Long-term outcomes of single-incision plus one-port laparoscopic surgery versus conventional laparoscopic surgery for rectosigmoid cancer: a randomized controlled trial.

BACKGROUND: Though our previous study has demonstrated that the single-incision plus one-port laparoscopic surgery (SILS + 1) is safe and feasible for sigmoid colon and upper rectal cancer and has better short-term outcomes compared with conventional laparoscopic surgery (CLS), the long-term outcomes of SILS + 1 remains uncertain and are needed to evaluated by an RCT. METHODS: Patients with clinical stage T1-4aN0-2M0 rectosigmoid cancer were enrolled. The participants were randomly assigned to either SILS + 1 (n = 99) or CLS (n = 99). The 3-year DFS, 5-year OS, and recurrence patterns were analyzed. RESULTS: Between April 2014 and July 2016, 198 patients were randomly assigned to either the SILS + 1 group (n = 99) or CLS group (n = 99). The median follow-up in the SILS + 1 group was 64.0 months and in CLS group was 65.0 months. The 3-year DFS was 87.8% (95% CI, 81.6-94.8%) in SILS + 1 group and 86.9% (95% CI, 81.3-94.5%) in CLS group (hazard ratio: 1.09 (95% CI, 0.48-2.47; P = 0.84)). The 5-year OS was 86.7% (95% CI,79.6-93.8%) in the SILS + 1 group and 80.5% (95% CI,72.5-88.5%) in the CLS group (hazard ratio: 1.53 (95% CI, 0.74-3.18; P = 0.25)). There were no significant differences in the recurrence patterns between the two groups. CONCLUSIONS: We found no significant difference in 3-year DFS and 5-year OS of patients with sigmoid colon and upper rectal cancer treated with SILS + 1 vs. CLS. SILS + 1 is noninferior to CLS when performed by expert surgeons. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02117557 (registered on 21/04/2014).

Single-cell transcriptomic architecture and intercellular crosstalk of human intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (ICC) is the second most common liver malignancy. ICC typically features remarkable cellular heterogeneity and a dense stromal reaction. Therefore, a comprehensive understanding of cellular diversity and the interplay between malignant cells and niche cells is essential to elucidate the mechanisms driving ICC progression and to develop therapeutic approaches. METHODS: Herein, we performed single-cell RNA sequencing (scRNA-seq) analysis on unselected viable cells from 8 human ICCs and adjacent samples to elucidate the comprehensive transcriptomic landscape and intercellular communication network. Additionally, we applied a negative selection strategy to enrich fibroblast populations in 2 other ICC samples to investigate fibroblast diversity. The results of the analyses were validated using multiplex immunofluorescence staining, bulk transcriptomic datasets, and functional in vitro and in vivo experiments. RESULTS: We sequenced a total of 56,871 single cells derived from human ICC and adjacent tissues and identified diverse tumor, immune, and stromal cells. Malignant cells displayed a high degree of inter-tumor heterogeneity. Moreover, tumor-infiltrating CD4 regulatory T cells exhibited highly immunosuppressive characteristics. We identified 6 distinct fibroblast subsets, of which the majority were CD146-positive vascular cancer-associated fibroblasts (vCAFs), with highly expressed microvasculature signatures and high levels of interleukin (IL)-6. Functional assays indicated that IL-6 secreted by vCAFs induced significant epigenetic alterations in ICC cells, particularly upregulating enhancer of zeste homolog 2 (EZH2) and thereby enhancing malignancy. Furthermore, ICC cell-derived exosomal miR-9-5p elicited high expression of IL-6 in vCAFs to promote tumor progression. CONCLUSIONS: Our single-cell transcriptomic dataset delineates the inter-tumor heterogeneity of human ICCs, underlining the importance of intercellular crosstalk between ICC cells and vCAFs, and revealing potential therapeutic targets. LAY SUMMARY: Intrahepatic cholangiocarcinoma is an aggressive and chemoresistant malignancy. Better understanding the complex transcriptional architecture and intercellular crosstalk of these tumors will help in the development of more effective therapies. Herein, we have identified important interactions between cancer cells and cancer-associated fibroblasts in the tumor stroma, which could have therapeutic implications.

The RUNX1 +24 enhancer and P1 promoter identify a unique subpopulation of hematopoietic progenitor cells derived from human pluripotent stem cells.

Derivation of hematopoietic stem cells (HSCs) from human pluripotent stem cells remains a key goal for the fields of developmental biology and regenerative medicine. Here, we use a novel genetic reporter system to prospectively identify and isolate early hematopoietic cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (iPSCs). Cloning the human RUNX1c P1 promoter and +24 enhancer to drive expression of tdTomato (tdTom) in hESCs and iPSCs, we demonstrate that tdTom expression faithfully enriches for RUNX1c-expressing hematopoietic progenitor cells. Time-lapse microscopy demonstrated the tdTom(+) hematopoietic cells to emerge from adherent cells. Furthermore, inhibition of primitive hematopoiesis by blocking Activin/Nodal signaling promoted the expansion and/or survival of the tdTom(+) population. Notably, RUNX1c/tdTom(+) cells represent only a limited subpopulation of the CD34(+) CD45(+) and CD34(+) CD43(+) cells with a unique genetic signature. Using gene array analysis, we find significantly lower expression of Let-7 and mir181a microRNAs in the RUNX1c/tdTom(+) cell population. These phenotypic and genetic analyses comparing the RUNX1c/tdTom(+) population to CD34(+) CD45(+) umbilical cord blood and fetal liver demonstrate several key differences that likely impact the development of HSCs capable of long-term multilineage engraftment from hESCs and iPSCs.

Pelvic Floor Peritoneum Closure Reduces Severe Postoperative Complications in Rectal Cancer Patients After Laparoscopic Anterior Rectal Resection.

BACKGROUND: Laparoscopic anterior rectal resection (LAR) is a commonly performed surgery for rectal cancer patients. Pelvic floor peritoneum closure (PC), a vital procedure in conventional anterior rectal resection, is not routinely performed in LAR. STUDY DESIGN: A total of 1118 consecutive patients with rectal cancer receiving LAR were included in this retrospective study. Patients were allocated into the PC group and the non-PC group. The occurrence of postoperative complications was compared between the 2 groups. Influential factors in anastomotic leakage (AL) were explored using univariate and multivariate logistic regression. RESULTS: There was no difference between the groups in terms of baseline characteristics. The occurrence of postoperative complications was similar between the groups. The PC group had significantly shorter postoperative hospitalization and longer operation duration compared with the non-PC group. The occurrences of Clavien-Dindo (CD) III-IV complications, CD III-IV AL, and reoperation were significantly lower in the PC group than the non-PC group. PC and a protective ileostomy were independent protective factors for CD III-IV AL. CONCLUSION: PC could reduce the occurrence of CD III-IV complications, especially CD III-IV AL, and the rate of secondary surgery, especially in patients with a lower body mass index and patients who did not receive protective ileostomies.

PROTAC-mediated vimentin degradation promotes terminal erythroid differentiation of pluripotent stem cells.

BACKGROUND: Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), can undergo erythroid differentiation, offering a potentially invaluable resource for generating large quantities of erythroid cells. However, the majority of erythrocytes derived from hPSCs fail to enucleate compared with those derived from cord blood progenitors, with an unknown molecular basis for this difference. The expression of vimentin (VIM) is retained in erythroid cells differentiated from hPSCs but is absent in mature erythrocytes. Further exploration is required to ascertain whether VIM plays a critical role in enucleation and to elucidate the underlying mechanisms. METHODS: In this study, we established a hESC line with reversible vimentin degradation (dTAG-VIM-H9) using the proteolysis-targeting chimera (PROTAC) platform. Various time-course studies, including erythropoiesis from CD34+ human umbilical cord blood and three-dimensional (3D) organoid culture from hESCs, morphological analysis, quantitative real-time PCR (qRT-PCR), western blotting, flow cytometry, karyotyping, cytospin, Benzidine-Giemsa staining, immunofluorescence assay, and high-speed cell imaging analysis, were conducted to examine and compare the characteristics of hESCs and those with vimentin degradation, as well as their differentiated erythroid cells. RESULTS: Vimentin expression diminished during normal erythropoiesis in CD34+ cord blood cells, whereas it persisted in erythroid cells differentiated from hESC. Depletion of vimentin using the degradation tag (dTAG) system promotes erythroid enucleation in dTAG-VIM-H9 cells. Nuclear polarization of erythroblasts is elevated by elimination of vimentin. CONCLUSIONS: VIM disappear during the normal maturation of erythroid cells, whereas they are retained in erythroid cells differentiated from hPSCs. We found that retention of vimentin during erythropoiesis impairs erythroid enucleation from hPSCs. Using the PROTAC platform, we validated that vimentin degradation by dTAG accelerates the enucleation rate in dTAG-VIM-H9 cells by enhancing nuclear polarization.

Decoding human in vitro terminal erythropoiesis originating from umbilical cord blood mononuclear cells and pluripotent stem cells.

Ex vivo red blood cell (RBC) production generates unsatisfactory erythroid cells. A deep exploration into terminally differentiated cells is required to understand the impairments for RBC generation and the underlying mechanisms. Here, we mapped an atlas of terminally differentiated cells from umbilical cord blood mononuclear cells (UCBMN) and pluripotent stem cells (PSC) and observed their dynamic regulation of erythropoiesis at single-cell resolution. Interestingly, we detected a few progenitor cells and non-erythroid cells from both origins. In PSC-derived erythropoiesis (PSCE), the expression of haemoglobin switch regulators (BCL11A and ZBTB7A) were significantly absent, which could be the restraint for its adult globin expression. We also found that PSCE were less active in stress erythropoiesis than in UCBMN-derived erythropoiesis (UCBE), and explored an agonist of stress erythropoiesis gene, TRIB3, could enhance the expression of adult globin in PSCE. Compared with UCBE, there was a lower expression of epigenetic-related proteins (e.g., CASPASE 3 and UBE2O) and transcription factors (e.g., FOXO3 and TAL1) in PSCE, which might restrict PSCE's enucleation. Moreover, we characterized a subpopulation with high proliferation capacity marked by CD99high in colony-forming unit-erythroid cells. Inhibition of CD99 reduced the proliferation of PSC-derived cells and facilitated erythroid maturation. Furthermore, CD99-CD99 mediated the interaction between macrophages and erythroid cells, illustrating a mechanism by which macrophages participate in erythropoiesis. This study provided a reference for improving ex vivo RBC generation.

Enhancing terminal erythroid differentiation in human embryonic stem cells through TRIB3 overexpression.

Tribbles pseudokinase 3 (TRIB3) expression significantly increases during terminal erythropoiesis in vivo. However, we found that TRIB3 expression remained relatively low during human embryonic stem cell (hESC) erythropoiesis, particularly in the late stage, where it is typically active. TRIB3 was expressed in megakaryocyte-erythrocyte progenitor cells and its low expression was necessary for megakaryocyte differentiation. Thus, we proposed that the high expression during late stage of erythropoiesis could be the clue for promotion of maturation of hESC-derived erythroid cells. To our knowledge, the role of TRIB3 in the late stage of erythropoiesis remains ambiguous. To address this, we generated inducible TRIB3 overexpression hESCs, named TRIB3tet-on OE H9, based on a Tet-On system. Then, we analyzed hemoglobin expression, condensed chromosomes, organelle clearance, and enucleation with or without doxycycline treatment. TRIB3tet-on OE H9 cells generated erythrocytes with a high proportion of orthochromatic erythroblast in flow cytometry, enhanced hemoglobin and related protein expression in Western blot, decreased nuclear area size, promoted enucleation rate, decreased lysosome and mitochondria number, more colocalization of LC3 with LAMP1 (lysosome marker) and TOM20 (mitochondria marker) and up-regulated mitophagy-related protein expression after treatment with 2 µg/mL doxycycline. Our results showed that TRIB3 overexpression during terminal erythropoiesis may promote the maturation of erythroid cells. Therefore, our study delineates the role of TRIB3 in terminal erythropoiesis, and reveals TRIB3 as a key regulator of UPS and downstream mitophagy by ensuring appropriate mitochondrial clearance during the compaction of chromatin.

Hydrogel Microneedle Patches Loaded with Stem Cell Mitochondria-Enriched Microvesicles Boost the Chronic Wound Healing.

Rescuing or compensating mitochondrial function represents a promising therapeutic avenue for radiation-induced chronic wounds. Adult stem cell efficacies are primarily dependent on the paracrine secretion of mitochondria-containing extracellular vesicles (EVs). However, effective therapeutic strategies addressing the quantity of mitochondria and mitochondria-delivery system are lacking. Thus, in this study, we aimed to design an effective hydrogel microneedle patch (MNP) loaded with stem cell-derived mitochondria-rich EVs to gradually release and deliver mitochondria into the wound tissues and boost wound healing. We, first, used metformin to enhance mitochondrial biogenesis and thereby increasing the secretion of mitochondria-containing EVs (termed "Met-EVs") in adipose-derived stem cells. To verify the therapeutic effects of Met-EVs, we established an in vitro and an in vivo model of X-ray-induced mitochondrial dysfunction. The Met-EVs ameliorated the mitochondrial dysfunction by rescuing mitochondrial membrane potential, increasing adenosine 5'-triphosphate levels, and decreasing reactive oxygen species production by transferring active mitochondria. To sustain the release of EVs into damaged tissues, we constructed a Met-EVs@Decellularized Adipose Matrix (DAM)/Hyaluronic Acid Methacrylic Acid (HAMA)-MNP. Met-EVs@DAM/HAMA-MNP can load and gradually release Met-EVs and their contained mitochondria into wound tissues to alleviate mitochondrial dysfunction. Moreover, we found Met-EVs@DAM/HAMA-MNP can markedly promote macrophage polarization toward the M2 subtype with anti-inflammatory and regenerative functions, which can, in turn, enhance the healing process in mice with skin wounds combined radiation injuries. Collectively, we successfully fabricated a delivery system for EVs, Met-EVs@DAM/HAMA-MNP, to effectively deliver stem cell-derived mitochondria-rich EVs. The effectiveness of this system has been demonstrated, holding great potential for chronic wound treatments in clinic.

Nanoengineered Red Blood Cells Loaded with TMPRSS2 and Cathepsin L Inhibitors Block SARS-CoV-2 Pseudovirus Entry into Lung ACE2+ Cells.

The enzymatic activities of Furin, Transmembrane serine proteinase 2 (TMPRSS2), Cathepsin L (CTSL), and Angiotensin-converting enzyme 2 (ACE2) receptor binding are necessary for the entry of coronaviruses into host cells. Precise inhibition of these key proteases in ACE2+ lung cells during a viral infection cycle shall prevent viral Spike (S) protein activation and its fusion with a host cell membrane, consequently averting virus entry to the cells. In this study, dual-drug-combined (TMPRSS2 inhibitor Camostat and CTSL inhibitor E-64d) nanocarriers (NCs) are constructed conjugated with an anti-human ACE2 (hACE2) antibody and employ Red Blood Cell (RBC)-hitchhiking, termed "Nanoengineered RBCs," for targeting lung cells. The significant therapeutic efficacy of the dual-drug-loaded nanoengineered RBCs in pseudovirus-infected K18-hACE2 transgenic mice is reported. Notably, the modular nanoengineered RBCs (anti-receptor antibody+NCs+RBCs) precisely target key proteases of host cells in the lungs to block the entry of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), regardless of virus variations. These findings are anticipated to benefit the development of a series of novel and safe host-cell-protecting antiviral therapies.

Protective effects of resveratrol on the ethanol-induced disruption of retinogenesis in pluripotent stem cell-derived organoids.

Prenatal alcohol exposure-induced fetal alcohol syndrome (FAS) can lead to serious maldevelopment in many organ systems, including the eyes. In the present study, the effects of alcohol exposure on early development of the human retina and the therapeutic effects of resveratrol on alcohol-induced neural retinal damage were observed for the first time in an in vitro retinal organoid model. We report that the number of proliferating and apoptotic cells decreased and increased, respectively, following ethanol treatment. In addition, the number of PAX6+ cells and migrating TUJ1+ cells decreased after ethanol exposure. However, pretreatment with resveratrol prevented all of these negative effects. Using RNA sequencing and immunofluorescence, we identified activation of the PI3K-AKT signalling pathway as the possible mechanism through which resveratrol protects the retina from alcohol-induced damage. These results suggest that while ethanol exposure can restrict the growth of the human retina and impede the development of specific retinal cells, pretreatment with resveratrol may be a feasible method for preventing these effects.

Generating Neural Retina from Human Pluripotent Stem Cells.

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of retinopathy. Unfortunately, ethical barriers hinder the collection of evidence from humans. Recently, numerous studies have shown that human pluripotent stem cells (PSCs) can be differentiated into retinal organoids (ROs) using different induction protocols, which have enormous potential in retinopathy for disease modeling, drug screening, and stem cell-based therapies. This study describes an optimized induction protocol to generate neural retina (NR) that significantly reduces the probability of vesiculation and fusion, increasing the success rate of production until day 60. Based on the ability of PSCs to self-reorganize after dissociation, combined with certain complementary factors, this new method can specifically drive NR differentiation. Furthermore, the approach is uncomplicated, cost-effective, exhibits notable repeatability and efficiency, presents encouraging prospects for personalized models of retinal diseases, and supplies a plentiful cell reservoir for applications such as cell therapy, drug screening, and gene therapy testing.

Influence of hypoxia on retinal progenitor and ganglion cells in human induced pluripotent stem cell-derived retinal organoids.

AIM: To observe the effect of low oxygen concentration on the neural retina in human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs). METHODS: The hiPSC and a three-dimensional culture method were used for the experiments. Generated embryoid bodies (EBs) were randomly and equally divided into hypoxic and normoxic groups. Photographs of the EBs were taken on days 38, 45, and 52, and the corresponding volume of EBs was calculated. Simultaneously, samples were collected at these three timepoints, followed by fixation, sectioning, and immunofluorescence. RESULTS: The proportion of Ki67-positive proliferating cells increased steadily on day 38; this proliferation-promoting effect tended to increase tissue density rather than tissue volume. On days 45 and 52, the two groups had relatively similar ratios of Ki67-positive cells. Further immunofluorescence analysis showed that the ratio of SOX2-positive cells significantly increased within the neural retina on day 52 (P<0.05). In contrast, the percentage of PAX6- and CHX10-positive cells significantly decreased following hypoxia treatment at all three timepoints (P<0.01), except for CHX10 at day 45 (P>0.05). Moreover, the proportion of PAX6-/TUJ1+ cells within the neural retinas increased considerably (P<0.01, <0.05, <0.05 respectively). CONCLUSION: Low oxygen promotes stemness and proliferation of neural retinas, suggesting that hypoxic conditions can enlarge the retinal progenitor cell pool in hiPSC-derived ROs.

Development and Validation of a Novel Survival Model for Cutaneous Melanoma Based on Necroptosis-Related Genes.

Background: Necroptosis is crucial for organismal development and pathogenesis. To date, the role of necroptosis in skin cutaneous melanoma (SKCM) is yet unveiled. In addition, the part of melanin pigmentation was largely neglected in the bioinformatic analysis. In this study, we aimed to construct a novel prognostic model based on necroptosis-related genes and analysis the pigmentation phenotype of patients to provide clinically actionable information for SKCM patients. Methods: We downloaded the SKCM data from the TCGA and GEO databases in this study and identified the differently expressed and prognostic necroptosis-related genes. Patients' pigmentation phenotype was evaluated by the GSVA method. Then, using Lasso and Cox regression analysis, a novel prognostic model was constructed based on the intersected genes. The risk score was calculated and the patients were divided into two groups. The survival differences between the two groups were compared using Kaplan-Meier analysis. The ROC analysis was performed and the area under curves was calculated to evaluate the prediction performances of the model. Then, the GO, KEGG and GSEA analyses were performed to elucidate the underlying mechanisms. Differences in the tumor microenvironment, patients' response to immune checkpoint inhibitors (ICIs) and pigmentation phenotype were analyzed. In order to validate the mRNA expression levels of the selected genes, quantitative real-time PCR (qRT-PCR) was performed. Results: Altogether, a novel prognostic model based on four genes (BOK, CD14, CYLD and FASLG) was constructed, and patients were classified into high and low-risk groups based on the median risk score. Low-risk group patients showed better survival status. The model showed high accuracy in the training and the validation cohort. Pathway and functional enrichment analysis indicated that immune-related pathways were differently activated in the two groups. In addition, immune cells infiltration patterns and sensitivity of ICIs showed a significant difference between patients from two risk groups. The pigmentation score was positively related to the risk score in pigmentation phenotype analysis. Conclusion: In conclusion, this study established a novel prognostic model based on necroptosis-related genes and revealed the possible connections between necroptosis and melanin pigmentation. It is expected to provide a reference for clinical treatment.

A Functional Screening Identifies a New Organic Selenium Compound Targeting Cancer Stem Cells: Role of c-Myc Transcription Activity Inhibition in Liver Cancer.

Cancer stem cells (CSCs) are reported to play essential roles in chemoresistance and metastasis. Pathways regulating CSC self-renewal and proliferation, such as Hedgehog, Notch, Wnt/ß-catenin, TGF-ß, and Myc, may be potential therapeutic targets. Here, a functional screening from the focused library with 365 compounds is performed by a step-by-step strategy. Among these candidate molecules, phenyl-2-pyrimidinyl ketone 4-allyl-3-amino selenourea (CU27) is chosen for further identification because it proves to be the most effective compound over others on CSC inhibition. Through ingenuity pathway analysis, it is shown CU27 may inhibit CSC through a well-known stemness-related transcription factor c-Myc. Gene set enrichment analysis, dual-luciferase reporter assays, expression levels of typical c-Myc targets, molecular docking, surface plasmon resonance, immunoprecipitation, and chromatin immunoprecipitation are conducted. These results together suggest CU27 binds c-Myc bHLH/LZ domains, inhibits c-Myc-Max complex formation, and prevents its occupancy on target gene promoters. In mouse models, CU27 significantly sensitizes sorafenib-resistant tumor to sorafenib, reduces the primary tumor size, and inhibits CSC generation, showing a dramatic anti-metastasis potential. Taken together, CU27 exerts inhibitory effects on CSC and CSC-associated traits in hepatocellular carcinoma (HCC) via c-Myc transcription activity inhibition. CU27 may be a promising therapeutic to treat sorafenib-resistant HCC.

The splicing regulatory factor hnRNPU is a novel transcriptional target of c-Myc in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common liver cancer with high mortality. Here, we found that hnRNPU is overexpressed in HCC tissues and is correlated with the poor prognosis of HCC patients. Besides, hnRNPU is of high significance in regulating the proliferation, apoptosis, self-renewal, and tumorigenic potential of HCC cells. Mechanismly, c-Myc regulates hnRNPU expression at the transcriptional level, and meanwhile, hnRNPU stabilizes the mRNA of c-MYC. We found that the hnRNPU and c-Myc regulatory loop exerts a synergistic effect on the proliferation and self-renewal of HCC, and promotes the HCC progression. Taken together, hnRNPU functions as a novel transcriptional target of c-Myc and promotes HCC progression, which may become a promising target for the treatment of c-Myc-driven HCC.

Integrative single-cell transcriptome analysis reveals a subpopulation of fibroblasts associated with favorable prognosis of liver cancer patients.

Single-cell transcriptome analysis has provided detailed insights into the ecosystem of liver cancer. However, the changes of the cellular and molecular components of liver tumors in comparison with normal livers have not been described at single-cell level. Here, we performed an integrative single-cell analysis of both normal livers and liver cancers. Principal component analysis was firstly performed to delineate the cell lineages in liver tissues. Differential gene expression within major cell types were then analyzed between tumor and normal samples, thus resolved the cell type-specific molecular alterations in liver cancer development. Moreover, a comparison between liver cancer derived versus normal liver derived cell components revealed that two subpopulations of fibroblasts were exclusively expanded in liver cancer tissues. By further defining subpopulation-specific gene signatures, characterizing their spatial distribution in tumor tissues and investigating their clinical significance, we found that the SPARCL1 positive fibroblasts, representing a group of tumor vessel associated fibroblasts, were related to reduced vascular invasion and prolonged survival of liver cancer patients. Through establishing an in-vitro endothelial-to-mesenchymal transition model, we verified the conversion of the fetal liver sinusoidal endothelial cells into the fibroblast-like cells, demonstrating a possible endothelial cell origination of the SPARCL1 positive fibroblasts. Our study provides new insights into the cell atlas alteration, especially the expanded fibroblasts in liver cancers.

Human decellularized adipose matrix derived hydrogel assists mesenchymal stem cells delivery and accelerates chronic wound healing.

Biological scaffolds based stem cell delivery methods have emerged as a promising approach for tissue repair and regeneration. Here we developed a hydrogel biological scaffold from human decellularized adipose matrix (hDAM) for human adipose-derived stem cells (hASCs) delivery to accelerate chronic wound healing. The hDAM hydrogel was prepared by pepsin mediated digestion and pH controlled neutralization. The morphology, survival, proliferation, and angiogenic paracrine activity of hASCs cultured in the hydrogel were assessed. Moreover, the therapeutic efficacy of the hASCs-hydrogel composite for impaired wound healing was evaluated by using a full-thickness wound model on diabetic mouse. The developed hDAM hydrogel was a thermosensitive hydrogel, presented the biochemical complexity of native extracellular matrix and formed a porous nanofiber structure after gelation. The hydrogel can support hASCs adhesion, survival, and proliferation. Compared to standard culture condition, hASCs cultured in the hydrogel exhibited enhanced paracrine activity with increased secretion of hepatocyte growth factor. In the diabetic mice model with excisional full-thickness skin wounds, mice treated with the hASCs-hydrogel composite displayed accelerated wound closure and increased neovascularization. Our results suggested that the developed hDAM hydrogel can provide a favorable microenvironment for hASCs with augmented regeneration potential to accelerate chronic wound healing.

Large-scale generation of megakaryocytes from human embryonic stem cells using transgene-free and stepwise defined suspension culture conditions.

OBJECTIVES: Ex vivo engineered production of megakaryocytes (MKs) and platelets (PLTs) from human pluripotent stem cells is an alternative approach to solve shortage of donor-donated PLTs in clinics and to provide induced PLTs for transfusion. However, low production yields are observed and the generation of clinically applicable MKs and PLTs from human pluripotent stem cells without genetic modifications still needs to be improved. MATERIALS AND METHODS: We defined an optimal, stepwise and completely xeno-free culture protocol for the generation of MKs from human embryonic stem cells (hESCs). To generate MKs from hESCs on a large scale, we improved the monolayer induction manner to define three-dimensional (3D) and sphere-like differentiation systems for MKs by using a special polystyrene CellSTACK culture chamber. RESULTS: The 3D manufacturing system could efficiently generate large numbers of MKs from hESCs within 16-18 days of continuous culturing. Each CellSTACK culture chamber could collect on an average 3.4 × 108 CD41+ MKs after a three-stage orderly induction process. MKs obtained from hESCs via 3D induction showed significant secretion of IL-8, thrombospondin-1 and MMP9. The induced cells derived from hESCs in our culture system were shown to have the characteristics of MKs as well as the function to form proPLTs and release PLTs. Furthermore, we generated clinically applicable MKs from clinical-grade hESC lines and confirmed the biosafety of these cells. CONCLUSIONS: We developed a simple, stepwise, 3D and completely xeno-free/feeder-free/transgene-free induction system for the generation of MKs from hESCs. hESC-derived MKs were shown to have typical MK characteristics and PLT formation ability. This study further enhances the clinical applications of MKs or PLTs derived from pluripotent stem cells.

A new protocol for long-term culture of a specific subpopulation of liver cancer stem cells enriched by cell surface markers.

Liver cancer stem cells (L-CSCs) are considered to be an important therapeutic target for hepatocellular carcinoma (HCC). This study provides a new in vitro long-term culture model for a specific subpopulation of L-CSCs enriched by cell surface markers. We combined CD13, CD133 and EpCAM to selectively enrich L-CSCs, which we then cultured in modified chemically defined medium. The enriched L-CSCs exhibited enhanced proliferation, self-renewal and long-term clonal maintenance ability as compared with non-CSCs. Compared with wild-type hepatocellular carcinoma, the expression of stemness surface markers, oncogenes, drug resistance and tumorigenicity in enriched L-CSCs was significantly increased. In summary, the subpopulation of L-CSCs still maintains cancer stem cell-related phenotypes after 14 days of culture.

HGF Mediates Clinical-Grade Human Umbilical Cord-Derived Mesenchymal Stem Cells Improved Functional Recovery in a Senescence-Accelerated Mouse Model of Alzheimer's Disease.

Stem cells have emerged as a potential therapy for a range of neural insults, but their application in Alzheimer's disease (AD) is still limited and the mechanisms underlying the cognitive benefits of stem cells remain to be elucidated. Here, the effects of clinical-grade human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on the recovery of cognitive ability in SAMP8 mice, a senescence-accelerated mouse model of AD is explored. A functional assay identifies that the core functional factor hepatocyte growth factor (HGF) secreted from hUC-MSCs plays critical roles in hUC-MSC-modulated recovery of damaged neural cells by down-regulating hyperphosphorylated tau, reversing spine loss, and promoting synaptic plasticity in an AD cell model. Mechanistically, structural and functional recovery, as well as cognitive enhancements elicited by exposure to hUC-MSCs, are at least partially mediated by HGF in the AD hippocampus through the activation of the cMet-AKT-GSK3ß signaling pathway. Taken together, these data strongly implicate HGF in mediating hUC-MSC-induced improvements in functional recovery in AD models.