Mature Cells Research Articles

Lgr5+ intestinal stem cells are required for organoid survival after genotoxic injury.

Progenitors and mature cells can maintain the intestinal epithelium by dedifferentiation and facultative intestinal stem cell (fISC) function when active ISCs (aISCs) are lost to damage. Here, we modeled fISC activation in intestinal organoids with doxorubicin (DXR), a chemotherapeutic known to ablate Lgr5+ aISCs in vivo. Similar fISC gene activation was observed between organoids treated with low vs high DXR, despite significantly decreased survival at the higher dose. aISCs exhibit dose-dependent loss after DXR but survive at doses compatible with organoid survival. We ablated residual aISCs after DXR using a Lgr52A-DTR allele and observed that aISC survival of the initial genotoxic insult is required for organoid survival following DXR. These results suggest that while typical fISC genes are activated by DXR injury in organoids, functional stemness remains dependent on the aISC pool. Finally, we show that human intestinal organoids require higher doses of DXR to induce loss of survival and downregulation of LGR5. Our data establish a reproducible model of DXR injury in intestinal organoids and reveal differences in in vitro responses to an established in vivo damage modality.

In Vitro Spermatogenesis on Human Decellularized Testicular Matrix Plates Following Exosome Treatment in a Dynamic Culture System.

Testicular tissue engineering for in vitro spermatogenesis aims to restore fertility, focusing on challenges like efficiency, ethical concerns, and the need for a deeper biological understanding. The use of decellularized scaffolds led to better cell seeding and differentiation, and exosomes led to enhanced spermatogenesis. Also, the dynamic culture systems are being explored to replicate in vivo conditions more accurately. In this study, we aimed to utilize a perfusion mini-bioreactor for the dynamic culture of mouse spermatogonial stem cells on decellularized testicular matrix plates supplemented with exosomes. Our goal was to assess the progression of the spermatogenesis process through histological, immunohistochemical, and molecular analyses over four weeks. Human testicular tissues were decellularized using 1% sodium dodecyl sulfate and were then fabricated into thin plates using a cryostat. Sertoli and spermatogonial stem cells were isolated from neonate mouse testis and seeded onto the decellularized testicular matrix plates. A mini-perfusion bioreactor was employed to create dynamic culture conditions. Also, MSCs-derived exosomes were introduced to the culture medium, alone or in combination with a spermatogenic medium containing numerous chemical factors. The histological, IHC, and molecular analyses were performed at the end of the experiment. Our decellularization procedure successfully preserved the ECM components, while eliminating native cells. The isolated cells expressed PLZF and VIMENTIN markers, confirming the presence of SSCs and Sertoli cells. The seeded scaffolds exhibited proper homing, viability, proliferation, and differentiation of the cells towards in vitro spermatogenesis. Also, exosome treatment is capable of enhancing the spermatogenic potential of SSCs. Our findings indicate that the dynamic culture system significantly promoted the proliferation and differentiation of SSCs into mature spermatozoa. The use of exosomes further enhanced these effects, as evidenced by improved cellular viability, reduced apoptosis, and advanced spermatogenesis to the elongated spermatid stage. The combined treatment of exosomes and spermatogenic medium showed a synergistic effect, yielding superior outcomes in terms of sperm cell maturity and functionality. This study underscores the potential of combining decellularized testicular matrices with exosome therapy in a dynamic culture set up to advance the field of reproductive biology and fertility restoration.

Dendritic cell maturation is induced by p53-armed oncolytic adenovirus via tumor-derived exosomes enhancing systemic antitumor immunity.

Dendritic cells (DCs) are crucial in cancer immunity, because they activate cytotoxic T cells by presenting tumor antigens. Recently, oncolytic virus therapy has been recognized as a systemic immune stimulator. We previously developed a telomerase-specific oncolytic adenovirus (OBP-301) and a p53-armed OBP-301 (OBP-702), demonstrating that these viruses strongly activate systemic antitumor immunity. However, their effects on DCs remained unclear. In the present study, the aim was to elucidate the mechanisms of DC activation by OBP-702, focusing particularly on tumor-derived exosomes. Exosomes (Exo53, Exo301, or Exo702) were isolated from conditioned media of human or murine pancreatic cancer cell lines (Panc-1, MiaPaCa-2, and PAN02) after treatment with Ad-p53, OBP-301, or OBP-702. Exo702 derived from Panc-1 and MiaPaCa-2 cells significantly upregulated CD86, CD80, CD83 (markers of DC maturation), and IFN-γ in DCs in vitro. Similarly, Exo702 derived from PAN02 cells upregulated CD86 and IFN-γ in bone marrow-derived DCs in a bilateral PAN02 subcutaneous tumor model. This DC maturation was inhibited by GW4869, an inhibitor of exosome release, and anti-CD63, an antibody targeting the exosome marker. Intratumoral injection of OBP-702 into PAN02 subcutaneous tumors significantly increased the presence of mature DCs and CD8-positive T cells in draining lymph nodes, leading to long-lasting antitumor effects through the durable activation of systemic antitumor immunity. In conclusion, tumor-derived exosomes play a significant role in DC maturation following OBP-702 treatment and are critical for the systemic activation of antitumor immunity, leading to the abscopal effect.

Effect of Astaxanthin in Cytoprotection and Maturation in Vitro Differentiation Process to Insulin-Producing Cells

Asx is a fat-soluble xanthophyll carotenoid, one of its most relevant properties is its antioxidant activity. Depending on the dose and environment, it participates in an adequate regulation of ROS/RNS at optimal levels, either directly scavenger or indirectly through the stimulation of “antioxidant” pathways such as NRF2. This regulation and interactions within the signaling pathways are studied here in order to find information that provides data to elucidate the effects that may influence the processes of obtaining IPC and the study of the effect of molecules that increase the efficiency of the cellular response during the trans differentiation process. We found that adding Asx to a cocktail of differentiation molecules to obtain IPC from DPSC increases the efficiency of insulin production, as well as the expression of important markers for the maturity and identity of pancreatic β-type cells (NGN3, PDX1, MAFA). It was also revealed that Asx favors the proper regulation of oxidative stress caused during the process of cellular trans differentiation of DPSC towards IPC, through the direct inactivation of ROS and the increase in the expression of NRF2 as well as the decrease of their principal inhibitor KEAP1, favoring the maturity of the IPC. These results suggest the potential use of Asx to deepen the knowledge of its interaction with other signaling pathways that favor the generation of IPC and other cells sensitive to oxidative stress, and thereby lay the foundations for a possible cell replacement therapy as in DM1.

Homeostatic self-MHC-I recognition regulates anti-metastatic function of mature lung natural killer cells

Natural killer (NK) cells are important innate immune effector cells for controlling tumor growth and metastasis. Differentiated mature NK cells preferentially reside in the peripheral tissues and express higher levels of self-major histocompatibility complex class I (MHC-I)-recognizing inhibitory receptors. MHC-I recognition by NK cells are known to be important for their development and maturation processes, however, the role of homeostatic MHC-I recognition in maintaining effector functions of mature NK cells in the peripheral tissues needs to be elucidated. In this study, we utilized a pan anti-MHC-I blocking monoclonal antibody (anti-MHC-I) to examine the role of homeostatic MHC-I recognition in the response of pulmonary mature NK cells in an experimental lung metastasis model of B16F10 melanoma. Anti-MHC-I treatment showed significant inhibition of the lung metastasis of B16F10 melanoma in NK cell- and IFN-γ-dependent mechanisms. The blockade of homeostatic MHC-I recognition increased mature lung NK cell responsiveness, such as direct cytotoxicity and IFN-γ production, rather than the number of lung NK cells. Mechanistically, the gene expression of activating receptors including DNAX accessory molecule-1 (DNAM-1) was upregulated in NK cells treated with anti-MHC-I, and further the enhanced NK cell cytotoxicity against B16F10 cells was DNAM-1-dependent. Collectively, homeostatic self-MHC-I recognition regulates anti-metastatic function of mature lung NK cells by restraining the expression of activating receptors.

Depletion of conventional CD4+ T cells is required for robust priming and dissemination of tumor antigen-specific CD8+ T cells in the setting of anti-CD4 therapy

BackgroundOvercoming immune suppression is a major barrier to eliciting potent CD8+ T cell responses against cancer. Treatment with anti-CD4 monoclonal antibody is an effective means for eliminating CD4+Foxp3+ regulatory (Treg)...

Expression pattern of Calbindin-D28k, NeuN, ATOH1 and EN2 genes in the human fetal cerebellum

BackgroundCertain molecules are expressed specific to cerebellar neurons and certain genes play specific role in the development of cerebellum. MethodsThis is a descriptive observational study carried out in human fetal cerebellum, fluorescent immunohistochemistry was used to study the expression of Calbindin-D28k and neuronal nuclease (NeuN), and while the expression of ATOH1 (Atonal homolog 1) and EN2 (Engrailed -2) genes were quantified with the help of qPCR. ResultsCalbindin-D28k was highly immunoreactive in the Purkinje cells and located in their cytoplasm, nucleus and dendrites whereas absent in their axons. NeuN was expressed weakly in the perinuclear cytoplasm and nucleus of granule cells whereas absent in their dendrites and axons. ATOH1 gene was upregulated during third trimester whereas EN2 gene was upregulated during second as well as third trimesters. ConclusionDistribution and intensity of Calbindin-D28k and NeuN proteins in the human fetal cerebellum increased with the increase of fetal age. Expression of ATOH1 and EN2 genes indicated that second and third trimesters are the crucial periods for the proliferation, migration and maturation of granule cells and essential for the establishment of normal morphology of human fetal cerebellum and its development.

Bioengineering human heavy-chain nanoferritin for glioblastoma multiforme-specific delivery and efficient immunotherapy

The blood–brain barrier (BBB) caused limited drug accumulation and programmed death-ligand 1 (PD-L1) compromised immunosuppressive microenvironment are bottlenecks in glioblastoma multiforme (GBM) treatment. Herein, we bioengineered a human heavy chain nanoferritin (HTE NPs) for GBM-specific delivery and efficient immunotherapy. To achieve targeted mitochondrial metabolic regulation, we connected triphenylphosphonium (TPP) with the photosensitive agent IR780 to form T780, which self-assembles with esomeprazole magnesium (EM) and human H-ferritin (HFn) to obtain HTE NPs. HFn endows HTE NPs with BBB penetration and specific site-targeting capabilities, thereby enhancing tumor accumulation and pH-responsive release within GBM cells. T780 subsequently induces mitochondrial damage, activating the AMP-dependent protein kinase (AMPK) pathway and blocking the transmission of T-cell inhibitory signals through the phosphorylation of PD-L1, thereby promoting T-cell activation. Moreover, EM inhibits the release of extracellular vesicles (EVs), reducing the inhibitory effect of PD-L1 on circulating T cells. Furthermore, mitochondrial impairment-induced mitochondrial DNA (mtDNA) leakage activates the cGAS-STING signaling pathway, inducing the maturation of dendritic cells (DCs), recruiting supportive immune cells to clear GBM cells, and improving the immunosuppressive microenvironment. Therefore, HTE NPs not only achieve efficient tumor accumulation but also facilitate the activation immune response, providing a promising strategy for the clinical treatment of GBM.

A theranostic photosensitizer-conjugated albumin co-loading with resiquimod for cancer-targeted imaging and robust photo-immunotherapy

Cancer immunotherapy remains a low immune response rate in clinic because of dominant immunosuppressive tumor microenvironment (TME) and lack of effective drug to specifically remodel the TME. In this work, we introduced a tumor-seeking human serum albumin (HSA) based delivery platform by covalently conjugating with a tumor-targeting near-infrared (NIR) photosensitizer (IR-DBI) and non-covalently loading of immune modulator Resiquimod (R848). HSA exhibited tumor-preferential accumulation after covalent conjugation with IR-DBI. Meanwhile, HSA restricted the rotation of IR-DBI, narrowed the HOMO-LUMO energy gap, significantly enhanced fluorescent intensity and dual-modal phototherapy (PTT/PDT). The enhanced phototherapeutic effect further induced robust ICD effect. More importantly, non-covalent loading of R848 could be released from HSA at tumor sites by laser irradiation-induced heat. The in-situ release of R848 in TME efficiently promoted the maturation of DC cells and repolarized M2 macrophages to M1 macrophages. Consequently, robust photo-induced antitumor immunity was triggered in the different mice models bearing primary and distant tumors or lung metastasis, which was further enhanced by combining with CTLA-4 blockade therapy. Taken together, this work may present a versatile albumin composite which exhibits tumor-preferential accumulation and imaging-guided PDT/PTT/immunotherapy.

Impact of Porosity and Stiffness of 3D Printed Polycaprolactone Scaffolds on Osteogenic Differentiation of Human Mesenchymal Stromal Cells and Activation of Dendritic Cells.

Despite the potential of extrusion-based printing of thermoplastic polymers in bone tissue engineering, the inherent nonporous stiff nature of the printed filaments may elicit immune responses that influence bone regeneration. In this study, bone scaffolds made of polycaprolactone (PCL) filaments with different internal microporosity and stiffness was 3D-printed. It was achieved by combining three fabrication techniques, salt leaching and 3D printing at either low or high temperatures (LT/HT) with or without nonsolvent induced phase separation (NIPS). Printing PCL at HT resulted in stiff scaffolds (modulus of elasticity (E): 403 ± 19 MPa and strain: 6.6 ± 0.1%), while NIPS-based printing at LT produced less stiff and highly flexible scaffolds (E: 53 ± 10 MPa and strain: 435 ± 105%). Moreover, the introduction of porosity by salt leaching in the printed filaments significantly changed the mechanical properties and degradation rate of the scaffolds. Furthermore, this study aimed to show how these variations influence proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hBMSC) and the maturation and activation of human monocyte-derived dendritic cells (Mo-DC). The cytocompatibility of the printed scaffolds was confirmed by live-dead imaging, metabolic activity measurement, and the continuous proliferation of hBMSC over 14 days. While all scaffolds facilitated the expression of osteogenic markers (RUNX2 and Collagen I) from hBMSC as detected through immunofluorescence staining, the variation in porosity and stiffness notably influenced the early and late mineralization. Furthermore, the flexible LT scaffolds, with porosity induced by NIPS and salt leaching, stimulated Mo-DC to adopt a pro-inflammatory phenotype marked by a significant increase in the expression of IL1B and TNF genes, alongside decreased expression of anti-inflammatory markers, IL10 and TGF1B. Altogether, the results of the current study demonstrate the importance of tailoring porosity and stiffness of PCL scaffolds to direct their biological performance toward a more immune-mediated bone healing process.

Fiber-based Biomaterial Scaffolds for Cell Support towards the Production of Cultivated Meat

The in vitro production of animal-derived foods via cellular agriculture is emerging as a key solution to global food security challenges. Here, the potential for fiber-based scaffolds, including silk and cotton, in the cultivation of muscle cells for tissue formation was pursued. Mechanical properties and cytocompatibility with the mouse myoblast cell line C2C12 and immortalized bovine muscle satellite cells (iBSCs) were assessed, as well as pre-digestion options for the materials due to their resilience within the human digestive track. The fibers supported cell adhesion, proliferation, and guided muscle cell orientation, facilitating myotube formation per differentiation. A progressive increase in biomass was also documented. Interestingly, iBSC proliferation was enhanced with coatings of recombinant proteins while C2C12 cells showed minimal response. Thus, both cotton and silk yarns were suitable as fiber-based scaffolds towards cell supportive goals, suggesting an alternative path toward structured protein-rich foods via this initial stage of textile engineering for food. Biomass prediction models were generated, enabling forecasts of cell growth and maturation across various scaffold conditions and cell types. This capability enhances the precision of the cultivation process towards an engineering approach, building on the inherent benefits of hierarchical muscle tissue structure, but here via textile engineering with these initial muscle-coated edible fibers. Further, the approach offers to reduce costs by optimizing cultivation time and media needs. These approaches are part of a foundation for future scalable and sustainable cultivated meat production. Statement of SignificanceThis research investigates the use of one-dimensional fiber-based scaffolds for cultivated meat production, contributing to advancements in cellular agriculture. It introduces a method to measure changes in biomass and scaffold degradation throughout the cultivation process. Additionally, our development of biomass prediction models improves the precision and predictability of cultivated meat production. This research not only aids in scaling up cultivated meats but also enhances the use of textile engineering techniques in tissue engineering, paving the way for producing complex, three-dimensional meat structures more sustainably.

A Thorough Navigation of miRNA's Blueprint in Crafting Cardiovascular Fate.

Cardiovascular diseases contribute significantly to global morbidity and mortality. MicroRNAs are crucial in the development and progression of these diseases by regulating gene expression in various cells and tissues. Their roles in conditions like atherosclerosis, heart failure, myocardial infarction, and arrhythmias have been widely researched. The present study provides an overview of existing evidence regarding miRNAs' role in cardiovascular disease pathogenesis. Furthermore, the study examines current state-of-the-art technologies used in the study of miRNAs in cardiovascular disease. As a final point, we examine how miRNAs may serve as disease biomarkers, therapeutic targets, and prognostic indicators. In cardiology, microRNAs, small noncoding RNA molecules, are crucial to the posttranscriptional regulation of genes. Their role in regulating cardiac cell differentiation and maturation is critical during the development of the heart. They maintain the cardiac function of an adult heart by contributing to its electrical and contractile activity. By binding to messenger RNA molecules, they inhibit protein translation or degrade mRNA. Several cardiovascular diseases are associated with dysregulation of miRNAs, including arrhythmias, hypertension, atherosclerosis, and heart failure. miRNAs can be used as biomarkers to diagnose and predict diseases as well as therapeutic targets. A variety of state-of-the-art technologies have aided researchers in discovering, profiling, and analyzing miRNAs, including microarray analysis, next-generation sequencing, and others. Developing new diagnostics and therapeutic approaches is becoming more feasible as researchers refine their understanding of miRNA function. Ultimately, this will reduce the burden of cardiovascular disease around the world.

Restoring sweat gland function in mice using regenerative sweat gland cells derived from chemically reprogrammed human epidermal keratinocytes

The regeneration of sweat glands (SwGs) plays a pivotal role in the functional recovery of extensive skin wounds. Recent research has illuminated the possibility of reprogramming human epidermal keratinocytes (HEKs) into induced SwG cells through the ectopic expression of ectodysplasin A. However, the clinical application of this genetic manipulation approach is inherently limited. In this study, we present findings demonstrating that a combination of six compounds can effectively and speedily reprogram HEKs in culture into fully functional SwG cells. These chemically induced SwG-like cells (ciSGCs) closely resemble the morphology, phenotypes, and functional properties of human primary SwG ductal cells. Furthermore, ciSGCs can be stimulated to differentiate into mature SwG cell types in vitro. In a 3D culture system, they can also generate SwG organoids that exhibit structural and biological features akin to native SwGs. Upon transplantation into scalded mouse paw skin, ciSGCs significantly expedited cutaneous wound healing and completely restored the structural and functional aspects of the SwGs. In conclusion, the small molecule cocktail-directed SwG reprogramming offers a non-transgenic and controllable strategy for producing high-quality, clinical-grade SwG cells, showing immense potential for the treatment of burn patients.

1α,25-hydroxyvitamin D3 alleviated rotavirus infection induced ferroptosis in IPEC-J2 cells by regulating the ATF3-SLC7A11-GPX4 axis

Rotavirus (RV) mainly infects mature intestinal epithelial cells and impairs intestinal absorption function, which leads to the death of infected cells and eventually fatal diarrhea. Ferroptosis is a novel regulatory cell death pattern, which can be caused by virus infection. 1α,25-hydroxyvitamin D3 (1,25D3) has an anti-RV infection effect and can regulate ferroptosis. However, whether RV infection can induce ferroptosis, and whether 1,25D3 can inhibit RV infection by regulating ferroptosis has not yet been studied. Present study shows that RV infection or erastin treatment induces IPEC-J2 cell death, which results in mitochondrial shrinkage, decreased mitochondrial membrane potential (MMP) and glutathione (GSH) content, increased MMP, intracellular Fe2+, reactive oxygen species (ROS), and malondialdehyde (MDA) contents. Meanwhile, ferrostatin-1 (Fer-1), liproxstatin-1 (Lip-1), and deferoxamine (DFO) treatment can effectively reverse the increase of intracellular Fe2+, ROS and MDA levels induced by RV infection. Moreover, RV infection increases activating transcription factor 3 (ATF3) mRNA and protein expressions, and inhibited SLC7A11 and glutathione peroxidase 4 (GPX4) expressions, which was partially alleviated by siATF3. 1,25D3 treatment significantly eliminates RV induced ferroptosis via ATF3-SLC7A11-GPX4 axis. Therefore, our results reveals that RV infection induces ferroptosis in IPEC-J2 cell and 1,25D3 alleviates RV induced ferroptosis by regulating the ATF3-SLC7A11-GPX4 axis.

A multifunctional cascade gas-nanoreactor with MnO2 as a gatekeeper to enhance starvation therapy and provoke antitumor immune response

Glucose oxidase (GOx)-mediated starvation therapy is an effective tumor treatment that blocks energy and activates the immune response. However, the insufficient tumor immunogenicity and immunosuppressive tumor microenvironment (TME) limited its therapeutic efficacy. To address this, we have designed a multifunctional cascade gas-nanoreactor with a MnO2 coating, which serves as an out gatekeeper to encapsulate both GOx and a carbon monoxide (CO) donor (denoted as GCM). Due to the protective effect of MnO2 coating, GCM maintains better stability in normal physiological environments, enhancing the catalytic activity of GOx and minimizing toxic side effects. Upon accumulation in the tumor, the degradation of MnO2 coating exposes the GOx enzyme, thereby initiating a cascade catalysis reaction to generate hydrogen peroxide (H2O2) and release CO in the hypoxic conditions. Additionally, the released Mn2+ reacts with H2O2 to generate toxic hydroxyl radical (•OH) as chemodynamic therapy (CDT). The synergistic treatments of starvation therapy, CO gas therapy and CDT effectively kill cancer cells and amplify immunogenic cell death (ICD), maturing DC cells and activating anti-tumor immune response. Furthermore, the released CO increases M1 macrophages infiltration and reduces myeloid-derived suppressor cells (MDSCs) infiltration, thus reversing the immunosuppressive TME. This multifunctional gas-nanoreactor provides a strategy for CO gas generation to trigger a robust anti-tumor immune response and has the potential for clinical application in cancer immunotherapy. Statement of significanceA multifunctional cascade gas-nanoreactor with a MnO2 gatekeeper was developed to perform synergistic treatments involving starvation therapy, CO gas therapy and chemodynamic therapy (CDT) for tumor elimination. The MnO2 gatekeeper enhanced the catalytic activity of GOx within the nanoreactor by generating oxygen, thereby minimizing toxic side effects after intravenous injection. The gas-nanoreactor amplified ICD through synergistic treatments to mature DC cells and activate anti-tumor immune response. Furthermore, the released CO could reverse the immunosuppression of the TME to enhance cancer immunotherapy. The combination strategy utilizing the gas-nanoreactor demonstrates clinical potential for facilitating cancer immunotherapy.

PTEN deletion in the adult dentate gyrus induces epilepsy.

Embryonic and early postnatal promotor-driven deletion of the phosphatase and tensin homolog (PTEN) gene results in neuronal hypertrophy, hyperexcitable circuitry and development of spontaneous seizures in adulthood. We previously documented that focal, vector-mediated PTEN deletion in mature granule cells of adult dentate gyrus triggers dramatic growth of cell bodies, dendrites, and axons, similar to that seen with early postnatal PTEN deletion. Here, we assess the functional consequences of focal, adult PTEN deletion, focusing on its pro-epileptogenic potential. PTEN deletion was accomplished by injecting AAV-Cre either bilaterally or unilaterally into the dentate gyrus of double transgenic PTEN-floxed, ROSA-reporter mice. Hippocampal recording electrodes were implanted for continuous digital EEG with concurrent video recordings in the home cage. Electrographic seizures and epileptiform spikes were assessed manually by two investigators, and corelated with concurrent videos. Spontaneous electrographic and behavioral seizures appeared after focal PTEN deletion in adult dentate granule cells, commencing around 2 months post-AAV-Cre injection. Seizures occurred in the majority of mice with unilateral or bilateral PTEN deletion and led to death in several cases. PTEN-deletion provoked epilepsy was not associated with apparent hippocampal neuron death; supra-granular mossy fiber sprouting was observed in a few mice. In summary, focal, unilateral deletion of PTEN in the adult dentate gyrus suffices to provoke time-dependent emergence of a hyperexcitable circuit generating hippocampus-origin, generalizing spontaneous seizures, providing a novel model for studies of adult-onset epileptogenesis.

Enhanced Purification of Uterine Smooth Muscle Cells from Adenomyosis Using a Novel Dual- Enzyme Digestion Method

Uterine adenomyosis causing attention to the abnormal smooth muscle layer has recently received increasing attention, which has created the need for a method that can efficiently collect high purity uterine smooth muscle cells (USMC) in a laboratory setting. In this study, we explored the composition ratios of the digestion solution to obtain the optimal digestion solution (DM4). Furthermore, we tested the superiority of the two methods of obtaining adenomyotic uterine smooth muscle by comparing DM4 with the conventional tissue adhesion method by growth rate, single-cell RNA sequencing, and purity of fluorescence identification. The results demonstrated that USMCs produced by the DM4 digestion method exhibited significantly higher rates of proliferation and were more effective in generating mature smooth muscle cells of high purity compared to those obtained using tissue adherence methods, which is more inclined to isolate the progenitor cell population. This study presents a reliable method for isolating USMCs and provides a solid foundation for future studies on the etiology and mechanism of adenomyosis.

Novel features of Drosophila hematopoiesis uncovered by long-term live imaging

Stem cells are subject to continuous regulation to ensure that the correct balance between stem cell differentiation and self-renewal is maintained. The dynamic and ongoing nature of stem cell regulation, as well as the complex signaling microenvironment in which stem cells are typically found, means that studying them in their endogenous environment in real time has multiple advantages over static fixed-sample approaches. We recently described a method for long-term, ex-vivo, live imaging of the blood progenitors in the Drosophila larval hematopoietic organ, the Lymph Gland (LG). This methodology has allowed us to analyze multiple aspects of fly hematopoiesis, in real time, in a manner that could not be carried out previously. Here, we describe novel insights derived from our quantitative live imaging approach. These insights include: the identification of extensive filopodia in the progenitors and description of their morphology and dynamics; visualization and quantitative analysis of JAK/STAT signaling in progenitors by the simultaneous tracking of thousands of vesicles containing internalized Domeless receptors; quantitative analysis of the location, morphology, and dynamics of mitochondria in blood progenitors; long-term tracking of patterns of cell division and migration of mature blood cell in the LG; long-term tracking of multiple cell behaviors in the distal committed progenitors; analysis of Ca2+ signaling of blood progenitors in the secondary lobes of the LG. Together, these observations illustrate the power of imaging fly hematopoiesis in real time and identify many previously undescribed processes and behaviors in the LG that are likely to play important roles in the regulation of progenitor differentiation and self-renewal.

CAR-NK cells derived from cord blood originate mainly from CD56-CD7+CD34-HLA-DR-Lin- NK progenitor cells

Cord blood (CB)–derived chimeric antigen receptor (CAR)–natural killer (NK) cells targeting CD19 have been shown to be effective against B-cell malignancies. While human CD56+ NK cells can be expanded in vitro, NK cells can also be differentiated from hematopoietic progenitor cells. It is still unclear whether CAR-NK cells originate from mature NK cells or NK progenitor cells in CB. Here we determined that CAR-NK cells were predominantly derived from CD56- NK progenitor cells. We first found that substantial numbers of CD19 CAR-NK cells were produced from CD56- CB mononuclear cells after in vitro culture for 2 weeks. Single-cell RNA sequencing analysis of CD56-CD3-CD14-CD19- CB mononuclear cells revealed that these cells could be subdivided into three subpopulations based on the expression of CD34 and HLA-DR. NK cells originated primarily from CD34-HLA-DR- cells. In addition, among the CD34-HLA-DR- cells, only CD7+ cells could differentiate into NK cells. These results indicate that CD56-CD7+CD34- HLA-DR- lineage marker (Lin)- cells are the major origin of human CB-derived CAR-NK cells, indicating the importance of developing methods to enhance the quality and quantity of NK cells produced from these NK progenitor cells.

Human Dendritic Cell Differentiation in Hematopoietic Stem Cell-Transplanted NOG hFLT3L Tg/mFlt3 KO Humanized Mice

Human immune system-reconstituted humanized mice are useful animal models to study human immunology in vivo. Human hematopoietic stem cell-transferred NOG mice are well recognized as humanized immune system models with reconstitution of mature lymphoid lineage cells such as T and B cells. However, human myeloid lineage cells including dendritic cells (DCs) do not fully differentiate in conventional NOG mice. DCs play a crucial role in adaptive immunity through antigen presentation to T cells to acquire antigen specificity. In this study, we established a novel humanized mouse with human DC differentiation. To induce DCs, we generated human Fms-like tyrosine kinase 3 ligand (hFLT3L) transgenic NOG (hFLT3L-Tg) mice and transferred human CD34+ hematopoietic stem cells (HSC) into them. Unexpectedly, low frequency of human cell engraftment was observed in the hFLT3L-Tg mice after HPC reconstitution. In the Tg mice, mouse CD11b+Gr1− myeloid cells were markedly expanded in the bone marrow due to the cross-reaction between hFLT3L and mouse Flt3 receptor, and these myeloid leukemia-like cells interfered with the engraftment of human hematopoietic cells in hFLT3L-Tg mice. To avoid this cross-reaction, we further generated NOG FLT3 receptor KO (mFlt3 KO) mice by CRISPR/Cas9 technique, and the KO mice combined with hFLT3L Tg mice to create hFLT3L Tg/mFlt3 KO (FL Tg/KO) mice. Mouse CD11b+Gr1− leukemia-like cells did not proliferate in FL Tg/KO mice due to blockade of the FLT3 signals in mouse leukocytes. After human HSC transplantation, human CD45+ cells were successfully engrafted in FL Tg/KO mice. Furthermore, major subsets of human DC populations, cDC1, cDC2, and pDC, and skin Langerhans cells were significantly differentiated in FL Tg/KO mice. Therefore, these humanized mouse models are potentially valuable in the investigation of DC-mediated human adaptive immune responses in vivo.