Cell Sheets Research Articles

Female Adnexal Tumor of Probable Wolffian Origin (Wolffian Tumor): A Potential Mimic of Peritoneal Mesothelioma.

Wolffian tumor and its nosologic relative, the recently defined STK11 adnexal tumor are rare neoplasms thought to arise from mesonephric remnants. These tumors typically arise in the broad ligament, fallopian tube, and ovarian hilum and although most are associated with a good prognosis, up to 50% of STK11 adnexal tumors demonstrate aggressive clinical behavior. The chief differential diagnoses include endometrioid adenocarcinoma and sex cord stromal tumors. However, the morphologic and immunohistochemical features of these tumors exhibit considerable overlap with peritoneal mesothelioma. To fully characterize their immunophenotypic signature, we examined a total of 21 cases (18 Wolffian and 3 STK11 adnexal tumors) with standard markers used in the diagnosis of mesothelioma. Morphologic and immunohistochemical (IHC) features were reviewed and additional IHC performed for cases with available material. Patient age ranged from 25 to 73 (mean: 51) years. Sites included adnexa/broad ligament (6, 28%), paratubal (5, 24%), ovary/paraovarian (5, 24%), tubal (intraluminal) (2, 9.5%), pelvis (2, 9.5%), and liver (1, 5%). The mean tumor size was 9.3cm (range: 0.2 to 22cm). The histomorphology in most cases (14/21, 66%) consisted of tubular to solid sheets of neoplastic cells lined by columnar to cuboidal cells containing uniform round to oval nuclei. Compressed tubules with slit-like lumens and sieve-like pattern were also seen in at least 7 (33%) cases. Three cases demonstrated interanastomosing cords and trabeculae of epithelioid cells with cribriform and microacinar patterns growing within prominent myxoid stroma as described in STK11 adnexal tumors. In the cases with available IHC for 3 mesothelial markers (calretinin, WT1, D2-40), 55.5% (5 of 9) showed reactivity with all 3 markers. In cases with at least 2 available mesothelial markers, 69% (11/16) were positive for 2 markers (mostly calretinin and WT1). Claudin-4, MOC31, and BER-EP4 were negative in most cases tested (78% [7/9], 71.4% [5/7], and 100% [6/6], respectively). Given the resemblance to mesothelioma, there was initial strong consideration and/or actual misdiagnosis of mesothelioma in 3 cases (14%). In summary, the morphologic and immunohistochemical features of Wolffian tumor and its recently defined relative, STK11 adnexal tumor, can lead to misdiagnosis of mesothelioma, particularly when encountered in the disseminated or metastatic setting. Wolffian tumor and STK11 adnexal tumor should be considered in the differential diagnosis of all pelvic and peritoneal mesotheliomas.

Fine-needle aspiration cytology of retroperitoneal myoepithelial carcinoma: A rare encounter with diagnostic dilemmas.

Myoepithelial carcinoma (MC) arises from the myoepithelial cells. It is a rare tumor with a predilection for salivary glands. MC in soft tissue is uncommon. Soft tissue MC exhibits dual epithelial and smooth muscle phenotype. The extremities and limb girdles are commonly affected. We present cytological findings of retroperitoneal MC with an accurate diagnosis being rendered with the aid of immunocytochemistry on the cell block and demonstration of EWSR1 rearrangements by fluorescence in situ hybridization on cytology smear. The smears were cellular, showing loose clusters and sheets of tumor cells embedded in dense eosinophilic to myxoid matrix material. The cells were oval to polygonal, with focal areas showing moderate nuclear pleomorphism, vesicular to coarse chromatin, and vacuolated cytoplasm with clearing. On immunocytochemistry, tumor cells were positive for epithelial membrane antigen, pan-cytokeratin, calponin, smooth muscle actin, and S-100. A literature review shows only a handful of cases of soft tissue MC. The current report emphasizes the need for cytomorphological awareness with the employment of ancillary testing for accurately diagnosing this rare tumor at an uncommon location. We also discuss the diagnostic challenges and troubleshooting.

Transplantation of Neural Progenitor Cells Derived from Stem Cells from Apical Papilla Through Small-Molecule Induction in a Rat Model of Sciatic Nerve Injury

Background:Stem cell-based transplantation therapy holds promise for peripheral nerve injury treatment, but adult availability is limited. A cell culture protocol utilizing a small-molecule cocktail effectively reprogrammed stem cells from apical papilla (SCAPs) into neural progenitor cells, subsequently differentiating into neuron-like cells. This study aims to evaluate neural-induced SCAPs, with and without small-molecule cocktail, for sciatic nerve repair potential.Methods:A scaffold-free cell sheet technique was used to construct a three-dimensional cell sheet. Subsequently, this cell sheet was carefully rolled into a tube and seamlessly inserted into a collagen conduit, which was then transplanted into a 5 mm sciatic nerve injury rat model. Functional sciatic nerve regeneration was evaluated via toe spread test, walking track analysis and gastrocnemius muscle weight. Additionally, degree of sciatic nerve regeneration was determined based on total amount of myelinated fibers.Results:Small-molecule cocktail induced SCAPs enhanced motor function recovery, evident in improved sciatic function index and gastrocnemius muscle retention. We also observed better host myelinated fiber retention than undifferentiated SCAPs or neural-induced SCAPs without small-molecule cocktail. However, clusters of neuron-like cell bodies (surrounded by sparse myelinated fibers) were found in all cell sheet-implanted groups in the implantation region. This suggests that while the implanted cells likely survived transplantation, integration was poor and would likely hinder long-term recovery by occupying the space needed for host nerve fibers to project through.Conclusion:Neural-induced SCAPs with small-molecule cocktail demonstrated promising benefits for nerve repair; further research is needed to improve its integration and optimize its potential for long-term recovery.

Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts.

Tendons and ligaments (T/L) are strong hierarchically organized structures unitingthemusculoskeletal system. These tissues have a strictly arranged collagen typeI-rich extracellular matrix (ECM) and T/L-lineage cells mainly positioned in parallel rows. After injury, T/L require a long time for rehabilitation with high failure risk and often unsatisfactory repair outcomes. Despite recent advancements in T/L biology research, one of the remaining challenges is that the T/L field still lacks a standardized differentiation protocol that is able to recapitulate T/L formation process in vitro. For example, bone and fat differentiation of mesenchymal precursor cells require just standard two-dimensional (2D) cell culture and the addition of specific stimulation media. For differentiation to cartilage, three-dimensional (3D) pellet culture and supplementation of TGFß is necessary. However, cell differentiation to tendon needs a very orderly 3D culture model, which ideally should also be subjectable to dynamic mechanical stimulation. We have established a 3-step (expansion, stimulation, and maturation) organoid model to form a 3D rod-like structure out of a self-assembled cell sheet, which delivers a natural microenvironment with its own ECM, autocrine, and paracrine factors. These rod-like organoids have a multi-layered cellular architecture within rich ECM and can be handled quite easily for exposure to static mechanical strain. Here, we demonstrated the 3-step protocol by using commercially available dermal fibroblasts. We could show that this cell type forms robust and ECM-abundant organoids. The described procedure can be further optimized in terms of culture media and optimized toward dynamic axial mechanical stimulation. In the same way, alternative cell sources can be tested for their potential to form T/L organoids and thus undergo T/L differentiation. In sum, the established 3D T/L organoid approach can be used as a model for tendon basic research and even for scaffold-free T/L engineering.

ATRT-08. ATYPICAL TERATOID RHABDOID TUMOR IN AN ADULT PATIENT WITH DOWN SYNDROME

Abstract BACKGROUND Atypical teratoid/rhabdoid tumor (ATRT) is a malignant, highly aggressive embryonal tumor of the central nervous system. Long-term survival remains dismal despite aggressive multimodal therapy, including a maximal safe surgical resection and intensive systemic chemotherapy +/- radiation therapy. While these tumors typically present in infancy or early childhood, there are scattered case reports of adult-onset ATRT. Making prognostic conclusions or therapeutic decisions for this older patient population remains challenging due to the paucity of these reports. CASE REPORT A 25-year-old female with Down syndrome presented with nausea, vomiting, dysphagia, and facial droop and was found to have an avidly enhancing, left cerebellopontine angle mass. There was initial clinical suspicion for leukemic chloroma given the patient’s Down syndrome status and known association with higher rates of hematologic malignancy concurrent with a dramatic scarcity of solid tumors in the Down syndrome population. However, histology demonstrated sheets of high-grade rhabdoid cells with loss of INI1 expression, pathognomonic for ATRT. Further sequencing defined a frameshift mutation in SMARCB1 (p.C298), and methylation profiling matched with high confidence to the MYC subclass of ATRT. The patient was treated with subtotal surgical resection and focal proton radiation, followed by chemotherapy on a modified regimen based on the Children’s Oncology Group protocol, ACNS0333. Systemic methotrexate and all consolidative high-dose chemotherapy with stem cell rescue were omitted due to concern for heightened risk of treatment-related toxicity. The patient remains alive and clinically asymptomatic 19 months from diagnosis. CONCLUSION This report represents the first known case of ATRT in an adult patient with Down syndrome, offering unique mechanistic insight into the tumorigenesis of ATRT. Adult ATRT is a rare entity without standardized treatment or established prognostic understanding. Further studies are needed to define an appropriate risk-adapted therapeutic approach for this patient population.

Microstripe pattern substrate consisting of alternating planar and nanoprotrusive regions improved hiPSC-derived cardiomyocytes' unidirectional alignment and functional properties.

The alignment of each cell in human myocardium is considered critical for the efficient movement of cardiac tissue. We investigated 96-well microstripe-patterned plates to align human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs), which resemble fetal myocardium. The aligned CMs (ACMs) cultured on the microstripe-patterned plates exhibited pathology, motor function, gene expression, and drug response that more closely resembled those of adult cells than did unaligned CMs cultured on a flat plate (FCMs). We used these ACMs to evaluate drug side effects and efficacy, and to determine whether these were similar to adult-like responses. When CMs from patients with hypertrophic cardiomyopathy (HCMs) were seeded and cultured on the microstripe-patterned plates or layered on top of the ACMs, both sets of HCMs showed increased heart rate and synchronized contractions, indicating improved cardiac function. It is suggested that the ACMs could be used for drug screening as cells representative of adult-like CMs and be transplanted in the form of a cell sheet for regenerative treatment of heart failure.

Breathing new life into tissue engineering: exploring cutting-edge vascularization strategies for skin substitutes.

Tissue-engineered skin substitutes (TESS) emerged as anew therapeutic option to improve skin transplantation. However, establishing an adequate and rapid vascularization in TESS is a critical factor for their clinical application and successful engraftment in patients. Therefore, several methods have been applied to improve the vascularization of skin substitutes including (i) modifying the structural and physicochemical properties of dermal scaffolds; (ii) activating biological scaffolds with growth factor-releasing systems or gene vectors; and (iii) developing prevascularized skin substitutes by loading scaffolds with capillary-forming cells. This review provides a detailed overview of the most recent and important developments in the vascularization strategies for skin substitutes. Onthe one hand, we present cell-based approaches using stem cells, microvascular fragments, adipose tissue derived stromal vascular fraction, endothelial cells derived from blood and skin as well as other pro-angiogenic stimulation methods. On the other hand, we discuss how distinct 3D bioprinting techniques and microfluidics, miRNA manipulation, cell sheet engineering and photosynthetic scaffolds like GelMA, can enhance skin vascularization for clinical applications. Finally, we summarize and discuss the challenges and prospects of the currently available vascularization techniques that may serve as a steppingstone to a mainstream application of skin tissue engineering.

Automated xeno-free chondrogenic differentiation from human embryonic stem cells: Enhancing efficiency and ensuring high-quality mass production

IntroductionRepairing damaged cartilage poses significant challenges, particularly in cases of congenital cartilage defects such as microtia or congenital tracheal stenosis, or as a consequence of traumatic injury, as the regenerative potential of cartilage is inherently limited. Stem cell therapy and tissue engineering offer promising approaches to overcome these limitations in cartilage healing. However, the challenge lies in the size of cartilage-containing organs, which necessitates a large quantity of cells to fill the damaged areas. Therefore, pluripotent stem cells that can proliferate indefinitely are highly desirable as a cell source. This study aims to delineate the differentiation conditions for cartilage derived from human embryonic stem cells (ESCs) and to develop an automated cell culture system to facilitate mass production for therapeutic applications. MethodsCartilage cell sheets were derived from human ESCs (SEES2, clinical trial-compatible line) by forming embryoid bodies (EBs) with either conventional manual culture or a benchtop multi-pipetter and an automated medium exchange integrated cell incubator, using xeno-free media. Cell sheets were implanted into the subcutaneous tissue of immunodeficient NOG mice to obtain cartilage tissue. The properties of cartilage tissues were examined by histological staining and quantitative PCR analysis. ResultsWe have optimized an efficient xeno-free system for cartilage production with the conventional culture method and successfully transitioned to an automated system. Differentiated cartilage was histologically uniform with cartilage-specific elasticity and strength. The cartilage tissues were stained by Alcian blue, safranin O, and toluidine blue, and quantitative PCR showed an increase in differentiation markers such as ACAN, COL2A1, and Vimentin. Automation significantly enhanced the efficiency of human ESC-derived chondrocyte differentiation. The number of constituent cells within EBs and the seeding density of EBs were identified as key factors influencing chondrogenic differentiation efficiency. By automating the process of chondrogenic differentiation, we achieved scalable production of chondrocytes. ConclusionsBy integrating the differentiation protocol with an automated cell culture system, there is potential to produce cartilage of sufficient size for clinical applications in humans. The resulting cartilage tissue holds promise for clinical use in repairing organs such as the trachea, joints, ears, and nose.

An analysis of treatment trends in sclerosing epithelioid fibrosarcoma.

e23547 Background: Sclerosing Epithelioid Fibrosarcoma (SEF) is a rare, malignant, soft tissue sarcoma found in the arms, legs, head, and neck. Histologically, SEF is characterized by a distinctive architecture composed of cords, nests, and/or sheets of uniform epithelioid cells. Previous studies have demonstrated that surgery with wide surgical margins is associated with superior outcomes in SEF patients. However, on account of the rare nature of SEF, insufficient research has been conducted on the association of treatment types with long-term survival. Using the National Cancer Database (NCDB), this study analyzed the receipt of different types of surgery and adjuvant treatments on the overall survival of patients with SEF. Methods: The National Cancer Database (NCDB) was used to identify patients diagnosed with SEF from 2004 to 2020 using histology code 8840 as assigned by the Commission on Cancer Accreditation program. Kaplan-Meier, ANOVA Chi-Square, and Logistic Regression tests were performed, and data were analyzed using SPSS version 29. Statistical significance was set at α = 0.05. Results: Out of the 1226 patients with SEF, 1088 patients ( 88.7%) received a tumor resection at the primary site. Receipt of surgery was associated with a greater median overall survival compared to not receiving surgery ( 123.6 months vs 45.7 months; p < 0.05). Of the surgical patients, 419 patients received a wedge/segmental resection ( 38.5%) and 609 patients received a lobectomy ( 56%). Wedge/segmental resections and lobectomy had improved outcomes compared to other surgery types ( p < 0.001). In addition to tumor resection, 140 patients ( 12.9%) received adjuvant chemotherapy, 21 patients ( 1.9%) received adjuvant radiation, and 11(1%) patients received adjuvant chemoradiation. Furthermore, the receipt of adjuvant therapies including adjuvant chemo, adjuvant radiation, and adjuvant chemoradiation, was associated with improved overall survival compared to receiving surgery alone ( p = 0.03; p = 0.005; p < 0.001, respectively). Patients who received adjuvant chemoradiation experienced 9.5 months longer median survival time than patients who received adjuvant chemotherapy ( p < 0.001). Conclusions: SEF patients who received tumor resection had a marked increase in overall survival. Receiving wedge/segmental and lobectomy surgery types were associated with improved patient outcomes compared to other surgery types. Furthermore, receiving any adjuvant therapy was associated with improved overall survival compared to receiving surgery alone. Receiving adjuvant chemoradiation was associated with superior outcomes in SEF patients compared to receiving other adjuvant therapies and surgery alone. To improve overall survival in SEF patients, further research is needed to assess what factors influence treatment trends.

The effects of metabolites from three vaginal bacteria on the Syndecan-1 of cervical epithelial cells

This study aims to explore the impact of metabolites from three vaginal bacteria on the expression of Syndecan 1 (SDC-1). Human cervical epithelial cells (HcerEpic) were separately incubated with the cell-free supernatants of Lactobacillus crispatus (LCS group), Gardnerella vaginalis (GVS group), and Atopobium vaginalis (AVS group). LCS showed a proliferative effect on HcerEpic, with the most significant effect observed at a concentration of 30 % (P < 0.001). GVS and AVS exhibited some cytotoxicity, with significant growth inhibitory effects observed at concentrations of 30 % and 40 % (P < 0.01). Therefore, subsequent experiments were conducted using 30 % LCS, 40 % GVS, and 40 % AVS. In terms of cellular morphology, compared to the Control group, the LCS group showed more frequent fusion of cell sheets, with no obvious changes in the morphology of individual cells. In the GVS and AVS groups, some individual cells became round and smaller, with reduced protrusions and even a small amount of floating cells. The metabolic products of the three vaginal bacteria significantly upregulated the expression of IL-1β, IL-6, and TNF-α in HcerEpic (P < 0.05). In the GVS and AVS groups, the level of SDC-1 on the surface of HcerEpic was significantly decreased (P < 0.01), while the concentration of SDC-1 in the cell culture supernatant was significantly increased (P < 0.0001). Additionally, the level of SDC-1 mRNA was significantly downregulated (P < 0.01). In the LCS group, no significant changes were observed in SDC-1 protein and mRNA expression (P > 0.05). LCS promotes HcerEpic proliferation, without significant impact on SDC-1 expression and shedding. This provides molecular evidence for LCS as a protective factor against human papillomavirus infection in the cervix. Metabolites of GV and AV inhibit HcerEpic proliferation, induce cytokine secretion, suppress SDC-1 transcription and expression, and promote SDC-1 shedding.

Long-term prognosis and DNA damage status after oral mucosal epithelial cell sheet transplantation following esophageal endoscopic submucosal dissection for squamous cell carcinoma: A case series

Autologous oral mucosal epithelial cell sheet (AOMECS) transplantation has recently been applied in human patients to prevent postprocedural stenosis following endoscopic submucosal dissection (ESD) for esophageal squamous cell carcinoma. However, the long-term safety of AOMECS transplantation remains unclear. We evaluated the long-term outcomes of 10 patients who participated in a clinical trial of AOMECS transplantation after esophageal ESD. Additionally, we assessed the local DNA damage response in the esophageal epithelium using p53 binding protein 1 (53BP1) immunofluorescence in post-AOMECS biopsy specimens. The median follow-up period was 118.5 months (range: 46–130 months). Two patients developed primary esophageal cancer near the AOMECS site and successfully underwent additional ESD. One patient developed lymph node metastasis and underwent chemotherapy. None of the patients died from the original disease, although one patient died from unrelated causes. The rate of abnormal 53BP1 nuclear foci, indicative of increased genome instability, increased with the progression of neoplasia in patients post AOMECS. Our case series suggests that AOMECS transplantation provides an acceptable long-term prognosis and 53BP1 foci may serve as a useful marker for assessing DNA instability in the post-AOMECS esophageal epithelium.

Current Strategies and Therapeutic Applications of Mesenchymal Stem Cell-Based Drug Delivery.

Mesenchymal stem cells (MSCs) have emerged as a promising approach for drug delivery strategies because of their unique properties. These strategies include stem cell membrane-coated nanoparticles, stem cell-derived extracellular vesicles, immunomodulatory effects, stem cell-laden scaffolds, and scaffold-free stem cell sheets. MSCs offer advantages such as low immunogenicity, homing ability, and tumor tropism, making them ideal for targeted drug delivery systems. Stem cell-derived extracellular vesicles have gained attention for their immune properties and tumor-homing abilities, presenting a potential solution for drug delivery challenges. The relationship between MSC-based drug delivery and the self-renewal and differentiation capabilities of MSCs lies in the potential of engineered MSCs to serve as effective carriers for therapeutic agents while maintaining their intrinsic properties. MSCs exhibit potent immunosuppressive functions in MSC-based drug delivery strategies. Stem cell-derived EVs have low immunogenicity and strong therapeutic potential for tissue repair and regeneration. Scaffold-free stem cell sheets represent a cutting-edge approach in regenerative medicine, offering a versatile platform for tissue engineering and regeneration across different medical specialties. MSCs have shown great potential for clinical applications in regenerative medicine because of their ability to differentiate into various cell types, secrete bioactive factors, and modulate immune responses. Researchers are exploring these innovative approaches to enhance drug delivery efficiency and effectiveness in treating various diseases.

Warthin-like Variant of Mucoepidermoid Carcinoma of the Parotid Gland

A 57-year-old woman with a 2-year history of a left infra-auricular mass with no associated symptoms presented with a 6.0 cm ´ 4.0 cm ´ 3.0 cm firm, non-tender, movable mass. No imaging was done. Fine needle aspiration biopsy (FNAB) revealed sheets of epithelial cells that had abundant dense grayish-blue cytoplasm in a mucinous background with abundant lymphocytes (Figure 1), suggestive of salivary gland neoplasm with oncocytic or oncocytoid features (Category IVB, Salivary Gland Neoplasm of Uncertain Malignant Potential).1 Total parotidectomy revealed a 4.3 X 3.2 X 3.0 cm deep lobe lesion with a tan-grey to dark brown, smooth and dull external surface. Cut sections showed a cream-white to pink, lobulated, heterogenous cut surfaces. Microscopically, the lesion was unencapsulated with poorly demarcated borders. The neoplastic cells were arranged in haphazard sheets and surrounded by abundant lymphocytes. The tumor cells had abundant eosinophilic and granular cytoplasm compatible with oncocytes with mild to moderate nuclear atypia. There were occasional cystic spaces that contained mucin though mucocytes were not readily apparent. (Figure 2) Necrosis, perineural and lymphovascular space invasion or anaplasia were not evident.

Cellularized Biomaterials Used as Gingival Connective Tissue Substitutes In Vivo: A Systematic Review.

Developing an in vitro model of gingival connective tissue that mimics the original structure and composition of gingiva for clinical grafting is relevant for personalized treatment of missing gingiva. Using tissue engineering techniques allows bypassing limitations encountered with existing solutions to increase oral soft tissue volume. This review aims to systematically analyze the different currently existing cellularized materials and technologies used to engineer gingival substitutes for invivo applications. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed. An electronic search on PubMed, Scopus, Web of Science, and Cochrane Library databases was conducted to identify suitable studies. In vivo studies about gingival substitutes and grafts containing oral cells compared with a control to investigate the graft remodeling were included. Risk of bias in the included studies was assessed using the Systematic Review Center for Laboratory animal Experimentation (SYRCLE) 10-item checklist. Out of 631 screened studies, 19 were included. Animal models were mostly rodents, and the most used implantation was subcutaneous. According to the SYRCLE tool, low-to-unclear risk of bias was prevalent. Studies checked vascularization and extracellular remodeling up to 60 days after implantation of the cellularized biomaterial. Cells used were mostly fibroblasts and stem cells from oral origin. Grafts presenting vascularization potential after implantation were produced by tissue engineering technologies including cell seeding or embedding for 14, cell sheets for 2, microsphere for 1, and extrusion 3D bioprinting for 2. Components used to build the scaffold containing the cells are all naturally derived and are mainly fibrin, gelatin, collagen, agarose, alginate, fibroin, guar gum, hyaluronic acid, and decellularized extracellular matrix. The most recurring crosslinking method was using chemicals. All studies except one reported vascularization of the graft after implantation, and some detailed extracellular matrix remodeling. Current solutions are not efficient enough. By assessing the relevant studies on the subject, this systematic review showed that a diversity of cellularized biomaterials substituting gingival connective tissue enables vascularization and extracellular remodeling. Taking the results of this review into account could help improve current bio-inks used in 3D bioprinting for invivo applications compensating for gingival loss.

Dual-color FISH analyses of xenogeneic human fibroblast sheets transplanted to repair lung pleural defects in an immunocompromised rat model

BackgroundPulmonary air leaks (PALs) due to visceral pleura injury during surgery is frequently observed after pulmonary resections and the complication is difficult to avoid in thoracic surgery. The development of postoperative PALs is the most common cause of prolonged hospitalization. Previously, we reported that PALs sealants using autologous dermal fibroblast sheets (DFSs) harvested from temperature-responsive culture dishes successfully closed intraoperative PALs during lung resection.ObjectiveIn this study, we investigated the fate of human DFSs xenogenetically transplanted onto lung surfaces to seal PALs of immunocompromised rat. Dual-color FISH analyses of human fibroblast was employed to detect transplantation human cells on the lung surface.ResultsOne month after transplantation, FISH analyses revealed that transplanted human fibroblasts still composed a sheet-structure, and histology also showed that beneath the sheet’s angiogenesis migrating into the sheets was observed from the recipient tissues. FISH analyses revealed that even at 3 months after transplantation, the transplanted human fibroblasts still remained in the sheet. Dual-color FISH analyses of the transplanted human fibroblasts were sparsely present as a result of the cells reaching the end of their lifespan, the cells producing extracellular matrix, and remained inside the cell sheet and did not invade the lungs of the host.ConclusionsDFS-transplanted human fibroblasts showed that they are retained within cell sheets and do not invade the lungs of the host.

Cell spray printing combined with Lycium barbarum glycopeptide promotes repair of corneal epithelial injury

The corneal epithelium, located as the outermost layer of the cornea, is inherently susceptible to injuries that may lead to corneal opacities and compromise visual acuity. Rapid restoration of corneal epithelial injury is crucial for maintaining the transparency and integrity of the cornea. Cell spray treatment emerges as an innovative and effective approach in the field of regenerative medicine. In our study, a cell spray printing platform was established, and the optimal printing parameters were determined to be a printing air pressure of 5 PSI (34.47 kPa) and a liquid flow rate of 30 ml/h. Under these conditions, the viability and phenotype of spray-printed corneal epithelial cells were preserved. Moreover, Lycium barbarum glycopeptide (LBGP), a glycoprotein purified from wolfberry, enhanced proliferation while simultaneously inhibiting apoptosis of the spray-printed corneal epithelial cells. We found that the combination of cell spray printing and LBGP facilitated the rapid construction of multilayered cell sheets on flat and curved collagen membranes in vitro. Furthermore, the combined cell spray printing and LBGP accelerated the recovery of the rat corneal epithelium in the mechanical injury model. Our findings offer a therapeutic avenue for addressing corneal epithelial injuries and regeneration.

Extracellular Vesicles Derived from Neutrophils Accelerate Bone Regeneration by Promoting Osteogenic Differentiation of BMSCs.

The reconstruction of bone defects has been associated with severe challenges worldwide. Nowadays, bone marrow mesenchymal stem cell (BMSC)-based cell sheets have rendered this approach a promising way to facilitate osteogenic regeneration in vivo. Extracellular vesicles (EVs) play an essential role in intercellular communication and execution of various biological functions and are often employed as an ideal natural endogenous nanomedicine for restoring the structure and functions of damaged tissues. The perception of polymorphonuclear leukocytes (neutrophils, PMNs) as indiscriminate killer cells is gradually changing, with new evidence suggesting a role for these cells in tissue repair and regeneration, particularly in the context of bone healing. However, the role of EVs derived from PMNs (PMN-EVs) in bone regeneration remains largely unknown, with limited research being conducted on this aspect. In the current study, we investigated the effects of PMN-EVs on BMSCs and the underlying molecular mechanisms as well as the potential application of PMN-EVs in bone regeneration. Toward this end, BMSC-based cell sheets with integrated PMN-EVs (BS@PMN-EVs) were developed for bone defect regeneration. PMN-EVs were found to significantly enhance the proliferation and osteogenic differentiation of BMSCs in vitro. Furthermore, BS@PMN-EVs were found to significantly accelerate bone regeneration in vivo by enhancing the maturation of the newly formed bone in rat calvarial defects; this is likely attributable to the effect of PMN-EVs in promoting the expression of key osteogenic proteins such as SOD2 and GJA1 in BMSCs. In conclusion, our findings demonstrate the crucial role of PMN-EVs in promoting the osteogenic differentiation of BMSCs during bone regeneration. Furthermore, this study proposes a novel strategy for enhancing bone repair and regeneration via the integration of PMN-EVs with BMSC-based cell sheets.

Graft cell expansion from hiPSC-RPE strip after transplantation in primate eyes with or without RPE damage

Clinical studies using suspensions or sheets of human pluripotent cell-derived retinal pigment epithelial cells (hiPSC-RPE) have been conducted globally for diseases such as age-related macular degeneration. Despite being minimally invasive, cell suspension transplantation faces challenges in targeted cell delivery and frequent cell leakage. Conversely, although the RPE sheet ensures targeted delivery with correct cell polarity, it requires invasive surgery, and graft preparation is time-consuming. We previously reported hiPSC-RPE strips as a form of quick cell aggregate that allows for reliable cell delivery to the target area with minimal invasiveness. In this study, we used a microsecond pulse laser to create a local RPE ablation model in cynomolgus monkey eyes. The hiPSC-RPE strips were transplanted into the RPE-ablated and intact sites. The hiPSC-RPE strip stably survived in all transplanted monkey eyes. The expansion area of the RPE from the engrafted strip was larger at the RPE injury site than at the intact site with no tumorigenic growth. Histological observation showed a monolayer expansion of the transplanted RPE cells with the expression of MERTK apically and collagen type 4 basally. The hiPSC-RPE strip is considered a beneficial transplantation option for RPE cell therapy.

Combination of mesenchymal stem cell sheet with poly-caprolactone nanofibrous mat and Gelfoam increased osteogenesis capacity in rat calvarial defect

Introduction: To date, different strategies have been used for co-transplantation of cell-loaded biomaterials for bone tissue regeneration. This study aimed to investigate the osteogenic properties of adipose-derived-mesenchymal stem cell (AD-MSC) sheets combined with nanofibrous poly-caprolactone (PCL) mat and Gelfoam in rats with calvarial bone defect. Methods: Calvarial critical-size defects were induced in male rats. Animals were classified into Control, Gelfoam, Gelfoam/PCL nanofiber, Gelfoam/AD-MSC sheet, and Gelfoam/PCL nanofiber/AD-MSC sheet groups. After 3 months, rats were sacrificed and the regeneration rate was evaluated. Results: Almost all groups showed bone regeneration properties, but the volume of newly formed bone was higher in groups that received Gelfoam/AD-MSC and Gelfoam/PCL nanofiber/AD-MSC sheets (P &lt; 0.05). The application of Gelfoam/PCL nanofiber/AD-MSC sheets not only increased bone thickness, bone volume/total bone volume (BV/TV) ratio, strong Hounsfield Unit (HU), but also led to the formation of ossified connective tissue with wrinkled patterns. Conclusion: The current study indicated that the Gelfoam/PCL nanofiber/AD-MSC sheet provides a suitable platform for effective osteogenesis in calvarial bone defects.

A rare case of neuroendocrine cell tumor mixed with a mucinous component in the ampulla of Vater

BackgroundA rare case of neuroendocrine cell tumor (NET) having both conventional and mucinous components was reported. Mucinous NET is rarely encountered in the pathological diagnosis of gastrointestinal (GI) tumors. Here we examined the mechanism for transformation of conventional NETs into mucinous NETs. Case presentation: Macroscopic examination revealed a tumor with ulceration in the ampulla of Vater that measured 1.7 cm in its largest diameter. Histologically, the tumor comprised two components: a tubular/ribbon-like feature and small nests floating in a mucinous lake. The tumor nests showed sheet, nest and ribbon-like structures of small cells having eosinophilic cytoplasm as well as small-sized nuclei with dense hyperchromatin. Immunohistochemical analysis showed tumor cells positive for pan-endocrine markers (synaptophysin, CD56, INSM1 and chromogranin). Based on the histological findings, the solid and mucinous components were diagnosed as conventional and mucinous NETs, respectively. Grading was NET G2 based on 12.8% and 13.2% Ki-67-positive cells in the solid and mucinous components, respectively. Immunohistochemically, the mucin phenotype of this tumor was gastric and intestinal. Only the mucinous NET component had cytoplasmic CD10 expression. Examination using a customized gene panel detected only a DPC4 mutation, which was limited to the mucinous component. Conclusions: Coexistence of conventional and mucinous NETs could provide important insight into evaluating the NET subtype histogenesis. Moreover, molecular alterations including cytoplasmic expression of CD10 and the DPC4 mutation can contribute to interpretation of tumor pathogenesis.