James Alexander Thomson, V.M.D., Ph.D., Diplomate A.C.V.P. - US grants

OBJECTIVE To define the developmental potential of marmoset ES cells in vivo, and to test the potential of marmoset ES cell nuclei to support development when transferred to enucleated oocytes. RESULTS During 1998, we tested a series constructs for use as lineages markers to identify marmoset ES cells in chimeras. We identified ovarian hyperstimulations protocols that allow the recovery of multiple oocytes per female marmoset, and developed IVF and culture conditions that allow in vitro development of marmoset embryos to the blastocyst stage.. FUTURE DIRECTIONS We will use expression constructs to analyze the tissue distribution of ES cell derivatives in chimeric marmoset fetuses, and thus define the developmental potential of marmoset ES cells. We will begin nuclear transfer experiments to produce genetically identical marmosets from ES cells. KEY WORDS Callithrix jacchus, chimera, embryonic stem cells, blastocyst FUNDING NIH 1R24RR11571 PUBLICATIONS Thomson, J.A. and Marshall, V.S. 1998. Primate Embryonic Stem Cells. Curr. Top. in Dev. Biol. 38:133-160.

animal tissue; reproductive system; growth factor; genetics; Primates;

OBJECTIVE: To define the developmental potential of marmoset ES cells in vivo, and to test the potential of marmoset ES cell nuclei to support development when transferred to enucleated oocytes. RESULTS During 1997, we tested a series of promoter-green fluorescent protein (GFP) constructs for use as lineages markers to identify marmoset ES cells in chimeras. We identified ubiquitin and b-actin promoters as providing the most consistently high levels of GFP expression in primate ES cells. FUTURE DIRECTIONS We will use these GFP expression constructs to analyze the tissue distribution of ES cell derivatives in chimeric marmoset fetuses, and thus define the developmental potential of marmoset ES cells. KEY WORDS Callithrix jacchus, chimera, embryonic stem cells, blastocyst

OBJECTIVE: To test various nuclear transfer strategies to generate genetically identical rhesus monkeys. RESULTS The role of Dr. Thomson's laboratory in this project is to derive new rhesus ES cell lines and provide them to the Oregon Regional Primate Center for nuclear transfer experiments. During 1997, we began weekly shipments of early passage rhesus ES cell lines, and derived a new rhesus ES cell line. FUTURE DIRECTIONS We anticipate that we will derive 6 new rhesus ES cell lines per year and provide them to the Oregon Regional Primate Research Center.

OBJECTIVE To derive new primate ES or EG cell lines; to expand neural precursor cells from primate ES cells; to generate primate ES cells MHC-matched to a specific individual. RESULTS We have demonstrated neural differentiation of rhesus ES cells in vitro in response to retinoic acid. FUTURE DIRECTIONS We will focus on genetic methods of purifying and expanding neural precursor cells more efficiently from primate ES cells, as a future source for transplantation. FUNDING Geron Corporation 133-BU-18 PUBLICATIONS Thomson, J.A., Marshall, V.S., and Trojanowski, J.Q. 1998. Neural differentiation of rhesus embryonic stem cells. AMPHIS 106 149-157.

DESCRIPTION (Adapted from the applicant's abstract): The applicants' recent derivation of NHP (rhesus monkey and common marmoset) and human ES cells has widespread implications for human developmental biology, drug discovery, drug testing, and transplantation medicine. The requirement by primate ES cells for a fibroblast feeder layer is the most important limiting factor in the routine large-scale expansion of NHP ES cells, and identifying the specific fibroblast-produce factor(s) that mediate NHP ES cell self-renewal is a critical research need. The applicants' preliminary results demonstrate that the essential factor(s) produced by fibroblasts are not secreted, and thus are either membrane bound or are tightly attached to the cell extracellular matrix. Functional strategies for identifying the essential signaling molecules produced by the fibroblasts are hampered by the fact that the molecules are not secreted; therefore novel approaches are required. The applicants' underlying hypothesis is that the ligands and receptors mediating NHP ES cell self-renewal are fibroblast and ES cell integral membrane proteins. To identify these essential signaling molecules, the investigators will accomplish the following specific aims: 1) They will use phage T7 display of fibroblast cDNAs to screen for fibroblast-produced polypeptides that specifically bind rhesus ES cell surface molecules. Polypeptides that specifically bind rhesus ES cells will be quantitatively assayed for stimulatory effects on rhesus ES cell self-renewal. Cloned fibroblast- produced polypeptides will be used to identify the ES cell surface receptor to which they bind. 2) The investigators will use a phagemid (M13) library to isolate high affinity single chain Fv antibodies that specifically bind to the cell surface of rhesus ES cells. Antibodies that bind rhesus ES cells will be quantitatively assayed for inhibitory effects on rhesus ES cell self-renewal. Antibodies that act as antagonists will be used to identify the ES cell surface receptors to which they bind.

[unreadable] DESCRIPTION (provided by applicant): ES cells can proliferate without limit, and even after prolonged culture, retain the ability to form cells ranging from cardiac muscle to nerve to blood; potentially any cell type that makes up the body. The recent derivation of nonhuman primate (NHP), i.e. rhesus monkey and common marmoset, and human ES cells has widespread implications for human developmental biology, drug discovery, drug testing, and transplantation medicine. Because of the close embryological similarities between humans and old world monkeys, rhesus monkey ES cells and rhesus monkeys provide an extremely accurate, necessary model system for developing human ES cell-based therapies. [unreadable] [unreadable] The efficient genetic manipulation of primate ES cells is essential to: 1) elucidate gene function both during differentiation and in specific differentiated cells; 2) direct the differentiation of ES cells to specific lineages by the manipulation of transcription factors; 3) purify desired differentiated cell types from a mixed population of ES cell derivatives by introducing selectable markers; 4) use the differentiated derivatives of primate ES cells as vehicles for gene therapy; and 5) modulate the immune response to transplanted ES cell derivatives. Unfortunately, primate ES cells are extremely difficult to transfect. Transfection methods routinely used for mouse ES cells fail for primate ES cells. Recently, a series of pseudotyped, self-inactivating lentiviral vectors with internal promoters were tested. These are the first vectors tested that allow the derivation of rhesus and marmoset ES cell lines stably expressing a foreign gene. To improve these vectors for primate ES cells, and to use them as tools to dissect mechanisms of primate ES cell self-renewal, the following Specific Aims will be accomplished to: 1) generate lentiviral vectors with improved long term, site independent expression in primate ES cells and their differentiated derivatives by testing the effects of insulator sequences and scaffold attachment regions; 2) use a functional ES cell screen to isolate novel genomic DNA fragments that promote long term, integration site independent expression of lentiviral vectors; 3) use lentiviral vectors to test the role of specific genes involved in the self-renewal of other stem cells (Beta-catenin, ID-1, N-myc, Notch, STAT-3) in promoting the self-renewal of primate ES cells; and 4) use a lentiviral cDNA expression cloning strategy to identify novel genes produced by fibroblasts, or by the ES cells themselves that promote ES cell self-renewal.

stem cells; Primates; animal colony;

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Note: Project Units is Stem Cell and Immunology.[unreadable] [unreadable] To differentiate human stem cells into multiple immune functions within an in vitro 3D culture system. The goal of [unreadable] this research is to ultimately develop the technologies and science for efforts leading to the creation of a 3D ex vivo [unreadable] human immune system. This system would be used to test new vaccine constructs and immunomodulators that [unreadable] would provide superior protection against bioterrorism threat agents.[unreadable] [unreadable] We developed homologous recombination vectors using recombineering technology and BAC clones. The targeting [unreadable] construct included a drug resistant Neo-resistant gene and a red fluorescent protein (RFP) gene as a reporter. We also used [unreadable] SAGE technology to identify differentially expressed genes in human ES cells and differentiated derivatives. This project [unreadable] ended 5/31/06.[unreadable] [unreadable] This research used WNPRC Stem Cell Resources, IS services and federally approved human ES cell lines.[unreadable]

embryonic stem cell; tissue /cell culture; Primates; animal colony;

Lentivirus; transfection /expression vector; embryonic stem cell; animal colony; Primates; biotechnology;

ATP[{..}]kanamycin 3'-O-phosphotransferase; Adenosine, 3'-((2-amino-3-(4-methoxyphenyl)-1-oxopropyl)amino)-3'-deoxy-N,N-dimethyl-, (S)-; Adenosine, 3'-(alpha-amino-p-methoxyhydrocinnamamido)-3'-deoxy-N,N-dimethyl-, L-; Amikacin 3'-Phosphotransferase; Aminocyclitol Phosphotransferase; Aminoglycoside 3'-Phosphotransferase Type VIII; Aminoglycoside Phosphotransferase; Back; CRISP; Cells; Cloning; Computer Retrieval of Information on Scientific Projects Database; Dorsum; Drugs; EC 2; ES cell; Funding; Genes; Genetic Markers; Grant; Institution; Investigators; Kanamycin Kinase; Kanamycin-Neomycin Phosphate Transferase; Lentiviral Vector; Lentivirus Vector; Mammals, Primates; Medication; Mother Cells; NIH; National Institutes of Health; National Institutes of Health (U.S.); Neomycin Phosphotransferase; Pharmaceutic Preparations; Pharmaceutical Preparations; Primates; Progenitor Cells; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Protocol; Protocols documentation; Puromicina; Puromycin; Puromycine; Puromycinum; Research; Research Personnel; Research Resources; Researchers; Resources; Role; Services; Site; Skin; Source; Stem cells; System; System, LOINC Axis 4; Testing; Transferase; United States National Institutes of Health; Viral; drug/agent; embryonic stem cell; expression vector; hESC; human ES cell; human ES cell lines; human ESC; human embryonic stem cell; human embryonic stem cell line; improved; particle; pluripotency; self-renewal; size; social role; stem cell of embryonic origin; transgene expression; vector

Antibodies; Body Tissues; Brachyury; Brachyury protein; CRISP; Cell Differentiation; Cell Differentiation process; Cell Isolation; Cell Segregation; Cell Separation; Cell Separation Technology; Computer Retrieval of Information on Scientific Projects Database; Condition; DNA; Deoxyribonucleic Acid; Funding; Genes; Grant; Institution; Investigators; Magnetism; Mesoderm; Mesoderm Cell; Mesodermal Cell; Methods and Techniques; Methods, Other; Mother Cells; NIH; National Institutes of Health; National Institutes of Health (U.S.); Neomycin; Progenitor Cells; Research; Research Personnel; Research Resources; Researchers; Resistance; Resources; Source; Stem Cell Research; Stem cells; T Brachyury protein; Techniques; Tissues; United States National Institutes of Health; Wisconsin; cell sorting; hESC; homologous recombination; human ES cell; human ES cell lines; human ESC; human embryonic stem cell; human embryonic stem cell line; magnetic; resistant; vector

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To develop a media formulation and protocol for the human ES cell culture that will improve human ES cell viability [unreadable] and promote uniformity, consistency and ease of use across a variety of users in different locations. Some of the [unreadable] specific objectives necessary to accomplish this goal are to: 1) optimize the physiochemical environment, 2) [unreadable] optimize the basal media formulation and 3) eliminate undefined media components.[unreadable] [unreadable] Human embryonic stem (ES) cells already provide a powerful research tool for understanding the human body, the [unreadable] current sub-optimal conditions for human ES cell culture place significant limitations on their use in basic research and [unreadable] on their large-scale expansion and distribution. The labor required to continuously prepare MEF feeder layers is a major [unreadable] limitation in large-scale production of human ES cells. Combined with concerns surrounding cross-species [unreadable] contamination that may arise from growth of human ES cells on murine feeder layers, the elimination of fibroblast [unreadable] feeder cells would greatly improve the efficiency and consistency of ES cell culture. We have previously reported that [unreadable] high bFGF concentrations support feeder-independent growth of human ES cells, but those conditions included poorly [unreadable] defined serum and matrix components. In the previous budget year we reported a feeder-independent human ES cell [unreadable] culture that includes protein components solely derived from recombinant sources or purified from human material [unreadable] (Nature Biotechnology, February 2006). While this culture system is efficient and effect at propagating human ES cells, [unreadable] it is cost prohibitive for most research laboratories. In this budget year, we developed and reported on (Nature Methods, [unreadable] August 2006) a modified version of our feeder-independent medium (mTeSR1) that is appropriate for use in standard [unreadable] human ES cell research laboratories. In applications where completely humanized media is not essential, the use of [unreadable] animal sourced proteins and zebrafish bFGF (zbFGF) significantly reduces the cost of preparation and enables the [unreadable] widespread adaptation of feeder-independent culture systems for human ES cell research. We also continue to optimize [unreadable] the medium to improve efficiency, conduct research to improve cloning efficiencies and identify alternate matricies to [unreadable] future improve human ES cell culture overall. This research used WNPRC stem cell resources and federally approved hES [unreadable] cell lines.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To maintain and further develop a specialized resource for studies relating to pluripotent stem cell research. Allocation of Resource Access: To date, the stem cell resource unit at the Wisconsin National Primate Research Center provides frozen rhesus and marmoset ES cells to interested investigators. No request has been denied. Additionally, the stem cell resource unit provides zebrafish bFGF for culturing primate ES cells. Since last submission 3 new investigators have received the recombinant protein bringing the total number of investigators receiving zbFGF on a regular basis to 21. Over 330mg of zbFGF has been distributed to date. This has saved investigators over $1,000,000. The DNA plasmid used to purify the protein itself is now available through Addgene (www.addgene.com) and was sent to 13 new investigators in 2008 bringing the total number of investigators to receive this plasmid to 39. Lastly, in 2009, 2 investigators requested and received rhesus ES cell provided by the stem cell resources unit and distributed by the WiCell Research Institute. Dissemination: Knowledge is disseminated to the scientific community via publications in peer reviewed journals and scientific meeting attendance. The Wisconsin National Primate Research Center also holds quarterly research retreats to create increased communication between the various service and resource units. Training: Training in culture techniques of primate embryonic stem cells is available. Many new investigators have taken advantage of this resource in previous reporting periods however there have been no new investigators trained this year. Progress: Past and present members of stem cell resources developed a method for generating iPS cells without vector transgene sequences;this work is referenced below in the highlights portion. We have provided ips cell derivation for disease specific cell lines for several UW investigators. To date, 4 disease cell lines have been used for ips cell generation and a total of 55 clones have been cultured and frozen for future use. We are also beginning to start reprogramming experiments on rhesus macaque fibroblasts. Other groups have successfully reprogrammed primate cells and we will be following similar protocols. Highlights: Past and present members of stem cell resources were authors on a paper detailing a vector free method of adult cell reprogramming: Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences Junying Yu, Kejin Hu, Kim Smuga-Otto, Shulan Tian, Ron Stewart, Igor I. Slukvin, James A. Thomson Science 8 May 2009 Vol 324. No. 5928, pp 797-801. Challenges: Due to funding shortages we were unable to receive cynomologous embryos and the project came to a halt. We look forward to working with CPI to receive Mauritian cynomolougous embryos to create new cynomologous ES cell lines as described in our P51 proposal. Concerns: No concerns at this time. Stem Cell Resource support is involved in numerous journal articles that depend in part or in full on WNPRC resources.

DESCRIPTION: (provided by applicant): Understanding how human embryonic stem (ES) cells can proliferate without limit and yet retain the ability to differentiate to any cell type is the theme that links the three individual projects of this proposal. Project 1 will identify novel histone modifications in human ES cells using a new mass spectrometry technique that allows an unprecedented ability to identify and map posttranslational protein modifications. Histone modifications in human ES cells will be identified globally, at promoters, and at select genes directly regulated by critical pluripotency factors. We will also examine the role of histone H3 variants in establishing long-term epigenetic memory. These studies will determine whether there is a novel histone code in pluripotent cells and determine how histone modifications change dynamically during differentiation. Project 2 examines the critical events that occur in the window of time during which human ES cells commit eprogram differentiated cells to a pluripotent state. We have previously reported that when myeloid cells are fused with human ES cells, the myeloid nucleus is reprogrammed to an ES cell state, indicating that transacting factors in ES cells are sufficient to mediate nuclear reprogramming. Our preliminary results suggest that over expressing combinations of human ES cell-enriched genes can reprogram myeloid cells, and this project will optimize this reprogramming. The combination of these projects will provide an increased understanding of the pluripotent state and the basic processes by which a cell can leave or return to that state. Such an understanding will be important to transplantation and regenerative medicine. Lay Description: Human ES cells are special because they can grow without limit and can give rise to all other cell types. Here we will try to understand why human ES cells have this remarkable developmental potential, and develop conditions to convert a cell with a more limited potential to an ES cell. Such reprogramming has implications for transplantation and regenerative medicine.

Project 2: Self-Renewal and Differentiation: Molecular events that commit ES cells to exit the pluripotent state. (James Thomson, PI) A. SPECIFIC AIMS In low (4 ng/ml) or absent exogenous bFGF, BMP4 induces human ES cells to form a homogenous population of trophoblast, the outer layer of the placenta. However, we have recently found that in the presence of high (100 ng/ml) concentrations of bFGF, BMP4 instead induces human ES cells to form a population of cells that no longer express trophoblast markers, but instead transiently express brachyury, a mesoderm or mesendoderm marker, and subsequently express a mixture of endoderm and mesoderm markers. In this project, we will study how BMPs induce human ES cells to exit the pluripotent state and commit to differentiation, and study how FGF mediates these divergent developmental outcomes in response to the same inducer. Understanding how ES cells exit the pluripotent state and why this exit is generally irreversible is central to achieving efficient reprogramming (Project 3), and understanding these key early lineage decisions will allow more efficient differentiation to specific clinically-relevant lineages. We will accomplish the following aims: Aim 1. We will establish a detailed time course of gene expression in human ES cells upon BMP4- induced differentiation, both in the presence (brachyury positive result) and absence (chorionic gonadotropin positive result) of bFGF, and correlate these changes with the commitment to exit the pluripotent state. Commitment will be measured by adding BMP4 for successively longer time periods, removing BMP4, and then examining how many cells retain markers of pluripotency several days later. The hypothesis of this aim is that the quantitative commitment curve will be most closely correlated with the expression levels of the genes that directly control these differentiation events. We will subsequently overexpress the transcription factors most closely associated with commitment, and we will downregulate genes by RNA interference that are downregulated during commitment to identify those which are sufficient to mediate differentiation to a brachyury-positive population. Preliminary results demonstrate that GATA2 and GATA3 are both individually sufficient to mediate trophoblast differentiation in the absence of bFGF, so we are optimistic that a single transcription factor will be sufficient to mediate human ES cell differentiation to a brachyury positive population in the presence of bFGF. Aim 2. We will use whole genome chromatin immunoprecipitation on chip (ChlP-chip) to map the genomic binding sites of TGFp/Activin-activated Smad 2/3 and BMP-activated Smad 1/5/8 during BMP4- induced differentiation. The central hypothesis of this aim is that Smad 2/3 directly activates the expression of key pluripotency factors in human ES cells, directly suppresses the expression of genes that would otherwise promote differentiation, and competes at the same promoters with Smad 1/5/8 which has the opposing effects. A second hypothesis is that bFGF will change the DNA binding sites of Smad 1/5/8 during BMP-induced differentiation, leading to the different developmental outcomes observed. Aim 3. We will use whole genome chromatin immunoprecipitation on chip (ChlP-chip) to map genomic binding sites of GATA2 and GATA3 during BMP4-induced differentiation. The hypothesis of this aim is that GATA2 and GAT A3 directly negatively regulate the transcription of key pluripotency genes, and directly positively regulate their own transcription, so that once they are induced by Smads, GAT A expression becomes self-sustaining and BMP-independent, and thus differentiation continues even upon BMP removal. A second hypothesis is that bFGF will change the DNA binding sites of GATA2/GATA3 during BMP-induced differentiation, leading to the different developmental outcomes observed. Aim 4. We will identify proteins that are differentially phosphorylated between BMP4-induced trophoblast differentiation (no bFGF) and BMP-induced brachyury positive cellular differentiation (high bFGF) to identify mediators of FGF signaling that cause the switch between these divergent developmental outcomes. The hypothesis of this aim is that differential phosphorylation of a limited number of transcription factors is casually related to the different developmental outcomes observed after BMP induction in the presence or absence of bFGF. Differentiation commitment curves will again become key for focusing attention to relevant phosphorylation events.

DESCRIPTION (provided by applicant): The Midwest Progenitor Ciell Consortium is a collaboration between the UW-Madison and the UMN which will serve as a research hub within the NHLBI's Progenitor Cell Biology Consortium. The goal is to develop new strategies to convert human pluripotent stem and somatic cells into hematopoietic stem cells (HSCs) and selfrenewing cardiac progenitor cells through identification of genetic program leading to formation of pre-HSCs and cardiac progenitors and activation of self-renewal program in lineage-restricted cells. Project 1, The De Novo Generation of Hematopoietic Stem CeUs from Human ES/iPS Cells and Somatic Cells, aims are: Identify the hierarchy of mesodermal progenitors of vascular endothelial and hematopoietic lineages using human ES cell lines with targeted FOXFl, GATA-2, GATA-3, RUNXl, and SCL genes; Determine the most critical molecular events leading to formation of hematopoietic cells and HSCs during embryogenesis in mouse aind human, and following in vitro differentiation of human pluripotent stem cells; Develop technologies for de novo generation of HSCs from human ES/iPS cells and somatic cells. Project 2, Cardiovascular Progenitors and Cardiomyocytes from Human ES/iPS Cells and Somatic Cells, aims are: Isolate and define human embryonic cardiovascular progenitors and ventricular myocytes derived from pluripotent stem cells; and reprogram human pluripotent stem cells and somatic cells using transcription factors to generate cardiovascular progenitors and ventricular myocytes. Project 3, Artificial Transcription Factors for Reprogramming/Transdifferentation, aims are: Design ATFs that activate expression of Oct4, Sox2 or Nanog in primary fibroblasts; Perform genome-wide location analysis, CSI and transcriptome analysis to determine gene targets and specificity ofthe designed ATFs; and combine ATFs to induce pluripotency in primary fibroblasts. Project 4, Rapid In Vitro Generation of Affinity Reagents for Hematopoietic and Cardiovascular Precursors, aims are: Develop methods for rapidly generating aptamers that bind to transmembrane protein targets with high affinity and specificity; Develop methods to accelerate discovery of aptamers with sub nanomolar affinities using high-throughput DNA sequencing technologies; and generate high affinity aptamers for surface markers of cardiovascular and blood precursors.

Nucleic acid aptamers possess many useful features as affinity reagents, including facile chemical synthesis, reversible folding, thermal stability and low cost, making them a powerful alternative to antibodies and other protein-based reagents. However, over the past two decades, aptamers have suffered from the fact that 1) the conventional method of aptamer generation (SELEX) is lengthy, labor intensive and often does not yield aptamers with sufficient affinity (<1 nM) and specificity;2) there is no "standard protocol" that can be generally applied to most protein targets to generate aptamers;and 3) the characterization steps to measure the affinity and specificity of candidate aptamers are lengthy and resource-intensive, because each aptamer must be measured individually. We believe that these challenges arise from deficiencies in the conventional methodology of performing the selection, which has not changed significantly since its initial description 20 years ago. We also believe that these problems can be solved, by systematically taking fundamentally different approaches towards the three central stages of the process - selection, analysis and characterization of the aptamers. We propose here the development of such a system. We will combine three distinctly novel technologies -microfluidic selection, next-generation aptamer sequencing, and SPR Imaging - to develop the Quantitative Parallel Aptamer Selection System (QPASS) platform. The QPASS platform will generate specific aptamers with sub-nanomolar affinities (Kd) for a wide range of protein targets within 3 rounds of selection, identify a pool of the best candidates by next generation DNA sequencing and bioinformatic analysis, and home in on the optimal aptamer sequence by the parallel synthesis and measurement of the affinities of thousands of aptamer candidates. Individually, each component represents a significant technological advance. Combined this integrated approach offers an opportunity to revolutionize the process of aptamer generation.

DESCRIPTION (provided by applicant): This proposal brings together leading experts in human pluripotent stem cell biology (Thomson), tissue engineering (Murphy), and machine learning (Page) to develop improved human cellular models for predicting developmental neural toxicity. Dramatic progress has been made in the derivation of many of the basic cellular components of the brain from human pluripotent stem cells (ES and iPS cells), but these advances have yet to be applied to predictive toxicology. The major components of the brain are derived from diverse embryological origins, including the neural plate (neurons, oligodendrocytes, and astrocytes), yolk sac myeloid progenitors (microglia), migratory mesodermal angioblasts (endothelial cells), and neural crest (vascular smooth muscle and pericytes). Because of their diverse origins, these components have very different inductive signaling histories. This means that deriving them all at once under the same conditions is not currently possible. For this reason, we will differentiate human pluripotent stem cells to early precursors of the major neural, glial, and vascular components of the cerebral cortex separately, cryopreserve the precursors, and subsequently combine them in 3D hydrogel assemblies to allow increased physiological interactions and maturation. Specifically, we will embed committed precursors for endothelial cells, pericytes, and microglia into hydrogels displaying combinations of peptide motifs that promote capillary network formation. We will then overlay this mesenchymal layer with neural and glial precursors to mimic the normal interactions between the cephalic mesenchyme and the neural epithelium, and promote the formation of the polarized layers of the cerebral cortex. After drug exposure, we will assess temporal changes in gene expression by these cerebral neural- vascular assemblies using highly multiplexed, deep RNA sequencing. Then, using safe drugs and known neural/developmental toxins from the NIH Clinical Collection, the University of Washington Teratogen Information System Database, and the EPA's Toxicity Reference Database as training sets, we will develop machine learning algorithms to predict neural toxicity of blinded drugs known to have failed in late stage animal testing or human clinical trials. This predictive, developmental neural toxicity model will be implemented on liquid handling robots and sequencers in widespread use, and will be readily adaptable to platforms being developed in complementary efforts by DARPA. The developmental potential of human pluripotent stem cells, the modular nature of the tunable hydrogels, and the discriminatory power of machine learning tools also makes the general approaches proposed readily applicable to predictive toxicity models for other tissue types throughout the body. PUBLIC HEALTH RELEVANCE: This project will develop three-dimensional constructs of human neural tissue to better predict the neural toxicity of drugs prior to clinical trials. To accomplish this, experts in human pluripotent stem cell biology will grow the required neural components in the laboratory, experts in tissue engineers will assemble those cells into multicellular constructs, and experts in machine learning will use changes in gene expression after drug exposure to predict whether a test compound is toxic.

Project Summary/Abstract for, ?Transplantation of MHC homozygous vascular progenitors in primates.? For tissue engineered arteries, the use of patient specific iPS cells would be severely limited by time constraints and cost. Banking iPS cells from rare individuals homozygous for HLA alleles has been proposed as a strategy to allow economies of scale, while still reducing rejection of iPS cell-derived transplanted tissues. Only a few hundred such cell lines would provide matches for the majority of the U.S. population, and the Waisman Clinical Biomanufacturing facility here on the University of Wisconsin has already produced cGMP HLA homozygous iPS cell lines. However, the immunological value of such an approach remains untested in an animal model with an immune system similar to the human immune system. Here we will use a unique population of MHC defined cynomolgus monkeys to test the immune response to MHC homozygous cynomolgus iPS cell-derived vascular cells transplanted to MHC haploidentical recipients. Using the MHC defined cynomolgus monkeys, we will use a limb ischemia model to determine the ability of iPS cell-derived arterial endothelial cells to contribute to collateral circulation when transplanted by themselves, in combination with iPS cell-derived smooth muscle cells, or when combined into a fully tissue engineered artery. A central premise of this proposal is that properly specified early arterial endothelial cells will robustly recruit, expand, and mature endogenous or co-transplanted smooth muscle cell progenitors to increase arteriogenesis in vivo, and that these arterial endothelial cells will be critical to producing tissue engineered arteries ex vivo that remain functional long after transplantation. The final goal of this proposal is to produce cGMP vascular progenitors from HLA homozygous human iPS cell lines for the pre-clinical animal studies required to file an IND for critical limb ischemia. With extensive human and primate pluripotent stem cell expertise, a strong bioengineering department, a National Primate Research Center with an MHC typing facility, and a GMP cell manufacturing facility, the environment at the University of Wisconsin is uniquely suited for completing the goals of this proposal.  

DESCRIPTION (provided by applicant): This proposal brings together leading experts in human pluripotent stem cell biology (Thomson), tissue engineering (Murphy), and machine learning (Page) to develop improved human cellular models for predicting developmental neural toxicity. Dramatic progress has been made in the derivation of many of the basic cellular components of the brain from human pluripotent stem cells (ES and iPS cells), but these advances have yet to be applied to predictive toxicology. The major components of the brain are derived from diverse embryological origins, including the neural plate (neurons, oligodendrocytes, and astrocytes), yolk sac myeloid progenitors (microglia), migratory mesodermal angioblasts (endothelial cells), and neural crest (vascular smooth muscle and pericytes). Because of their diverse origins, these components have very different inductive signaling histories. This means that deriving them all at once under the same conditions is not currently possible. For this reason, we will differentiate human pluripotent stem cells to early precursors of the major neural, glial, and vascular components of the cerebral cortex separately, cryopreserve the precursors, and subsequently combine them in 3D hydrogel assemblies to allow increased physiological interactions and maturation. Specifically, we will embed committed precursors for endothelial cells, pericytes, and microglia into hydrogels displaying combinations of peptide motifs that promote capillary network formation. We will then overlay this mesenchymal layer with neural and glial precursors to mimic the normal interactions between the cephalic mesenchyme and the neural epithelium, and promote the formation of the polarized layers of the cerebral cortex. After drug exposure, we will assess temporal changes in gene expression by these cerebral neural- vascular assemblies using highly multiplexed, deep RNA sequencing. Then, using safe drugs and known neural/developmental toxins from the NIH Clinical Collection, the University of Washington Teratogen Information System Database, and the EPA's Toxicity Reference Database as training sets, we will develop machine learning algorithms to predict neural toxicity of blinded drugs known to have failed in late stage animal testing or human clinical trials. This predictive, developmental neural toxicity model will be implemented on liquid handling robots and sequencers in widespread use, and will be readily adaptable to platforms being developed in complementary efforts by DARPA. The developmental potential of human pluripotent stem cells, the modular nature of the tunable hydrogels, and the discriminatory power of machine learning tools also makes the general approaches proposed readily applicable to predictive toxicity models for other tissue types throughout the body.

DESCRIPTION (provided by applicant): This proposal brings together leading experts in human pluripotent stem cell biology (Thomson), tissue engineering (Murphy), and machine learning (Page) to develop improved human cellular models for predicting developmental neural toxicity. Dramatic progress has been made in the derivation of many of the basic cellular components of the brain from human pluripotent stem cells (ES and iPS cells), but these advances have yet to be applied to predictive toxicology. The major components of the brain are derived from diverse embryological origins, including the neural plate (neurons, oligodendrocytes, and astrocytes), yolk sac myeloid progenitors (microglia), migratory mesodermal angioblasts (endothelial cells), and neural crest (vascular smooth muscle and pericytes). Because of their diverse origins, these components have very different inductive signaling histories. This means that deriving them all at once under the same conditions is not currently possible. For this reason, we will differentiate human pluripotent stem cells to early precursors of the major neural, glial, and vascular components of the cerebral cortex separately, cryopreserve the precursors, and subsequently combine them in 3D hydrogel assemblies to allow increased physiological interactions and maturation. Specifically, we will embed committed precursors for endothelial cells, pericytes, and microglia into hydrogels displaying combinations of peptide motifs that promote capillary network formation. We will then overlay this mesenchymal layer with neural and glial precursors to mimic the normal interactions between the cephalic mesenchyme and the neural epithelium, and promote the formation of the polarized layers of the cerebral cortex. After drug exposure, we will assess temporal changes in gene expression by these cerebral neural- vascular assemblies using highly multiplexed, deep RNA sequencing. Then, using safe drugs and known neural/developmental toxins from the NIH Clinical Collection, the University of Washington Teratogen Information System Database, and the EPA's Toxicity Reference Database as training sets, we will develop machine learning algorithms to predict neural toxicity of blinded drugs known to have failed in late stage animal testing or human clinical trials. This predictive, developmental neural toxicity model will be implemented on liquid handling robots and sequencers in widespread use, and will be readily adaptable to platforms being developed in complementary efforts by DARPA. The developmental potential of human pluripotent stem cells, the modular nature of the tunable hydrogels, and the discriminatory power of machine learning tools also makes the general approaches proposed readily applicable to predictive toxicity models for other tissue types throughout the body.