Trophoblast stem cells, trophoblast invasion, and organoids - advancements in gynecology.

The human placenta serves as a vital barrier between the mother and the developing fetus during pregnancy. A defect in the early development of the placenta is associated with severe pregnancy disorders. Despite its complex development, various molecular processes control placental development, and the specialization of trophoblast cells is still not fully understood. One primary obstacle is the lack of suitable cell model systems. Traditional two-dimensional (2D) cell cultures fail to mimic in vivo conditions and do not capture the intricate intercellular interactions vital for studying placental development. However, three-dimensional (3D) organoid models derived from stem cells that replicate natural cell organization and architecture have greatly improved our understanding of trophoblast behavior and its medicinal applications. Organoids with relevant phenotypes provide a valuable platform to model both placental physiology and pathology, including the modeling of placental disorders. They hold great promise for personalized medicine, improved diagnostics, and the evaluation of pharmaceutical drug efficacy and safety. This article provides a concise overview of trophoblast stem cells, trophoblast invasion, and the evolving role of organoids in gynecology.

Directed biomechanical compressive forces enhance fusion efficiency in model placental trophoblast cultures.

The syncytiotrophoblast is a multinucleated structure that arises from fusion of mononucleated cytotrophoblasts, to sheath the placental villi and regulate transport across the maternal-fetal interface. Here, we ask whether the dynamic mechanical forces that must arise during villous development might influence fusion, and explore this question using in vitro choriocarcinoma trophoblast models. We demonstrate that mechanical stress patterns arise around sites of localized fusion in cell monolayers, in patterns that match computational predictions of villous morphogenesis. We then externally apply these mechanical stress patterns to cell monolayers and demonstrate that equibiaxial compressive stresses (but not uniaxial or equibiaxial tensile stresses) enhance expression of the syndecan-1 and loss of E-cadherin as markers of fusion. These findings suggest that the mechanical stresses that contribute towards sculpting the placental villi may also impact fusion in the developing tissue. We then extend this concept towards 3D cultures and demonstrate that fusion can be enhanced by applying low isometric compressive stresses to spheroid models, even in the absence of an inducing agent. These results indicate that mechanical stimulation is a potent activator of cellular fusion, suggesting novel avenues to improve experimental reproductive modelling, placental tissue engineering, and understanding disorders of pregnancy development.

In vitro proliferation and differentiation of mouse spermatogonial stem cells in decellularized human placenta matrix.

Utilizing natural scaffold production derived from extracellular matrix components presents a promising strategy for advancing in vitro spermatogenesis. In this study, we employed decellularized human placental tissue as a scaffold, upon which neonatal mouse spermatogonial cells (SCs) were cultured three-dimensional (3D) configuration. To assess cellular proliferation, we examined the expression of key markers (Id4 and Gfrα1) at both 1 and 14 days into the culture. Our quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed a notable increase in Gfrα1 gene expression, with the 3D culture group exhibiting the highest levels. Furthermore, the relative frequency of Gfrα1-positive cells significantly rose from 38.1% in isolated SCs to 46.13% and 76.93% in the two-dimensional (2D) and 3D culture systems, respectively. Moving forward to days 14 and 35 of the culture period, we evaluated the expression of differentiating markers (Sycp3, acrosin, and Protamine 1). Sycp3 and Prm1 gene expression levels were upregulated in both 2D and 3D cultures, with the 3D group displaying the highest expression. Additionally, acrosin gene expression increased notably within the 3D culture. Notably, at the 35-day mark, the percentage of Prm1-positive cells in the 3D group (36.4%) significantly surpassed that in the 2D group (10.96%). This study suggests that the utilization of placental scaffolds holds significant promise as a bio-scaffold for enhancing mouse in vitro spermatogenesis.

NBR1-dependent autophagy activation protects against environmental cadmium-evoked placental trophoblast senescence.

Cadmium (Cd), a well-established developmental toxicant, accumulates in the placentae and disrupts its structure and function. Population study found adverse pregnancy outcomes caused by environmental Cd exposure associated with cell senescence. However, the role of autophagy activation in Cd-induced placental cell senescence and its reciprocal mechanisms are unknown. In this study, we employed animal experiments, cell culture, and case-control study to investigate the above mentioned. We have demonstrated that exposure to Cd during gestation induces placental senescence and activates autophagy. Pharmacological and genetic interventions further exacerbated placental senescence induced by Cd through the suppression of autophagy. Conversely, activation of autophagy ameliorated Cd-induced placental senescence. Knockdown of NBR1 exacerbated senescence in human placental trophoblast cells. Further investigations revealed that NBR1 facilitated the degradation of p21 via LC3B. Our case-control study has demonstrated a positive correlation between placental senescence and autophagy activation in all-cause fetal growth restriction (FGR). These findings offer a novel perspective for mitigating placental aging and placental-origin developmental diseases induced by environmental toxicants.

Cell type-dependent response to benzo(a)pyrene exposure of human placental cell lines under normoxic, hypoxic, and pro-inflammatory conditions.

Benzo(a)pyrene (BaP) can be detected in the human placenta. However, little is known about the effects of BaP exposure on different placental cells under various conditions. In this study, we aimed to investigate the effects of BaP on mitochondrial function, pyrin domain-containing protein 3 (NLRP3) inflammasome, and apoptosis in three human trophoblast cell lines under normoxia, hypoxia, and inflammatory conditions. JEG-3, BeWo, and HTR-8/SVneo cell lines were exposed to BaP under normoxia, hypoxia, or inflammatory conditions for 24 h. After treatment, we evaluated cell viability, apoptosis, aryl hydrocarbon receptor (AhR) protein and cytochrome P450 (CYP) gene expression, mitochondrial function, including mitochondrial DNA copy number (mtDNAcn), mitochondrial membrane potential (ΔΨm), intracellular adenosine triphosphate (iATP), and extracellular ATP (eATP), nitric oxide (NO), NLPR3 inflammasome proteins, and interleukin (IL)-1ß. We found that BaP upregulated the expression of AhR or CYP genes to varying degrees in all three cell lines. Exposure to BaP alone increased ΔΨm in all cell lines but decreased NO in BeWo and HTR-8/SVneo, iATP in HTR-8/SVneo, and cell viability in JEG-3, without affecting apoptosis. Under hypoxic conditions, BaP did not increase the expression of AhR and CYP genes in JEG-3 cells but increased CYP gene expression in two others. Pro-inflammatory conditions did not affect the response of the 3 cell lines to BaP with respect to the expression of CYP genes and changes in the mitochondrial function and NLRP3 inflammasome proteins. In addition, in HTR-8/SVneo cells, BaP increased IL-1ß secretion in the presence of hypoxia and poly(I:C). In conclusion, our results showed that BaP affected mitochondrial function in trophoblast cell lines by increasing ΔΨm. This increased ΔΨm may have rescued the trophoblast cells from activation of the NLRP3 inflammasome and apoptosis after BaP treatment. We also observed that different human trophoblast cell lines had cell type-dependent responses to BaP exposure under normoxia, hypoxia, or pro-inflammatory conditions.

Human Placenta-Derived Mesenchymal Stem Cells Improve Neurological Function in Rats with Intrauterine Hypoxic-Ischaemic Encephalopathy by Reducing Apoptosis and Inflammatory Reactions.

BACKGROUND: Hypoxic-ischaemic encephalopathy (HIE) is a major cause of neonatal disability and mortality. Although hypothermia therapy offers some neuroprotection, the recovery of neurological function is limited. Therefore, new synergistic therapies are necessary to improve the prognosis. Mesenchymal stem cell-based therapy is emerging as a promising treatment option for HIE. In this study, we studied the therapeutic efficacy of human placenta-derived mesenchymal stem cells (PD-MSCs) in the HIE rat model and analyzed the underlying therapeutic mechanisms. METHODS: Rats were divided into 6 groups (n = 9 for each) as follows: control, HIE model, HIE + normal saline, and HIE + PD-MSC transplantation at days 7, 14 and 28 postpartum. Following PD-MSC transplantation, neurological behavior was evaluated using rotarod tests, traction tests, and the Morris water maze test. The degree of brain tissue damage was assessed by histological examination and Nissl staining. Expression levels of apoptosis-related proteins and inflammatory factors were quantified by Western blotting and enzyme-linked immunosorbent assays. Immunofluorescence was used to investigate the ability of PD-MSCs to repair the morphology and function of hippocampal neurons with hypoxic-ischaemic (HI) injury. RESULTS: PD-MSC transplantation enhanced motor coordination and muscle strength in HIE rats. This treatment also improved spatial memory ability by repairing pathological damage and preventing the loss of neurons in the cerebral cortex. The most effective treatment was observed in the HIE + PD-MSC transplantation at day 7 group. Expression levels of microtubule-associated protein-2 (MAP-2), B-cell lymphoma-2 (BCL-2), interleukin (IL)-10, and transforming growth factor (TGF -ß1) were significantly higher in the HIE + PD-MSC treatment groups compared to the HIE group, whereas the levels of BCL-2-associated X protein (BAX), BCL-2-associated agonist of cell death (BAD), IL-1ß and tumour necrosis factor α (TNF-α) were significantly lower. CONCLUSIONS: We demonstrated that intravenous injection of PD-MSC at 7, 14 and 28 days after intrauterine HI damage in a rat model could improve learning, memory, and motor function, possibly by inhibiting apoptosis and inflammatory damage. These findings indicate that autologous PD-MSC therapy could have potential application for the treatment of HIE.

Utah flouts FDA with new placental stem cell law.

Measure declares that patients can be given unapproved treatments.

PL-hMSC and CH-hMSC derived soluble factors inhibit proliferation but improve hGBM cell migration by activating TGF-ß and inhibiting Wnt signaling.

Glioblastoma multiforme (GBM) is one of the most common and aggressive brain tumors. GBM resists most chemotherapeutic agents, resulting in a high mortality rate in patients. Human mesenchymal stem cells (hMSCs), which are parts of the cancer stroma, have been shown to be involved in the development and progression of GBM. However, different sources of hMSCs might affect GBM cells differently. In the present study, we established hMSCs from placenta (PL-hMSC) and chorion (CH-hMSC) to study the effects of their released soluble factors on the proliferation, migration, invasion, gene expression, and survival of human GBM cells, U251. We found that the soluble factors derived from CH-hMSCs and PL-hMSCs suppressed the proliferation of U251 cells in a dose-dependent manner. In contrast, soluble factors derived from both hMSC sources increased U251 migration without affecting their invasive property. The soluble factors derived from these hMSCs decreased the expression levels of CyclinD1, E2Fs and MYC genes that promote GBM cell proliferation but increased the expression level of TWIST gene, which promotes EMT and GBM cell migration. The functional study suggests that both hMSCs might exert their effects, at least in part, by activating TGF-ß and suppressing Wnt/ß-catenin signaling in U251 cells. Our study provides a better understanding of the interaction between GBM cells and gestational tissue-derived hMSCs. This knowledge might be used to develop safer and more effective stem cell therapy that improves the survival and quality of life of patients with GBM by manipulating the interaction between hMSCs and GBM cells.

Human Placenta Decellularized Extracellular Matrix Hydrogel Promotes the Generation of Human Spinal Cord Organoids with Dorsoventral Organization from Human Induced Pluripotent Stem Cells.

Spinal cord organoids are of significant value in the research of spinal cord-related diseases by simulating disease states, thereby facilitating the development of novel therapies. However, the complexity of spinal cord structure and physiological functions, along with the lack of human-derived inducing components, presents challenges in the in vitro construction of human spinal cord organoids. Here, we introduce a novel human decellularized placenta-derived extracellular matrix hydrogel (DPECMH) and, combined with a new induction protocol, successfully construct human spinal cord organoids. The human placenta-sourced decellularized extracellular matrix (dECM), verified through hematoxylin and eosin staining, DNA quantification, and immunofluorescence staining, retained essential ECM components such as elastin, fibronectin, type I collagen, laminin, and so forth. The temperature-sensitive hydrogel made from human placenta dECM demonstrated good biocompatibility and promoted the differentiation of human induced pluripotent stem cell (hiPSCs)-derived spinal cord organoids into neurons. It displayed enhanced expression of laminar markers in comparison to Matrigel and showed higher expression of laminar markers compared to Matrigel, accelerating the maturation process of spinal cord organoids and demonstrating its potential as an organoid culture substrate. DPECMH has the potential to replace Matrigel as the standard additive for human spinal cord organoids, thus advancing the development of spinal cord organoid culture protocols and their application in the in vitro modeling of spinal cord-related diseases.

Pan-microscopic examination of monkeypox virus in trophoblasts cells reveals new insights into virions release through filopodia-like projections.

Vertical transmission has been described following monkeypox virus (MPXV) infection in pregnant women. The presence of MPXV has been reported in the placenta from infected women, but whether pathogens colonize placenta remains unexplored. We identify trophoblasts as a target cell for MPXV replication. In a pan-microscopy approach, we decipher the specific infectious cycle of MPXV and inner cellular structures in trophoblasts. We identified the formation of a specialized region for viral morphogenesis and replication in placental cells. We also reported infection-induced cellular remodeling. We found that MPXV stimulates cytoskeleton reorganization with intercellular extensions for MPXV cell spreading specifically to trophoblastic cells. Altogether, the specific infectious cycle of MPXV in trophoblast cells and these protrusions that were structurally and morphologically similar to filopodia reveal new insights into the infection of MPXV.

Human trophoblast stem cells restrict human cytomegalovirus replication.

Placental infection plays a central role in the pathogenesis of congenital human cytomegalovirus (HCMV) infections and is a cause of fetal growth restriction and pregnancy loss. HCMV can replicate in some trophoblast cell types, but it remains unclear how the virus evades antiviral immunity in the placenta and how infection compromises placental development and function. Human trophoblast stem cells (TSCs) can be differentiated into extravillous trophoblasts (EVTs), syncytiotrophoblasts (STBs), and organoids, and this study assessed the utility of TSCs as a model of HCMV infection in the first-trimester placenta. HCMV was found to non-productively infect TSCs, EVTs, and STBs. Immunofluorescence assays and flow cytometry experiments further revealed that infected TSCs frequently only express immediate early viral gene products. Similarly, RNA sequencing found that viral gene expression in TSCs does not follow the kinetic patterns observed during lytic infection in fibroblasts. Canonical antiviral responses were largely not observed in HCMV-infected TSCs and TSC-derived trophoblasts. Rather, infection dysregulated factors involved in cell identity, differentiation, and Wingless/Integrated signaling. Thus, while HCMV does not replicate in TSCs, infection may perturb trophoblast differentiation in ways that could interfere with placental function. IMPORTANCE: Placental infection plays a central role in human cytomegalovirus (HCMV) pathogenesis during pregnancy, but the species specificity of HCMV and the limited availability and lifespan of primary trophoblasts have been persistent barriers to understanding how infection impacts this vital organ. Human trophoblast stem cells (TSCs) represent a new approach to modeling viral infection early in placental development. This study reveals that TSCs, like other stem cell types, restrict HCMV replication. However, infection perturbs the expression of genes involved in differentiation and cell fate determination, pointing to a mechanism by which HCMV could cause placental injury.

Establishment and comparison of human term placenta-derived trophoblast cells†.

Research on the biology of fetal-maternal barriers has been limited by access to physiologically relevant cells, including trophoblast cells. In this study, we describe the development of a human term placenta-derived cytotrophoblast immortalized cell line (hPTCCTB) derived from the basal plate. Human-term placenta-derived cytotrophoblast immortalized cell line cells are comparable to their primary cells of origin in terms of morphology, marker expression, and functional responses. We demonstrate that these can transform into syncytiotrophoblast and extravillous trophoblasts. We also compared the hPTCCTB cells to immortalized chorionic trophoblasts (hFM-CTC), trophoblasts of the chorionic plate, and BeWo cells, choriocarcinoma cell lines of conventional use. Human-term placenta-derived cytotrophoblast immortalized cell line and hFM-CTCs displayed more similarity to each other than to BeWos, but these differ in syncytialization ability. Overall, this study (1) demonstrates that the immortalized hPTCCTB generated are cells of higher physiological relevance and (2) provides a look into the distinction between the spatially distinct placental and fetal barrier trophoblasts cells, hPTCCTB and hFM-CTC, respectively.

Increased Hepatocyte Growth Factor Secretion by Placenta-Derived Mesenchymal Stem Cells Improves Ovarian Function in an Ovariectomized Rat Model via Vascular Remodeling by Wnt Signaling Activation.

The vascular network contributes to the development of follicles. However, the therapeutic mechanism between vascular remodeling and ovarian functions is still unclear. Therefore, we demonstrated whether increased HGF by placenta-derived mesenchymal stem cells (PD-MSCs) improves ovarian function in an ovariectomized rat model via vascular remodeling by Wnt signaling activation. We established a half-ovariectomized rat model in which damaged ovaries were induced by ovariectomy of half of each ovary, and PD-MSCs (5 × 105 cells) were transplanted by intravenous injection. Three weeks after transplantation, rats in all groups were sacrificed. We examined the secretion of HGF by PD-MSCs through culture medium. The vascular structure in injured ovarian tissues was restored to a greater extent in the PD-MSC transplantation (Tx) group than in the nontransplantation (NTx) group (* p < 0.05). The expression of genes related to Wnt signaling (e.g., LRP6, GSK3ß, ß-catenin) was significantly increased in the Tx group compared to the NTx group (* p < 0.05). However, the expression of genes related to vascular permeability (e.g., Asef, ERG3) was significantly decreased in the Tx group compared to the NTx group (* p < 0.05). Follicular development was improved in the Tx group compared to the NTx group (* p < 0.05). Furthermore, to evaluate vascular function, we cocultivated PD-MSCs after human umbilical vein endothelial cells (HUVECs) with lipopolysaccharide (LPS), and we analyzed the vascular formation assay and dextran assay in HUVECs. Cocultivation of PD-MSCs with injured HUVECs enhanced vascular formation and decreased endothelial cell permeability (* p < 0.05). Also, cocultivation of PD-MSCs with explanted ovarian tissues improved follicular maturation compared to cocultivation of the Wnt inhibitor-treated PD-MSCs with explanted ovarian tissues. Therefore, HGF secreted by PD-MSCs improved ovarian function in rats with ovarian dysfunction by decreasing vascular permeability via Wnt signaling.

Core conserved transcriptional regulatory networks define the invasive trophoblast cell lineage.

The invasive trophoblast cell lineages in rat and human share crucial responsibilities in establishing the uterine-placental interface of the hemochorial placenta. These observations have led to the rat becoming an especially useful animal model for studying hemochorial placentation. However, our understanding of similarities or differences between regulatory mechanisms governing rat and human invasive trophoblast cell populations is limited. In this study, we generated single-nucleus ATAC-seq data from gestation day 15.5 and 19.5 rat uterine-placental interface tissues, and integrated the data with single-cell RNA-seq data generated at the same stages. We determined the chromatin accessibility profiles of invasive trophoblast, natural killer, macrophage, endothelial and smooth muscle cells, and compared invasive trophoblast chromatin accessibility with extravillous trophoblast cell accessibility. In comparing chromatin accessibility profiles between species, we found similarities in patterns of gene regulation and groups of motifs enriched in accessible regions. Finally, we identified a conserved gene regulatory network in invasive trophoblast cells. Our data, findings and analysis will facilitate future studies investigating regulatory mechanisms essential for the invasive trophoblast cell lineage.

Self-patterning of human stem cells into post-implantation lineages.

Investigating human development is a substantial scientific challenge due to the technical and ethical limitations of working with embryonic samples. In the face of these difficulties, stem cells have provided an alternative to experimentally model inaccessible stages of human development in vitro1-13. Here we show that human pluripotent stem cells can be triggered to self-organize into three-dimensional structures that recapitulate some key spatiotemporal events of early human post-implantation embryonic development. Our system reproducibly captures spontaneous differentiation and co-development of embryonic epiblast-like and extra-embryonic hypoblast-like lineages, establishes key signalling hubs with secreted modulators and undergoes symmetry breaking-like events. Single-cell transcriptomics confirms differentiation into diverse cell states of the perigastrulating human embryo14,15 without establishing placental cell types, including signatures of post-implantation epiblast, amniotic ectoderm, primitive streak, mesoderm, early extra-embryonic endoderm, as well as initial yolk sac induction. Collectively, our system captures key features of human embryonic development spanning from Carnegie stage16 4-7, offering a reproducible, tractable and scalable experimental platform to understand the basic cellular and molecular mechanisms that underlie human development, including new opportunities to dissect congenital pathologies with high throughput.

Endogenous retrovirus-derived enhancers confer the transcriptional regulation of human trophoblast syncytialization.

Endogenous retroviruses (ERVs) have been proposed as a driving force for the evolution of the mammalian placenta, however, the contribution of ERVs to placental development and the underlying regulatory mechanism remain largely elusive. A key process of placental development is the formation of multinucleated syncytiotrophoblasts (STBs) in direct contact with maternal blood, through which constitutes the maternal-fetal interface critical for nutrient allocation, hormone production and immunological modulation during pregnancy. We delineate that ERVs profoundly rewire the transcriptional program of trophoblast syncytialization. Here, we first determined the dynamic landscape of bivalent ERV-derived enhancers with dual occupancy of H3K27ac and H3K9me3 in human trophoblast stem cells (hTSCs). We further demonstrated that enhancers overlapping several ERV families tend to exhibit increased H3K27ac and reduced H3K9me3 occupancy in STBs relative to hTSCs. Particularly, bivalent enhancers derived from the Simiiformes-specific MER50 transposons were linked to a cluster of genes important for STB formation. Importantly, deletions of MER50 elements adjacent to several STB genes, including MFSD2A and TNFAIP2, significantly attenuated their expression concomitant to compromised syncytium formation. Together, we propose that ERV-derived enhancers, MER50 specifically, fine-tune the transcriptional networks accounting for human trophoblast syncytialization, which sheds light on a novel ERV-mediated regulatory mechanism underlying placental development.

Placental Mesenchymal Stem Cells Alleviate Podocyte Injury in Diabetic Kidney Disease by Modulating Mitophagy via the SIRT1-PGC-1alpha-TFAM Pathway.

The use of mesenchymal stem cells (MSCs) has become a new strategy for treating diabetic kidney disease (DKD). However, the role of placenta derived mesenchymal stem cells (P-MSCs) in DKD remains unclear. This study aims to investigate the therapeutic application and molecular mechanism of P-MSCs on DKD from the perspective of podocyte injury and PINK1/Parkin-mediated mitophagy at the animal, cellular, and molecular levels. Western blotting, reverse transcription polymerase chain reaction, immunofluorescence, and immunohistochemistry were used to detect the expression of podocyte injury-related markers and mitophagy-related markers, SIRT1, PGC-1α, and TFAM. Knockdown, overexpression, and rescue experiments were performed to verify the underlying mechanism of P-MSCs in DKD. Mitochondrial function was detected by flow cytometry. The structure of autophagosomes and mitochondria were observed by electron microscopy. Furthermore, we constructed a streptozotocin-induced DKD rat model and injected P-MSCs into DKD rats. Results showed that as compared with the control group, exposing podocytes to high-glucose conditions aggravated podocyte injury, represented by a decreased expression of Podocin along with increased expression of Desmin, and inhibited PINK1/Parkin-mediated mitophagy, manifested as a decreased expression of Beclin1, the LC3II/LC3I ratio, Parkin, and PINK1 associated with an increased expression of P62. Importantly, these indicators were reversed by P-MSCs. In addition, P-MSCs protected the structure and function of autophagosomes and mitochondria. P-MSCs increased mitochondrial membrane potential and ATP content and decreased the accumulation of reactive oxygen species. Mechanistically, P-MSCs alleviated podocyte injury and mitophagy inhibition by enhancing the expression of the SIRT1-PGC-1α-TFAM pathway. Finally, we injected P-MSCs into streptozotocin-induced DKD rats. The results revealed that the application of P-MSCs largely reversed the markers related to podocyte injury and mitophagy and significantly increased the expression of SIRT1, PGC-1α, and TFAM compared with the DKD group. In conclusion, P-MSCs ameliorated podocyte injury and PINK1/Parkin-mediated mitophagy inhibition in DKD by activating the SIRT1-PGC-1α-TFAM pathway.

Spatial multiomics map of trophoblast development in early pregnancy.

The relationship between the human placenta-the extraembryonic organ made by the fetus, and the decidua-the mucosal layer of the uterus, is essential to nurture and protect the fetus during pregnancy. Extravillous trophoblast cells (EVTs) derived from placental villi infiltrate the decidua, transforming the maternal arteries into high-conductance vessels1. Defects in trophoblast invasion and arterial transformation established during early pregnancy underlie common pregnancy disorders such as pre-eclampsia2. Here we have generated a spatially resolved multiomics single-cell atlas of the entire human maternal-fetal interface including the myometrium, which enables us to resolve the full trajectory of trophoblast differentiation. We have used this cellular map to infer the possible transcription factors mediating EVT invasion and show that they are preserved in in vitro models of EVT differentiation from primary trophoblast organoids3,4 and trophoblast stem cells5. We define the transcriptomes of the final cell states of trophoblast invasion: placental bed giant cells (fused multinucleated EVTs) and endovascular EVTs (which form plugs inside the maternal arteries). We predict the cell-cell communication events contributing to trophoblast invasion and placental bed giant cell formation, and model the dual role of interstitial EVTs and endovascular EVTs in mediating arterial transformation during early pregnancy. Together, our data provide a comprehensive analysis of postimplantation trophoblast differentiation that can be used to inform the design of experimental models of the human placenta in early pregnancy.

Combining topical dermal infused exosomes with injected calcium hydroxylapatite for enhanced tissue biostimulation.

BACKGROUND: Exosome research continues to flourish. Subsequent knowledge surrounding indications, dose-response, safety, efficacy, and the ability to combine exosome treatment as a "skin primer"-for biostimulation modalities such as calcium hydroxylapatite (CaHA), platelet-rich plasma (PRP), and platelet-rich fibrin matrix (PRFM) is growing rapidly. The objective of this study was to develop safe, reproducible methods of improving topical exosome absorption to enhance the quality of skin either by themselves, or in combination with injectable CaHA. METHODS: Under IRB Approval (International Cell Surgical Society: ICSS-2022-007), 40 patients were enrolled in this study. Twenty patients underwent facial biostimulatory dermal infusion alone, to determine if this method allowed adequate exosome absorption. Five patients underwent facial biostimulatory infusion followed immediately by Dilute CaHA injection (1:1 dilution) to the face. Five patients underwent exosome biostimulatory dermal infusion followed immediately by hyperdilute CaHA (dilution 1:4) injection to the neck. Five patients underwent Facial Dilute CaHA injection (1:1 dilution) alone, without dermal infusion. Five patients underwent neck hyperdilute CaHA injection (1:4 dilution) alone, without dermal infusion. All patients had pretreatment Quantificare 3-D photo-documentation and skin analysis (Quantificare, France). In all patients, the skin was first cleansed with a gentle glycolic acid facial wash (Gregory MD). To induce a "homing inflammatory environment" for the exosomes, sea salt exfoliation was performed (SaltFacial®, SaltMed, Cardiff, CA). A nitric oxide-generating serum (N101 Pneuma Nitric Oxide, Austin, TX) was then applied to act as an enhanced vehicle for absorption. A 3 MHz ultrasound (SaltFacial®, SaltMed, Cardiff, CA) was then utilized to further deepen the absorption of the nitric oxide serum. A topical emulsion containing equal volumes (1.0 cc containing 1 million) of exosomes (Kimera Labs, Miramar, FL), 25 units of botulinum toxin (Xeomin, Merz Aesthetics, Raleigh, NC) and hyaluronic acid (Belatero, Merz Aesthetics, Raleigh, NC) was mixed via back-and-forth propulsion in a 3-cc syringe. When adequately mixed, the emulsion was then applied to the treatment areas. The cavitating ultrasound was then used to aid in the absorption of the emulsion. The patients were then treated with high-intensity LED therapy (SaltFacial®, SaltMed, Cardiff, CA), utilizing the collagen restoration preset program of combination red (660 nm) near-infrared (930 nm) wavelength for 20 min. Post-treatment Quantificare analysis was performed at 15 and 30 days after treatment. RESULTS: Without exception, all dermal infusion alone and CaHA injection alone patients showed an improvement in the tone, quality, and texture of their skin. Quantificare results showed consistent improvement in wrinkles, pores, skin evenness, improved vascularity, and a reduction in oiliness and unwanted pigment. When employed as a skin primer prior to injections (CaHA), enhanced and more rapid results were seen. CONCLUSIONS: Biostimulatory dermal infusion can be achieved utilizing topical placental mesenchymal stem cell-derived exosomes. These exosomes can be used alone, or mixed with ancillary ingredients such as botulinum toxin, hyaluronic acid dermal filler, and CaHA to customize and personalize treatments based upon individual patient needs. Topical absorption is enhanced with sea salt exfoliation, a topical nitric oxide-generating serum, and 3 MHz cavitating ultrasound. Post-absorption activity is enhanced with high-intensity LED treatment. The addition of CaHA injections after the topical exosome "priming of the skin" yielded enhanced skin quality faster than exosomes or CaHA alone.