Grantee Research Project Results
Final Report: Formative Center for the Evaluation of Environmental Impacts on Fetal Development
EPA Grant Number: R834594Center: Formative Center for the Evaluation of Environmental Impacts on Fetal Development
Center Director: Boekelheide, Kim
Title: Formative Center for the Evaluation of Environmental Impacts on Fetal Development
Investigators: Boekelheide, Kim , De Paepe, Monique E , Thanos, Christopher , Phipps, Maureen , Brown, Phil , Gruppuso, Phillip
Institution: Brown University , Women & Infants Hospital of Rhode Island
EPA Project Officer: Hahn, Intaek
Project Period: February 15, 2010 through February 14, 2014
Project Amount: $1,189,575
RFA: Children's Environmental Health and Disease Prevention Research Centers: Formative Centers (with NIEHS) (2009) RFA Text | Recipients Lists
Research Category: Children's Health , Human Health
Objective:
Our "Formative Center for the Evaluation of Environmental Impacts on Fetal Development" studied mechanisms by which environmental toxicants alter normal fetal development. We were unique in studying HUMAN fetal tissues directly, thereby obviating the need for extrapolating findings in fetal rodent models to humans. We brought together clinicians and scientists from different backgrounds, creating an interdisciplinary, cooperative and synergistic enterprise. This collaboration allowed us to collect human fetal tissues from spontaneous abortions of 12–22 weeks gestation and perform xenotransplantation into immunodeficient rodent hosts.
The P20 funding enabled us to make a major investment in developing this consortium and the team approach needed for rapid and ethical acquisition and xenotransplanting of human fetal tissues. It allowed us to establish new models for testing developmental toxicity. As demonstrated by our publications, presentations, and preliminary data, exposure of the rodent host to an environmental toxicant and assessment of the response of xenotransplanted human fetal tissues allowed for mechanistic dissection of pathway alterations, identification of adverse effects, and evaluation of dose response. Through our maternal, fetal, and children's health experts, and our community outreach activities, we translated these laboratory observations into clinically relevant and community-appropriate interventions with the goal of improving public health. As a result, our Formative Center became a well-known and valuable resource for the Rhode Island children’s environmental health community.
Significance
Early life exposures to environmental chemicals produce an enormous disease burden in children and a high societal cost. Our model of human fetal tissue xenotransplantation allowed us to study human fetal tissues in a rodent host that is subject to manipulation. Because of rapid cell proliferation and the simultaneous expression of many developmental pathways, the fetus is uniquely sensitive to environmental exposure. Our Research Projects focused on human fetal tissues—liver/adipose tissue, prostate, and lung—for which prenatal exposures are strongly associated with childhood diseases. Each project chose relevant environmental chemicals to evaluate based on the available data. Our research results made an important contribution to the ability of physicians, other healthcare professionals, and regulators to make informed decisions about the safety and effects of environmental exposures on fetal development and their contribution to health and disease later in life.
Relevance
This Center addressed the following broadly defined research topics:
- The effects of in utero and/or perinatal exposures to environmental agents and the role they play by lifestage in biological changes associated with adverse childhood health outcomes.
- Identification of potential mechanisms that influence susceptibility to environmental exposures and contribute to disease onset or course in children, and the role of epigenetics in gene-environment interactions.
The primary stakeholders for our Center were fetuses, children, parents, communities, healthcare professionals, the Children’s Environmental Health Centers, children’s health researchers broadly defined, and the funding agencies NIEHS and the U.S. Environmental Protection Agency (EPA). The Administrative Core, Health Specialist, and Community Outreach and Translation Core (COTC) worked together to communicate our research findings to our stakeholders and to engage our stakeholders in helping to shape our work. Through COTC activities, we enhanced the understanding of children’s environmental health in our communities and promoted health-conscious practices with our hospital's partners.
Summary/Accomplishments (Outputs/Outcomes):
The P20 Formative Center funding provided the opportunity to establish the complex infrastructure needed to support the coordinated and ethical collection of human fetal tissues and their xenotransplantation into immunodeficient rodent hosts. As demonstrated by our publications and data, this novel approach was successful in creating unique human-relevant models for mechanistic research on the developmental origins of childhood diseases.
The following activities, events and accomplishments reflect the close interactions, connectivity and impact of our team efforts:
- Program faculty and staff meetings. The entire program faculty and staff met monthly since the inception of the program. Topics of discussion included ethics and consent, patient recruitment, xenotransplant techniques and host models, human fetal tissues of interest and pilot projects, community engagement, content of the Grand Rounds presentations for professional-to-professional communication, and identification of seminar speakers.
- Seminar series. Overall, 13 seminars were held featuring prominent invited speakers.
- Brainstorming session. A half-day brainstorming session (held 1/24/2011) included the External Advisory Committee (EAC) and was organized at the beginning of the second year of funding to reflect on progress to date and to plan for the future. This also provided an opportunity to catalyze further connections with a broader range of researchers across campus.
- Retreats. A full-day retreat was held May 2, 2011, included the EAC, and was focused on evaluating the accomplishments of the research projects and cores. Another retreat was held December 11, 2012, and featured an overview by the Director, Dr. Kim Boekelheide, and reports by each of the project leaders. There also was a presentation by the Community Outreach and Translation Core (COTC) and a talk by Monica Anderson (Miriam Hospital) entitled “Hospitals for a Healthy Environment in Rhode Island.” Dr. Mary-Elizabeth Patti from the Joslin Diabetes Center, Boston, MA, attended the retreat as an External Advisor in addition to presenting her research in a talk titled “Multigenerational Effects of Intrauterine Nutritional Exposures Promoting Obesity and Diabetes Risk.”
- Workshop. A very successful half-day workshop entitled “Epigenetics and Fetal Origins of Health and Disease” was held on October 3, 2011, with 83 attendees, including 23 nurses who received continuing education credit.
- Presentations at meetings and seminars. In total, program faculty, postdoctoral fellows, students, and staff made 32 presentations of their work.
- Publications. To date, there are eight publications based on the work of the P20 Formative Center projects and cores.
- Outreach. Outreach strategies have been developed and a wide range of activities have been conducted, including two annual conferences entitled Hospitals for a Healthy Environment in Rhode Island.
Our P20 Formative Center embraced and implemented the three required Essential Elements (Community Engagement, Pediatric Health Specialist, and Career Development) and supported one Research Project and two Pilot Projects. The achievements of these programmatic efforts is described below.
Community Engagement
Dr. Brown formed a Community Advisory Board to help guide the COTC. This Board was comprised of 10 members from Rhode Island community-based organizations, disease-based charities, state agencies, and education, who were actively involved in children’s health and welfare. Dr. Brown led the Center’s team in evaluating its approach to detailed informed consent in which patients were told specifics about how their miscarriage tissue would be used in our research, a reflexive ethics approach that has resulted in a publication that contributes to a growing literature on bioethics and advanced forms of informed consent. With postdoctoral fellow Dr. Bindu Pannikar, the COTC conducted two statewide workshops on children’s environmental health for Meeting Street early intervention staff and Bradley Research Center home visitors who work with developmentally disabled children and their families. The COTC developed Hospitals for a Healthy Environment in Rhode Island (H2ERI), a large coalition of providers and professionals who work on environmental health and related improvements in healthcare. This originated from our work in partnership with Women and Infants Hospital, based on their Neonatal Intensive Care Unit removing phthalate- and PVC-containing medical equipment; this effort rapidly spread statewide. Two annual H2ERI conferences have made a strong mark on the state, fostering much “greening” work in healthcare settings among its 80 and 100 attendees. At the 2012 Annual Meeting of the Children’s Environmental Health Centers and Partnerships for Environmental Health, Dr. Brown led a workshop to help Centers in other locations build similar coalitions. The COTC has worked with the Rhode Island Department of Environmental Management on pharmaceutical waste reduction efforts, including supervision of an Environmental Studies Master’s thesis that provided groundwork for new regulatory policies and practices. The COTC has joined with the Rhode Island Medical Society and other health groups in an Alliance for a Healthy Rhode Island to curtail sugar-sweetened beverages in schools, hospitals and public facilities. The COTC has worked with the HUD-funded Green and Healthy Housing Initiative to address residential environmental health issues.
Pediatric Health Specialist
Dr. Gruppuso put much effort into grand rounds for pediatricians, OB-GYNs, and others, where he provided fundamental information on fetal origins of later life disease. Communicating this new knowledge was very valuable because physicians are largely unaware of these important scientific developments. Dr. Gruppuso, in collaboration with COTC Director Dr. Brown, supervised a medical student carrying out research on physicians’ knowledge and understanding of the developmental origins of health and disease.
Career Development and Training
Monique DePaepe, M.D., Leader of Pilot Project 3, was a new investigator mentored by Dr. Joshua Hamilton. Dr. DePaepe was extraordinarily successful in maturing her pilot project research. Overall, the P20 Formative Center had 11 undergraduates, 4 graduate students, 2 medical students, and 1 postdoctoral fellow participate as trainees in the various research projects and cores.
R834594C001: Human Fetal Liver and the Metabolic Syndrome; Project Leader, Philip Gruppuso, M.D.
Despite an extensive effort and multiple transplant trials in various immunodeficient rodent hosts, human fetal liver xenotransplants consistently led to a rapid loss of hepatocytes and the selective development within the xenografts of maturing biliary duct structures. Since the goal of Research Project 1 was to study toxicant-induced metabolic syndrome, institutional funds were used to explore xenotransplantation of human fetal adipose tissue as a model to study adipogenesis and obesogens. This approach has been successful, and has led Dr. Gruppuso to seek independent R01 funding to support a research program focused on human adipogenesis and the molecular response to the potential obesogens.
R834594C002: Human Fetal Prostate and Endocrine Disruption; Project Leader, Kim Boekelheide, M.D., Ph.D.
This was a highly successful Pilot Project. Previous studies of early life exposure to xenoestrogens in rats showed induction of prostatic epithelial and stromal hyperplasia, inflammation, and prostatic intraepithelial neoplasia (PIN) lesions. We used our human fetal prostate xenograft model to evaluate the response of human fetal prostate implants to estrogen during both an early acute exposure, as well as an additional later exposure. Interestingly, in response to estrogen, the human prostate xenografts demonstrated primarily basal cell hyperplasia, while the endogenous rat prostate exhibited atypical hyperplasia.
R834594C003: Human Fetal Lung, Arsenic Exposure, and Tissue Remodeling; Project Leader, Monique DePaepe, M.D.
Pilot Project 3 established the human fetal lung xenograft as an invaluable model system to study the effects of antenatal toxicant exposure on postcanalicular fetal lung development. In a first set of studies, it was determined that human fetal lung tissues retain the capacity to proliferate and differentiate into functional lung tissue, even when harvested and transplanted more than 24 hours after spontaneous miscarriage. This unanticipated degree of resilience of human fetal lung tissue supported the use of fetal lungs from stillbirths (as opposed to lungs obtained from induced abortions) for xenotransplant purposes. In a second set of experiments, it was demonstrated that the human fetal lung xenograft, especially in renal subcapsular position and in severely immune suppressed rodent hosts, faithfully replicates not only epithelial, but also microvascular postcanalicular development in situ. After optimization of the human fetal lung xenograft model in these preliminary studies, experiments involving exposure of human fetal lung xenografts to clinically relevant levels of arsenic (0-10-100 ppb) were initiated.
R834594C001: Human Fetal Liver and the Metabolic Syndrome
The original goals of this project were to develop a model in which human fetal liver is transplanted into nude rats. We intended to demonstrate that manipulation of the host environment in the adult rat xenograft recipients will induce changes in fetal liver, then go on to characterize the effect of host arsenic exposure, rapamycin administration and dietary restriction on the xenograft epigenome. However, the goals changed significantly by the end of the second year of the project, by which time we had concluded that the fetal liver transplantation model had not succeeded. At that point, we transferred our efforts to the xenotransplantation of human fetal white adipose tissue (WAT).
In focusing on WAT, we have made progress on the original overall goal of the application, to assess the effect of environmental factors on the fetus with an emphasis on fetal origins of metabolic syndrome in the offspring. Initial feasibility studies involved the transplantation of WAT derived from perigonadal fat of the rat into SCID beige mice, and WAT from mice into Rowett Nude (RNU) rats. At time points up to 7 days after transplantation, there was a marked and progressive loss of adipocytes. This was associated with the appearance of extracellular fat droplets and hemorrhage. However, at periods from 14 to 28 days, there developed a healthy appearing, growing fat pad. Given that mature adipocytes lack the ability to proliferate, the only potential explanation for this observation was de novo adipogenesis.
Having established the rodent-to-rodent xenotransplant model, we moved on to perform human to SCID-Beige mouse xenotransplantation. WAT tissue from spontaneous fetal loss at 18 to 22 weeks gestation yielded consistent results. At 2 and 8 days, the xenografts had the gross appearance of connective tissue with no obvious WAT at the transplant sites. Small foci of WAT development were present at 14 days. These progressed to obvious fat pads at the longer time points. Histology showed small WAT foci at 14 days with evidence for active adipogenesis. The markedly expanded WAT at 28, 35 and 60 days also contained adipocytes that were extremely heterogeneous in size. There also was evidence in all samples for numerous, small pericytes in close proximity to blood vessels. Immunohistochemical staining using an antibody specific for the human endothelial cell adhesion molecule CD31 demonstrated rich vascularity of the transplanted tissue. At later time points (28 and 35 days), there were xenograft-derived blood vessels in the host renal parenchyma, indicating extensive angiogenesis.
We have gone on to perform extensive characterization of gene expression and immunostaining for markers of preadipocytes, mature adipocytes and angiogenesis. In the aggregate, our studies have established human fetal WAT xenografts as a model for the long term (up to 3 months) modulation of fetal WAT tissue development by manipulation of the host environment. The work establishing and validating this model is in preparation for publication.
We pursued the one aspect of the liver project that was available to us, a comparison of gene expression and DNA methylation in mid-gestation human fetal liver versus adult liver. Triplicate samples of each were used to prepare total RNA and DNA, which were analyzed for gene expression (Affymetrix GeneChip Human Gene 1.0 ST Arrays) and DNA methylation (Illumina Infinium HumanMethylation450 BeadChips). Genes overexpressed in fetal liver were ordered based on fold-differences in expression. We selected 94 genes that were overexpressed by > 3-fold in fetal relative to adult liver and 46 genes that were overexpressed by > 3-fold in adult relative to fetal liver. These genes were submitted for analysis of methylation state by gene region (within 200 or 1500 bp of the transcription start site; within the 5’ and 3’ UTRs; within the 1st exon and gene body). Results were generated using R software for statistical computing (http://www.r- project.org/). More specifically, we examined the frequency (“density”) of methylation rates associated with the selected genes. The fetal overexpressed genes showed minimal differences from their adult counterparts in methylation adjacent to the transcription start site and within the 5’UTR. However, there were marked differences in 1st exon methylation, with fetal overexpressed genes showing lower methylation in this region. This differed from results with the genes overexpressed in adult liver. For these genes, there appeared to be a reduction in methylation in the transcription start site, 5’UTR and 3’UTR regions. These findings support the novel observation that 1st exon methylation is functionally associated with fetal, but not adult, hepatic gene expression. We will be preparing a publication on this work soon. We will extend this same approach to analysis of fetal and adult WAT and to the WAT xenografts at extended time points.
R834594C002: Human Fetal Prostate and Endocrine Disruption
Project 2 has been guided by the following working hypothesis: Exposure of the human fetal prostate to estrogenic chemicals during development results in epigenetic modifications that alter differentiation and predispose to carcinogenesis. This hypothesis was tested by xenotransplanting human fetal prostate into immunodeficient rodent hosts and studying the effects of estradiol exposure on maturation and development. Specific Aims 1 and 2 were completed, clearly demonstrating an effect of estradiol exposure on maturation and development. Specific Aim 3 has been partially fulfilled, evaluating epigenetic alterations induced by estradiol exposure on the developing human fetal prostate. The evaluation of other endocrine disrupting chemicals (genistein, bisphenol A) was not achieved because of there was not enough time. The originally stated Specific Aims, shown below, provided a roadmap to guide the project:
Specific Aim 1: Characterize the differentiation of human fetal prostate xenotransplants using immunohistochemical markers and gene expression following a time-course study.
Specific Aim 2: Examine the differentiation and carcinogenesis of human fetal prostate xenotransplants following estrogenic exposures.
Specific Aim 3: Investigate the epigenetic alterations induced by estradiol, genistein and bisphenol A exposure during human fetal prostate development using a genome-wide approach.
Significant Results (Positive and Negative):
- Human fetal prostate can be xenotransplanted into either rat or mouse immunodeficient hosts and remains viable.
- The xenotransplanted fetal prostate tissue undergoes rapid proliferation and differentiation when exposed to an adult male hormonal host environment.
- Immunohistochemical and gene expression analyses provide detailed information about the differentiation and maturation of the xenotransplanted prostate tissue.
- Prostate maturation and differentiation follow a predictable histological and molecular sequence that is reminiscent of normal prostate development in the intact human, although much accelerated.
- Estradiol exposure alters normal differentiation and maturation, producing histopathological and molecular alterations consistent with hyperplasia, including significantly increased proliferation in the epithelium.
- Epigenetic effects of estradiol exposure are most prominent in the stroma, as determined by laser capture microdissection separation of stromal and epithelial prostate tissue.
- The predominant stromal epigenetic effect was DNA hypomethylation.
- In addition to the published manuscripts listed under publications, a manuscript titled “Developmental exposure to estrogen alters differentiation and epigenetic programming in a human fetal prostate xenograft model” currently is under review with Endocrinology.
R834594C003: Human Fetal Lung, Arsenic Exposure, and Tissue Remodeling
This project was guided by the hypothesis that arsenic exposure of human fetal lung xenotransplants results in disrupted postglandular lung remodeling mediated by molecular and epigenetic alterations.
The Specific Aims of the original application were to:
- Develop a human fetal lung xenograft model of in utero arsenic exposure.
- Determine the effects of arsenic exposure on growth dynamics and gene expression in the developing human lung.
- Determine arsenic-induced epigenetic changes in the developing human lung.
Significant Results (Positive and Negative):
Optimization and validation of the human fetal lung xenograft model
We optimized and validated the human fetal lung xenograft model (Specific Aim 1). Human fetal lung tissues, derived from 13–22 weeks’ gestation stillbirths (n = 12), were implanted below the kidney capsule and/or subcutaneously in immune-suppressed mice or rats. The animals were sacrificed and grafts dissected at 2 and 4 weeks post-transplantation. Lung grafts were preserved in RNAlater for molecular genetic/epigenetic studies and formalin-fixed for morphologic studies.
Results
- According to the original protocol, immune-suppressed nude rats were used as recipients for the first 11 human lung harvests (n = 38 recipient rats). Using this recipient model, varying degrees of inflammatory response were detected in the xenografts, in some cases replicating the allograft rejection reaction ("bronchiolitis obliterans") seen in human transplant lungs. This host-versus-graft immune reaction had variable effects on graft growth and vascularization, which might confound interpretation of the effects of arsenic exposure on such lungs. For this reason, we transitioned to the SCID-beige immune-suppressed mouse as recipient model from the ninth human lung harvest onward (n = 21 recipient mice). In contrast to the nude rat, the SCID-beige mouse does not contain natural killer cells, and thus is more profoundly immune suppressed. Following adoption of the SCID-beige mouse as model, host-derived inflammation in the grafts was virtually absent, and graft growth and vascularization were significantly enhanced.
- In the original experiments, fetal lung tissues were implanted in renal subcapsular as well as subcutaneous sites. While the subcutaneous grafts showed higher levels of lung growth and proliferation, they displayed a non-physiologic type of lung growth, characterized by an abnormal dissociation of epithelial and vascular growth (epithelial overgrowth).
Conclusion:
- Renal subcapsular grafts in SCID-beige mice provide the best model of normal postcanalicular human fetal lung development and were used in subsequent experiments.
- Fetal lung tissues derived from stillbirths show unanticipated resilience and are capable of restoring proliferative activity even when obtained up to 48 hours after death (potential implications for organ transplantation/pulmonary regenerative medicine).
- Analysis of effects of arsenic exposure on growth dynamics, gene expression and epigenetic changes in the developing lung
Following optimization and validation of the fetal lung xenograft model, studies were performed to determine the effects of arsenic exposure on growth dynamics and gene expression in the developing human lung (Specific Aim 2) as well as epigenetic changes (DNA methylation) (Specific Aim 3). Animals were exposed to 10 or 100 ppb inorganic arsenic as sodium arsenite, added to drinking water, for 2 or 4 weeks post-transplantation. Controls received arsenic-free water. Arsenic levels were determined in host tail at 4 weeks post-transplantation (Dartmouth). Lung grafts were preserved in RNAlater for gene expression (microarray and real-time RT-PCR) or DNA methylation studies and formalin-fixed for morphologic studies.
Results
- Arsenic exposure was well tolerated and did not lead to weight loss or other systemic effects.
- Exposure to 100 ppb sodium arsenite resulted in significantly increased tissue (tail) arsenic levels in host mice, indicating arsenic addition to drinking water at time of xenografts provides effective delivery.
- Histopathologic analysis revealed architectural maturation in arsenic-exposed and control xenografts. Qualitatively, and based on the first arsenic experiments only, the airspaces of arsenic-exposed grafts appeared enlarged and simplified compared with those of controls. In addition, the microvasculature of arsenic-exposed lungs appeared more tortuous than that of controls. Morphometric studies of alveolar and microvascular development of the xenografts as well as proliferation analyses are pending.
- Gene expression studies were initially performed by Affymetrix microarray analysis on three control and four arsenic-exposed xenografts (post-transplantation week 4). Various differentially regulated genes were identified, including the angiogenesis regulators ANGPT2, ESM1 (endocan) and LYVE1. Upregulation of ANGPT2 and ESM1 in arsenic-exposed lungs was confirmed by real-time RT-PCR analysis. Additional larger Affymetrix microarray analyses failed to confirm significant differential expression of these or other genes. Prominent batch effect was noted (inter-graft variability, likely attributable to clinical conditions). Pathway analyses are in progress. Similarly, DNA methylation studies failed to reveal significant differences in DNA methylation between grafts exposed to 100 ppb arsenic and controls.
Conclusion:
- Exposure of human fetal lung xenografts to 100 ppb arsenic appears to result in morphologic alterations (including the microvasculature).
- Arsenic exposure, at the doses studied and with the sample sizes available, did not have significant effects on gene expression or DNA methylation.
- Of note, microvascular growth patterns noted in some grafts were reminiscent of the dysangiogenesis characteristic of new bronchopulmonary dysplasia (chronic lung disease of preterm newborn), suggesting the xenografts may serve as a model of this disease.
Administrative Core
The operations of the Administrative Core achieved full functionality in meeting its stated Specific Aims:
Specific Aim 1: Provide, support and develop processes that sustain and accommodate the growth of interdisciplinary basic and applied research and training.
Specific Aim 2: Maintain a communication structure that strengthens team building and facilitates the dissemination of information.
Specific Aim 3: Implement mechanisms that will optimally promote the program’s objectives and ensure the best and most responsible use of the available resources.
Significant Results (Positive and Negative):
The Administrative Core delivered a high level of support in furthering the research, educational, training and outreach goals of the Formative Center. Monthly group meetings were scheduled to promote strong team building and to ensure excellent communication regarding the sharing of information, planning events, ensuring best scientific practices and procedures for tissue procurement, meeting IRB and IACUC approvals, maintaining the highest ethical standards for the recruitment and participation of research participants, and achieving the goals and objectives of the Formative Center. The Administrative Core also was responsible for building and maintaining the Formative Center’s website. Website updates were timely and served as a valuable tool for expanding networking and outreach capabilities. Additionally, the Administrative Core organized a monthly seminar series and an Annual Retreat that served as a forum for sharing and evaluating our research and outreach activities. The Administrative Core provided oversight of all activities, ensuring that the Terms and Conditions, and all reporting requirements of the grant awarded by NIEHS and EPA were properly fulfilled.
Community Outreach and Translation CoreThe Community Outreach and Translation Core made excellent progress toward achieving its stated specific aims:
Specific Aim 1: Develop ethics model for the research use of fetal tissue and provide ethics education and consultation to Formative Center staff.
Specific Aim 2: Provide ethics education, outreach, and consultation for hospital staff and the IRB.
Specific Aim 3: Provide education and capacity building for patients.
Specific Aim 4: Provide education and capacity building for the broad public, both in Rhode Island and nationally.
Specific Aim 5: Provide education for the overall Brown University community.
Specific Aim 6: Publish research articles on ethics of fetal tissue research.
Significant Results (Positive and Negative):
The Community Outreach and Translation Core (COTC) served three important functions for the Center: (1) established partnerships with healthcare, government and community organizations; (2) facilitated educational and outreach opportunities to build capacity among faculty, healthcare practitioners, and Rhode Islanders; and (3) investigated ethical issues regarding informed consent in fetal tissue research as well as healthcare practitioners’ knowledge of the (fetal) developmental origins of health and disease (DOHaD).
The COTC employed a dynamic multi-tiered community engagement strategy to target key audiences. The COTC formed two active coalitions: the Community Advisory Board (CAB) to engage RI organizations and agencies, and Hospitals for a Healthy Environment in Rhode Island (H2ERI) to engage the healthcare community. Additionally, COTC staff served as active board members for a number of coalitions and organizations that address children’s health issues. The COTC organized successful conferences, workshops and presentations on environmental health topics and encouraged environmentally sustainable practices to reduce environmental exposures detrimental to children’s health. In addition to outreach, COTC has played a key role in setting rigorous ethical standards for fetal tissue research and interacting with study participants.
The COTC was shaped by its highly involved Community Advisory Board (CAB) and community-based partners. The CAB met monthly during the planning phase of the Center’s formation, then quarterly to guide this Core. The CAB had 10 members from Rhode Island community-based organizations, disease-based charities, state agencies and education who were actively involved in children’s health and welfare: Environmental Justice League of Rhode Island, Childhood Lead Action Project, Meeting Street, Ready to Learn, Rhode Island Department of Health (HEALTH), March of Dimes, American Lung Association, Lincoln School, and Project HEALTH. CAB members were invited to the Center’s monthly seminars and its annual retreat. They received notifications of all Center-related activities. In turn, some CAB members presented at the Center’s annual retreat and epigenetics conferences. The CAB provided one avenue by which the COTC assessed the effectiveness of their developed products.
The COTC formed the H2ERI Coalition. This Coalition began as a COTC partnership with nurses at Women and Infants Hospital to remove DEHP- and PVC-based medical equipment from its Neonatal Intensive Care Unit. H2ERI’s formation was deeply influenced by the expressed needs and suggestions of Rhode Island-based hospital managers, healthcare professionals, union leaders, and members of food policy groups and community-based health organizations. With assistance from Health Care Without Harm, Practice GreenHealth, and the Maryland Hospitals for a Healthy Environment, efforts were expanded to encompass environmental sustainability in healthcare settings. This initiative was widely successful. Three annual conferences on best practices for environmental excellence in healthcare were held. There were 80, 100 and 150 attendees, respectively. Brown students were recruited to help organize conferences as a way to educate them and to spread word of the Center’s work throughout the Brown campus. The first event featured topics on environmentally preferable purchasing, hospital waste management, and innovative models of care. The second year introduced such topics as metrics for sustainability, sugar-sweetened beverages, sustainable landscaping and composting, green cleaning, and establishing green teams. In 2013, the Blue Wrap Blue Jean Ball created awareness on blue wrap recycling in healthcare. This event featured an exhibit by the Northeastern University School of Nursing entitled Blue Wrap Cradle-to-Grave. It raised funds to sponsor one employee from each Rhode Island hospital to attend CleanMed, a national healthcare conference on environmental sustainability. Additionally, H2ERI presented the Rhode Island Environmental Sustainability Award to Kent Hospital and Sylvia Weber, lobbyist for state of Rhode Island.
In 2012, in partnership with Health Care Without Harm and Farm Fresh Rhode Island, H2ERI formed a program Healthy Food in Healthcare to promote the procurement of fresh, local and sustainable produce in healthcare. In 2013, this healthy food program instituted a local seafood pilot program in five of 13 Rhode Island hospitals. Dr. Panikkar provided testimony to Senator Whitehouse’s office about the Animal Drug User Fee Reauthorization of 2013 (ADUFA), and Prevention of Antibiotics for Medical Treatment Act (PAMTA). This legislation prohibits use of antibiotics in animal agriculture unless there is a medical emergency.
Today, the coalition has 50 members from 31 different organizations representing hospitals, hospital associations, state and federal agencies, and NGOs. The coalition met bimonthly to plan activities and showcase successful programs and initiatives. COTC postdoctoral fellow Dr. Panikkar represented the H2ERI coalition at the national CleanMed conference and helped Miriam Hospital in Providence to appoint an environmental sustainability coordinator. Dr. Panikkar gave presentations on topics including DEHP/PVC in medical devices, green team building, and environmental sustainability practices in healthcare. At the 2012 annual CEHC/Partnerships in Environmental Public Health meeting, Dr. Brown led a workshop to help other Centers build similar Healthy Hospital coalitions.
The COTC was actively involved in community partnerships. The COTC participated in environmental health policy and public health campaign initiatives through the Environmental Justice League of RI and Childhood Lead Action Project. Additionally, Core staff became members of the Alliance for a Healthier RI (AHRI); LAUNCH, a federal mental health and substance abuse program; and the HUD-funded Green and Healthy Housing Initiative. The COTC was very involved with AHRI’s campaign to impose an excise tax on sugar-sweetened beverages and curtail distribution in schools, hospitals and public facilities. Dr. Panikkar and other AHRI members briefed RI Attorney General Peter Kilmartin on this issue. Additionally, Dr. Panikkar established a healthy beverage work group. COTC members advised RIDOH’s Maternal, Infant and Early Childhood Home Visiting Program and the Local Wellness Council (LWC), an organization that addresses the needs of developmentally disabled children ages 0–8. Dr. Thompson served as an advisor to Rhode Island’s Strategic Plan for Preconception Health to address preconception health through clinical practice, health policy and public health initiatives. The COTC co-sponsored the state’s Preconception Health Summit. Information about the Center’s research was distributed to attendees.
The COTC was involved in pharmaceutical waste reduction and medical waste management. The COTC joined Rhode Island Department of Environmental Management’s (RIDEM) task force, developed related undergraduate student projects in nursing, and conducted Master’s thesis research in environmental studies under the guidance of Drs. Thompson and Brown, respectively. These activities have provided groundwork for new RIDEM-related policies and practices. Additionally, Dr. Thompson (Health Specialist) briefed members of the RI State Senate on these issues at their request.
The COTC held seminars on Epigenetics and Fetal Origins of Health and Disease. In 2011, a 2-hour workshop held for 83 healthcare professionals, faculty and students from Rhode Island and Massachusetts. In 2013, Dr. Thompson presented a teleconference hosted by webinar, Alaska Collaborative on Health and the Environment (50 attendees), and a webinar hosted by the American Society of Safety Engineers for 37 attendees about the intergenerational effects of maternal exposures to multiple environmental hazards. Additionally, Dr. Thompson was the featured speaker at the Sigma Theta Tau International Nursing Honor Society Delta Upsilon Chapter-at-Large Fall Meeting, Is it Genetic or Is it Environmental? Through the efforts of Dr. Thompson and the Rhode Island State Nurses Association, Registered Nurse (RN) attendees received continuing education credit not only for this workshop, but the second H2ERI conference as well.
The COTC provided Environmental Health Training for Home Visitors. With CAB members, Drs. Brown and Panikkar developed a training program and conducted three statewide workshops on children’s environmental health for Meeting Street early intervention staff, the Bradley Research Center home visitors who work with developmentally disabled children and their families, and Miriam Hospital youth green team members. These workshops highlighted how children are uniquely susceptible to environmental hazards during their earliest years. These workshops illustrated ways to avoid or reduce residential exposures. Topics presented included developmental impacts of environmental exposures on infants and toddlers, environmental hazards of concern to young children, and best practices for providing child-safe environments.
The COTC organized Science Cafes. The COTC organized a series of Science Cafes in local coffee shops and restaurants on relevant environmental health topics in conjunction with the Silent Spring Institute.
The COTC co-sponsored Community Environmental College and ECO-Youth. These summer and after-school train-the-trainer programs allowed 80 high school students to teach others about environmental health issues, including asthma, obesity, and reproductive health. Since 2009, the COTC has co-sponsored these programs with Brown’s Superfund Research Program and the Environmental Justice League of RI.
The COTC conducted Ethics Research. Ethics is an integral part of the Center’s work. The Center’s team acknowledged that fetal tissue xenotransplantation research could be a publically contentious issue. To ensure that ethical guidelines would be well integrated with the Center’s research projects, Dr. Brown held a series of meetings with the former Health Specialist and the Tissue Procurement Core Leader to discuss specific project-related ethical issues. These meetings shaped the ethical issues that the Center addresses. Many ethical issues arose in designing these research projects. Dr. Brown led the Center’s team in evaluating the informed consent process through participant interviews. This preliminary work resulted in a manuscript that contributed to the literature on bioethics and advanced forms of informed consent (Panikkar et al., 2012). The results were based on the interviews we conducted with all of the key research personnel in the Center, including project staff from the Administrative Core (n = 1), Tissue Procurement Core (n = 3), and Xenotransplant Core or laboratory scientists (n = 6). In addition, we interviewed the recruiter and the parents who agreed to participate in the research and donated their fetal tissues. These interviews were done 1 or 2 weeks following their miscarriage. The parents who donated the fetal tissues were enrolled in the study after the research team decided on offering detailed informed consent to the parents who join the study. We reached out to 16 women to join the follow up ethics research and seven of them consented and were interviewed.
The COTC assisted with Research on Evidence-Based Practices. Drs. Gruppuso and Thompson (Center’s Health Specialists) and Alisha Lakhani, MPH conducted a descriptive survey of 539 nurse midwives through the Association of Certified Nurse Midwives (ACNM). This online survey indicated that DOHaD is an area that is relatively unfamiliar to these caregivers and that some key aspects of DOHaD, such as exposure to environmental and occupational toxicants and the effects of maternal stress, may warrant greater emphasis in the initial and continuing education of nurse midwives. A manuscript titled “Knowledge and attitudes regarding ‘fetal origins’” currently is under going peer review with Journal of Midwifery & Women’s Health.
Tissue Procurement CoreThe Tissue Procurement Core achieved its stated specific aims:
Specific Aim 1: Facilitate the identification, inclusion and rapid procurement of spontaneous abortions (miscarriages) to be evaluated for enrollment in the Formative Center research protocol.
Specific Aim 2: Evaluate, procure and process selected fetal tissues to ensure successful use of the tissues in the proposed research projects. These tissues include: fetal lung, liver, ovary, uterus, mammary gland, testes, adipose and prostate tissue.
Specific Aim 3: Provide opportunities for clinical fellows, including Pediatric, Neonatology, Pathology, and Maternal Fetal Medicine fellows, to engage in research related to the Formative Center.
Significant Results (Positive and Negative):
The tissue Procurement Core has submitted all human subjects research materials for IRB review and approval. Women & Infants Hospital IRB approval was obtained (WIH IRB 09-007). Six amendments to the protocol were submitted and approved by the IRB. These included adding the Ambulatory Surgery Unit as a site for subject recruitment, adding six more fetal tissues for procurement (mammary gland, ovary, uterus, testes, adipose tissue, and penile and clitoral corporal tissue), and several clarifications regarding fetal tissue eligibility requirements. In addition, two amendments to the consent form were submitted and approved by the IRB. These were related to contacting patients for follow-up questions, issues addressing privacy and confidentiality of questionnaire information, and offering a gift card for participation. During the course of the study, procedures for enhancing participant recruitment involving clinical staff were developed and implemented. Training for consent procedures was developed and implemented. Procedures for facilitating tissue collections and processing were established. A total of 112 patients was approached for participation in the study. Of those approached, 30 consented and 28 were enrolled and tissues successfully delivered to the Xenotransplant Core. Two patients withdrew consent.
Xenotransplant Core
The overall goal of the Xenotransplantation Core has been to provide specialized, on-demand surgical services for the transplantation of human fetal tissues into immunodeficient animals in support of the research projects carried out by the Formative Center. The Encrustation Core achieved its stated specific aims:
Specific Aim 1: Provide animal surgery and model development services.
Specific Aim 2: Interact with research investigators to initiate animal experiments in support of the Formative Center’s research projects.
Specific Aim 3: Implement current and emerging surgical techniques in xenotransplant model development and training of technical staff.
Significant Results (Positive and Negative):
Specific Aim 1:
Since the inception of the P20 Formative Center, ~474 surgeries have been performed. A list of human fetal tissue specimens implanted to date is provided below in Table 1.
Model characterization studies helped to define the host species/strain, implantation location, and various parameters required for proper tissue proliferation and development for each of the research projects. Project 1 centered on liver and metabolic syndrome. In this project, human fetal hepatocytes failed to proliferate and grow following xenotransplantation. The Xenotransplantation Core played a central role in helping to characterize this model by employing a range of host strains (Nude rat, NOD SCID mouse, SCID Beige mouse), and implant locations (renal subcapsular, intra-abdominal, liver subcapsular). A novel technique employed by our core included the subcutaneous implantation of a mesh-based vascularization cage device 2 weeks prior to tissue xenografting, in an effort to provide a microenvironment conducive to angiogenesis. In spite of these efforts, the host environment was not sufficiently supportive for hepatocyte growth, and Project 1 was redirected to examine human fetal adipose tissue using the renal subcapsular location in the SCID Beige mouse host. Xenotransplanted adipose tissue exhibited excellent growth and development within the renal subcapsular location up to 60 days post-transplantation.
For Project 3, host-derived inflammatory infiltrates were observed at the interface between lung xenografts and host kidney tissue by 2 weeks with use of the Nude rat host and xenotransplantation of tissue into a dorsal subcutaneous location showed poor growth, which was attributed to underdeveloped and dysmorphic vascularization. To address these concerns, the SCID Beige mouse, which lacks B cell immunity, was substituted for the Nude rat and larger-sized implants (2–3mm3) were used. The result was excellent growth and development of xenotransplanted fetal lung tissue, due to the improved vascular environment and elimination of B cell infiltration.
Table 1. Human fetal tissue specimens to date
ID |
Date |
Sex |
Age (weeks) |
Notes |
Clinical Observations |
1 |
6/22/10 |
F |
21 |
|
No gross abnormalities |
2 |
8/18/10 |
M |
22 |
|
No gross abnormalities |
3 |
9/9/10 |
M |
17 |
|
No gross abnormalities |
4 |
9/28/10 |
F |
16.3 |
|
Premature membrane rupture |
5 |
10/25/10 |
M |
13.4 |
|
Maternal bleeding |
6 |
12/1/10 |
M |
22 |
|
No gross abnormalities |
7 |
1/7/11 |
F |
20.5 |
|
No gross abnormalities |
8 |
3/8/11 |
F |
19 |
|
Multiple fetal anomalies |
9 |
3/17/11 |
F |
15 |
|
Home delivery |
10 |
3/18/11 |
M |
17 |
twin A |
Chorioamnionitis |
11 |
3/18/11 |
M |
17 |
twin B |
Chorioamnionitis |
12 |
7/20/11 |
M |
15 |
|
No gross abnormalities |
13 |
1/9/12 |
M |
21 |
|
No gross abnormalities |
14 |
3/9/12 |
M |
20 |
|
No gross abnormalities |
15 |
4/13/12 |
M |
20.6 |
|
No gross abnormalities |
16 |
4/23/12 |
M |
21.5 |
|
Premature membrane rupture |
17 |
5/18/12 |
M |
22 |
twin A |
No gross abnormalities |
18 |
5/18/12 |
M |
22 |
twin B |
No gross abnormalities |
19 |
9/11/12 |
M |
20 |
|
Premature membrane rupture |
20 |
10/12/12 |
M |
16 |
|
No gross abnormalities |
21 |
10/15/12 |
M |
22.5 |
|
No gross abnormalities |
22 |
10/17/12 |
M |
15 |
|
No gross abnormalities |
23 |
11/29/12 |
M |
20 |
|
No gross abnormalities |
24 |
12/11/12 |
F |
21 |
|
No gross abnormalities |
25 |
12/11/12 |
M |
18 |
twin A |
No gross abnormalities |
26 |
3/1/13 |
M |
18 |
twin B |
No gross abnormalities |
27 |
1/19/13 |
M |
20 |
|
Premature membrane rupture |
28 |
3/1/13 |
M |
20.6 |
|
No gross abnormalities |
Specific Aim 2:
Throughout the duration of the P20 Formative Center, the Xenotransplantation Core worked closely with the leaders of the individual research projects to facilitate model characterization. Our expertise expanded beyond technical surgical skills to encompass the generation and implementation of IACUC amendments necessary to explore various characteristics of the models used in each project. Successful IACUC amendments were authored by the Xenotransplantation Core in consultation with project leaders to use various mouse strains (Nude mouse, NOD SCID, SCID Beige, athymic Nude), different implantation locations (dorsal subcutaneous, renal subcapsular, liver subcapsular, intra-abdominal), to use angiogenesis-promoting implant cages, and for approval of pilot projects to explore additional tissues (mammary, ovary). For each amendment, the Xenotransplantation Core researched the necessary surgical procedures, trained the surgical staff performing the techniques, and worked closely with the research project leaders to discuss any special considerations as each model was developed.
Specific Aim 3:
The Xenotransplantation Core grew from two to four surgical stations, which necessitated training additional surgical technicians to be available on demand. Training of new technicians included attending Animal Welfare and Biosafety training, as well as intense one-on-one training sessions with senior technicians to acquire the surgical skills and animal handling techniques necessary. In 2012, the surgical technicians and Core manager attended a webinar entitled “Aseptic and Precise Surgical Technique in Rodents,” presented by Dr. Marcel Perret-Gentil, D.V.M., of the University of Texas at San Antonio, to reinforce our current skillset. Since the inception of the P20 Formative Center in 2010, the Xenotransplantation Core has trained nine students, postdoctoral fellows, and research assistants and is currently comprised of four surgical technicians.Journal Articles: 8 Displayed | Download in RIS Format
| Other center views: | All 13 publications | 8 publications in selected types | All 8 journal articles |
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Boekelheide K, Blumberg B, Chapin RE, Cote I, Graziano JH, Janesick A, Lane R, Lillycrop K, Myatt L, States JC, Thayer KA, Waalkes MP, Rogers JM. Predicting later-life outcomes of early-life exposures. Environmental Health Perspectives 2012;120(10):1353-1361. |
R834594 (Final) R834594C002 (Final) |
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De Paepe ME, Chu S, Hall S, Heger NE, Thanos C, Mao Q. The human fetal lung xenograft: validation as model of microvascular remodeling in the postglandular lung. Pediatric Pulmonology 2012;47(12):1192-1203. |
R834594 (2012) R834594 (Final) R834594C003 (Final) |
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De Paepe ME, Chu S, Heger N, Hall S, Mao Q. Resilience of the human fetal lung following stillbirth: potential relevance for pulmonary regenerative medicine. Experimental Lung Research 2012;38(1):43-54. |
R834594 (2012) R834594 (Final) R834594C003 (Final) |
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Heger NE, Hall SJ, Sandrof MA, McDonnell EV, Hensley JB, McDowell EN, Martin KA, Gaido KW, Johnson KJ, Boekelheide K. Human fetal testis xenografts are resistant to phthalate-induced endocrine disruption. Environmental Health Perspectives 2012;120(8):1137-1143. |
R834594 (Final) R834594C002 (Final) |
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Panikkar B, Smith N, Brown P. Reflexive research ethics in fetal tissue xenotransplantation research. Accountability in Research 2012;19(6):344-369. |
R834594 (2012) R834594 (Final) |
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Saffarini CM, McDonnell EV, Amin A, Spade DJ, Huse SM, Kostadinov S, Hall SJ, Boekelheide K. Maturation of the developing human fetal prostate in a rodent xenograft model. The Prostate 2013;73(16):1761-1775. |
R834594 (2012) R834594 (Final) R834594C002 (Final) |
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Spade DJ, Hall SJ, Saffarini CM, Huse SM, McDonnell EV, Boekelheide K. Differential response to abiraterone acetate and di-n-butyl phthalate in an androgen-sensitive human fetal testis xenograft bioassay. Toxicological Sciences 2014;138(1):148-160. |
R834594 (Final) R834594C002 (Final) |
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Thompson MR, Boekelheide K. Multiple environmental chemical exposures to lead, mercury and polychlorinated biphenyls among childbearing-aged women (NHANES 1999-2004): body burden and risk factors. Environmental Research 2013;121:23-30. |
R834594 (Final) |
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Supplemental Keywords:
Health, Scientific Discipline, ENVIRONMENTAL MANAGEMENT, Health Risk Assessment, Risk Assessments, Biology, Risk Assessment, biological pathways, children's health, fetal exposure, bioavailability, developmental effects, perinatal exposure, biochemical researchProgress and Final Reports:
Original Abstract Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R834594C001 Liver and the Metabolic Syndrome
R834594C002 Prostate and Endocrine Disruption
R834594C003 Lung, Arsenic Exposure, and Tissue Remodeling
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.