Stem Cell Research & Therapy最新文献

Circadian rhythms are present in almost all cells, but their existence in stem cells has remains not well established. Circadian clock appears to be closely associated with differentiated mature cells and rarely detected in immature embryonic stem cells. Recent evidence reveals the presence of circadian genes and rhythmic physiologic activities in stem cells as well as stem cell-derived extracellular vesicle (EV) characteristics. The circadian clock entails diverse physiologic and pathological mechanisms underlying cell fate. Integration of circadian rhythm to clinical applications, such as chronotherapy, chrono-biomarker, and environment modification, may facilitate therapeutic outcomes of stem cell-based regenerative medicine. Understanding circadian rhythms in stem cells can optimize stem cell-based therapies by determining the best times for harvesting and administering stem cells, thereby enhancing therapeutic efficacy. Further research into the circadian properties of stem cells will refine stem cell-based therapies, contributing to advancements in regenerative medicine.

Background: Despite numerous studies addressing the molecular mechanisms by which pluripotent stem cells (PSCs) maintain self-renewal and pluripotency under normal culture conditions, the fundamental question of how PSCs manage to survive stressful conditions remains largely unresolved. Post-transcriptional/translational regulation emerges to be vital for PSCs, but how PSCs coordinate and balance their survival and differentiation at translational level under extrinsic and intrinsic stress conditions is unclear.

Methods: The high-throughput sequencing of cross-linking immunoprecipitation cDNA library (HITS-CLIP) was employed to decipher the genome-wide OCT4-RNA interactome in human PSCs, a combined RNC-seq/RNA-seq analysis to assess the role of OCT4 in translational regulation of hypoxic PSCs, and an OCT4-protein interactome to search for OCT4 binding partners that regulate cap-independent translation initiation. By taking the Heterozygous Knocking In N-terminal Tags (HKINT) approach that specifically disrupts the 5'-UTR secondary structure and tagging its protein product of the mRNA from one allele while leaving that from the other allele intact, we examined the effect of disrupting the OCT4/5'-UTR interaction on translation of AKT1 mRNA.

Results: We revealed OCT4 as a bona fide RNA-binding protein (RBP) in human PSCs that bound to the 5'-UTR, 3'-UTR and CDS regions of mRNAs. Multiple known proteins participating in IRES-mediated translation initiation were detected in the OCT4-protein interactome, and a combined RNC-seq/RNA-seq analysis further confirmed a crucial role of OCT4 in translational regulation of PSCs in response to hypoxic stress. Remarkably, OCT4 bound to the GC-rich elements in the 5'-UTR of AKT1 and multiple PI3K/AKT-pathway-gene mRNAs, and promoted their translation initiation via IRES-mediated pathways under stress conditions. Specifically disrupting the AKT1 mRNA 5'-UTR structure and the OCT4/5'-UTR interaction by the HKINT approach significantly reduced the translation level of AKT1 that led to a higher susceptibility of PSCs to oxidative stress-induced apoptotic death and prioritized differentiation toward ectoderm and endoderm.

Conclusions: Our results reveal OCT4 as an anti-stress RBP for translational regulation that critically coordinates the survival and differentiation of PSCs in response to various stressors.

Background: Hemophilia B is an inherited disorder caused by a mutation in the FIX gene, which results in insufficient blood clotting factor IX (FIX) production from hepatocytes. Currently, there are no treatments for hemophilia B patients. The patients should be continuously administrated with clotting factor concentrates 2-3 times a month to prevent bleeding. Therefore, this study aimed to develop an engineered FIX-secreting hepatocyte sheet that can release FIX for an extended period. Within this study, the engineered FIX-secreting hepatocyte sheet was developed by integrating two core technologies, including a gene editing platform to generate FIX-secreting cells and cell sheet technology to improve cell delivery efficacy.

Methods: The human FIX gene was inserted into the APOC3 site of iPSCs by CRISPR/Cas9, which secretes the target protein after differentiation into hepatocytes. FIX-secreting hepatocyte sheets were obtained by temperature-responsive polymer grafted cell culture dishes (TRCD). Immunohistochemical and functional tests were performed for hepatocyte-like cells differentiated from FIX KI-iPSCs and wild-type iPSCs (WT-iPSCs). After validating the functional activity and secretion of FIX protein, the engineered hepatocyte-like cell sheets were transplanted to NOD/SCID mice for the in vivo experiments.

Results: The insertion of the human FIX gene into the APOC3 site demonstrated a significant increase in FIX secretion in hepatocyte-like cells differentiated from FIX KI-iPSCs compared with those obtained from WT-iPSCs. Among the iPSCs to hepatocyte differentiation stages, the hepatic endoderm stage was most suitable for seeding the cells on TRCD and generating cell sheets by temperature changes from 37^oC to room temperature when the hepatocyte-like cells have reached maturity. The engineered FIX-secreting cell sheets showed intensive expression of the FIX proteins without losing hepatocyte morphology for 20 days. Furthermore, an in vivo study showed that engineered FIX-secreting cell sheets retained their FIX secretion functions for two weeks, whereas single-cell injected traditionally were barely detected in the experimental animals.

Conclusions: The engineered FIX-secreting cell sheets fabricated from functionally improved iPSCs with practical cell delivery tools could be a promising tool for clinically treating Hemophilia B.

Background: Gastric cancer is the malignant disease. The problems associated with cancer stemness and chemotherapy resistance in gastric cancer therapy remain unresolved. Glucose-regulated protein 78 (GRP78) is a biomarker of gastric cancer and modulates cancer stemness and chemoresistance. Previous studies have shown that mitochondrial transplantation from healthy cells is a promising method for treating various diseases and that the regulation of mitochondrial metabolism is crucial for modulating the stemness and chemoresistance of cancer cells. The aim of this study was to investigate the therapeutic effect of mitochondrial transplantation from normal gastric epithelial cells into gastric cancer and the associated mechanisms.

Methods: The expression of cancer stemness markers, intracellular oxidative stress, or apoptotic-related proteins were evaluated via flow cytometry. Western blotting was used to investigate the molecular mechanism involved in MKN45 or AGS human gastric cancer cells after transplantation with human gastric epithelial mitochondria. The mitochondrial metabolic function of gastric cancer cells was determined via a Seahorse bioanalyzer, and extracellular lactate was evaluated via bioluminescent assay. The viability of 5-fluorouracil (5-FU)-treated gastric cancer cells was detected via a CCK-8 assay. Furthermore, a xenograft tumor animal study was performed to validate the therapeutic effects of human gastric epithelial mitochondrial transplantation in gastric cancer. Immunohistochemistry and Western blotting were then used to assess the expressions related to cancer stemness and mitochondrial metabolism-related proteins in tumor tissues.

Results: Transplanting human gastric epithelial mitochondria downregulates gastric cancer mitochondrial biogenesis, glycolysis, GRP78-mediated cancer stemness, and increases oxidative stress, cell apoptosis under hypoxic conditions and chemosensitivity in response to 5-FU treatment. Moreover, the transplantation of epithelial mitochondria into gastric tumors inhibited the tumor growth in vivo tumor graft animal models. Therefore, mitochondrial transplantation can be considered for the treatment of gastric cancer.

Background: Induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) have greater potential for generating chondrocytes without hypertrophic and fibrotic phenotypes compared to bone marrow-derived mesenchymal stem/stromal cells (BMSCs). However, there is a lack of research demonstrating the use of autologous iMSCs for repairing articular chondral lesions in large animal models. In this study, we aimed to evaluate the effectiveness of autologous miniature pig (minipig) iMSC-chondrocyte (iMSC-Ch)-laden implants in comparison to autologous BMSC-chondrocyte (BMSC-Ch)-laden implants for cartilage repair in porcine femoral condyles.

Methods: iMSCs and BMSCs were seeded into fibrin glue/nanofiber constructs and cultured with chondrogenic induction media for 7 days before implantation. To assess the regenerative capacity of the cells, 19 skeletally mature Yucatan minipigs were randomly divided into microfracture control, acellular scaffold, iMSC, and BMSC subgroups. A cylindrical defect measuring 7 mm in diameter and 0.6 mm in depth was created on the articular cartilage surface without violating the subchondral bone. The defects were then left untreated or treated with acellular or cellular implants.

Results: Both cellular implant-treated groups exhibited enhanced joint repair compared to the microfracture and acellular control groups. Immunofluorescence analysis yielded significant findings, showing that cartilage treated with iMSC-Ch implants exhibited higher expression of COL2A1 and minimal to no expression of COL1A1 and COL10A1, in contrast to the BMSC-Ch-treated group. This indicates that the iMSC-Ch implants generated more hyaline cartilage-like tissue compared to the BMSC-Ch implants.

Conclusions: Our findings contribute to filling the knowledge gap regarding the use of autologous iPSC derivatives for cartilage repair in a translational animal model. Moreover, these results highlight their potential as a safe and effective therapeutic strategy.

Background: Considerable evidence suggests that tumor initiation, malignancy, metastasis and recurrence occur due to emergence of cancer stem cells (CSCs). Fas binding factor 1 (FBF1) is a multifunctional protein that plays essential roles in the regulation of development and cell fate decisions. However, the function in maintaining stem cell-like properties of breast cancer remains elusive.

Methods: Tissue microarray was used to evaluate FBF1 expression. Cancer stemness assays were performed in FBF1 silencing and overexpressing cells in vitro and in a xenograft model in vivo. RNA sequencing, immunofluorescence and immunoprecipitation assays were performed to explore the underlying mechanism. Clinical expression and significance of FBF1 and stemness-associated factors were explored by analyzing datasets.

Results: We report that FBF1 was highly expressed in breast cancer and significantly correlated with clinical progression. Silencing FBF1 in MDA-MB-231 cells restrained CSCs properties, including side population, sphere formation and migration, whereas ectopic FBF1 expression increased the side population proportion, enhanced the sphere formation ability, and promoted the expression of core stemness genes, such as SOX2, OCT4, KLF4 and NANOG, as well as facilitated metastasis of T47D breast cancer cells. Furthermore, mice bearing FBF1-overexpressed T47D xenografts had higher tumorigenic frequency and stronger metastasis potential. In addition, exploration of the underlying mechanism indicated that FBF1 binds PI3K which then activates PI3K-AKT phosphorylation cascades. Then the activated p-AKT interacts with stemness marker SOX2, elevates SOX2 and OCT4 activity, and finally forms PI3K/AKT/SOX2 axis, which mediates stem cell-like identities. Moreover, PI3K inhibitors abolished FBF1-mediated signaling pathway and diminished breast cancer stemness in vitro and in vivo. In 24 human breast cancer samples, we found a good positive correlation between the expression of FBF1 and p-AKT, as well as between FBF1 and SOX2 as determined by IHC. Clinical data showed that FBF1 expression was positively correlated with the expression of POU5F1 (OCT4), AKT1 and was negatively correlated with PTEN, which is a negative regulator of PI3K/AKT signaling.

Conclusion: Collectively, we identified a potential CSCs regulator and suggested a novel mechanism by which FBF1 governs cancer cell stemness. This study thus introduces an effective target for the diagnosis and treatment of breast cancer.

Background: There's a scarcity of drugs effective against nonalcoholic steatohepatitis (NASH). Exosomes from Human umbilical cord mesenchymal stem cells (huc-MSCs) show potential in managing glycolipid metabolism and the immune response. Therefore, further investigations are required to explore their application in NASH and the underlying mechanisms.

Methods: C57BL/6J mice were fed with a western diet for 12 weeks to induce NASH, and huc-MSCs exosomes (MSCs-exo) were administered during the feeding period. The effect of MSCs-exo was evaluated by monitoring changes in body weight, fat distribution, blood glucose, and insulin levels, and analyzing pathological alterations in liver tissue. Mechanism investigations were carried out using flow cytometry, immunofluorescence staining, and other experimental techniques.

Results: MSCs-exo could reduce liver fat, inflammation, fibrosis, and improved metabolism to alleviate the progression of NASH. Besides, MSCs-exo could decrease macrophage accumulation in the liver, encouraging M2 over M1 macrophage polarization. Furthermore, our study found that MSCs-exo had a high expression of miR-24-3p, which may regulate macrophage polarization by targeting the interferon-stimulated genes (STING) gene in macrophages, with its overexpression amplifying MSCs-exo's NASH benefits.

Conclusions: These findings suggest that the therapeutic effect of MSCs-exo on NASH may be attributed to the regulation of macrophage M2 polarization through miR-24-3p targeting STING. This provides a scientific basis for future clinical application.

Background: To explore the therapeutic effects and mechanisms of the exosomes derived from dental follicle stem cells (DFSC-Exos) in reducing osteoclastogenesis and root resorption (RR) by inhibiting periodontal ligament cell (PDLC) pyroptosis.

Methods: DFSC-Exos, with force stimulation (Force-Exos) or without (Ctrl-Exos), were co-cultured with human PDLCs in vitro and injected into the periodontal ligament (PDL) of rats following the establishment of RR models in vivo. Subsequently, resorption volume, PDLC pyroptotic ratio, and NLRP3-mediated pyroptosis pathway activation were performed to investigate the therapeutic effects of DFSC-Exos on PDLC pyroptosis during RR. Furthermore, the number of M1/M2 macrophages, osteoclast formation, and transwell polarization elucidated the role of Force-Exo treatment in macrophage polarization and osteoclastogenesis by inhibiting pyroptosis. Exosomal miRNA sequencing and bioinformatic analysis were used to identify differentially abundant exosome-derived miRNAs, as well as the dominant biological processes and pathways modulated by miRNA. The administration of miRNA inhibitors further verified the regulation of exosomal miRNA on RR via modulating pyroptosis. Moreover, the potential mechanisms involving candidate miRNAs and relevant pathways were explored.

Results: Exosomes released by force-stimulated DFSCs (Force-Exos) inhibited NOD-like receptor 3 (NLRP3)-mediated PDLC pyroptosis, which impacted M1 macrophage activation and osteoclast formation. Based on exosomal miRNA sequencing, miR-140-3p in Force-Exos were transferred to PDLCs, and the administration of miR-140-3p inhibitors significantly reversed the reduction in PDLC pyroptosis, M1 macrophage polarization, osteoclast number, and resorption volume caused by Force-Exos. More importantly, mechanistic studies demonstrated that miR-140-3p mediated the function of Force-Exos by targeting DNA methyltransferase 1 (DNMT1) to alter the DNA methylation of suppressor of cytokine signaling (SOCS1) and the downstream nuclear factor κB (NF-κB) signaling pathway in PDLCs. Blocking the DNMT1/SOCS1/NFκB axis with DFSC-derived exosomal miR-140-3p downregulated NLRP3-mediated PDLC pyroptosis to impact M1 polarization and osteoclast formation, thereby alleviating RR.

Conclusion: DFSC-Exos downregulated NLRP3-mediated PDLC pyroptosis via miR-140-3p to block DNMT1/SOCS1/NFκB axis, which impacted M1 polarization and osteoclast formation, thereby alleviating RR.

Hair follicles are essential appendages of human skin that function in protection, sensation, thermoregulation and social interactions. The multicellular components, particularly the dermal papilla, matrix and bulge housing stem cells, enable cyclic hair growth postnatally. However, miniaturization and loss of hair follicles can occur in the context of ageing, trauma and various alopecia-related diseases. Conventional treatments involve the redistribution of existing follicles, which may not be viable in patients lacking follicular resources. Recent progress in the comprehension of morphogenesis and the development of biomaterials has significantly advanced follicle reconstruction, incorporating organ germ assembling, stem cell induction and bioprinting techniques. Despite these advancements, fully restoring hair follicles remains challenging due to the complexities of replicating embryonic signals and sustaining growth cycles. Identifying suitable cell sources for clinical applications also presents a hurdle. Here, we retrospect the progress made in the field of hair follicle regeneration, aiming to offer an exhaustive analysis on the benefits and limitations of these methods, and to foster the development of innovative solutions.

Background: Vascular insufficiency is associated with the pathogenesis and therapeutic outcomes of diabetic foot ulcers (DFU). While mesenchymal stem cells (MSCs) hold potential for DFU treatment, further enhancement in promoting angiogenesis in the challenging DFU wounds is imperative.

Methods: The differential expression of pro- and anti-angiogenic factors during both normal and diabetic wound healing was compared using quantitative PCR. MSCs derived from the umbilical cord was prepared, and the engineered MSC (MSC^ANG1) overexpressing both the candidate pro-angiogenic gene, angiopoietin-1 (ANG1), and green fluorescent protein (GFP) was constructed using a lentiviral system. The pro-vascular stabilizing effects of MSC^ANG1 were assessed in primary endothelial cell cultures. Subsequently, MSC^ANG1 was transplanted into streptozotocin (STZ)-induced diabetic wound models to evaluate therapeutic effects on angiogenesis and wound healing. The underlying mechanisms were further examined both in vitro and in vivo.

Results: The comprehensive analysis of the temporal expression of pro- and anti-angiogenic factors revealed a consistent impairment in ANG1 expression throughout diabetic wound healing. MSC^ANG1 exhibited robust EGFP expression in 80% of cells, with overexpression and secretion of the ANG1 protein. MSC^ANG1 notably enhanced the survival and tubulogenesis of endothelial cells and promoted the expression of junction proteins, facilitating the establishment of functional vasculature with improved vascular leakage. Although MSC^ANG1 did not enhance the survival of engrafted MSCs in diabetic wounds, it significantly promoted angiogenesis in diabetic wound healing, fostering the establishment of stable vasculature during the healing process. Activation of the protein kinase B (Akt) pathway and suppression of proto-oncogene tyrosine kinase Src (Src) activity in MSC^ANG1-treated diabetic wounds confirmed efficient angiogenesis process. Consequently, epidermal and dermal reconstruction, as well as skin appendage regeneration were markedly accelerated in MSC^ANG1-treated diabetic wounds compared to MSC-treated wounds.

Conclusion: Treatment with MSCs alone promotes angiogenesis and DFU healing, while the engineering of MSCs with ANG1 provides substantial additional benefits to this therapeutic process. The engineering of MSCs with ANG1 presents a promising avenue for developing innovative strategies in managing DFU.