Micropropagation and production of Veratrum californicum is most successful when using a premixed Murishage and Skoog basal medium with vitamins and a 5-week subculture cycle at 16 °C for multiplication. These culture conditions provide the best percent survival after acclimatization in the greenhouse. However, clone response to temperature and light quality within culture conditions varies. Micropropagated plants have mass and morphology similar to 2- or 3-year-old seedlings. Acclimatized plantlets can then be grown in the greenhouse using sub-irrigation (ebb and flood) to maintain substrate volumetric water content > 44 %. Growth cycle in the greenhouse must be about 100 days, followed by dormancy for 5 months at 5 °C.

Cell models are becoming more complex to better mimic the in vivo environment and provide greater predictivity for compound efficacy and toxicity. There is an increasing interest in exploring the use of three-dimensional (3D) spheroids for modeling developmental and tissue biology with the goal of accelerating translational research in these areas. Accordingly, the development of high-throughput quantitative assays using 3D cultures is an active area of investigation. In this study, we have developed and optimized methods for the formation of 3D liver spheroids derived from human iPS cells and used those for toxicity assessment. We used confocal imaging and 3D image analysis to characterize cellular information from a 3D matrix to enable a multi-parametric comparison of different spheroid phenotypes. The assay enables characterization of compound toxicities by spheroid size (volume) and shape, cell number and spatial distribution, nuclear characterization, number and distribution of cells expressing viability, apoptosis, mitochondrial potential, and viability marker intensities. In addition, changes in the content of live, dead, and apoptotic cells as a consequence of compound exposure were characterized. We tested 48 compounds and compared induced pluripotent stem cell (iPSC)-derived hepatocytes and HepG2 cells in both two-dimensional (2D) and 3D cultures. We observed significant differences in the pharmacological effects of compounds across the two cell types and between the different culture conditions. Our results indicate that a phenotypic assay using 3D model systems formed with human iPSC-derived hepatocytes is suitable for high-throughput screening and can be used for hepatotoxicity assessment in vitro.

During the past 20 years liver transplantation has become the definitive treatment for most severe types of liver failure and hepatocellular carcinoma, in both children and adults. In the U.S., roughly 16,000 individuals are on the liver transplant waiting list. Only 38% of them will receive a transplant due to the organ shortage. This paper explores another option: bioengineering an autologous liver graft. We developed a 20-year model projecting future demand for liver transplants, along with costs based on current technology. We compared these cost projections against projected costs to bioengineer autologous liver grafts. The model was divided into: 1) the epidemiology model forecasting the number of wait-listed patients, operated patients and postoperative patients; and 2) the treatment model forecasting costs (pre-transplant-related costs; transplant (admission)-related costs; and 10-year post-transplant-related costs) during the simulation period. The patient population was categorized using the Model for End-Stage Liver Disease score. The number of patients on the waiting list was projected to increase 23% over 20 years while the weighted average treatment costs in the pre-liver transplantation phase were forecast to increase 83% in Year 20. Projected demand for livers will increase 10% in 10 years and 23% in 20 years. Total costs of liver transplantation are forecast to increase 33% in 10 years and 81% in 20 years. By comparison, the projected cost to bioengineer autologous liver grafts is $9.7M based on current catalog prices for iPS-derived liver cells. The model projects a persistent increase in need and cost of donor livers over the next 20 years that’s constrained by a limited supply of donor livers. The number of patients who die while on the waiting list will reflect this ever-growing disparity. Currently, bioengineering autologous liver grafts is cost prohibitive. However, costs will decline rapidly with the introduction of new manufacturing strategies and economies of scale.

Drug-induced liver injury (DILI) remains a great challenge and a major concern during late-stage drug development. Induced pluripotent stem cells (iPSC) represent an exciting alternative in vitro model system to explore the role of genetic diversity in DILI, especially when derived from patients who have experienced drug-induced hepatotoxicity. The development and validation of the iPSC-derived hepatocytes as an in vitro cell-based model of DILI is an essential first step in creating more predictive tools for understanding patient-specific hepatotoxic responses to drug treatment. In this study, we performed extensive morphological and functional analyses on iPSC-derived hepatocytes from a commercial source. iPSC-derived hepatocytes exhibit many of the key morphological and functional features of primary hepatocytes, including membrane polarity and production of glycogen, lipids, and key hepatic proteins, such as albumin, asialoglycoprotein receptor and alpha 1-antitrypsin. They maintain functional activity for many drug-metabolizing enzyme pathways and possess active efflux capacity of marker substrates into bile canalicular compartments. Whole genome-wide array analysis of multiple batches of iPSC-derived cells showed that their transcriptional profiles are more similar to those from neonatal and adult hepatocytes than those from fetal liver. Results from experiments using prototype DILI compounds, such as acetaminophen and trovafloxacin, indicate that these cells are able to reproduce key characteristic metabolic and adaptive responses attributed to the drug-induced hepatotoxic effects in vivo. Overall, this novel system represents a promising new tool for understanding the underlying mechanisms of idiosyncratic DILI and for screening new compounds for DILI-related liabilities.

Steroid alkaloids have been shown to elicit a wide range of pharmacological effects that include anticancer and antifungal activities. Understanding the biosynthesis of these molecules is essential to bioengineering for sustainable production. Herein, we investigate the biosynthetic pathway to cyclopamine, a steroid alkaloid that shows promising antineoplastic activities. Supply of cyclopamine is limited, as the current source is solely derived from wild collection of the plant Veratrum californicum. To elucidate the early stages of the pathway to cyclopamine, we interrogated a V. californicum RNA-seq dataset using the cyclopamine accumulation profile as a predefined model for gene expression with the pattern-matching algorithm Haystack. Refactoring candidate genes in Sf9 insect cells led to discovery of four enzymes that catalyze the first six steps in steroid alkaloid biosynthesis to produce verazine, a predicted precursor to cyclopamine. Three of the enzymes are cytochromes P450 while the fourth is a γ-aminobutyrate transaminase; together they produce verazine from cholesterol.

The need for more predictive in vitro toxicity models is a critical deficit in current preclinical pipeline safety evaluations. Current models employing tumor-derived cancer cell lines and isolated primary human hepatocytes (PHHs) afford an approximation of overt cytotoxicity but do not provide hepatotoxicity prediction owing to liabilities in metabolic activity along with phenotypic variability and instability in culture. Induced pluripotent stem cell-derived hepatocytes (iPSC-HCs) offer a long-term solution to accessing liver tissue from representative diverse as well as idiosyncratic patient populations and can be sourced indefinitely. iPSC-HCs are currently being evaluated as potential replacements for the existing cell models, but they have yet to prove superiority. It is acknowledged that iPSC-HCs are not functionally equivalent to PHHs and are somewhat mixed in terms of their gene expression profile, simultaneously displaying mature and immature markers in vitro. Combining iPSC-HCs with organotypic culture systems affords an opportunity to maximize the potential of both technologies where the cells benefit from more complex culture conditions while unlocking the potential of the culture systems by affording stability and reproducibility to provide the future of predictive in vitro toxicity models.

Drug-induced liver injury (DILI) remains a great challenge and a major concern during late-stage drug development. Induced pluripotent stem cells (iPSC) represent an exciting alternative in vitro model system to explore the role of genetic diversity in DILI, especially when derived from patients who have experienced drug-induced hepatotoxicity. The development and validation of the iPSC-derived hepatocytes as an in vitro cell-based model of DILI is an essential first step in creating more predictive tools for understanding patient-specific hepatotoxic responses to drug treatment. In this study, we performed extensive morphological and functional analyses on iPSC-derived hepatocytes from a commercial source. iPSC-derived hepatocytes exhibit many of the key morphological and functional features of primary hepatocytes, including membrane polarity and production of glycogen, lipids, and key hepatic proteins, such as albumin, asialoglycoprotein receptor and α1-antitrypsin. They maintain functional activity for many drug-metabolizing enzyme pathways and possess active efflux capacity of marker substrates into bile canalicular compartments. Whole genome-wide array analysis of multiple batches of iPSC-derived cells showed that their transcriptional profiles are more similar to those from neonatal and adult hepatocytes than those from fetal liver. Results from experiments using prototype DILI compounds, such as acetaminophen and trovafloxacin, indicate that these cells are able to reproduce key characteristic metabolic and adaptive responses attributed to the drug-induced hepatotoxic effects in vivo. Overall, this novel system represents a promising new tool for understanding the underlying mechanisms of idiosyncratic DILI and for screening new compounds for DILI-related liabilities.

Endothelial cell (EC) morphogenesis during angiogenesis is a highly dynamic process tightly regulated by growth factor activity in the extracellular milieu. Current in vitro models of EC morphogenesis employ endpoint analyses of primary endothelial cells, such as HUVECs. These vascular cells can exhibit functional variability between donors and methods for measuring morphogenesis in response to soluble cues typically employ potentially destructive labeling and thus fail to capture the full complexity of processes involved in EC morphogenesis. Here, we describe use of human induced pluripotent stem cell derived endothelial cells (iPSC-ECs) in a real-time electronic cell sensor impedance array technology to monitor invasion and migration behavior. Additionally, an image processing algorithm was developed to assess sprouting behavior from aggregated iPSC-EC organoid cultures. iPSC-EC responses on the impedance platform assessed the impact of serum, growth factors ( e.g. VEGF, FGF-2), and small-molecule angiogenesis inhibitors ( e.g. Sunitinib, SU5402) on the proliferation, migration, and invasion of iPSC- ECs compared to primary HUVECs. Our results indicated that iPSC-ECs were more sensitive to inhibition of VEGF mediated invasion by SU5402 than HUVECs with EC50s of 4.4nM and 99nM, respectively. In addition, matrigel invasion toward VEGF was not observed appreciably with HUVECs, whereas iPSC-ECs demonstrated a marked invasive phenotype that could be inhibited by Nocodazole at 1nM. Using a novel method to monitor the sprouting behavior of the iPSC-ECs in an aggregated organoid into matrigel by HCA and image processing, IC50s for SU5402 (2.7nM) and Nocodazole (0.4nM) were determined. The use of iPSC-derived endothelial cells provides a robust and reproducible source of cells that outperform HUVECs. The real-time monitoring of cellular processes offers important advantages over traditional end-point assays - specifically, the ability to measure receptor activation along with morphologic and adhesive remodeling of EC upon growth factor activation and inhibition. The combination of iPSC- ECs together with real time monitoring systems provides a more biologically relevant human model system for vasculogenesis investigations.

Factors affecting the micropropagation of Veratrum californicum, a slow-growing species that is a potentially valuable source of cyclopamine, were investigated. Sterile cultures were initiated on modified Murashige and Skoog medium, and clones from individual donor plants were assigned to experimental conditions when approximately 100 shoots of each clone were available. The effects of temperature, light quality, and plant growth regulators on multiplication and survival were assessed. Four clones from which large greenhouse populations were obtained were selected for in-depth analysis. When shoots were cultured at 10°C and 16°C, multiplication ratios consistently >1 were observed from three of four clones and two of four clones, respectively, during the five-subculture cycles. None of the clones stably increased when cultured at 24°C, and plants from this treatment did not survive acclimatization in the greenhouse. Only one clone showed increased multiplication ratios in response to plant growth regulator treatments, with maximum multiplication when shoots were cultured with 9 μM benzyladenine and 0.5 μM naphthaleneacetic acid. Light quality in the laboratory did not affect multiplication ratio but did affect subsequent greenhouse survival. The size of plants derived from culture was most often equivalent (65% of 1,271) to 3-yr-old seed-derived plants. Although the growth of clones during acclimatization differed, plants derived from cultures incubated at 16°C had the best rates of overall greenhouse survival. Temperature and light treatments in vitro critical to long-term plant survival were demonstrated and will assist the establishment of a mass propagation system for V. californicum.

Here we describe a strategy to model blood vessel development using a well-defined induced pluripotent stem cell-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats.

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Endothelial cell (EC) morphogenesis during the early stages of angiogenesis is a highly dynamic process tightly regulated by growth factor activity in the extracellular milieu. Other in vitro models for the analysis of EC morphogenesis have previously been developed to understand the cellular processes and the impact of soluble cues and pharmacological inhibitors. However, these assays typically utilize end-point analysis of primary endothelial cells (eg. HUVEC), which are a physiologically relevant vascular cell type but do exhibit variability from the diversity of donors. Additionally, current methods for measuring morphogenesis (eg. proliferation and migration) only capture a snapshot of cell function owing to potentially destructive labeling of the cells and do not capture the complexity of cellular processes involved in EC morphogenesis Here, we describe use of a real-time system for monitoring of cellular processes using electronic cell sensor array technology. The cellular model tested on this impedance-based platform was a well-defined human induced pluripotent stem cell (iPSC)-derived endothelial cells. Following assay optimization and workflow improvement, we assessed the impact of serum, growth factors (e.g. VEGF, EGF, FGF-2), and small-molecule angiogenesis inhibitors (e.g. Sunitinib, SU1498) on the proliferation, migration, and invasion of iPSC-derived ECs compared to primary cells. We observed that real-time monitoring of such cellular processes offers distinct and important advantages over traditional end-point assays - specifically, the ability to measure receptor activation and quantify morphologic and adhesive remodeling of EC upon growth factor activation or signaling inhibition. By better understanding cellular morphogenesis in vitro, we can generate a picture of what soluble cues regulate the process and how pharmacological agents can modulate the different cell fates underlying morphogenesis. The use of iPSC-derived endothelial cells provides a robust and reproducible source of cells that perform equivalently to the standard that is HUVEC. The combination of iPSC-derived EC together with a real time monitoring system provides a biologically relevant human model system. Citation Format: David Mann, David Belair, Coby Carlson, Arne Thompson, Yama Abassi, Jeff Irelan. Label-free, real-time analysis of endothelial cell morphogenesis using iPSC-derived endothelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 27. doi:10.1158/1538-7445.AM2014-27

Corn lily or California false hellebore (Veratrum californicum Durand), a perennial species native to the western United States, produces several alkaloid compounds. A derivative of these alkaloid compounds, primarily veratramine and cyclopamine, shows promise as a therapeutic agent for treatment of a variety of tumor types. Here we report the first study of corn lily cultivated in greenhouse. Growth response of corn lily was examined under two light levels (ambient and supplemental), two fertilization types (20 N-4.4 P16.6 K Peat-lite special and 15N-2.2P-12.5K CalMag special) at 100-mg·L total nitrogen, and three irrigation cycles [sub-irrigation every day (wet), every third day (dry), and hand watering]. Net CO2 assimilation rate (Pn) and transpiration rate (ET) of corn lily grown under supplemental light were 11.0% and 44.7%, respectively, higher than those under ambient light. The Pn and ET of corn lily grown with the wet irrigation cycle increased by 15.2% and 29.4%, respectively, when compared with the Pn and ET of plants grown under the dry irrigation cycle. Corn lily grown wet with supplemental light had the highest average Pn of 8.55 ± 0.36 μmol·m·s, while plants grown under ambient light with hand watering had the lower average Pn of 6.52 ± 0.48 μmol·m·s. The highest mean ET recorded for corn lily was 4.97 ± 0.17 mmol·m·s when plants were grown dry with supplemental light, while the lowest ET recorded was 2.51 ± 0.18 mmol ms when plants were grown under ambient light and hand with supplemental light and when volumetric water content remained above 44%. The water use efficiency of corn lily may be low, as water is not normally limiting in the natural environment where corn lily grows. INTRODUCTION Corn lily (Veratrum californicum Durand; Melanthiaceae family) is a poisonous, herbaceous perennial, facultative wetland species in its native habitat range within the Rocky Mountains and mountains of western North America (Niehaus et al., 1984; USDA, 2011). The Veratrum genus consists of 27 species (Ferguson, 2010; Liao et al., 2007; Zomlefer et al., 2003). The corn lily plant, also known as California false hellebore, can grow from 1 to 2 m in height with a cornstalk-like stem (Niehaus et al., 1984). The plant is attractive with large, broadly ovateelliptical leaves and with dense panicles of creamywhite flowers (James et al., 2004; Keeler and Binns, 1971). Corn lily has been used in herbal medicine for a relatively long time and has potential pharmaceutical uses (Boericke, 1927). The activity of the plant alkaloids, such as cyclopamine, manifests as birth defects, such as cyclopia and holoprosencephaly on grazing pregnant animals (James et al., 2004; Keeler and Binns, 1971). Recently, cyclopamine and its derivatives have been examined as promising therapeutic agents for the treatment of tumors arising from activation of the hedgehogsignaling pathway (Berman et al., 2002; Chen et al., 2002; James et al., 2004; Taipale and Beachy, 2001; Wang, 1990; Watkins et al., 2003). In particular, watered. In corn lily, photosynthesis was increased Sun et al.: Photosynthetic Characteristics of Veratrum californicum in Varied