Biology subjects

Meads, M.

Publications and source records attributed to Meads, M..

5 recordsLinked to original sources

bioRxiv2026

Creatine kinase regulates energy metabolism and growth of trophoblasts

Study questionDoes the human placenta utilize the creatine phosphagen system for energy homeostasis during development? Summary answerComponents of the creatine (Cr)-creatine kinase (CK)-phosphocreatine (PCr) system are dynamically expressed by the trophoblast and mesenchymal compartments throughout gestation wherein creatine kinase is required for cellular ATP metabolism, cell cycle, and proliferation of trophoblast cells. What is known alreadyThe Cr-CK-PCr system maintains ATP homeostasis in tissues with high energy demand and is required for proliferation, migration, and invasion of tumor cells. The term human placenta can synthesize and transport creatine locally. Early placental development involves trophoblast proliferation, an event requiring ATP, but the role of the creatine phosphagen system during early placental development remains unknown. Study design, size, durationWe performed immunohistochemistry (IHC) and immunofluorescence (IF) for different components (biosynthesis, transport, utilization) of the Cr-Ck-PCr system in human placentae (n=3/group) across gestation including first trimester, second trimester, and term. Using primary human trophoblast stem cells (hTSCs) and trophoblast organoids (TO), we determined the role of the creatine phosphagen system in trophoblast growth by functional inhibition of creatine kinase. Participants/materials, setting, methodsIHC/IF were performed in human placentae across gestation for proteins involved in biosynthesis (AGAT and GAMT), transport (SLC6A8, SLC22A15, and SLC6A13) and utilization (CKB and CKMT1) of creatine to determine the presence of the creatine phosphagen system locally in the placenta. For delineating the functional importance of this system in placental development, cyclocreatine (cCr), a creatine analogue, was used for functional inhibition of CK. Primary hTSCs were culture in medium containing 0 (control), 1, 10, 20 mM cCr for 48 hours followed by analysis of cell growth (cell count), cell cycle (EdU incorporation assay), apoptosis (Annexin V/PI flow cytometry), energy metabolism (Sea horse mito-stress and glycolytic stress tests), and gene expression (qPCR). Primary TO were also treated with 20mM cCr for 6 days in vitro to determine the role of Cr-CK-PCr system in placental development. Main results and the role of chanceAGAT localized to the fetal villous mesenchyme, while GAMT was broadly expressed in the trophoblast and fetal mesenchyme compartments across gestation. CKB localized primarily to fetal mesenchyme with strongest expression at term. CKMT1 was broadly expressed in all trophoblast subtypes. SLC6A8 was abundant in early syncytiotrophoblast but absent at term, where its expression shifted to fetal blood vessels. SLC22A15 was expressed in the endothelial cells of fetal capillaries across gestation. In primary hTSCs, cyclocreatine (20mM) treatment reduced proliferation (P<0.001), decreased expression of trophoblast epithelial marker EGFR (P<0.05), induced G0/G1 and G2/M arrests (P<0.0001), enhanced early and late apoptosis (P<0.0001), and downregulated GPX8 expression (P<0.05). Seahorse analysis revealed marked reductions (P<0.01) in mitochondrial (basal, maximal, and ATP-linked) and glycolytic (rate, capacity, and reserve) function compared to controls. In primary human TO, cyclocreatine treatment reduced the growth of organoids (P<0.05) as well the expression of EGFR (P<0.05). Large scale dataN/A Limitations, reasons for cautionFurther experiments assessing apoptosis, cellular stress and redox imbalance may provide more mechanistic role of the creatine phosphagen system in trophoblast metabolism and function. Since the functional role of the Cr-CK-PCr system was investigated in vitro, findings of this study should be taken with caution for implications of in vivo placental development. Nevertheless, reproducible results of reduced growth of trophoblast cells using both 2D and 3D cultures is highly suggestive of the importance of the creatine phosphagen system in early placental development. Wider implications of the findingsThis study provides foundational knowledge that the placenta contains the creatine phosphagen system, known for ATP homeostasis, and that this system ensures proper cell division, survival and placental development. Dysregulation of components of Cr-CK-PCr system in placenta has been observed in pregnancy disorders such as preeclampsia and fetal growth restriction warranting continued investigation into mechanisms and potential remediation using creatine supplementation. Stem cells share similar metabolic features so findings of this study can be implicated in other stem cells models as well. Study funding/competing interest(s)This work was supported by CIRM EDUC4-12804 Interdisciplinary Stem Cell Training Grant and a Lalor Foundation Postdoctoral Fellowship awarded to NS, and by the California Institute for Regenerative Medicine (DISC0-13757) and the National Institute of Child Health and Human Development (R01-HD096260) award to FS. The authors have no competing interest to declare.

physiology↗

bioRxiv2025

Derivation of functional early gestation decidual natural killer cell subtypes from induced pluripotent stem cells

Abnormal decidual natural killer cell (dNK) function is linked to pregnancy complications occurring in both early and late gestation, including recurrent pregnancy loss, preeclampsia, and preterm birth. Exploration of dNK heterogeneity as it relates to function is an active area of research; however, most of this work has focused on early gestation. Using flow cytometric and transcriptomic single-cell definitions of dNK subtypes, we characterized dNK heterogeneity in term dNK within both chorioamniotic membranes and basal plate. We also applied aptamer-based secretome profiling to first trimester and term dNK, and found dNK-specific proteins - VEGF and PLGF - to be reduced at term. We further determined that, compared to first trimester dNK, term dNK have reduced cytoxicity against target cells. Finally, we applied this knowledge to establish a protocol for differentiation of induced pluripotent stem cells (iPSC) into functional dNK. We found that treatment with TGF{beta} enriched for dNK2 subtype, while inducing dNK markers, CD9 and CD103. We evaluated function using cytokine and degranulation assays, aptamer-based secretome profiling, and cytotoxicity assays. We found that iPSC-dNK are functionally most similar to primary dNK. Further, TGF{beta} iPSC-dNK had reduced GM-CSF in response to PMA/I and increased secretion of VEGF and other first trimester-specific proteins - supportive of a shift towards an early gestation, dNK2-dominant, phenotype. We conclude that changes in dNK function across gestation reflect shifts in dNK subtypes that can be reproducibly derived from iPSC, providing a new method for modeling dNK and laying the foundation for cell-based therapeutics for reproductive disease. Significance StatementAlterations in maternal decidual natural killer cells (dNK) are associated with pregnancy complications - from recurrent pregnancy loss to preeclampsia and preterm birth. We found dNK from different regions of the term placenta to be distinct from peripheral blood NK and early gestation dNK, based on gene and surface marker expression, subtype composition, secretome, and cytotoxicity. We report a novel, reproducible protocol to generate dNK resembling the most abundant dNK subtype in early gestation from induced pluripotent stem cells. Our study lays the foundation for in vitro modeling of the maternal-fetal interface and therapeutic development for reproductive disease.

cell biology↗

bioRxiv2025

Miniaturized Workflow for Transcriptomic Profiling of Urinary Extracellular RNA during Pregnancy

Urine contains extracellular RNA (exRNA) carried by extracellular vesicles (EVs) and other biomolecular complexes. There is currently a need for studies focused on female cohorts to develop new methods for non-invasive analysis of biofluids to create reference profiles and for identification of biomarkers of reproductive and pregnancy disorders. The objective of this study was therefore to identify optimal methods for transcriptomic profiling of urine by testing different exRNA isolation and scalable library preparation methods that enable detection of biomarkers that reflect pregnancy-associated changes in the placenta and maternal tissues. RNA was extracted from pooled and individual urine samples obtained from normal non-pregnant and pregnant females, as well as males, using input volumes of either 0.6 mL, 1 mL, or 4 mL. Samples were extracted using methods that focused either on isolating vesicular (EV-associated) or total (EV-associated and non EV-associated) exRNA. Small RNA libraries (n=208) were prepared using the NEBNext Small RNA Library Prep kit and long RNA libraries (n=97) were prepared using the SMART-Seq v4 Ultra Low Input RNA or the SMARTer Stranded Total RNA-Seq Kit v2 Pico Input kits (Takara). Principal component analysis showed that the greatest source of variance amongst technical replicates of small RNA libraries (n=176 which passed quality control) was exRNA isolation method, and amongst long RNA libraries (n=97 which passed quality control) was library preparation method. Long RNA libraries prepared from exRNA extracted using miRCURY showed that the SMART-Seq v4 method yielded significantly more uniquely mapped reads compared to the Pico v2 method (p<0.05). We have established a scalable pipeline for small and long RNA-Seq profiling of exRNA in urine in a reproducible manner, which we used to identify differentially expressed urinary exRNAs in pregnancy, and will enable transcriptomic profiling of urinary exRNA in disorders of pregnancy, including preeclampsia.

genetics↗

bioRxiv2024

Single-cell transcriptomics reveal differences between chorionic and basal plate cytotrophoblasts and trophoblast stem cells

Cytotrophoblast (CTB) of the early gestation human placenta are bipotent progenitor epithelial cells, which can differentiate into invasive extravillous trophoblast (EVT) and multinucleated syncytiotrophoblast (STB). Trophoblast stem cells (TSC), derived from early first trimester placentae, have also been shown to be bipotential. In this study, we set out to probe the transcriptional diversity of first trimester CTB and compare TSC to various subgroups of CTB. We performed single-cell RNA sequencing on six normal placentae, four from early (6-8 weeks) and two from late (12-14 weeks) first trimester, of which two of the early first trimester cases were separated into basal (maternal) and chorionic (fetal) fractions prior to sequencing. We also sequenced three TSC lines, derived from 6-8 week placentae, to evaluate similarities and differences between primary CTB and TSC. CTB clusters displayed notable distinctions based on gestational age, with early first trimester placentae showing enrichment for specific CTB subtypes, further influenced by origin from the basal or chorionic plate. Differential expression analysis of CTB from basal versus chorionic plate highlighted pathways associated with proliferation, unfolded protein response, and oxidative phosphorylation. We identified trophoblast states representing initial progenitor CTB, precursor STB, precursor and mature EVT, and multiple CTB subtypes. CTB progenitors were enriched in early first trimester placentae, with basal plate cells biased toward EVT, and chorionic plate cells toward STB, precursors. Clustering and trajectory inference analysis indicated that TSC were most like EVT precursor cells, with only a small percentage of TSC on the pre-STB differentiation trajectory. This was confirmed by flow cytometric analysis of 6 different TSC lines, which showed uniform expression of proximal column markers ITGA2 and ITGA5. Additionally, we found that ITGA5+ CTB could be plated in 2D, forming only EVT upon spontaneous differentiation, but failed to form self-renewing organoids; conversely, ITGA5-CTB could not be plated in 2D, but readily formed organoids. Our findings suggest that distinct CTB states exist in different regions of the placenta as early as six weeks gestation and that current TSC lines most closely resemble ITGA5+ CTB, biased toward the EVT lineage.

developmental biology↗

bioRxiv2024

TLCellClassifier: Machine Learning Based Cell Classification for Bright-Field Time-Lapse Images

Immunotherapies have shown promising results in treating patients with hematological malignancies like multiple myeloma, which is an incurable but treatable bone marrow-resident plasma cell cancer. Choosing the most efficacious treatment for a patient remains a challenge in such cancers. However, pre-clinical assays involving patient-derived tumor cells co-cultured in an ex vivo reconstruction of immune-tumor micro-environment have gained considerable notoriety over the past decade. Such assays can characterize a patients response to several therapeutic agents including immunotherapies in a high-throughput manner, where bright-field images of tumor (target) cells interacting with effector cells (T cells, Natural Killer (NK) cells, and macrophages) are captured once every 30 minutes for upto six days. Cell detection, tracking, and classification of thousands of cells of two or more types in each frame is bound to test the limits of some of the most advanced computer vision tools developed to date and requires a specialized approach. We propose TLCellClassifier (time-lapse cell classifier) for live cell detection, cell tracking, and cell type classification, with enhanced accuracy and efficiency obtained by integrating convolutional neural networks (CNN), metric learning, and long short-term memory (LSTM) networks, respectively. State-of-the-art computer vision software like KTH-SE and YOLOv8 are compared with TLCellClassifier, which shows improved accuracy in detection (CNN) and tracking (metric learning). A two-stage LSTM-based cell type classification method is implemented to distinguish between multiple myeloma (tumor/target) cells and macrophages/monocytes (immune/effector cells). Validation of cell type classification was done both using synthetic datasets and ex vivo experiments involving patient-derived tumor/immune cells. Availability and implementationhttps://github.com/QibingJiang/cell classification ml

cancer biology↗