Challenges of Cell Counting in Cell Therapy Products.

Cell counting is a common and fundamental cell measurement technique that plays a crucial role in the development and quality control of cell therapy products. However, accurate and reliable cell counting can be challenging owing to the complexity of cell preparations, diverse counting purposes, and various counting methods. This review summarizes the challenges encountered in cell counting for cell therapy products and provides strategies to improve the cell counting accuracy, thereby guiding the counting process and ensuring the quality of cell therapy products.

Utilization of convolutional neural networks to analyze microscopic images for high-throughput screening of mesenchymal stem cells.

This work investigated the high-throughput classification performance of microscopic images of mesenchymal stem cells (MSCs) using a hyperspectral imaging-based separable convolutional neural network (CNN) (H-SCNN) model. Human bone marrow mesenchymal stem cells (hBMSCs) were cultured, and microscopic images were acquired using a fully automated microscope. Flow cytometry (FCT) was employed for functional classification. Subsequently, the H-SCNN model was established. The hyperspectral microscopic (HSM) images were created, and the spatial-spectral combined distance (SSCD) was employed to derive the spatial-spectral neighbors (SSNs) for each pixel in the training set to determine the optimal parameters. Then, a separable CNN (SCNN) was adopted instead of the classic convolutional layer. Additionally, cultured cells were seeded into 96-well plates, and high-functioning hBMSCs were screened using both manual visual inspection (MV group) and the H-SCNN model (H-SCNN group), with each group consisting of 96 samples. FCT served as the benchmark to compare the area under the curve (AUC), F1 score, accuracy (Acc), sensitivity (Sen), specificity (Spe), positive predictive value (PPV), and negative predictive value (NPV) between the manual and model groups. The best classification Acc was 0.862 when using window size of 9 and 12 SSNs. The classification Acc of the SCNN model, ResNet model, and VGGNet model gradually increased with the increase in sample size, reaching 89.56 ± 3.09, 80.61 ± 2.83, and 80.06 ± 3.01%, respectively at the sample size of 100. The corresponding training time for the SCNN model was significantly shorter at 21.32 ± 1.09 min compared to ResNet (36.09 ± 3.11 min) and VGGNet models (34.73 ± 3.72 min) (P < 0.05). Furthermore, the classification AUC, F1 score, Acc, Sen, Spe, PPV, and NPV were all higher in the H-SCNN group, with significantly less time required (P < 0.05). Microscopic images based on the H-SCNN model proved to be effective for the classification assessment of hBMSCs, demonstrating excellent performance in classification Acc and efficiency, enabling its potential to be a powerful tool in future MSCs research.

Mendelian randomization study of inflammatory bowel disease and type 1 diabetes.

PURPOSE: Our purpose was to investigate and test the causal relationship between type 1 diabetes (T1D) and inflammatory bowel disease (IBD) and its major phenotypes, including ulcerative colitis (UC) and Crohn's disease (CD), in two large datasets. METHODS: We obtained IBD samples from the largest publicly available genome-wide association study (GWAS), as well as the FinnGen database and the publicly accessible IEU GWAS database of T1D. We employed a two-sample Mendelian randomization approach to assess bidirectional causality using the inverse variance weighting (IVW) method as the primary outcome. RESULTS: Genetic predisposition to T1D was associated with reduced risk of IBD (IVW: odds ratio (OR), 0.867; 95% confidence interval (CI), [0.852, 0.883]; P < 0.001), UC (OR = 0.879 [0.823, 0.939], P < 0.001), and CD (OR = 0.925 [0.872, 0.981], P = 0.009). The republication results found IBD genetically possessed negative association with T1D (OR = 0.781 [0.684, 0.891], P < 0.001). Additionally, a meta-analysis of results was conducted to prove the strong evidence between T1D and CD (OR = 0.95 [0.91, 0.98]; p = 0.01). CONCLUSIONS: This study first demonstrated a causal effect of TID on the reduced risk of CD in the mendelian randomization study.

A GMP-compliant manufacturing method for Wharton's jelly-derived mesenchymal stromal cells.

BACKGROUND: Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) hold great therapeutic potential in regenerative medicine. Therefore, it is crucial to establish a Good Manufacturing Practice (GMP)-compliant methodology for the isolation and culture of WJ-MSCs. Through comprehensive research, encompassing laboratory-scale experiments to pilot-scale studies, we aimed to develop standardized protocols ensuring the high yield and quality of WJ-MSCs manufacturing. METHODS: Firstly, optimization of parameters for the enzymatic digestion method used to isolate WJ-MSCs was conducted. These parameters included enzyme concentrations, digestion times, seeding densities, and culture media. Additionally, a comparative analysis between the explant method and the enzymatic digestion method was performed. Subsequently, the consecutive passaging of WJ-MSCs, specifically up to passage 9, was evaluated using the optimized method. Finally, manufacturing processes were developed and scaled up, starting from laboratory-scale flask-based production and progressing to pilot-scale cell factory-based production. Furthermore, a stability study was carried out to assess the storage and use of drug products (DPs). RESULTS: The optimal parameters for the enzymatic digestion method were a concentration of 0.4 PZ U/mL Collagenase NB6 and a digestion time of 3 h, resulting in a higher yield of P0 WJ-MSCs. In addition, a positive correlation between the weight of umbilical cord tissue and the quantities of P0 WJ-MSCs has been observed. Evaluation of different concentrations of human platelet lysate revealed that 2% and 5% concentrations resulted in similar levels of cell expansion. Comparative analysis revealed that the enzymatic digestion method exhibited faster outgrowth of WJ-MSCs compared to the explant method during the initial passage. Passages 2 to 5 exhibited higher viability and proliferation ability throughout consecutive passaging. Moreover, scalable manufacturing processes from the laboratory scale to the pilot scale were successfully developed, ensuring the production of high-quality WJ-MSCs. Multiple freeze-thaw cycles of the DPs led to reduced cell viability and viable cell concentration. Subsequent thawing and dilution of the DPs resulted in a significant decrease in both metrics, especially when stored at 20-27 °C. CONCLUSION: This study offers valuable insights into optimizing the isolation and culture of WJ-MSCs. Our scalable manufacturing processes facilitate the large-scale production of high-quality WJ-MSCs. These findings contribute to the advancement of WJ-MSCs-based therapies in regenerative medicine.

Heterozygous Spink1 c.194+2T>C mutation promotes chronic pancreatitis after acute attack in mice.

BACKGROUND & AIMS: Mutations in genes, including serine protease inhibitor Kazal-type 1 (SPINK1), influence disease progression following sentinel acute pancreatitis event (SAPE) attacks. SPINK1 c.194+2T > C intron mutation is one of the main mutants of SPINK1，which leads to the impairment of SPINK1 function by causing skipping of exon 3. Research on the pathogenesis of SAPE attacks would contribute to the understanding of the outcomes of acute pancreatitis. Therefore, the aim of the study was to clarify the role of SPINK1 c.194+2T > C mutation in the CP progression after an AP attack. METHODS: SAPE attacks were induced in wildtype and SPINK mutant (Spink1 c.194+2T > C) mice by cerulein injection. The mice were sacrificed at 24 h, 14 d, 28 d, and 42 d post-SAPE. Data-independent acquisition (DIA) proteomic analysis was performed for the identification of differentially expressed protein in the pancreatic tissues. Functional analyses were performed using THP-1 and HPSCs. RESULTS: Following SAPE attack, the Spink1 c.194+2T > C mutant mice exhibited a more severe acute pancreatitis phenotype within 24 h. In the chronic phase, the chronic pancreatitis phenotype was more severe in the Spink1 c.194+2T > C mutant mice after SAPE. Proteomic analysis revealed elevated IL-33 level in Spink1 c.194+2T > C mutant mice. Further in vitro analyses revealed that IL-33 induced M2 polarization of macrophages and activation of pancreatic stellate cells. CONCLUSION: Spink1 c.194+2T > C mutation plays an important role in the prognosis of patients following SAPE. Heterozygous Spink1 c.194+2T > C mutation promotes the development of chronic pancreatitis after an acute attack in mice through elevated IL-33 level and the induction of M2 polarization in coordination with pancreatic stellate cell activation.

Association between dietary inflammation index and inflammatory bowel disease in adults: Results from National Health and Nutrition Examination Survey 2009-2010.

Previous study has demonstrated that the Dietary Inflammation Index (DII) played a role in the risk of inflammatory bowel disease (IBD), however, the prevalence and risk factors for IBD are distinct across locations and groups, and therefore, the findings are debatable and warrant further investigation. A total of 4363 participants were calculated in the National Health and Nutrition Examination Survey (NHANES) 2009 to 2010, of whom 1.21% self-reported a history of IBD. DII values were performed as a good predictor of dietary inflammation based on data from two 24-h dietary reviews in the NHANES database. Comparing the multifarious effects along with variations of the whole population by grouping populations according to DII quartiles, dietary inflammation levels increased progressively from DII quartile 1(Q1) to quartile 4(Q4). The association between DII and IBD was tested with multi-variable logistic regression models, subgroup analyses and weighted generalized additive models. Participants in the Q4 group showed the highest levels of C-reactive protein and reduced haemoglobin and albumin levels. Logistic regression confirmed the odds ratios (95% confidence intervals) of IBD for DII were 0.99 (0.86, 1.15), 0.97 (0.84, 1.13) and 0.80 (0.66, 0.98) in models 1, 2 and 3, respectively. The negative correlation between DII and IBD among United States adults from the NHANES database became increasingly apparent as covariates were adjusted. Subgroup analyses and smoothed curve fitting confirmed the inverse results. The study revealed that DII was correlated with the overall physical well-being of participants. However, there was no significant association between DII and IBD.

Pancreas-directed AAV8-hSPINK1 gene therapy safely and effectively protects against pancreatitis in mice.

OBJECTIVE: Currently, there is no cure for chronic pancreatitis (CP). Germline loss-of-function variants in SPINK1 (encoding trypsin inhibitor) are common in patients with CP and are associated with acute attacks and progression of the disease. This preclinical study was conducted to explore the potential of adeno-associated virus type 8 (AAV8)-mediated overexpression of human SPINK1 (hSPINK1) for pancreatitis therapy in mice. DESIGN: A capsid-optimised AAV8-mediated hSPINK1 expression vector (AAV8-hSPINK1) to target the pancreas was constructed. Mice were treated with AAV8-hSPINK1 by intraperitoneal injection. Pancreatic transduction efficiency and safety of AAV8-hSPINK1 were dynamically evaluated in infected mice. The effectiveness of AAV8-hSPINK1 on pancreatitis prevention and treatment was studied in three mouse models (caerulein-induced pancreatitis, pancreatic duct ligation and Spink1 c.194+2T>C mouse models). RESULTS: The constructed AAV8-hSPINK1 vector specifically and safely targeted the pancreas, had low organ tropism for the heart, lungs, spleen, liver and kidneys and had a high transduction efficiency (the optimal expression dose was 2×1011 vg/animal). The expression and efficacy of hSPINK1 peaked at 4 weeks after injection and remained at significant level for up to at least 8 weeks. In all three mouse models, a single dose of AAV8-hSPINK1 before disease onset significantly alleviated the severity of pancreatitis, reduced the progression of fibrosis, decreased the levels of apoptosis and autophagy in the pancreas and accelerated the pancreatitis recovery process. CONCLUSION: One-time injection of AAV8-hSPINK1 safely targets the pancreas with high transduction efficiency and effectively ameliorates pancreatitis phenotypes in mice. This approach is promising for the prevention and treatment of CP.

Assessment of the triglyceride glucose index in adult patients with chronic diarrhea and constipation.

BACKGROUND: Accumulating evidence suggests that the gut microbiome is involved in the pathogenesis of insulin resistance (IR). However, the link between two of the most prevalent bowel disorders, chronic diarrhea and constipation, and the triglyceride glucose (TyG) index, a marker of IR, has not yet been investigated. AIM: To investigate the potential association between TyG and the incidence of chronic diarrhea and constipation. METHODS: This cross-sectional study enrolled 2400 participants from the National Health and Nutrition Examination Survey database from 2009-2010. TyG was used as an exposure variable, with chronic diarrhea and constipation as determined by the Bristol Stool Form Scale used as the outcome variables. A demographic investigation based on TyG quartile subgroups was performed. The application of multivariate logistic regression models and weighted generalized additive models revealed potential correlations between TyG, chronic diarrhea, and constipation. Subgroup analyses were performed to examine the stability of any potential associations. RESULTS: In the chosen sample, chronic diarrhea had a prevalence of 8.00%, while chronic constipation had a prevalence of 8.04%. In multiple logistic regression, a more prominent positive association was found between TyG and chronic diarrhea, particularly in model 1 (OR = 1.45; 95%CI: 1.17-1.79, P = 0.0007) and model 2 (OR = 1.40; 95%CI: 1.12-1.76, P = 0.0033). No definite association was observed between the TyG levels and chronic constipation. The weighted generalized additive model findings suggested a more substantial positive association with chronic diarrhea when TyG was less than 9.63 (OR = 1.89; 95%CI: 1.05-3.41, P = 0.0344), and another positive association with chronic constipation when it was greater than 8.2 (OR = 1.74; 95%CI: 1.02-2.95, P = 0.0415). The results of the subgroup analyses further strengthen the extrapolation of these results to a wide range of populations. CONCLUSION: Higher TyG levels were positively associated with abnormal bowel health.

Investigation on the correlation factors of positive Streptococcus pneumoniae antibody and IgG antibody level of Streptococcus pneumoniae in the elderly over 60 years old in Shenzhen.

BACKGROUND: Pneumococcal Polysaccharide Vaccine (PPV-23), designed to protect against the most common serotype of Streptococcus pneumoniae, is intended to protect the elderly and other high-risk groups. However, the immunogenicity of all 23 pneumococcal polysaccharide vaccines in older adults has not been thoroughly studied. OBJECTIVE: The purpose of this study is to look into the factors that influence the effect of the pneumonia vaccine on the elderly over 60 years old in Shenzhen, as well as their IgG antibody level against Streptococcus pneumoniae. METHODS: To determine the immune effectiveness of pneumococcal vaccination in older adults over 60 years old, we used the 3rd generation enzyme-linked immunosorbent assay to detect the antibody level of older adults to all 23 pneumococcal polysaccharide vaccines following pneumococcal immunization. RESULTS: Vaccination, the number of physical examinations, pneumonia knowledge, and the pneumonia vaccination policy of the elderly in Shenzhen were all positively correlated with Streptococcus pneumoniae antibody positivity. The distribution of subtypes did not differ between elderly adults (over 65) and younger adults (under 65). The GMCs of IgG antibodies to PPS were significantly lower in males than in females for types 7f, 18c and 19a. At the same time, we found that people with chronic respiratory disease have lower type 9n than people without chronic respiratory disease. Other chronic diseases, such as hypertension and diabetes, had no difference in subtype distribution. CONCLUSION: There was a statistically significant difference in antibody positivity rates for older people with more frequent medical check-ups in Shenzhen, indicating that publicity is playing a role. The effects of age, gender, and chronic diseases on naturally acquired anti-PPS IgG differ.

Targeting ZIP8 mediated ferroptosis as a novel strategy to protect against the retinal pigment epithelial degeneration.

The degeneration of retinal pigment epithelium (RPE) plays an important role in the development of age-related macular degeneration (AMD). However, the underlying mechanism remains elusive. In this study, we identified that ZIP8, a metal-ion transporter, plays a crucial role in the degeneration of RPE cells mediated by ferroptosis. ZIP8 was found to be upregulated in patients with AMD through transcriptome analysis. Upregulated ZIP8 was also observed in both oxidative-stressed RPE cells and AMD mouse model. Importantly, knockdown of ZIP8 significantly inhibited ferroptosis in RPE cells induced by sodium iodate-induced oxidative stress. Blocking ZIP8 with specific antibodies reversed RPE degeneration and restored retinal function, improving visual loss in a mouse model of NaIO3-induced. Interestingly, the modification of the N-glycosylation sites N40, N72 and N88, but not N273, was essential for the intracellular iron accumulation mediated by ZIP8, which further led to increased lipid peroxidation and RPE death. These findings highlight the critical role of ZIP8 in RPE ferroptosis and provide a potential target for the treatment of diseases associated with retinal degeneration, including AMD.

Interleukin-18-primed human umbilical cord-mesenchymal stem cells achieve superior therapeutic efficacy for severe viral pneumonia via enhancing T-cell immunosuppression.

Coronavirus disease 2019 (COVID-19) treatments are still urgently needed for critically and severely ill patients. Human umbilical cord-mesenchymal stem cells (hUC-MSCs) infusion has therapeutic benefits in COVID-19 patients; however, uncertain therapeutic efficacy has been reported in severe patients. In this study, we selected an appropriate cytokine, IL-18, based on the special cytokine expression profile in severe pneumonia of mice induced by H1N1virus to prime hUC-MSCs in vitro and improve the therapeutic effect of hUC-MSCs in vivo. In vitro, we demonstrated that IL-18-primed hUC-MSCs (IL18-hUCMSC) have higher proliferative ability than non-primed hUC-MSCs (hUCMSCcon). In addition, VCAM-1, MMP-1, TGF-ß1, and some chemokines (CCL2 and CXCL12 cytokines) are more highly expressed in IL18-hUCMSCs. We found that IL18-hUCMSC significantly enhanced the immunosuppressive effect on CD3+ T-cells. In vivo, we demonstrated that IL18-hUCMSC infusion could reduce the body weight loss caused by a viral infection and significantly improve the survival rate. Of note, IL18-hUCMSC can also significantly attenuate certain clinical symptoms, including reduced activity, ruffled fur, hunched backs, and lung injuries. Pathologically, IL18-hUCMSC transplantation significantly enhanced the inhibition of inflammation, viral load, fibrosis, and cell apoptosis in acute lung injuries. Notably, IL18-hUCMSC treatment has a superior inhibitory effect on T-cell exudation and proinflammatory cytokine secretion in bronchoalveolar lavage fluid (BALF). Altogether, IL-18 is a promising cytokine that can prime hUC-MSCs to improve the efficacy of precision therapy against viral-induced pneumonia, such as COVID-19.

Identification of novel factors that affect the onset of idiopathic chronic pancreatitis: The role for microRNA-323b-5p.

BACKGROUND: The c.194+2 T>C variant of serine protease inhibitor Kazal type 1 (SPINK1) is a known genetic risk factor found in Chinese patients with idiopathic chronic pancreatitis (ICP), but the early-onset mechanisms of ICP are still unclear. METHODS: Complementary experimental approaches were used to pursue other potential pathologies in the present study. The serum level of SPINK1 of ICP patients in the Han population in China was detected and verified by an enzyme-linked immunosorbent assay. Next, differentially expressed proteins and microRNAs from plasma samples of early-onset and late-onset ICP patients were screened by proteomic analysis and microarray, respectively. RESULTS: Combined with these advanced methods, the data strongly suggest that the regulatory effects of microRNAs were involved in the early-onset mechanism of the ICP by in vitro experiments. There was no significant difference in the plasma SPINK1 expression between the early-onset ICP and the late-onset patients. However, the expression of plasma glutathione peroxidase (GPx3) in early-onset ICP patients was markedly lower than that in late-onset ICP patients, although the level of hsa-miR-323b-5p was lower in late-onset patients compared to the early-onset ICP group. In vitro experiments confirmed that hsa-miR-323b-5p could increase apoptosis in caerulein-treated pancreatic acinar cells and inhibit the expression of GPx3. CONCLUSIONS: The up-regulated hsa-miR-323b-5p might play a crucial role in the early-onset mechanisms of ICP by diminishing the antioxidant activity through the down-regulation of GPx3.

Cell Recognition Using BP Neural Network Edge Computing.

This exploration is to solve the efficiency and accuracy of cell recognition in biological experiments. Neural network technology is applied to the research of cell image recognition. The cell image recognition problem is solved by constructing an image recognition algorithm. First, with an in-depth understanding of computer functions, as a basic intelligent algorithm, the artificial neural network (ANN) is widely used to solve the problem of image recognition. Recently, the backpropagation neural network (BPNN) algorithm has developed into a powerful pattern recognition tool and has been widely used in image edge detection. Then, the structural model of BPNN is introduced in detail. Given the complexity of cell image recognition, an algorithm based on the ANN and BPNN is used to solve this problem. The BPNN algorithm has multiple advantages, such as simple structure, easy hardware implementation, and good learning effect. Next, an image recognition algorithm based on the BPNN is designed and the image recognition process is optimized in combination with edge computing technology to improve the efficiency of algorithm recognition. The experimental results show that compared with the traditional image pattern recognition algorithm, the recognition accuracy of the designed algorithm for cell images is higher than 93.12%, so it has more advantages for processing the cell image algorithm. The results show that the BPNN edge computing can improve the scientific accuracy of cell recognition results, suggesting that edge computing based on the BPNN has a significant practical value for the research and application of cell recognition.

Reproductive and Developmental Toxicity Assessment of Human Umbilical Cord Mesenchymal Stem Cells in Rats.

Objective: Human umbilical cord mesenchymal stem cells (hUC-MSCs) have shown very attractive potential in clinical applications for the treatment of various diseases. However, the data about the reproductive and developmental toxicity of hUC-MSCs remains insufficient. Thus, we assessed the potential effects of intravenous injection of hUC-MSCs on reproduction and development in Sprague-Dawley rats. Methods: In the fertility and early embryonic development study, hUC-MSCs were administered at dose levels of 0, 6.0 × 106, 8.5 × 106, and 1.2 × 107/kg to male and female rats during the pre-mating, mating and gestation period. In the embryo-fetal development study, the pregnant female rats received 0, 6.0 × 106, 1.2 × 107, and 2.4 × 107/kg of hUC-MSCs from gestation days (GD) 6-15. Assessments made included mortality, clinical observations, body weight, food consumption, fertility parameters of male and female, litter, and fetus parameters, etc. Results: No hUC-MSCs-related toxicity was observed on the fertility of male and female rats, and no teratogenic effect on fetuses. hUC-MSCs at 1.2 × 107/kg caused a mildly decrease in body weight gain of male rats, transient listlessness, tachypnea, and hematuria symptoms in pregnant female rats. Death was observed in part of the pregnant females at a dose of 2.4 × 107/kg, which could be due to pulmonary embolism. Conclusion: Based on the results of the studies, the no-observed-adverse-effect levels (NOAELs) are 8.5 × 106/kg for fertility and early embryonic development, 1.2 × 107/kg for maternal toxicity and 2.4 × 107/kg for embryo-fetal development in rats intravenous injected with hUC-MSCs, which are equivalent to 8.5-fold, 12-fold, and 24-fold respectively of its clinical dosage in humans. These findings may provide a rational basis for human health risk assessment of hUC-MSCs.

Differential Effects of Dietary Macronutrients on the Development of Oncogenic KRAS-Mediated Pancreatic Ductal Adenocarcinoma.

KRAS mutations are prevalent in patients with pancreatic ductal adenocarcinoma (PDAC) and are critical to fostering tumor growth in part by aberrantly rewiring glucose, amino acid, and lipid metabolism. Obesity is a modifiable risk factor for pancreatic cancer. Corroborating this epidemiological observation, mice harboring mutant KRAS are highly vulnerable to obesogenic high-fat diet (HFD) challenges leading to the development of PDAC with high penetrance. However, the contributions of other macronutrient diets, such as diets rich in carbohydrates that are regarded as a more direct source to fuel glycolysis for cancer cell survival and proliferation than HFD, to pancreatic tumorigenesis remain unclear. In this study, we compared the differential effects of a high-carbohydrate diet (HCD), an HFD, and a high-protein diet (HPD) in PDAC development using a mouse model expressing an endogenous level of mutant KRASG12D specifically in pancreatic acinar cells. Our study showed that although with a lower tumorigenic capacity than chronic HFD, chronic HCD promoted acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) lesions with increased inflammation, fibrosis, and cell proliferation compared to the normal diet (ND) in KrasG12D/+ mice. By contrast, chronic HPD showed no significant adverse effects compared to the ND. Furthermore, ablation of pancreatic acinar cell cyclooxygenase 2 (Cox-2) in KrasG12D/+ mice abrogated the adverse effects induced by HCD, suggesting that diet-induced pancreatic inflammation is critical for promoting oncogenic KRAS-mediated neoplasia. These results indicate that diets rich in different macronutrients have differential effects on pancreatic tumorigenesis in which the ensuing inflammation exacerbates the process. Management of macronutrient intake aimed at thwarting inflammation is thus an important preventive strategy for patients harboring oncogenic KRAS.

Segmentation, Detection, and Tracking of Stem Cell Image by Digital Twins and Lightweight Deep Learning.

The current work aims to strengthen the research of segmentation, detection, and tracking methods of stem cell image in the fields of regenerative medicine and tissue damage restoration. Firstly, based on the relevant theories of stem cell image segmentation, digital twins (DTs), and lightweight deep learning, a new phase contrast microscope is introduced through the research of optical microscope. Secondly, the results of DTs method and phase contrast imaging principle are compared in stem cell image segmentation and detection. Finally, a lightweight deep learning model is introduced in the segmentation and tracking of stem cell image to observe the gray value and mean value before and after stem cell image movement and stem cell division. The results show that phase contrast microscope can increase the phase contrast and amplitude difference of stem cell image and solve the problem of stem cell image segmentation to a certain extent. The detection results of DTs method are compared with phase contrast imaging principle. It indicates that not only can DTs method make the image contour more accurate and clearer, but also its accuracy, recall, and F1 score are 0.038, 0.024, and 0.043 higher than those of the phase contrast imaging method. The lightweight deep learning model is applied to the segmentation and tracking of stem cell image. It is found that the gray value and mean value of stem cell image before and after movement and stem cell division do not change significantly. Hence, the application of DTs and lightweight deep learning methods in the segmentation, detection, and tracking of stem cell image has great reference significance for the development of biology and medicine.

Human Platelet Lysate Maintains Stemness of Umbilical Cord-Derived Mesenchymal Stromal Cells and Promote Lung Repair in Rat Bronchopulmonary Dysplasia.

Mesenchymal stromal cells (MSCs) show potential for treating preclinical models of newborn bronchopulmonary dysplasia (BPD), but studies of their therapeutic effectiveness have had mixed results, in part due to the use of different media supplements for MSCs expansion in vitro. The current study sought to identify an optimal culture supplement of umbilical cord-derived MSCs (UC-MSCs) for BPD therapy. In this study, we found that UC-MSCs cultured with human platelet lysate (hPL-UCMSCs) were maintained a small size from Passage 1 (P1) to P10, while UC-MSCs cultured with fetal bovine serum (FBS-UCMSCs) became wide and flat. Furthermore, hPL was associated with lower levels of senescence in UC-MSCs during in vitro expansion compared with FBS, as indicated by the results of ß-galactosidase staining and measures of senescence-related genes (CDKN2A, CDKN1A, and mTOR). In addition, hPL enhanced the proliferation and cell viability of the UC-MSCs and reduced their doubling time in vitro. Compared with FBS-UCMSCs, hPL-UCMSCs have a greater potential to differentiate into osteocytes and chondrocytes. Moreover, using hPL resulted in greater expression of Nestin and specific paracrine factors (VEGF, TGF-ß1, FGF2, IL-8, and IL-6) in UC-MSCs compared to using FBS. Critically, we also found that hPL-UCMSCs are more effective than FBS-UCMSCs for the treatment of BPD in a rat model, with hPL leading to improvements in survival rate, lung architecture and fibrosis, and lung capillary density. Finally, qPCR of rat lung mRNA demonstrated that hPL-UCMSCs had lower expression levels of inflammatory factors (TNF-α and IL-1ß) and a key chemokine (MCP-1) at postnatal day 10, and there was significant reduction of CD68+ macrophages in lung tissue after hPL-UCMSCs transplantation. Altogether, our findings suggest that hPL is an optimal culture supplement for UC-MSCs expansion in vitro, and that hPL-UCMSCs promote lung repair in rat BPD disease.

Human umbilical cord mesenchymal stromal cells attenuate pulmonary fibrosis via regulatory T cell through interaction with macrophage.

BACKGROUND: Pulmonary fibrosis (PF) is a growing clinical problem with limited therapeutic options. Human umbilical cord mesenchymal stromal cell (hucMSC) therapy is being investigated in clinical trials for the treatment of PF patients. However, little is known about the underlying molecular and cellular mechanisms of hucMSC therapy on PF. In this study, the molecular and cellular behavior of hucMSC was investigated in a bleomycin-induced mouse PF model. METHODS: The effect of hucMSCs on mouse lung regeneration was determined by detecting Ki67 expression and EdU incorporation in alveolar type 2 (AT2) and lung fibroblast cells. hucMSCs were transfected to express the membrane localized GFP before transplant into the mouse lung. The cellular behavior of hucMSCs in mouse lung was tracked by GFP staining. Single cell RNA sequencing was performed to investigate the effects of hucMSCs on gene expression profiles of macrophages after bleomycin treatment. RESULTS: hucMSCs could alleviate collagen accumulation in lung and decrease the mortality of mouse induced by bleomycin. hucMSC transplantation promoted AT2 cell proliferation and inhibited lung fibroblast cell proliferation. By using single cell RNA sequencing, a subcluster of interferon-sensitive macrophages (IFNSMs) were identified after hucMSC infusion. These IFNSMs elevate the secretion of CXCL9 and CXCL10 following hucMSC infusion and recruit more Treg cells to the injured lung. CONCLUSIONS: Our study establishes a link between hucMSCs, macrophage, Treg, and PF. It provides new insights into how hucMSCs interact with macrophage during the repair process of bleomycin-induced PF and play its immunoregulation function.

Selective killing of cancer cells harboring mutant RAS by concomitant inhibition of NADPH oxidase and glutathione biosynthesis.

Oncogenic RAS is a critical driver for the initiation and progression of several types of cancers. However, effective therapeutic strategies by targeting RAS, in particular RASG12D and RASG12V, and associated downstream pathways have been so far unsuccessful. Treatment of oncogenic RAS-ravaged cancer patients remains a currently unmet clinical need. Consistent with a major role in cancer metabolism, oncogenic RAS activation elevates both reactive oxygen species (ROS)-generating NADPH oxidase (NOX) activity and ROS-scavenging glutathione biosynthesis. At a certain threshold, the heightened oxidative stress and antioxidant capability achieve a higher level of redox balance, on which cancer cells depend to gain a selective advantage on survival and proliferation. However, this prominent metabolic feature may irrevocably render cancer cells vulnerable to concurrent inhibition of both NOX activity and glutathione biosynthesis, which may be exploited as a novel therapeutic strategy. In this report, we test this hypothesis by treating the HRASG12V-transformed ovarian epithelial cells, mutant KRAS-harboring pancreatic and colon cancer cells of mouse and human origins, as well as cancer xenografts, with diphenyleneiodonium (DPI) and buthionine sulfoximine (BSO) combination, which inhibit NOX activity and glutathione biosynthesis, respectively. Our results demonstrate that concomitant targeting of NOX and glutathione biosynthesis induces a highly potent lethality to cancer cells harboring oncogenic RAS. Therefore, our studies provide a novel strategy against RAS-bearing cancers that warrants further mechanistic and translational investigation.

Endoscopic submucosal dissection versus esophagectomy for early esophageal squamous cell carcinoma with tumor invasion to different depths.

Endoscopic submucosal dissection (ESD) is a minimally invasive alternative to esophagectomy for early esophageal squamous cell carcinoma (EESCC). The aim of this study was to compare the efficacy and safety of ESD and esophagectomy in EESCC with different depth of invasion. The data of EESCC patients who received ESD or esophagectomy between Jan 2011 to Dec 2018 at our center were retrospectively analyzed. Overall survival (OS), disease-specific survival (DSS), recurrence-free survival (RFS), and procedure-related variables were compared between ESD and esophagectomy patients. 222 EESCC patients underwent ESD, while 184 underwent esophagectomy. No significant differences were found between the two groups in OS (P=0.417), DSS (P=0.423), and RFS (P=0.726). Procedure duration, post-procedure hospital stay, and hospitalization cost were all lower in ESD patients. Oncologic outcomes were similar between the two groups in propensity score-matched analysis. The R0 resection rate was comparable between ESD and esophagectomy groups in the T1a-M1/M2 and M3/SM1 EESCC subgroups; no significant differences were found in OS, DSS and RFS. In the SM2/SM3 EESCC subgroup, although the prognosis of the two treatment groups was similar, the R0 resection rate was significantly lower in ESD patients than in esophagectomy patients. Thus, we concluded ESD could be a first-line treatment for T1a-M1/M2 and M3/SM1 EESCC as oncologic outcome is comparable to that achieved with esophagectomy with minimal invasion, lower cost and lower incidence of serious adverse events. However, in SM2/SM3 EESCC patients, esophagectomy may be preferable.