RNA interference is a powerful tool for dissecting gene function. In Caenorhabditis elegans, ingestion of double stranded RNA causes strong, systemic knockdown of target genes. Further insight into gene function can be revealed by tissue-specific RNAi techniques. Currently available tissue-specific C. elegans strains rely on rescue of RNAi function in a desired tissue or cell in an otherwise RNAi deficient genetic background. We attempted to assess the contribution of specific tissues to polyunsaturated fatty acid (PUFA) synthesis using currently available tissue-specific RNAi strains. We discovered that rde-1 (ne219), a commonly used RNAi-resistant mutant strain, retains considerable RNAi capacity against RNAi directed at PUFA synthesis genes. By measuring changes in the fatty acid products of the desaturase enzymes that synthesize PUFAs, we found that the before mentioned strain, rde-1 (ne219) and the reported germline only RNAi strain, rrf-1 (pk1417) are not appropriate genetic backgrounds for tissue-specific RNAi experiments. However, the knockout mutant rde-1 (ne300) was strongly resistant to dsRNA induced RNAi, and thus is more appropriate for construction of a robust tissue-specific RNAi strains. Using newly constructed strains in the rde-1(null) background, we found considerable desaturase activity in intestinal, epidermal, and germline tissues, but not in muscle. The RNAi-specific strains reported in this study will be useful tools for C. elegans researchers studying a variety of biological processes.

First-trimester placental development comprises many critical yet understudied cellular events that determine pregnancy outcomes. Improper placentation leads to a host of health issues that not only impact the fetal period but also influence later-life offspring health. Thus, an experimental paradigm for studying early placental development is necessary and has spurred the development of new in vitro models. Organoid model systems are three-dimensional structures comprising multiple differentiated cell types that originate from a progenitor population. Trophoblasts are the progenitor cells that serve as the proliferative base for the differentiation and maintenance of the placenta. Due to research constraints, experimental studies on the causal mechanisms underlying pathological pregnancies cannot readily be performed in human subjects. The nonhuman primate (NHP) offers a solution to this problem as it circumvents the limitations of human pregnancy sampling. Importantly, NHPs share many developmental features of human pregnancy, including placenta type and a similar fetal growth trajectory, making longitudinal pregnancy studies feasible and relevant. Since perturbations made in vivo can be validated in vitro, an NHP model of early pregnancy would facilitate mechanistic studies of pregnancy disorders. Herein, we describe the methodology for the establishment of a first-trimester NHP placenta trophoblast organoid model system.

The rhesus macaque (Macaca mulatta) is a valuable model for pregnancy research due to its physiological similarity to humans and the ability to conduct studies in a controlled environment. Our previous work used noninvasive imaging methods to assess placental hemodynamics across gestation with correlative tissue analysis post-delivery. Here, we expand access to longitudinal timepoints from ongoing pregnancies by obtaining placental biopsies using ultrasound-guided needle aspiration. This approach aligns with New Approach Methods (NAMs) and supports animal welfare by reducing the number of animals required. We describe a chorionic villus sampling (CVS) simulation model which facilitates training to gain proficiency in technical skills prior to performing the procedure in animals. We report outcomes from three rhesus macaques that underwent CVS three times between gestational days 40 to 106 (term: 165 days). Although biopsy samples are smaller than whole placenta, tissue yields were sufficient for multiple uses. We demonstrate 1) appropriate histology from aspirated samples, 2) good RNA quality and yield, and 3) the ability to isolate trophoblast organoids, a NAMs advancement that reduces the need for first-trimester surgical delivery. No adverse outcomes occurred following serial CVS procedures, supporting the use of this sampling to maximize animal utilization in longitudinal pregnancy studies.

The rhesus macaque (Macaca mulatta) is a valuable model for pregnancy research due to its physiological similarity to humans and the ability to conduct studies in a controlled environment. Our previous work used non-invasive imaging methods to assess placental hemodynamics across gestation with correlative tissue analysis post-delivery. Here, we expand access to longitudinal timepoints from ongoing pregnancies by obtaining placental biopsies using ultrasound-guided needle aspiration. This approach aligns with New Approach Methods (NAMs) and supports animal welfare by reducing the number of animals required. We describe a chorionic villus sampling (CVS) simulation model which facilitates training to gain proficiency in technical skills prior to performing the procedure on animals. We report outcomes from three rhesus macaques that underwent CVS three times between gestational days 40 to 106 (term: 165 days). Although biopsy samples are smaller than whole placenta, tissue yields were sufficient for multiple uses. We demonstrate (1) appropriate histology from aspirated samples, (2) good RNA quality and yield, and (3) the ability to isolate trophoblast organoids, an advancement in NAMs that reduces the need for first-trimester surgical delivery. No spontaneous preterm delivery occurred following serial CVS procedures, supporting the use of this sampling method to maximize animal utilization in longitudinal pregnancy studies.

First trimester placental development is comprised of many critical yet understudied cellular events that determine pregnancy outcomes. Improper placentation leads to a host of health issues that not only impact the fetal period but also influence later-life offspring health. Thus, an experimental paradigm to study early placental development is necessary and has spurred the development of new in vitro models. Organoid model systems are three-dimensional structures comprised of multiple differentiated cell types that originate from a progenitor population. Trophoblasts are the progenitor cells that serve as the proliferative base for differentiation and maintenance of the placenta. Due to research constraints, experimental studies on the causal mechanisms underlying pathological pregnancies cannot readily be performed in human subjects. The nonhuman primate (NHP) offers a solution to this problem as it circumvents the limitations of human pregnancy sampling. Importantly, NHPs share many developmental features of human pregnancy including placenta type and a similar fetal growth trajectory, making longitudinal pregnancy studies feasible and relevant. Since perturbations made in vivo can be validated in vitro, an NHP model of early pregnancy would facilitate mechanistic studies of pregnancy disorders. Herein we describe the methodology for establishment of a first trimester NHP placenta trophoblast organoid model system.