleus vary depending on species and laboratories. Hoechst staining has been used successfully for invasive enucleation in many animal studies, though it is known that Hoechst staining and ultraviolet (UV) light can damage oocyte mitochondrial DNA. More recently, noninvasive NT techniques that rely on polarized microscopic imaging systems have been used to visualize the meiotic spindle without DNA staining and UV illumination. This protocol describes a method for noninvasive human nuclear transfer by visualizing the oocyte spindle without DNA staining.

Preparation of mouse embryonic fibroblast feeder cells for human embryonic stem cell culture.

INTRODUCTIONEmbryonic stem cells (ESCs) are derived from the inner cell mass of day 5-6 blastocysts. ESCs are pluripotent, meaning that they are able to differentiate into all derivatives of the three primary germ layers (ectoderm, endoderm, and mesoderm). In order to maintain the undifferentiated status of human ESCs (hESCs), feeder cells are used to provide both a suitable attachment substrate and critical soluble factors. Since the first hESC lines were established on mouse embryonic fibroblasts (MEFs), mitotically inactivated MEFs have commonly been used for supporting the culture of undifferentiated hESCs. Some previous studies suggest that MEFs may support hESC growth better than the human feeder cells typically isolated from post-natal tissues. This protocol describes a method for isolation and irradiation of MEFs for use in hESC culture.

Culturing human embryonic stem cells with mouse embryonic fibroblast feeder cells.

INTRODUCTIONThe potential of human embryonic stem cells (hESCs) to differentiate into diverse cell types of all major tissue types has raised hope that hESCs can be used for novel cell-based therapies as well as fundamental studies of cell lineage differentiation. In order to support the growth of hESCs, mouse embryonic fibroblasts (MEFs), typically isolated from E13-E14 mouse embryos and between passages three and seven, are routinely used as feeder cells. Feeder density is a critical determinant of successful hESC culture. In the presence of too few feeder cells, hESCs may not maintain self-renewal and pluripotency properties; they may also fail to attach to feeder cells appropriately. At excessively high density, feeder cells may detach from the plate such that hESC colonies will be lost. Here we describe an efficient method for culturing and passaging hESCs by enzyme (collagenase) for culture on irradiated MEFs. The protocol can be used for routine hESC culture for basic research.

Culturing human embryonic stem cells in feeder-free conditions.

INTRODUCTIONHuman embryonic stem cells (hESCs) have the potential to differentiate into all three germ layers and proliferate in long-term culture in vitro. hESCs can provide a cell source for the testing of novel therapies, drug screening, and functional genomics applications. Undifferentiated hESCs can be maintained and proliferated on mouse embryonic fibroblasts (MEFs) or human feeder cells. In this protocol, we describe the culture of hESCs in feeder-free conditions on Matrigel with MEF-conditioned medium. This protocol can be used for applications such as genetic modification of hESCs without feeder cell contamination.