Yolk Sac Cells Research Articles

Abstract We085: Origin and fate of CX3CR1 + cells in the heart: their unique contribution to cardiovascular cells

Introduction: During development, various cells intricately collaborate to form embryonic and fetal hearts. We can harness this naturally occurring generative mechanism to advance regenerative medicine for cardiac repair. Studies reported that C-X3-C Motif Chemokine Receptor 1 (CX3CR1) + cells play vital roles in cardiac development and disease. However, the origin and the fate of CX3CR1 + cells during cardiogenesis remain unclear. Specifically, the existence of CX3CR1 + cells before E8.5 and their commitment other than to macrophages have not been investigated. Methods: We investigated the origin and fate of CX3CR1 + cells using various knock-in reporter mice with CX3CR1 drivers for spatiotemporal genetic lineage tracing of CX3CR1 + cells. In addition, we developed a method to generate CX3CR1 + cells from differentiating mouse embryonic stem cells (mESCs). mESC-derived CX3CR1 + cells were characterized in vitro, in the fetal mouse heart ex vivo , and in the adult heart in vivo . Their heterogeneity and trajectory were analyzed via single cell RNA sequencing (scRNA-seq). Results: Genetic fate mapping of CX3CR1 + cells identified epiblasts at E6.5, the parietal endoderm at E7.0, yolk sac cells at E8.0, and cardiomyocytes at E9.5. Temporal genetic labeling (Cx3cr1-CreERT2;R26-tdTomato) showed that CX3CR1 + cells at E6.5 can contribute to cardiomyocytes (CMs) and endothelial cells (ECs) during prenatal development via both de novo differentiation and fusion with pre-existing CMs or ECs, respectively. In the adult heart, cells that have ever expressed Cx3cr1 comprised 13.5 ± 0.2% of CMs and 30.9 ± 1.3% of ECs. In addition, CX3CR1 + cells, which were differentiated from mESCs generated CMs, ECs and macrophages in vitro, in the fetal mouse heart ex vivo , and in the adult heart in vivo . Single cell RNA sequencing showed that Cx3cr1 + cells represent an intermediate cell population differentiating into the mesoderm from pluripotent stem cells. Conclusions: Our data demonstrate that CX3CR1 serves as a marker for a unique subset of progenitors that contribute to the formation of CMs and ECs as well as macrophages, providing new insights into the versatile role of CX3CR1 + cells in cardiogenesis.

Fertility-sparing approach to malignant ovarian germ cell tumors - Oncologic and obstetric outocome: A retrospective study.

To evaluate oncologic (such as disease-free and overall survival) and obstetric outcomes in patients diagnosed with malignant ovarian germ cell tumors (MOGCTs). Patients diagnosed with MOGCTs between March 2007 and February 2022 were evaluated and patients who underwent fertility sparing surgery were included in this retrospective study. The obstetric and oncologic outcomes were evaluated by collecting data up until the patient's last follow-up visit from the hospital records and patient files. The study was approved by Başkent University Institutional Review Board (KA23/124). Seventy FSS patients were included in this study. The median age of the patients was 22.5 years (range: 11-37). The median follow-up time was 92.0 months (10-189). Immature teratoma was the most common histological subtype (32.9%). Bilateral involvement was detected in only one patient with immature teratoma (1.4%). The 5-year DFS rates of immature teratoma, dysgerminoma, yolk sac, and mixed germ cell histologic types were 91.1%, 94.1%, 82.4%, and 88.9%, respectively (P: 0.716). The 5-year OS rates of the same histologic types were 95.7%, 100%, 88.2%, and 88.9%, respectively (P = 0.487). All patients (100%) had a regular menstrual cycle after the completion of adjuvant treatment. The mean time between the last chemotherapy and menstruation was 4.38 months. To date, a total of 34 patients tried to conceive after the completion of disease treatment. A total of 23 (67.6%) patients conceived, resulting in 27 live births in 22 (100%) patients. Fertility preservation should be the first treatment option in MOGCTs in young patients due to the unilateral involvement of the disease and its chemosensitive nature.

Yolk sac cell atlas reveals multiorgan functions during human early development.

The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.

3D culture of mesenchymal stem cells from the yolk sac to generate intestinal organoid

Organoids are in vitro models that originated from the three-dimensional culture of stem cells with the ability to reproduce part of the in vivo structural and functional specificities of body organs. Intestinal organoids have great relevance in cell therapy, as they provide more accurate information about tissue composition and architecture in relation to two-dimensional culture, in addition to serving as a study model for host interaction and drug testing. The yolk sac (YS) is a promising source of mesenchymal stem cells (MSCs), which are multipotent stem cells with self-renewal ability and potential to differentiated into mesenchymal lineages. Besides this, the YS is responsible for the formation of intestinal epithelium during embryonic development. Thus, the aim of this study was to verify if the three-dimensional in vitro culture of stem cells derived from the canine YS is capable of developing intestinal organoids. MSCs from the canine YS and gut cells were isolated and characterized, then three-dimensionally cultured in Matrigel. In both cells lineages, spherical organoids were observed and after 10 days the gut cells formed crypt-like buds and villus-like structures. Despite having the same induction of differentiation process and having the expression of intestinal markers, the MSC from the YS morphology was not in the form of crypt budding. The hypothesis is that these cells could generate structures equivalent to the intestinal organoids of the colon that other studies showed formed only spherical structures. The culture of MSC from the YS, as well as the establishment of protocols for 3D cultivation of this tissue, is relevant, as it will serve as a tool in various applications in basic and scientific biology.

Initial Characterization of 3D Culture of Yolk Sac Tissue.

The role of the yolk sac (YS) in miscarriage is not yet clear, largely due to ethical reasons that make in vivo studies difficult to conduct. However, 3D cultures could provide a solution to this problem by enabling cells to be arranged in a way that more closely mimics the structure of the YS as it exists in vivo. In this study, three domestic species (porcine, canine, and bovine) were chosen as models to standardize 3D culture techniques for the YS. Two techniques of 3D culture were chosen: the Matrigel® and Hanging-Drop techniques, and the 2D culture technique was used as a standardized method. The formed structures were initially characterized using scanning electron microscopy (SEM), immunohistochemistry (IHC), and quantitative real-time PCR (RT-qPCR). In general, the 3D culture samples showed better organization of the YS cells compared to 2D cultures. The formed structures from both 3D methods assemble the mesothelial layer of YS tissue. Regarding the IHC assay, all in vitro models were able to express zinc and cholesterol transport markers, although only 3D culture techniques were able to generate structures with different markers pattern, indicating a cell differentiation process when compared to 2D cultures. Regarding mRNA expression, the 3D models had a greater gene expression pattern on the Hemoglobin subunit zeta-like (HBZ) gene related to the YS tissue, although no significant expression was found in Alpha-fetoprotein (AFP), indicating a lack of endodermal differentiation in our 3D model. With the initial technique and characterization established, the next step is to maintain the cultures and characterize the diversity of cell populations, stemness, functions, and genetic stability of each 3D in vitro model.

Abstract CT161: A phase II basket trial of dual anti-CTLA-4 and anti-PD-1 blockade in rare tumors (DART) SWOG S1609: the non-epithelial ovarian tumor cohort

Abstract Background: Dual checkpoint inhibition with Anti-PD-1 and anti-CTLA4 checkpoint inhibitors have proven to be effective in several malignancies but their potential role in numerous rare solid cancers is yet to be established. This study presents the first results of ipilimumab and nivolumab in the non-epithelial ovarian tumor cohort (#13) of the SWOG S1609 Dual Anti-CTLA-4 & Anti-PD-1 blockade in Rare Tumors (DART) trial. Methods: DART is a prospective, open-label, multicenter/multi-cohort phase 2 clinical trial of ipilimumab (1mg/kg intravenously every 6 weeks) plus nivolumab (240mg intravenously every 2 weeks). This cohort included several histologies grouped for statistical analysis. The primary endpoint was objective response rate (ORR) (RECIST v1.1) (confirmed complete (CR) and partial responses (PR)); progression-free survival (PFS), overall survival (OS), stable disease (SD) >6 months, and toxicity are secondary endpoints. Results: Seventeen evaluable patients (median age, 64 years) were analyzed. The subtypes of non-epithelial ovarian cancer were: granulosa cell (47%, n=8), carcinosarcoma or malignant mixed Mullerian tumor (MMMT; 35%, n=6), one each for Wolffian duct, yolk sac, and Sertoli-Leydig cell. There were 2 responses in the 8 patients with granulosa cell (ORR of 25% in the granulosa cell tumors): 1CR (79% regression [due to lymph node < 1.0cm], 59 month PFS, juvenile type) and 1 PR (79% regression, 51+ month PFS, adult type). 6/8 patients remain alive (PFS 52-1774 days). In contrast, carcinosarcomas showed no responses. One patient with Sertoli-Leydig cell tumor had a 22% response and 341 day PFS. Overall ORR was 12% (2/17), clinical benefit rate (CBR; no progression > 6months) of 29.4%. The median PFS was 3.5 months, median OS was 42.5 months. The most common adverse events were fatigue (52.9%, n=9) and hypothyroidism (35.3%, n=6). Grade 3-4 adverse events occurred in 47.1% of patients (n=8). There were 3 adverse events (17.6%) that led to discontinuation, of which 2 (11.8%) were grade 3-4. There were no grade 5 adverse events. Conclusion: Ipilimumab plus nivolumab in non-epithelial ovarian cancer resulted in an ORR of 12% (with 2 of 8 patients with granulosa ovarian tumors showing a durable CR and PR [both, >4 years)) and CBR of 29.4%, with durable responses seen. In contrast there were no responses in the carcinosarcoma group. One patient with Sertoli Leydig cell tumor suggested a possible benefit. Correlative studies to determine response and resistance markers are ongoing. Expanded prospective studies in granulosa tumor but not carcinosarcoma are warranted. Citation Format: Young Kwang Chae, Megan Othus, Sandip Pravin Patel, Kelly J. Wilkinson, Emily M. Whitman-Purves, Jayanthi Lea, John M. Schallenkamp, Nabil Adra, Leonard J. Appleman, Mitchell Alden, Jessica Thomes Pepin, John A. Ellerton, Andrew Poklepovic, Adam Walter, Murtuza M. Rampurwala, William R. Robinson, Liam Il-Young Chung, Christine M. McLeod, Helen X. Chen, Elad Sharon, Howard Streicher, Christopher W. Ryan, Charles D. Blanke, Razelle Kurzrock. A phase II basket trial of dual anti-CTLA-4 and anti-PD-1 blockade in rare tumors (DART) SWOG S1609: the non-epithelial ovarian tumor cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT161.

Long-term retroperitoneal lymph node dissection outcomes for nonseminomatous germ cell tumors with postchemotherapy retroperitoneal residual tumors, in a specialized hospital of Saudi Arabia

Abstract Background: In men under the age of 35 years, testicular cancer is the most prevalent solid tumor. Additional treatment is necessary for many people with nonseminomatus germ cell tumors (NSGCTs). Whether in conjunction with chemotherapy or as a stand-alone procedure, retroperitoneal lymph node dissection (RPLND) continues to play a critical role in the treatment of these patients. There is a lack of information in Saudi Arabian literature about the long-term oncologic outcome of NSGCT following RPLND surgery. Objective: The study’s goals were to identify clinical and pathological characteristics related with long-term survival following NSGCT for RPLND, as well as to assess the surgical and oncological results of this treatment. Methodology: From January 2010 to April 2021, nine adults who had had orchidectomy for testicular cancer and RPLND following treatment for the evidence of persistent NSGCT disease at our hospital were included in this retrospective research. Demographic information, laboratory results, radiological findings, staging, chemotherapy and radiotherapy status, surgical procedure details, perioperative problems, morbidity, and mortality were all acquired from patients’ medical records. Rates of disease return and overall survival. SPSS was utilized for the data analysis. Results: During the study period, nine patients with NSGCT underwent RPLND, with a mean age of 28 years (5.4: standard deviation). The most common diseases were yolk sac and embryonal cell cancer. In addition to receiving neoadjuvant chemotherapy, all patients had a Clavien–Dindo score of 2, and there was no mortality after RPLND. Overall median survival time was 26, and disease-free survival time was 21 months. Overall survival at 1 year was 86%, and at 5 years, it was 66%. The prognosis of embryonal cell cancer was more optimistic than that of a tumor of the yolk sac. Conclusion: We have shown that RPLND has an extremely low rate of morbidity and postoperative problems. In terms of oncology, it results in adequate overall and disease-free survival rates, with low morbidity and postoperative complications.

Microglia Regulating Neuronal Apoptosis Via Alpha7 Nicotinic Acetylcholine Receptor in HIV-Associated Neurocognitive Disorders

Background: Microglia, the innate immune cells of the central nervous system (CNS), originate from erythromyeloid progenitor cells in the embryonic yolk sac. As the innate immune cells of the CNS, microglia play an important role in innate immunity, neurogenesis, and brain tissue repair by activating the alpha7 nicotinic acetylcholine receptor (α7nAChR). They are the key target cells for HIV-1 infection in the CNS and have been shown to produce a chronic inflammatory response in the brain which underlies HIV-associated neurocognitive disorders (HAND). Apart from the microglial neuroinflammation, recent studies have reported that activated microglia could induce neuronal apoptosis through the release of neurotoxic factors (e.g., ROS and NLRP3 inflammasome) in HAND. In addition, α7nAChR was significantly up-regulated in the striatum which is the most serious part of neuron apoptosis in gp120 transgenic mice brains. However, the underlying mechanism of neuronal cell apoptosis by gp120-activated microglia remains unclear. Here, this study aimed to explore the mechanism by which HIV-1 gp120-activated microglia induce neuronal apoptosis. Materials and methods: The expression of α7nAChR was examined by immunohistochemistry in the brain of HIV-1 LAV gp120 transgenic (Tg) mice (C57BL/6J background, 12-month-old). Methyllycaconitine (MLA, specific α7nAChR agonists) and Nicotine (NT, specific α7nAChR antagonists) were used to evaluate the effect of α7nAChR on apoptosis induced by gp120. Immortalized microglial cells (BV2) were incubated for 24 hours with gp120, NT, and gp120+NT, respectively, with or without preincubation with MLA for 15 minutes to obtain their supernatants. Next, the neuronal cells (SH-SY5Y) were treated with the same as BV2 or with collected BV2 culture supernatants. The apoptosis effect of SH-SY5Y and the level of ROS in the BV2 culture supernatants were assessed via quantitative analysis of flow cytometry. Comparisons between supernatant groups and drug groups were conducted to evaluate the effects of the SH-SY5Y apoptosis induced by microglia. Western blotting of microglia cell lysates was employed for the analysis of cellular signaling pathways. Student's t-tests and ANOVA were applied to determine statistical significance. Results: The expression of α7nAChR in HIV-1 gp120 transgenic mice was significantly upregulated (P < 0.001). The effect of apoptosis was more significant in microglia culture supernatants groups compared to that in drug-treated groups (P < 0.001). Neuronal activity was not changed in the NT group or MLA group than in the control, but HIV-1 gp120 could increase the number of apoptotic neurons (P < 0.01). Significant neuronal apoptosis was observed when treated with BV2 supernatants from the group treated by combining the gp120 with NT (P < 0.001), and this effect was in turn inhibited by MLA (P < 0.001). These results suggested that gp120-activated microglia might induce neuronal apoptosis invitro owing to gp120 upregulating α7nAChR. In addition, the ROS level in microglia was significantly upregulated following gp120 exposure (P < 0.001), and this effect could be enhanced by NT (P < 0.001). Notably, there was a positive correlation between the levels of ROS in microglia culture supernatants and the levels of neuronal apoptosis treated in the same supernatants. This positive relevance indicated that microglia might induce neuronal apoptosis by producing excessive ROS. The expressions of p53, OMI, and caspase-3 significantly upregulated following gp120 exposure with the down-regulate expression of XIAP (P < 0.001). And this effect could be enhanced by NT (P < 0.001). The results of western blotting indicated that gp120 and NT can up-regulate the ROS to activate p53 by α7nAChR, which could lead to an increased OMI and decreased XIAP levels. (P < 0.001). Conclusions: Microglia may play a critical role in regulating neuronal apoptosis in HAND. HIV-1 gp120 and NT could increase the generation of ROS through α7nAChR in microglia, which leads to neuronal apoptosis. Moreover, our results showed that the combined action of NT and gp120 may upregulate p53 and Omi / HtrA2 protein expression, and the downregulation of XIAP protein may significantly activate the level of caspase-3. (Acknowledgements:Corresponding author: Hong Cao, gzhcao@smu.edu.cn; National Natural Science Foundation of China, No. 82172259 to H.C.) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Differential Etv2 threshold requirement for endothelial and erythropoietic development.

SUMMARYEndothelial and erythropoietic lineages arise from a common developmental progenitor. Etv2 is a master transcriptional regulator required for the development of both lineages. However, the mechanisms through which Etv2 initiates the gene-regulatory networks (GRNs) for endothelial and erythropoietic specification and how the two GRNs diverge downstream of Etv2 remain incompletely understood. Here, by analyzing a hypomorphic Etv2 mutant, we demonstrate different threshold requirements for initiation of the downstream GRNs for endothelial and erythropoietic development. We show that Etv2 functions directly in a coherent feedforward transcriptional network for vascular endothelial development, and a low level of Etv2 expression is sufficient to induce and sustain the endothelial GRN. In contrast, Etv2 induces the erythropoietic GRN indirectly via activation of Tal1, which requires a significantly higher threshold of Etv2 to initiate and sustain erythropoietic development. These results provide important mechanistic insight into the divergence of the endothelial and erythropoietic lineages.

Nutrient Transporter Gene Expression in the Early Conceptus-Implications From Two Mouse Models of Diabetic Pregnancy.

Maternal diabetes in early pregnancy increases the risk for birth defects in the offspring, particularly heart, and neural tube defects. While elevated glucose levels are characteristic for diabetic pregnancies, these are also accompanied by hyperlipidemia, indicating altered nutrient availability. We therefore investigated whether changes in the expression of nutrient transporters at the conception site or in the early post-implantation embryo could account for increased birth defect incidence at later developmental stages. Focusing on glucose and fatty acid transporters, we measured their expression by RT-PCR in the spontaneously diabetic non-obese mouse strain NOD, and in pregnant FVB/N mouse strain dams with Streptozotocin-induced diabetes. Sites of expression in the deciduum, extra-embryonic, and embryonic tissues were determined by RNAscope in situ hybridization. While maternal diabetes had no apparent effects on levels or cellular profiles of expression, we detected striking cell-type specificity of particular nutrient transporters. For examples, Slc2a2/Glut2 expression was restricted to the endodermal cells of the visceral yolk sac, while Slc2a1/Glut1 expression was limited to the mesodermal compartment; Slc27a4/Fatp4 and Slc27a3/Fatp3 also exhibited reciprocally exclusive expression in the endodermal and mesodermal compartments of the yolk sac, respectively. These findings not only highlight the significance of nutrient transporters in the intrauterine environment, but also raise important implications for the etiology of birth defects in diabetic pregnancies, and for strategies aimed at reducing birth defects risk by nutrient supplementation.

DOT1L Mediated Gene Repression in Extensively Self-Renewing Erythroblasts.

DOT1L is essential for embryonic hematopoiesis but the precise mechanisms of its action remain unclear. The only recognized function of DOT1L is histone H3 lysine 79 (H3K79) methylation, which has been implicated in both transcriptional activation and repression. We observed that deletion of the mouse Dot1L gene (Dot1L-KO) or selective mutation of its methyltransferase domain (Dot1L-MM) can differentially affect early embryonic erythropoiesis. However, both mutations result in embryonic lethality by mid-gestation and growth of hematopoietic progenitor cells (HPCs) is similarly affected in extensively self-renewing erythroblast (ESRE) cultures established from yolk sac cells. To understand DOT1L-mediated gene regulation and to clarify the role of H3K79 methylation, we analyzed whole transcriptomes of wildtype and Dot1L-mutant ESRE cells. We observed that more than 80% of the differentially expressed genes (DEGs) were upregulated in the mutant ESRE cells either lacking the DOT1L protein or the DOT1L methyltransferase activity. However, approximately 45% of the DEGs were unique to either mutant group, indicating that DOT1L possesses both methyltransferase-dependent and -independent gene regulatory functions. Analyses of Gene Ontology and signaling pathways for the DEGs were consistent, with DEGs that were found to be common or unique to either mutant group. Genes related to proliferation of HPCs were primarily impacted in Dot1L-KO cells, while genes related to HPC development were affected in the Dot1L-MM cells. A subset of genes related to differentiation of HPCs were affected in both mutant groups of ESREs. Our findings suggest that DOT1L primarily acts to repress gene expression in HPCs, and this function can be independent of its methyltransferase activity.

Primitive Erythropoiesis in the Mouse is Independent of DOT1L Methyltransferase Activity.

DOT1-like (DOT1L) histone methyltransferase is essential for mammalian erythropoiesis. Loss of DOT1L in knockout (Dot1l-KO) mouse embryos resulted in lethal anemia at midgestational age. The only recognized molecular function of DOT1L is its methylation of histone H3 lysine 79 (H3K79). We generated a Dot1l methyltransferase mutant (Dot1l-MM) mouse model to determine the role of DOT1L methyltransferase activity in early embryonic hematopoiesis. Dot1l-MM embryos failed to survive beyond embryonic day 13.5 (E13.5), similarly to Dot1l-KO mice. However, when examined at E10.5, Dot1l-MM embryos did not exhibit overt anemia like the Dot1l-KO. Vascularity and the presence of red blood cells in the Dot1l-MM yolk sacs as well as in the AGM region of Dot1l-MM embryos appeared to be similar to that of wildtype. In ex vivo cultures of yolk sac cells, Dot1l-MM primitive erythroblasts formed colonies comparable to those of the wildtype. Although ex vivo cultures of Dot1l-MM definitive erythroblasts formed relatively smaller colonies, inhibition of DOT1L methyltransferase activity in vivo by administration of EPZ-5676 minimally affected the erythropoiesis. Our results indicate that early embryonic erythropoiesis in mammals requires a DOT1L function that is independent of its intrinsic methyltransferase activity.

3107 – MULTI-ORGAN FUNCTIONS OF YOLK SAC DURING HUMAN EARLY DEVELOPMENT

Background Model organisms have provided valuable insights into the functions of yolk sac (YS), a conserved extraembryonic structure which provides haematopoietic and metabolic support, but species-specific differences demand a systematic examination of human YS. Methods We profiled n=6 human YS and matched liver samples (CS14-CS23) by droplet-based scRNAseq and leveraged additional data sets on YS at gastrulation (CS7) and CS10-11, aorta gonad mesonephros (AGM) at CS14-17, fetal liver and bone marrow (BM). We incorporated data from a time course of induced pluripotent stem cells (iPSCs) undergoing macrophage differentiation. Results The proportional representation of YS cell states varied over gestational age. Progenitors and megakaryocytes were over-represented early, while dendritic cells and microglia-like cells emerged later. B-lineage cells (including B1 cells) were exclusive to matched liver, while NFIL3, ACY3-expressing innate lymphoid progenitors and PTGS2, MSL1-expressing pre-macrophages were exclusive to YS. CS7-11 YS progenitors expressed SPINK2 and HLF in common with later YS (CS14-23), AGM, liver and BM samples. Liver and BM HSPCs expressed HOXA7, PROM1 and HLA-DRA. Monocytes emerged in YS only after CS14. In force-directed graph embedding, there were two routes to macrophages; a monocyte-independent route prior to CS14 and a monocyte-dependent trajectory after CS14. These pathways were recapitulated during macrophage differentiation from iPSCs. YS endoderm and matched liver shared functions in lipid and glucose metabolism and coagulation. YS endoderm cells also expressed EPO and THPO, critical for erythropoiesis and megakaryopoiesis, and F3-activated coagulation pathway components. Conclusions These findings support the concept of the human YS providing multiorgan functions, which are subsequently subsumed by the liver, kidney and BM. Model organisms have provided valuable insights into the functions of yolk sac (YS), a conserved extraembryonic structure which provides haematopoietic and metabolic support, but species-specific differences demand a systematic examination of human YS. We profiled n=6 human YS and matched liver samples (CS14-CS23) by droplet-based scRNAseq and leveraged additional data sets on YS at gastrulation (CS7) and CS10-11, aorta gonad mesonephros (AGM) at CS14-17, fetal liver and bone marrow (BM). We incorporated data from a time course of induced pluripotent stem cells (iPSCs) undergoing macrophage differentiation. The proportional representation of YS cell states varied over gestational age. Progenitors and megakaryocytes were over-represented early, while dendritic cells and microglia-like cells emerged later. B-lineage cells (including B1 cells) were exclusive to matched liver, while NFIL3, ACY3-expressing innate lymphoid progenitors and PTGS2, MSL1-expressing pre-macrophages were exclusive to YS. CS7-11 YS progenitors expressed SPINK2 and HLF in common with later YS (CS14-23), AGM, liver and BM samples. Liver and BM HSPCs expressed HOXA7, PROM1 and HLA-DRA. Monocytes emerged in YS only after CS14. In force-directed graph embedding, there were two routes to macrophages; a monocyte-independent route prior to CS14 and a monocyte-dependent trajectory after CS14. These pathways were recapitulated during macrophage differentiation from iPSCs. YS endoderm and matched liver shared functions in lipid and glucose metabolism and coagulation. YS endoderm cells also expressed EPO and THPO, critical for erythropoiesis and megakaryopoiesis, and F3-activated coagulation pathway components. These findings support the concept of the human YS providing multiorgan functions, which are subsequently subsumed by the liver, kidney and BM.

The Earliest T-Precursors in the Mouse Embryo Are Susceptible to Leukemic Transformation.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in pediatric patients. About 10–15% of pediatric ALL belong to T-cell ALL (T-ALL), which is characterized by aggressive expansion of immature T-lymphoblasts and is categorized as high-risk leukemia. Leukemia initiating cells represent a reservoir that is responsible for the initiation and propagation of leukemia. Its perinatal origin has been suggested in some childhood acute B-lymphoblastic and myeloblastic leukemias. Therefore, we hypothesized that child T-ALL initiating cells also exist during the perinatal period. In this study, T-ALL potential of the hematopoietic precursors was found in the para-aortic splanchnopleura (P-Sp) region, but not in the extraembryonic yolk sac (YS) of the mouse embryo at embryonic day 9.5. We overexpressed the Notch intracellular domain (NICD) in the P-Sp and YS cells and transplanted them into lethally irradiated mice. NICD-overexpressing P-Sp cells rapidly developed T-ALL while YS cells failed to display leukemia propagation despite successful NICD induction. These results suggest a possible role of fetal-derived T-cell precursors as leukemia-initiating cells.

Risk assessment of genetically modified zoysiagrasses (Zoysia japonica) in a zebrafish model

Genetically modified (GM) plants have been mainly advanced for the mass production of agricultural plants. However, there are concerns about GM crops which cause side effects on ecosystems and human beings. Zebrafish is a better in vivo model and possess the advantages; small size, large clutches, transparency, low cost, and physiological similarity to mammals. The objective of this study was to assess the safety of non-GM and GM grasses (Zoysia japonica) including JG21, JG21-MS1, JG21-MS2 in zebrafish embryos by examining the survival rates, heart-beating rate, yolk sac edema size, cell death, and morphology. The survival rates, heart-beating rates, and yolk sac edema sizes in non-GM and GM grass-treated zebrafish were similar change with the control group. And, all the groups did not show a significant difference within less than 1.0-fold in cell death by fluorescence levels. These results indicate that the zebrafish model is a useful and simple tool and can be used for risk assessment of GM plants.

Surveillance Only for High-risk FIGO Stage IA/IB Malignant Ovarian Germ Cell Tumors: Results From a National Cancer Database.

Investigate the use and outcomes of a surveillance only strategy for patients with high-risk stage I malignant ovarian germ cell tumors. Patients with International Federation of Gynecology and Obstetrics stage IA/IB grade 2 or 3 immature teratoma, yolk sac, or mixed germ cell tumor diagnosed between 2004 and 2014 who had at least 1 month of follow-up were drawn from the National Cancer Database. Overall survival (OS) was evaluated for each histologic subtype using Kaplan-Meier curves, and compared with the log-rank test. A total of 497 patients were identified; 115 (23.1%) with grade 2 immature teratoma, 157 (31.6%) with grade 3 immature teratoma, 101 (20.3%) with yolk sac tumor, 124 (25%) with mixed germ cell tumor. Rate of adjuvant chemotherapy was 68.2% (655 patients), while rate of lymph node biopsy/dissection was 55.2%. A total of 19 (3.8%) deaths were observed at a median of 29.8 months. There was no difference in OS between patients who did and did not receive adjuvant chemotherapy with grade 2 (P=0.35) and grade 3 immature teratoma (P=0.47) or mixed germ cell tumors (P=0.55). Patients with yolk sac tumors those who received chemotherapy had better OS compared with those who did not, P=0.019; 5-year OS rates were 92.7% and 79.6%, respectively. A surveillance only strategy for patients with stage I malignant ovarian germ cell tumors is associated with excellent survival outcomes for patients with grade 2 or 3 immature teratoma or mixed germ cell tumors.

Contributions of Embryonic HSC-Independent Hematopoiesis to Organogenesis and the Adult Hematopoietic System

During ontogeny, the establishment of the hematopoietic system takes place in several phases, separated both in time and location. The process is initiated extra-embryonically in the yolk sac (YS) and concludes in the main arteries of the embryo with the formation of hematopoietic stem cells (HSC). Initially, it was thought that HSC-independent hematopoietic YS cells were transient, and only required to bridge the gap to HSC activity. However, in recent years it has become clear that these cells also contribute to embryonic organogenesis, including the emergence of HSCs. Furthermore, some of these early HSC-independent YS cells persist into adulthood as distinct hematopoietic populations. These previously unrecognized abilities of embryonic HSC-independent hematopoietic cells constitute a new field of interest. Here, we aim to provide a succinct overview of the current knowledge regarding the contribution of YS-derived hematopoietic cells to the development of the embryo and the adult hematopoietic system.

3074 – SINGLE CELL TRANSCRIPTOMIC ANALYSIS COMBINED WITH MACHINE LEARNING IDENTIFIES HSC-LIKE CELLS DIFFERENTIATED IN VITRO FROM HUMAN IPSCS.

During embryonic development, haematopoietic stem and progenitor cells (HSPCs) develop in different waves at various anatomical sites. This process can be recapitulated in vitro by cytokine-mediated differentiation of human pluripotent stem cells (hPSCs) although the differentiation of haematopoietic stem cells (HSCs) remains a major challenge in the field. To depict the developmental dynamics and to study the heterogeneity of HSPCs that can be generated in vitro from hPSCs, we employed single cell RNA sequencing (scRNAseq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq). We identified the transcriptome and functionally validated naïve progenitors as well as erythroid, megakaryocyte and leukocyte-committed progenitors and we associated to these the expression of CD44, CD326, ICAM2/CD9 and CD18, respectively. To identify and compare the in vitro-generated cell types to those developed in vivo, we employed a machine learning approach. We trained an artificial neural network (ANN) using a human fetal liver scRNAseq dataset of hematopoietic cells from HSCs to mature cell types. We identified a wide range of hPSCs-derived phenotypes, including a small group classified as HSC/MPP. This transient HSC-like population decreased with the progression of the differentiation protocol and was not identified by the ANN in the datasets that we generated from sorted CD43+ cells. By comparing the transcriptome of these in vitro-generated HSC/MPP-like cells with those within the fetal liver we identified transcription factors and molecular pathways that can be exploited to improve the in vitro production of HSCs in the future. Finally, when these HSC-like cells were compared to yolk sac and AGM cells we observed that in vitro derived progenitors displayed signatures of high oxidative respiration, suggesting an overall divergent metabolic profile.

Megalin: A Novel Endocytic Receptor for Prorenin and Renin.

Megalin is an endocytic receptor contributing to protein reabsorption. Impaired expression or trafficking of megalin increases urinary renin and allowed the detection of prorenin, which normally is absent in urine. Here, we investigated (pro)renin uptake by megalin, using both conditionally immortalized proximal tubule epithelial cells and Brown Norway Rat yolk sac cells (BN16). To distinguish binding and internalization, cells were incubated with recombinant human (pro)renin at 4°C and 37°C, respectively. (Pro)renin levels were assessed by immunoradiometric assay. At 4°C, BN16 cells bound 3× more prorenin than renin, suggestive for a higher affinity of prorenin. Similarly, at 37°C, prorenin accumulated at 3- to 4-fold higher levels than renin in BN16 cells. Consequently, depletion of medium prorenin (but not renin) content occurred after 24 hours. No such differences were observed in conditionally immortalized proximal tubule epithelial cells, and M6P (mannose-6-phosphate) greatly reduced conditionally immortalized proximal tubule epithelial cells (pro)renin uptake, suggesting that these cells accumulate (pro)renin largely via M6P receptors. M6P did not affect (pro)renin uptake in BN16 cells. Yet, inhibiting megalin expression with siRNA greatly reduced (pro)renin binding and internalization by BN16 cells. Furthermore, treating BN16 cells with albumin, an endogenous ligand of megalin, also decreased binding and internalization of (pro)renin, while deleting the (pro)renin receptor affected the latter only. Exposing prorenin's prosegment with the renin inhibitor aliskiren dramatically increased prorenin binding, while after prosegment cleavage with trypsin prorenin binding was identical to that of renin. In conclusion, megalin might function as an endocytic receptor for (pro)renin and displays a preference for prorenin. Megalin-mediated endocytosis requires the (pro)renin receptor.

Decoding human fetal liver haematopoiesis.

SummaryDefinitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells/multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Using single cell transcriptome profiling of ~140,000 liver and ~74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the impact of tissue microenvironment on blood and immune cell development. We reveal physiological erythropoiesis in fetal skin and the presence of mast cells, NK and ILC precursors in the yolk sac. We demonstrate a shift in fetal liver haematopoietic composition during gestation away from being erythroid-predominant, accompanied by a parallel change in HSC/MPP differentiation potential, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a valuable reference for harnessing the therapeutic potential of HSC/MPPs.