ABSTRACT
Background: Efficiently increasing the production of clinical-grade mesenchymal stem cells (MSCs) is crucial for clinical applications. Challenges with the current planar culture methods include scalability issues, labour intensity, concerns related to cell senescence, and heterogeneous responses. This study aimed to establish a large-scale production system for MSC generation. In addition, a comparative analysis of the biological differences between MSCs cultured under various conditions was conducted. Methods and materials: We developed a GMP-grade three-dimensional hypoxic large-scale production (TDHLSP) system for MSCs using self-fabricated glass microcarriers and a multifunctional bioreactor. Different parameters, including cell viability, cell diameter, immunophenotype, morphology, karyotype, and tumourigenicity were assessed in MSCs cultured using different methods. Single-cell RNA sequencing (scRNA-seq) revealed pathways and genes associated with the enhanced functionality of MSCs cultured in three dimensions under hypoxic conditions (3D_Hypo MSCs). Moreover, CD142 knockdown in 3D_Hypo MSCs confirmed its in vitro functions. Results: Inoculating 2 × 108 MSCs into a 2.6 L bioreactor in the TDHLSP system resulted in a final scale of 4.6 × 109 3D_Hypo MSCs by day 10. The 3D_Hypo MSCs retained characteristics of the 2D MSCs, demonstrating their genomic stability and non-tumourigenicity. Interestingly, the subpopulations of 3D_Hypo MSCs exhibited a more uniform distribution and a closer relationship than those of 2D MSCs. The heterogeneity of MSCs was strongly correlated with 'cell cycle' and 'stroma/mesenchyme', with 3D_Hypo MSCs expressing higher levels of activated stroma genes. Compared to 2D MSCs, 3D_Hypo MSCs demonstrated enhanced capabilities in blood vessel formation, TGF-ß1 secretion, and inhibition of BV2 proliferation, with maintenance of Senescence-Associated ß-Galactosidase (SA-ß-gal) negativity. However, the enhanced functions of 3D_Hypo MSCs decreased upon the downregulation of CD142 expression. Conclusion: The TDHLSP system led to a high overall production of MSCs and promoted uniform distribution of MSC clusters. This cultivation method also enhanced key cellular properties, such as angiogenesis, immunosuppression, and anti-aging. These functionally improved and uniform MSC subpopulations provide a solid basis for the clinical application of stem cell therapies.
ABSTRACT
Background: Treatment of heart failure post myocardial infarction (post-MI HF) with mesenchymal stem/stromal cells (MSCs) holds great promise. Nevertheless, 2-dimensional (2D) GMP-grade MSCs from different labs and donor sources have different therapeutic efficacy and still in a low yield. Therefore, it is crucial to increase the production and find novel ways to assess the therapeutic efficacy of MSCs. Materials and methods: hUC-MSCs were cultured in 3-dimensional (3D) expansion system for obtaining enough cells for clinical use, named as 3D MSCs. A post-MI HF mouse model was employed to conduct in vivo and in vitro experiments. Single-cell and bulk RNA-seq analyses were performed on 3D MSCs. A total of 125 combination algorithms were leveraged to screen for core ligand genes. Shinyapp and shinycell workflows were used for deploying web-server. Result: 3D GMP-grade MSCs can significantly and stably reduce the extent of post-MI HF. To understand the stable potential cardioprotective mechanism, scRNA-seq revealed the heterogeneity and division-of-labor mode of 3D MSCs at the cellular level. Specifically, scissor phenotypic analysis identified a reported wound-healing CD142+ MSCs subpopulation that is also associated with cardiac protection ability and CD142- MSCs that is in proliferative state, contributing to the cardioprotective function and self-renewal, respectively. Differential expression analysis was conducted on CD142+ MSCs and CD142- MSCs and the differentially expressed ligand-related model was achieved by employing 125 combination algorithms. The present study developed a machine learning predictive model based on 13 ligands. Further analysis using CellChat demonstrated that CD142+ MSCs have a stronger secretion capacity compared to CD142- MSCs and Flow cytometry sorting of the CD142+ MSCs and qRT-PCR validation confirmed the significant upregulation of these 13 ligand factors in CD142+ MSCs. Conclusion: Clinical GMP-grade 3D MSCs could serve as a stable cardioprotective cell product. Using scissor analysis on scRNA-seq data, we have clarified the potential functional and proliferative subpopulation, which cooperatively contributed to self-renewal and functional maintenance for 3D MSCs, named as "division of labor" mode of MSCs. Moreover, a ligand model was robustly developed for predicting the secretory efficacy of MSCs. A user-friendly web-server and a predictive model were constructed and available (https://wangxc.shinyapps.io/3D_MSCs/).
Subject(s)
Heart Failure , Myocardial Infarction , Mice , Animals , Ligands , Myocardial Infarction/genetics , Heart , Heart Failure/etiology , Heart Failure/therapy , Stromal CellsABSTRACT
LAMIN A/C, encoded by the LMNA gene, supports the normal structure of the cell nucleus and regulates the connection between the nucleus and the cytoskeleton as a component of the nucleus envelope. The loss of expression and function of the LMNA gene would lead to the occurrence of congenital muscular dystrophy and Emery-Dreifuss muscular dystrophy which are collectively named as laminopathies. Here, we report a human induced pluripotent stem cell (iPSC) line (EHTJUi005-A-3) generated from a wild iPSC (EHTJUi005-A) with homozygous knockout of the gene LMNA through CRISPR/Cas9. This iPSC line provides a useful research model for studying laminopathies disease.
Subject(s)
Induced Pluripotent Stem Cells , Laminopathies , Muscular Dystrophy, Emery-Dreifuss , CRISPR-Cas Systems/genetics , Humans , Induced Pluripotent Stem Cells/metabolism , Lamin Type A/genetics , Lamin Type A/metabolism , Muscular Dystrophy, Emery-Dreifuss/genetics , Mutation , TechnologyABSTRACT
SUV39H1 is a histone methyltransferase involve numerous biological processes, including of aging, embryo development, tumor growth and mitosis via catalysis of dimethylation and trimethylation of lysine 9 of histone H3. Here we report a human induced pluripotent stem cell line (EHTJUi005-A-1) which is generated from a wildtype human iPSC previously established in our laboratory, and this iPSC has a homozygous knockout of 8 bp in Exon 2 of SUV39H1. This iPSC model provides a valuable resource to study epigenetic regulation in extensive biological processes as mentioned above.
Subject(s)
Induced Pluripotent Stem Cells , CRISPR-Cas Systems/genetics , Epigenesis, Genetic , Histone Methyltransferases , Histones/genetics , Histones/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Methyltransferases/genetics , Methyltransferases/metabolism , Repressor Proteins/geneticsABSTRACT
Hypertrophic cardiomyopathy (HCM) is an autosomal dominant heart disease. An induced pluripotent stem cell line (EHTJUi003-A) was generated from umbilical cord blood mononuclear cells (UCBMCs) of a female neonate with heterozygous mutation of p.L460Wfs (c.1377delC) in the MYBPC3 gene. This iPSC model offers a very valuable resource to study the pathological mechanism of HCM in vitro.
Subject(s)
Cardiomyopathy, Hypertrophic , Induced Pluripotent Stem Cells , Cardiomyopathy, Hypertrophic/genetics , Cytoskeletal Proteins , Female , Heterozygote , Humans , Infant, Newborn , MutationABSTRACT
Familial Arrhythmogenic Right Ventricular Dysplasia (ARVD) is a primary cardiomyopathy characterized by the abnormality of the right ventricular muscle. ARVD may be life-threatening due to the induction of paroxysmal refractory ventricular tachycardia or supraventricular arrhythmia. A human induced pluripotent stem cell line (EHTJUi004-A) was generated from human umbilical cord blood mononuclear cells (UCBMCs) of a female neonate with heterozygous mutation of p.Leu1563fs (c.4683_4684delCT) in the DSP gene. This iPS cell line resource provides an ideal in vitro model to study the pathological mechanism of ARVD.
Subject(s)
Arrhythmogenic Right Ventricular Dysplasia , Induced Pluripotent Stem Cells , Tachycardia, Ventricular , Arrhythmias, Cardiac , Arrhythmogenic Right Ventricular Dysplasia/genetics , Female , Humans , Infant, Newborn , MutationABSTRACT
Familial exudative vitreoretinopathy (FEVR) is an autosomal dominant genetic disease. An induced pluripotent stem cell line (EHTJUi002-A) was generated from umbilical cord blood mononuclear cells (UCBMCs) of a neonate with heterozygous mutation of p.W226X(c.678G>A) in the FZD4 gene. This iPSC model offers a very valuable resource to study the pathological mechanism of FEVR in vitro.
Subject(s)
Induced Pluripotent Stem Cells , Familial Exudative Vitreoretinopathies , Frizzled Receptors/genetics , Humans , Infant, Newborn , Mutation , PedigreeABSTRACT
Multifunctional nanomedical platforms have broad prospects in imaging-guided combination therapy in cancer precision medicine. In this work, metal-organic framework (MOF)-derived novel porous Fe3O4@C nanocomposites were developed as an intelligent cancer nanomedical platform for combined cancer therapy with MRI-guided magnetic-triggered hyperthermia and chemotherapy functions. The magnetic behavior, porous character and good surface modification endowed this smart nanoplatform with favorable biocompatibility, high-efficiency MRI imaging, magnetic-triggered on-demand DOX release function, and synergistic therapy of magnetic hyperthermia and chemotherapy, which proposed an all-in-one platform for cancer therapy. Additionally, in vivo animal experiments verified the significant suppression of malignant tumor growth with negligible side effects, which were attributed to the consecutive 13 day synergistic therapy of magnetic hyperthermia and chemotherapy in one. To be specific, Fe3O4@C-PVP@DOX significantly decreases the volume (2.5 to 0.44 of tumor volume ratio) and weight (0.49 g to 0.10 g) of tumors after magnetic-triggered hyperthermia and chemotherapy treatments. Moreover, no big difference of body weight and associated damage was observed among all major organs. Therefore, owing to its high-efficiency combined therapy of magnetic-triggered hyperthermia and chemotherapy, this smart nanoplatform holds great potential application in the precise treatments of clinical cancer.
Subject(s)
Antineoplastic Agents/therapeutic use , Drug Carriers/therapeutic use , Magnetite Nanoparticles/therapeutic use , Metal-Organic Frameworks/therapeutic use , Nanocomposites/therapeutic use , Neoplasms/drug therapy , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Combined Modality Therapy/methods , Doxorubicin/chemistry , Doxorubicin/therapeutic use , Drug Carriers/chemistry , Drug Carriers/toxicity , Drug Liberation , Drug Therapy/methods , Humans , Hyperthermia, Induced/methods , Magnetic Phenomena , Magnetite Nanoparticles/chemistry , Magnetite Nanoparticles/toxicity , Male , Metal-Organic Frameworks/chemistry , Metal-Organic Frameworks/toxicity , Mice , Mice, Inbred BALB C , Mice, Nude , NIH 3T3 Cells , Nanocomposites/chemistry , Nanocomposites/toxicity , Porosity , Povidone/chemistry , Povidone/toxicity , Xenograft Model Antitumor AssaysABSTRACT
At concentrations found in humans after ingestion of one to two cups of green tea, epicatechin-3-gallate (ECG) modulates Na/K-ATPase conformation and activity. Akin to ouabain, an archetypal Na/K-ATPase ligand of the cardiotonic steroid (CTS) family, ECG also activates protein kinase C epsilon type (PKCε) translocation and increases the force of contraction of the rat heart. This study evaluated whether, like ouabain, ECG also modulates Na/K-ATPase/Src receptor function and triggers pre- and postconditioning against ischemia/reperfusion (I/R) injury. In vitro, ECG activated the purified Na/K-ATPase/Src complex. In Langendorff-perfused rat hearts, submicromolar concentrations of ECG administered either before or after ischemia reduced infarct size by more than 40%, decreased lactate dehydrogenase release, and improved the recovery of cardiac function. ECG protection was blocked by PKCε inhibition and attenuated by mitochondrial KATP channel inhibition. In a unique mammalian cell system with depleted Na/K-ATPase α1 expression, ECG-induced PKCε activation persisted but protection against I/R was blunted. Taken together, these results reveal a Na/K-ATPase- and PKCε-dependent mechanism of protection by ECG that is also distinct from the mechanism of action of ouabain. These ECG properties likely contribute to the positive impact of green tea consumption on cardiovaascular health and warrant further investigation into the role of cardiac Na/K-ATPase signaling in the cardioprotective effect of green tea consumption. SIGNIFICANCE STATEMENT: Consumption of green tea, the richest dietary source of ECG, is associated with a reduced risk of cardiac mortality. Antioxidant effects of ECG and other tea polyphenols are well known, but reported for concentrations well above dietary levels. Therefore, the mechanism underlying the cardioprotective effect of green tea remains incompletely understood. This study provides experimental evidence that ECG concentrations commonly detected in humans after consumption of a cup of tea trigger the Na/K-ATPase/Src receptor in a cell-free system, activate a CTS-like signaling pathway, and provide PKCε-dependent protection against ischemia/reperfusion injury in rat hearts. Mechanistic studies in mammalian cells with targeted Na/K-ATPase depletion revealed that although Na/K-ATPase does not mediate ECG-induced PKCε activation, it is required for ECG-induced protection against ischemia/reperfusion injury.